InteractiveFly: GeneBrief

ß amyloid protein precursor-like:: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - ß amyloid protein precursor-like

Synonyms -

Cytological map position - 1B8

Function - transmembrane domain protein

Keywords - axonogenesis, neurogenesis

Symbol - Appl

FlyBase ID: FBgn0000108

Genetic map position - 1-0.0

Classification - beta-amyloid-like protein

Cellular location - transmembrane and extracellular

NCBI link: Entrez Gene

Appl orthologs: Biolitmine

Recent literature
Burnouf, S., Gorsky, M. K., Dols, J., Gronke, S. and Partridge, L. (2015). Aβ is neurotoxic and primes aggregation of Aβ in vivo. Acta Neuropathol. PubMed ID: 25862636
The involvement of Amyloid-β in the pathogenesis of Alzheimer's disease (AD) is well established. However, it is becoming clear that the amyloid load in AD brains consists of a heterogeneous mixture of Aβ peptides, implying that a thorough understanding of their respective role and toxicity is crucial for the development of efficient treatments. Besides the well-studied Aβ40 and Aβ42 species, recent data have raised the possibility that Aβ43 peptides might be instrumental in AD pathogenesis, because they are frequently observed in both dense and diffuse amyloid plaques from human AD brains and are highly amyloidogenic in vitro. However, whether Aβ43 is toxic in vivo is currently unclear. Using Drosophila transgenic models of amyloid pathology, this study showed that Aβ43 peptides are mainly insoluble and highly toxic in vivo, leading to the progressive loss of photoreceptor neurons, altered locomotion and decreased lifespan when expressed in the adult fly nervous system. In addition, it was demonstrated that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains. Altogether, this study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.

Jonson, M., Pokrzywa, M., Starkenberg, A., Hammarstrom, P. and Thor, S. (2015). Systematic Aβ analysis in Drosophila reveals high toxicity for the 1-42, 3-42 and 11-42 peptides, and emphasizes N- and C-Terminal residues. PLoS One 10: e0133272. PubMed ID: 26208119
Brain amyloid plaques are a hallmark of Alzheimer's disease (AD), and primarily consist of aggregated peptides. While Aβ 1-40 and Aβ 1-42 are the most abundant, a number of other Aβ peptides have also been identified. Studies have indicated differential toxicity for these various Aβ peptides, but in vivo toxicity has not been systematically tested. To address this issue, this study generated improved transgenic Drosophila UAS strains expressing 11 pertinent Aβ peptides. UAS transgenic flies were generated by identical chromosomal insertion, hence removing any transgenic position effects, and crossed to a novel and robust Gal4 driver line. Using this improved Gal4/UAS set-up, survival and activity assays revealed that Aβ 1-42 severely shortens lifespan and reduces activity. N-terminal truncated peptides were quite toxic, with 3-42 similar to 1-42, while 11-42 showed a pronounced but less severe phenotype. N-terminal mutations in 3-42 (E3A) or 11-42 (E11A) resulted in reduced toxicity for 11-42, and reduced aggregation for both variants. Strikingly, C-terminal truncation of Aβ were non-toxic. In contrast, C-terminal extension to 1-43 resulted in reduced lifespan and activity, but not to the same extent as 1-42. Mutating residue 42 in 1-42 greatly reduced Aβ accumulation and toxicity. Histological and biochemical analysis revealed strong correlation between in vivo toxicity and brain Aβ aggregate load, as well as amount of insoluble Aβ. This systematic Drosophila in vivo and in vitro analysis reveals crucial N- and C-terminal specificity for Aβ neurotoxicity and aggregation, and underscores the importance of residues 1-10 and E11, as well as a pivotal role of A42.

Bourdet, I., Lampin-Saint-Amaux, A., Preat, T. and Goguel, V. (2015). Amyloid-β peptide exacerbates the memory deficit caused by Amyloid Precursor Protein loss-of-function in Drosophila. PLoS One 10: e0135741. PubMed ID: 26274614
The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. APPL, the Drosophila APP ortholog, is required for associative memory in young flies. The present report provides the first analysis of the amyloidogenic pathway's influence on memory in the adult. Transient overexpression of the β-secretase, beta-site APP-cleaving enzyme, in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms-APPL loss-of-function and amyloid peptide toxicity-revealing in Drosophila a functional interaction between APPL and amyloid peptide.
Ramaker, J. M., Cargill, R. S., Swanson, T. L., Quirindongo, H., Cassar, M., Kretzschmar, D. and Copenhaver, P. F. (2016). Amyloid precursor proteins are dynamically trafficked and processed during neuronal development. Front Mol Neurosci 9: 130. PubMed ID: 27932950
Proteolytic processing of the Amyloid Precursor Protein (APP) produces β-amyloid (Aβ) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. Insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. This study investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, it was shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, it was found that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of the double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, these results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.
Turrel, O., Goguel, V. and Preat, T. (2017). Drosophila neprilysin 1 rescues memory deficits caused by amyloid-beta peptide. J Neurosci [Epub ahead of print]. PubMed ID: 28931572
Neprilysins are type-II metalloproteinases known to degrade and inactivate a number of small peptides, in particular the mammalian amyloid-beta peptide (Abeta). In Drosophila, several neprilysins expressed in the brain are required for middle-term (MTM) and long-term memory (LTM) in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the center of olfactory memory. These data indicate that one or several peptides need to be degraded for MTM and LTM. Previous work has shown that the fly amyloid precursor protein (APPL) is required for memory in the MB. This study shows that APPL is also required in adult DPM neurons for MTM and LTM formation. This finding prompted a search for an interaction between neprilysins and Drosophila Abeta (dAbeta), a cleavage product of APPL. To find out whether dAbeta was a neprilysin's target, inducible drivers were used to modulate neprilysin 1 (Nep1) and dAbeta expression in adult DPM neurons. Experiments were conducted either in both sexes or in females. The study shows that Nep1 inhibition makes dAbeta expression detrimental to both MTM and LTM. Conversely, memory deficits displayed by dAbeta-expressing flies are rescued by Nep1 overexpression. Consistent with behavioral data, biochemical analyses confirmed that Nep1 degrades dAbeta. Taken together, these findings establish that Nep1 and dAbeta expressed in DPM neurons are functionally linked for memory processes, suggesting that dAbeta is a physiological target for Nep1.
Hahm, E. T., Nagaraja, R. Y., Waro, G. and Tsunoda, S. (2018). Cholinergic homeostatic synaptic plasticity drives the progression of Abeta-induced changes in neural activity. Cell Rep 24(2): 342-354. PubMed ID: 29996096
Homeostatic synaptic plasticity (HSP) is the ability of neurons to exert compensatory changes in response to altered neural activity. How pathologically induced activity changes are intertwined with HSP mechanisms is unclear. This study shows that, in cholinergic neurons from Drosophila, beta-amyloid (Abeta) peptides Abeta40 and Abeta42 (see Drosophila β amyloid protein precursor-like) both induce an increase in spontaneous activity. In a transgenic line expressing Abeta42, it was observed that this early increase in spontaneous activity is followed by a dramatic reduction in spontaneous events, a progression that has been suggested to occur in cholinergic brain regions of mammalian models of Alzheimer's disease. Evidence is presented that the early enhancement in synaptic activity is mediated by the Drosophila alpha7 nicotinic acetylcholine receptor (nAChR) and that, later, Abeta42-induced inhibition of synaptic events is a consequence of Dalpha7-dependent HSP mechanisms induced by earlier hyperactivity. Thus, while HSP may initially be an adaptive response, it may also drive maladaptive changes and downstream pathologies.
Zhang, N., Parr, C., Birch, A. M., Goldfinger, M. H. and Sastre, M. (2018). The Amyloid Precursor Protein binds to beta-catenin and modulates its cellular distribution. Neurosci Lett. PubMed ID: 30176342
Accumulating evidence has shown that the processing of the amyloid precursor protein (APP) and the formation of amyloid-beta are associated with the canonical Wnt/ beta-catenin signalling pathway. It was recently published that the Drosophila homologue of APP (Appl) is a conserved modulator of Wnt PCP signalling, suggesting a potential regulation of this pathway by APP. The aim of this study was to investigate the potential interaction of APP with the canonical Wnt pathway. APP overexpression in N2a cells led to alterations in the subcellular distribution of beta-catenin by physically binding to it, preventing its translocation to the nucleus and precluding the transcription of Wnt target genes. In addition, studies in APP transgenic mice and human Alzheimer's disease (AD) brain tissue showed the cellular co-localization of APP and beta-catenin and binding of both proteins, suggesting the formation physical complexes of APP and beta-catenin, yet not present in healthy controls. Furthermore, a reduction in the levels of nuclear beta-catenin was detected in AD brains compared to controls as well as a decrease in the expression of the inactive phosphorylated Glycogen synthase kinase 3 (GSK3) isoform. Therefore, these findings indicate a reciprocal regulation of Wnt/beta-catenin signalling pathway and APP processing involving a physical interaction between APP and beta-catenin.
Penserga, T., Kudumala, S. R., Poulos, R. and Godenschwege, T. A. (2019). A role for Drosophila Amyloid precursor protein in retrograde trafficking of L1-Type cell adhesion molecule Neuroglian. Front Cell Neurosci 13: 322. PubMed ID: 31354437
The normal function of APP in the nervous system is poorly understood. This study characterized the role of the Drosophila homolog (APPL) in the adult giant fiber (GF) neurons. Endogenous APPL is transported from the synapse to the soma in the adult. Live-imaging revealed that retrograde moving APPL vesicles co-traffic with L1-type cell adhesion molecule Neuroglian (Nrg). In APPL null mutants, stationary Nrg vesicles were increased along the axon, and the number of Nrg vesicles moving in retrograde but not anterograde direction was reduced. In contrast, trafficking of endo-lysosomal vesicles, which did not co-localize with APPL in GF axons, was not affected. This suggests that APPL loss of function does not generally disrupt axonal transport but that APPL has a selective role in the effectiveness of retrograde transport of proteins it co-traffics with. While the GF terminals of Loss of function animals exhibited pruning defects. Cell-autonomous developmental expression of a secretion-deficient form of APPL (APPL-SD), lacking the alpha-, beta-, and, gamma-secretase cleavage sites, resulted in progressive retraction of the GF terminals. Conditional expression of APPL-SD in mature GFs caused accumulation of Nrg in normal sized synaptic terminals, which was associated with severely reduced retrograde flux of Nrg labeled vesicles in the axons. Albeit beta-secretase null mutants developed GF terminals they also exhibited Nrg accumulations. This suggests that cleavage defective APPL has a toxic effect on retrograde trafficking and that beta-secretase cleavage has a function in Nrg sorting in endosomal compartments at the synapse.
Kessissoglou, I. A., Langui, D., Hasan, A., Maral, M., Dutta, S. B., Hiesinger, P. R. and Hassan, B. A. (2020). The Drosophila amyloid precursor protein homologue mediates neuronal survival and neuroglial interactions. PLoS Biol 18(12): e3000703. PubMed ID: 33290404
The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer's disease (fAD), most research focuses on this aspect of APP biology. This study investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. This study reports that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, this study shows that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. It is proposes that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis.
Kaur, P., Kibat, C., Teo, E., Gruber, J., Mathuru, A. and Tolwinski, A. N. S. (2020). Use of Optogenetic Amyloid-beta to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans. Bio Protoc 10(23): e3856. PubMed ID: 33659494
Alzheimer's Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. This study developed an optogenetic method to induce Aβ aggregation, and its biological influence was tested in three model organisms-D. melanogaster, C. elegans and D. rerio. A fluorescently labeled, optogenetic Aβ peptide was generated that oligomerizes rapidly in vivo in the presence of blue light in all organisms. This paper details the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and metabolic assays were perfomred to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.
Rimal, S., Li, Y., Vartak, R., Geng, J., Tantray, I., Li, S., Huh, S., Vogel, H., Glabe, C., Grinberg, L. T., Spina, S., Seeley, W. W., Guo, S. and Lu, B. (2021). Inefficient quality control of ribosome stalling during APP synthesis generates CAT-tailed species that precipitate hallmarks of Alzheimer's disease. Acta Neuropathol Commun 9(1): 169. PubMed ID: 34663454
Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-β (Aβ) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aβ-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. Drosophila models were used to expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and β-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. This study found that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid β peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. The finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets (Rimal, 2021).
Sun, Z. D., Hu, J. X., Wu, J. R., Zhou, B. and Huang, Y. P. (2022). Toxicities of amyloid-beta and tau protein are reciprocally enhanced in the Drosophila model. Neural Regen Res 17(10): 2286-2292. PubMed ID: 35259851
Extracellular aggregation of amyloid-beta (Aβ) and intracellular tau tangles are two major pathogenic hallmarks and critical factors of Alzheimer's disease. A linear interaction between Aβ and tau protein has been characterized in several models. Aβ induces tau hyperphosphorylation through a complex mechanism; however, the master regulators involved in this linear process are still unclear. In a study with Drosophila melanogaster, was found to regulate tau hyperphosphorylation and toxicity by activating c-Jun N-terminal kinase. Importantly, Aβ toxicity was dependent on tau hyperphosphorylation, and flies with hypophosphorylated tau were insulated against Aβ-induced toxicity. Strikingly, tau accumulation reciprocally interfered with Aβ degradation and correlated with the reduction in mRNA expression of genes encoding Aβ-degrading enzymes, including dNep1, dNep3, dMmp2, dNep4, and dIDE. These results indicate that Aβ and tau protein work synergistically to further accelerate Alzheimer's disease progression and may be considered as a combined target for future development of Alzheimer's disease therapeutics.
Larsson, J. N. K., Nystrom, S. and Hammarstrom, P. (2022). HSP10 as a Chaperone for Neurodegenerative Amyloid Fibrils. Front Neurosci 16: 902600. PubMed ID: 35769706
Neurodegenerative diseases (NDs) are associated with accumulated misfolded proteins (MPs). MPs oligomerize and form multiple forms of amyloid fibril polymorphs that dictate fibril propagation and cellular dysfunction. Protein misfolding processes that impair protein homeostasis are implicated in onset and progression of NDs. A wide variety of molecular chaperones safeguard the cell from MP accumulation. A rather overlooked molecular chaperone is HSP10, known as a co-chaperone for HSP60. Due to the ubiquitous presence in human tissues and protein overabundance compared with HSP60, this work shows how HSP10 alone influences fibril formation in vitro of Alzheimer's disease-associated Aβ1-42 (see Appl). At sub-stoichiometric concentrations, eukaryotic HSP10s (human and Drosophila) significantly influenced the fibril formation process and the fibril structure of Aβ1-42, more so than the prokaryotic HSP10 GroES. Similar effects were observed for prion disease-associated prion protein HuPrP90-231. Paradoxically, for a chaperone, low concentrations of HSP10 appeared to promote fibril nucleation by shortened lag-phases, which were chaperone and substrate dependent. Higher concentrations of chaperone while still sub-stoichiometric extended the nucleation and/or the elongation phase. It is hypothesized that HSP10 by means of its seven mobile loops provides the chaperone with high avidity binding to amyloid fibril ends. The preserved sequence of the edge of the mobile loop GGIM(V)L (29-33 human numbering) normally dock to the HSP60 apical domain. Interestingly, this segment shows sequence similarity to amyloidogenic core segments of Aβ1-42, GGVVI (37-41), and HuPrP90-231 GGYML (126-130) likely allowing efficient competitive binding to fibrillar conformations of these MPs. Thee results propose that HSP10 can function as an important molecular chaperone in human proteostasis in NDs.
Chocron, E. S., Munkácsy, E., Kim, H. S., Karpowicz, P., Jiang, N., Van Skike, C. E., DeRosa, N., Banh, A. Q., Palavicini, J. P., Wityk, P., Kalinowski, L., Galvan, V., Osmulski, P. A., Jankowska, E., Gaczynska, M. and Pickering, A. M. (2022). Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models. Sci Adv 8(23): eabk2252. PubMed ID: 35675410
The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). Prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. This study developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, a set of TAT-based proteasome-activating peptidomimetics was developed that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.
Hewitt, V. L., Miller-Fleming, L., Twyning, M. J., Andreazza, S., Mattedi, F., Prudent, J., Polleux, F., Vagnoni, A. and Whitworth, A. J. (2022). Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ(42) toxicity. Life Sci Alliance 5(11). PubMed ID: 35831024
Mitochondria<>/a-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. Reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β(42) (Aβ(42)). Together, these results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.
Ataellahi, F., Masoudi, R. and Haddadi, M. (2022). Differential dysregulation of CREB and synaptic genes in transgenic Drosophila melanogaster expressing shaggy (GSK3), Tau(WT), or Amyloid-beta. Mol Biol Rep. PubMed ID: 36399243
Tau, Amyloid-beta (Aβ42), and Glycogen synthase kinase 3 (GSK3) contribute to synaptic dysfunction observed in Alzheimer's disease (AD), the most common form of dementia. In the current study, the effect of pan-neuronal expression of TauWT, Aβ42, or shaggy (orthologue of GSK3) in Drosophila melanogaster was assessed on the locomotor function, ethanol sensitivity, synaptic genes and CREB expression. The effect of TauWT and Aβ42 on the expression of shaggy was also determined. Gene expression analysis was performed using quantitative real-time RT-PCR method. While syt1, SNAP25 and CREB (upstream transcription factor of syt1 and SNAP25) were upregulated in flies expressing Tau(WT) or Aβ42, a prominent decline was observed in those genes in shaggy expressing flies. Although all transgenic flies showed climbing disability and higher sensitivity to ethanol, abnormality in these features was significantly more prominent in transgenic flies expressing shaggy compared to TauWT or Aβ42. Despite a significant upregulation of shaggy transcription in TauWT expressing flies, Aβ42 transgenic flies witnessed no significant changes. TauWT, A&beta42, and shaggy may affect synaptic plasticity through dysregulation of synaptic genes and CREB, independently. However shaggy has more detrimental effect on synaptic genes expression, locomotor ability and sensitivity to ethanol. It is important when it comes to drug discovery. It appears that CREB is a direct effector of changes in synaptic genes expression as they showed similar pattern of alteration and it is likely to be a part of compensatory mechanisms independent of the GSK3/CREB pathway in TauWT or Aβ(42) expressing flies.
Hao, Y., Shao, L., Hou, J., Zhang, Y., Ma, Y., Liu, J., Xu, C., Chen, F., Cao, L. H. and Ping, Y. (2023). Resveratrol and Sir2 Reverse Sleep and Memory Defects Induced by Amyloid Precursor Protein. Neurosci Bull. PubMed ID: 37041405
Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). This study tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. A mild but significant transcriptional increase was found of Drosophila Sir2 (dSir2) by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. It was further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, it was shown that Aβ aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). The data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.
Hao, Y., Shao, L., Hou, J., Zhang, Y., Ma, Y., Liu, J., Xu, C., Chen, F., Cao, L. H. and Ping, Y. (2023). Resveratrol and Sir2 Reverse Sleep and Memory Defects Induced by Amyloid Precursor Protein. Neurosci Bull. PubMed ID: 37041405
Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). This study, tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. A mild but significant transcriptional increase of Drosophila Sir2 (dSir2) was found by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. It was further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, this study showed that A&betal aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). These data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.
Huang, Z. (2023). A Function of Amyloid-β in Mediating Activity-Dependent Axon/Synapse Competition May Unify Its Roles in Brain Physiology and Pathology. J Alzheimers Dis. PubMed ID: 36710681
Amyloid-β protein precursor (AβPP) gives rise to amyloid-β (Aβ), a peptide at the center of Alzheimer's disease (AD). AβPP, however, is also an ancient molecule dating back in evolution to some of the earliest forms of metazoans. This suggests a possible ancestral function that may have been obscured by those that evolve later. Based on literature from the functions of Aβ/AβPP in nervous system development, plasticity, and disease, to those of anti-microbial peptides (AMPs) in bacterial competition as well as mechanisms of cell competition uncovered first by Drosophila genetics, this paper proposes that Aβ/AβPP may be part of an ancient mechanism employed in cell competition, which is subsequently co-opted during evolution for the regulation of activity-dependent neural circuit development and plasticity. This hypothesis is supported by foremost the high similarities of Aβ to AMPs, both of which possess unique, opposite (i.e., trophic versus toxic) activities as monomers and oligomers. A large body of data further suggests that the different Aβ oligomeric isoforms may serve as the protective and punishment signals long predicted to mediate activity-dependent axonal/synaptic competition in the developing nervous system and that the imbalance in their opposite regulation of innate immune and glial cells in the brain may ultimately underpin AD pathogenesis. This hypothesis can not only explain the diverse roles observed of Aβ and AβPP family molecules, but also provide a conceptual framework that can unify current hypotheses on AD. Furthermore, it may explain major clinical observations not accounted for and identify approaches for overcoming shortfalls in AD animal modeling.
Tan, F. H. P., Ting, A. C. J., Najimudin, N., Watanabe, N., Shamsuddin, S., Zainuddin, A., Osada, H. and Azzam, G. (2023). 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) is protective against Aβ42-induced toxicity in vitro and in an Alzheimer's Disease Drosophila. J Gerontol A Biol Sci Med Sci. PubMed ID: 37453137
Alzheimer's disease (AD) is the most prevalent type of dementia globally. The accumulation of amyloid-beta (Aβ) extracellular senile plaques in the brain is one of the hallmark mechanisms found in AD. Aβ42 is the most damaging and aggressively aggregating Aβ isomer produced in the brain. Although Aβ42 has been extensively researched as a crucial peptide connected to the development of the characteristic amyloid fibrils in AD, the specifics of its pathophysiology are still unknown. Therefore, the main objective was to identify novel compounds that could potentially mitigate the negative effects of Aβ42. 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) was identified as a ligand for Aβ42 and for reducing fibrillary Aβ42 aggregation. THICAPA also improved cell viability when administered to PC12 neuronal cells that were exposed to Aβ42. Additionally, this compound diminished Aβ42 toxicity in the current AD Drosophila model by rescuing the rough eye phenotype, prolonging the lifespan and enhancing motor functions. Through Next-generation RNA-sequencing, immune response pathways were downregulated in response to THICAPA treatment. Thus, this study suggests THICAPA as a possible disease-modifying treatment for AD.

The effects of amyloid precursor protein (APP) on neuronal development and function have been extensively studied because of its implication in Alzheimer's disease (for review, see Mattson, 1997; Small, 1998; Sinha, 1999). The Drosophila ß-amyloid precursor protein homolog ß amyloid protein precursor-like (Appl) is a pan-neural protein belonging to the conserved APP family. This family includes APP and amyloid precursor-like protein (APLP)1/2 in mammals and apl-1 in C. elegans. APP is synthesized as a transmembrane protein composed of extracellular, transmembrane, and cytoplasmic domains. In the normal process of APP function, APP undergoes a regulated proteolytic cleavage, releasing the long ectodomain (Torroja, 1999a and references therein).

A principal component of amyloid plaques in Alzheimer's disease (AD) is a 38-43-amino acid ß-amyloid peptide (A ß), which is derived from the C-terminal portion of the extracellular domain of APP and from the adjacent transmembrane domain. Production and accumulation of A ß is involved in the etiology of AD. APP is an integral membrane protein with a short intracellular C-terminus. Normal proteolytic processing of APP generates two fragments, a large soluble secreted amino-terminal domain (sAPPalpha) and a truncated carboxyl-terminal fragment (CTFalpha), both of which are products of cleavage within the A ß domain at approximately amino acid number 17: this site is termed the alpha cleavage site. Another form of proteolytic processing occurs at the ß-site, located at amino acid number 1 of the A ß peptide. This gives rise to production of carboxyl-terminal fragments (CTFß) and A ß in unidentified intracellular compartments of the protein secretory pathway. The protein Presenilin (see Drosophila Presenilin) directs the cleavage of APP at even a third site, gamma, located between amino acids 40 and 42, the C-terminus of the A ß peptide. The A ß peptide is generated by cleavages at both the beta site and the gamma site; cleavage at these two sites generate the 40 to 42 amino acid A ß peptide (Sinah, 1999 and references therein).

In vivo evidence supports a role for APP in synapse formation, maintenance, and plasticity. APP infusion into rat brains increases synaptic density and memory retention (Roch, 1994), and exposure of hippocampal slices to secreted APP enhances long-term potentiation and modifies the induction of long-term depression (Ishida, 1997). APP knock-out mice show impaired learning and memory (Müller; 1994; Zheng, 1995). Alternative mRNA splicing can generate isoforms of APP that contain a Kunitz protease inhibitor (KPI) domain. The KPI forms of APP are implicated in the regulation of extracellular cleavage of secreted APP by acting to inhibit the activity of a secreted APP-degrading protease (Caswell, 1999). A second extracellular target of the KPI domain is the proneuropeptide processing enzyme prohormone thiol protease (PTP), involved in the processing of active neurotransmitters and peptide hormones; for example the prohormone proenkephalin (Hook, 1999).

Neuronal overexpression of APPL, the Drosophila homologue of the amyloid precursor protein (APP), disrupts axonal transport

The two pathological hallmarks of Alzheimer's disease, amyloid plaques and neurofibrillary tangles, involve two apparently unrelated proteins, the amyloid precursor protein (APP) and Tau. Although it is known that aberrant processing of APP is associated with Alzheimer's disease, the definitive role of APP in neurons is not yet clear. Tau regulates microtubule stabilization and assembly in axons and is, thus, an essential component of the microtubule-associated organelle transport machinery. Although several groups have reported physical interaction between APP and Tau, and induction of Tau phosphorylation by APP and beta-amyloid peptide, the functional connection between APP and Tau is unclear. To explore the possibility that the functions of these two proteins may somehow converge on the same cellular process, Appl, the Drosophila homolog of APP, was overexpressed along with Tau in Drosophila neurons. Panneural coexpression of Appl and Tau results in adults that, upon eclosion, fail to expand wings and harden the cuticle, which is suggestive of neuroendocrine dysfunction. Axonal transport was analyzed when Tau and Appl were coexpressed and transport of axonal cargo was found to be disrupted, as evidenced by increased retention of synaptic proteins in axons and scarcity of neuropeptide-containing vesicles in the distal processes of peptidergic neurons. Demonstrated in an independent approach were genetic interaction and phenotypic similarity between APPL overexpression and mutations in the Kinesin heavy chain gene, the product of which is a motor for anterograde vesicle trafficking (Torroja, 1999a).

To investigate the role of APP at single, identified synapses, the Drosophila neuromuscular junction (NMJ) has been used as a model system. Appl overexpression in motoneurons results in a dramatic increase in the number of synaptic boutons. Conversely, NMJs of Appl null larvae exhibit a significant decrease in synaptic bouton number. The synapse-promoting function requires a conserved internalization sequence and a putative Go binding site at the cytoplasmic domain. This function is partially suppressed by mutations with increased excitability, which by themselves regulate synapse growth (Budnik, 1990) and Appl expression at the NMJ (Torroja, 1999a).

To determine whether Appl is transported to motoneuron synapses, antibodies directed against the Appl ectodomain were used to label larval body wall muscle preparations. Appl is found to be consistently distributed in a punctate pattern within the abdominal nerves, down to the point of contact with body wall muscles, consistent with axonal transport of Appl. NMJs show very weak Appl signal. No signal is detected in nerves or NMJs of the Appl null mutant (Appld) larvae, demonstrating that Appl immunoreactivity at nerves and NMJs is specific (Torroja, 1999a).

Appl signal at synaptic terminals is enhanced when Appl is overexpressed in the nervous system. Neural Appl overexpression was achieved using the UAS/Gal4 system for targeted gene expression. The UAS-Appl+ transgene was driven by pan-neural Gal4 drivers c155 or Appl-Gal4, or the motoneuron-specific Gal4 driver P[Gal4w+]C164. Expression of Appl and its influence on synapse structure was examined at muscles 6 and 7. These muscles are innervated by two glutamatergic motoneurons that give rise to morphologically and physiologically distinct synaptic boutons [type Ib (big) and type Is (small)]. At the NMJs of larvae overexpressing APPL+, Appl immunoreactivity is readily evident. Appl signal appears in a nonhomogenous punctate pattern, primarily inside synaptic boutons (colocalized with anti-HRP as a presynaptic marker) but also outside in the immediate vicinity of synaptic boutons. The Appl immunoreactivity outside the boutons may represent secreted Appl, because overexpressing a secretion-deficient Appl form (APPLsd) results in Appl immunoreactivity that is more restricted to synaptic boutons (Torroja, 1999a).

The presence of Appl in motor axons and their synaptic terminals suggested that it might be involved in synapse development and/or function. NMJs from Appld larvae were examined to ascertain the effects of the lack of Appl on synapse structure. NMJs from mutant larvae, examined in anti-HRP stained preparations, have a generally normal appearance but contain fewer boutons. Appld shows a significant decrease in the number of synaptic boutons (34%), as compared with wild type. When Appl is overexpressed in motoneurons, the increase in Appl signal is accompanied by a change in overall morphology of the NMJ. These profound structural changes include a dramatic increase in the number of synaptic boutons that result largely from numerous small synaptic boutons of abnormal appearance. In wild-type NMJs, type I synaptic boutons within a branch resemble a string of beads, with boutons connected to one another by a short neuritic process. In contrast, in larvae overexpressing Appl, many abnormal small 'satellite' boutons appear to bud off from a central bouton of normal appearance. This phenotype is associated with both type Ib and type Is endings. A fraction of satellite boutons (~24%) are also observed budding off from neuronal processes connecting two boutons (Torroja, 1999a).

To determine whether the satellite boutons have normal features of synaptic boutons, presynaptic and postsynaptic markers were used. Analysis of the presynaptic markers synapsin, synaptotagmin, and CSP reveals that each satellite bouton is immunopositive for these markers. Furthermore, analysis of Discs large immunoreactivity, a marker for postsynaptic subsynaptic reticulum (SSR), clearly demonstrates that virtually all satellite boutons are surrounded by SSR as is the case with normal boutons. Thus, at least some elements of the presynaptic apparatus and postsynaptic SSR are likely to be normal at the satellite synapses. The fine structure of the satellite boutons was analyzed at the EM level. As expected from the light microscopic appearance of these NMJs, clusters of boutons are common in APPL+ overexpressors, and larger boutons are often surrounded by many small satellite boutons. These satellite boutons appear to share a common SSR with the central boutons and are frequently observed budding off from a parent bouton. At the presynaptic site, the satellite boutons contain 40 nm clear synaptic vesicles and mitochondria and display one or more T-shaped presynaptic densities (active zones) of normal appearance. Quantification of the number of active zones per bouton midline reveals that boutons from Appl overexpressors and from wild type have a similar number of active zones. These observations suggest that, although small, satellite boutons have at least several normal type I bouton presynaptic and postsynaptic elements and therefore may form functional synaptic connections (Torroja, 1999a).

To quantify the increase of synaptic boutons observed in APPL+ overexpressing NMJs, the total number of synaptic boutons, the percentage of satellite boutons, and the number of normal boutons (parent boutons) at muscles 6 and 7 of abdominal segment 3 were counted at the light microscopic level. For this quantification, boutons were considered to be satellites if they were substantially smaller than normal type I boutons and were connected to a central (parent) bouton. Boutons that have a size and morphology similar to typical satellites but emerge directly from an NMJ process were also considered to be satellites. In larvae overexpressing APPL+, there was an ~2.5-fold increase in the total number of boutons, and an approximate threefold increase in the percentage of satellite boutons compared with wild type. No significant change in the number of synaptic boutons compared with wild type was observed in the control progeny. Thus, whereas the lack of Appl in Appld mutants leads to a relatively small decrease in the number of synaptic boutons, increasing Appl levels results in a dramatic rise in bouton number. This increase in bouton number resulting from both an increase in satellite boutons and an elevated number of nonsatellite boutons of normal appearance (parent boutons). Interestingly, expression of a human APP isoform, APP695, also results in a significant but comparatively smaller increase in the total number of synaptic boutons and, particularly, an increase in the percentage of satellite boutons (approximately twofold) (Torroja, 1999a).

All APP family members are proteolytically processed and can be found as transmembrane and soluble protein forms. Studies demonstrating extensive regulation of the cleavage and release of soluble APP suggest that the two protein forms may play specific roles. To determine whether the transmembrane or soluble Appl isoform can promote satellite formation, transgenes were used that express mutant Appl proteins upon Gal4 activation. UAS-Applsd transgene encodes a secretion-defective transmembrane form in which Appl lacks the proteolytic cleavage site and consequently is expressed only as a membrane-bound protein. UAS-Appls transgene encodes a constitutively secreted form because it lacks the transmembrane and cytoplasmic domains. Analysis of NMJs in larvae expressing APPLsd or APPLs demonstrates that APPLsd expression results in a phenotype similar to the phenotype observed in larvae overexpressing APPL+, increasing the percentage of satellite boutons (approximately fourfold) and, to a lesser extent, the number of parent boutons (~1.7-fold). In contrast, APPLs does not significantly differ from wild type with regard to the total number of synaptic boutons or satellites (Torroja, 1999a).

The lack of effect seen in APPLs may be because of differences in the amount of protein expressed. Therefore, the levels of expression of the different transgenic Appl forms were assessed in immunoblots probed with anti-APPL antibodies. Immunoblots of adult head extracts of wild type, Appld, and transgenic flies expressing different Appl constructs in an Appld background reveal that Gal4-driven expression of both APPL+ and APPLsd results in high levels of protein relative to the endogenous level of Appl. In contrast, the amount of APPLs generated from the transgene is only comparable with the amount of endogenous soluble Appl. It is suspected that this is attributable to the lability of the expressed soluble protein, because other Appl mutant constructs expressed with this system all result in robust expression. Attempts were made to increase the amount of APPLs by using two doses of the transgene. This manipulation does not alter the total number of boutons but significantly reduces the number of satellites compared with wild type. In conclusion, both overexpression of APPL+ holoprotein or transmembrane APPLsd can strongly promote satellite bouton formation, but APPLs does not affect the number of total boutons, even after doubling the number of copies of the transgene. However, two doses of APPLs decreases the number of satellite boutons (Torroja, 1999a).

These results suggest that the synaptic bouton-promoting activity associated with Appl overexpression is sensitive to Appl concentration. To test this possibility, the phenotypes of NMJs were examined in Appld larvae that overexpress APPLsd to eliminate wild-type contribution. The increase in the number of synaptic boutons, percentage of satellites, and number of parent boutons is intermediate but significantly different from wild-type and APPLsd overexpressing larvae, supporting a concentration effect of APPL on this activity (Torroja, 1999a).

Which domains of Appl are important for satellite and parent bouton-forming activity? APP family proteins have several conserved domains that have been initially recognized from APP and Appl comparison (Rosen, 1989). Among these are a perfectly conserved internalization sequence in the cytoplasmic domain (GYENPTY) and extracellular regions of high homology E1 and E2. Additionally, in the cytoplasmic domain of APP, a Go-protein binding site, which is conserved in Appl, has been demonstrated (Nishimoto, 1993; Okamoto, 1995). These defined domains have been used as a guide to construct a series of deletion mutants: DeltaE1, lacking the distal half of E1 and those amino acids (aa) c-terminal to it (85-321 aa); DeltaE2, lacking 75% of the distal E2 and amino acids c-terminal to it (449-740 aa); C100, lacking the entire ectodomain (21-787 aa); DeltaC, lacking the entire cytoplasmic domain (836-886 aa); DeltaCI, lacking the internalization sequence (872-883 aa); and DeltaCg, lacking the putative Go-protein binding site (845-855 aa). Applsd was chosen rather than Appl+ as a parent construct to avoid complications in interpretation that could arise if generated APPLs had an independent activity. Because several transformants were obtained with each construct, those lines were selected that showed strong and similar levels of protein expression in immunoblot analysis (Torroja, 1999a).

NMJs from larvae expressing the different constructs were double stained with anti-HRP and anti-APPL, and the total number of boutons, the percentage of satellite boutons, and the number of parent boutons were determined. Labeling with anti-APPL antibodies determined that, with the exception of C100, all Appl mutant proteins are transported at similar levels to the NMJs in which they are present. The salient observations from quantification of synaptic bouton number in larvae expressing APPLsd -deletion proteins, DeltaE1, DeltaE2, and DeltaC, are as follows. (1) APPLsd, further deleted for either of the two extracellular domains (DeltaE1 or DeltaE2), prevents an increase in the number of parent boutons, yet it still retains satellite bouton-forming activity. (2) In contrast, deletion of the entire cytoplasmic domain of APPLsd (DeltaC) completely abolishes both the satellite bouton-promoting activity and the increase in the number of parent boutons. Thus, the cytoplasmic domain is essential for both the satellite bouton-promoting activity and the formation of extra parent boutons (Torroja, 1999a).

To further dissect the likely cytoplasmic domain regions responsible for promoting synapse formation, smaller deletions in the cytoplasmic domain were overexpressed. Deletion of the putative Go-protein binding site (DeltaCg) retains the satellite bouton-promoting capacity, but the formation of extra parent boutons is suppressed. Conversely, larvae expressing APPL variants that lack the conserved internalization signal (DeltaCI) have NMJs with a wild-type appearance with regard to the number of satellite boutons. Interestingly, however, these larvae still show a dramatic increase in the number of parent boutons, which translates into an increase in the total number of boutons. Thus, the satellite bouton-promoting activity depends on the presence of the internalization signal, whereas the formation of extra parent boutons depends on the presence of the Go-protein binding site and the presence of an intact extracellular domain (Torroja, 1999a).

A role for neuronal activity in the development and maintenance of synapses has been documented in many systems. Neuronal activity has also been shown to regulate APP metabolism. It was therefore of interest to enquire whether Appl-mediated regulation of the NMJ growth is induced through neuronal activity. To explore this question, the effects of Appl overexpression in the hyperexcitable double mutant eag Sh was examined. Intriguingly, Appl immunoreactivity in eag Sh differs from wild type. In wild type, Appl signal at synaptic boutons is very low but is observed throughout the NMJ. In contrast, in eag Sh mutants, Appl signal is consistently brighter and more concentrated at the most distal bouton(s) of a given NMJ branch. In addition, Appl signal appears more concentrated at the bouton border, suggesting that there might be an increase in plasma membrane-associated Appl. Western analysis of adult heads has revealed no significant difference in the amount of total Appl between wild type and eag Sh (Torroja, 1999a).

In eag Sh mutants, both the number of boutons and NMJ branches are increased. However, the increase in synaptic boutons in eag Sh double mutants differs from that observed in NMJs with Appl overexpression. In NMJs with enhanced Appl levels, the increase in the number of synaptic boutons derives primarily from satellite boutons, whereas in eag Sh mutants the increase in the number of synaptic boutons results from an increased elongation of the presynaptic terminals and the formation of new secondary branches. In eag Sh larvae overexpressing APPLsd, a reduction in the percentage of satellite boutons is observed when compared with APPLlsd overexpressed in a wild-type background. However, the formation of extra parent boutons is still observed. Thus, eag Sh changes the pattern of endogenous Appl expression and partially attenuates the satellite bouton-promoting activity elicited by overexpression of Appl (Torroja, 1999a).

It is suggested that Appl is involved in synaptic plasticity, because Appl is nonessential for formation and maintenance of synapses but can promote synapse formation and appears to be affected by neuronal activity. NMJ phenotypes resulting from the expression of Appl proteins lacking specific domains suggest that Appl trafficking and Appl-dependent signal transduction are two processes that regulate Appl-induced synaptic growth. The evidence indicates that (1) APP can be rapidly internalized (Koo, 1996); (2) processing and trafficking of Appl and APP is affected by activity (Allinquant, 1994); and (3) plasma membrane APP, and possibly Appl, behave as Go-protein linked receptors (Nishimoto, 1993 and Torroja, 1999a).

It is proposed that NMJ expansion occurs by two consecutive steps: the formation of sprouts and the consolidation of some of these sprouts into differentiated boutons. Bouton differentiation entails the proper arrangement of presynaptic and postsynaptic components, as well as the enlargement of the sprout to accommodate all the elements required for synaptic transmission. Both sprouting and differentiation are modulated by Appl. It is suggested that plasma membrane Appl induces sprouting and that this response is independent of Appl signal transduction. In contrast, bouton differentiation is regulated by Appl signal transduction, which may involve Go. Internalization of Appl stops both activities. It is proposed that a satellite bouton is formed when Appl-induced sprouting is initiated, followed by rapid internalization of Appl, thus reducing Appl-dependent signal transduction and, therefore, bouton differentiation. As a result, some degree of differentiation, such as the formation of active zones and transport of vesicles, does occur, but other aspects, such as bouton enlargement, do not (Torroja, 1999a).

Several predictions from this model are in line with these findings. For instance, decreasing Appl internalization (DeltaCI) would reduce formation of satellites. A persistent increase in Appl activation, as a result of decreased internalization, is predicted to promote the differentiation of sprouts, thus effectively increasing the number of parent boutons. In contrast, overexpression of Appl variants that are unable to undergo ligand-dependent receptor activation (DeltaE1, DeltaE2, DeltaCg) would reduce bouton differentiation, preventing the increase of parents but not of satellites. Full-length Appl (Appl+, Applsd) would stimulate both sprouting and differentiation and could be rapidly internalized as in wild type, thus promoting both parent and satellite bouton formation (Torroja, 1999a).

Rapid Appl internalization appears to be key to the normally low level of plasma membrane Appl. Increases in plasma membrane-associated Appl result from increased neuronal activity, a factor shown to increase bouton number (Budnik, 1990). Strikingly, overexpression of Applsd in eag Sh results in reduction of satellites, with a concomitant increase in the number of parent boutons. This further supports the idea that neuronal activity can drive Appl-mediated bouton differentiation. Based on these observations, it is concluded that Appl has functional significance for the regulation of synapse formation. Moreover, this process involves the Appl domains that are likely to affect Appl signal transduction, suggesting a novel mechanism for the regulation of the size of synaptic arbors (Torroja, 1999a).

The Drosophila beta-amyloid precursor protein homolog promotes synapse differentiation at the neuromuscular junction

Although abnormal processing of beta-amyloid precursor protein (APP) has been implicated in the pathogenic cascade leading to Alzheimer's disease, the normal function of this protein is poorly understood. To gain insight into APP function, a molecular-genetic approach was taken to manipulate the structure and levels of the Drosophila APP homolog APPL. Wild-type and mutant forms of APPL were expressed in motoneurons to determine the effect of APPL at the neuromuscular junction (NMJ). APPL was transported to motor axons and that its overexpression caused a dramatic increase in synaptic bouton number and changes in synapse structure. In an Appl null mutant, a decrease in the number of boutons was found. Examination of NMJs in larvae overexpressing APPL revealed that the extra boutons had normal synaptic components and thus were likely to form functional synaptic contacts. Deletion analysis demonstrated that APPL sequences responsible for synaptic alteration reside in the cytoplasmic domain, at the internalization sequence GYENPTY and a putative G(o)-protein binding site. To determine the likely mechanisms underlying APPL-dependent synapse formation, hyperexcitable mutants, which also alter synaptic growth at the NMJ, were examined. These mutants with elevated neuronal activity changed the distribution of APPL at synapses and partially suppressed APPL-dependent synapse formation. A model is proposed by which APPL, in conjunction with activity-dependent mechanisms, regulates synaptic structure and number (Torroja, 1999b).

The Drosophila β-amyloid precursor protein (APP) homolog APPL is a pan-neural protein belonging to the conserved APP family. This family includes APP and APLP1/2 in mammals, apl-1 in nematodes, and APP747 in Xenopus. APP is synthesized as a transmembrane glycoprotein composed of extracellular, transmembrane, and cytoplasmic domains. APPs undergo proteolytic cleavage, releasing the ectodomain. Homology among APP members is paralleled by their ability to functionally substitute for each other: transgenes encoding Drosophila APPL or a human neural APP both rescue behavioral defects of Appl null flies (Torroja, 1999b).

Expression studies reveal a relationship between stages of APP synthesis and neurite outgrowth and synaptogenesis. In mammals, transmembrane APP is associated with elongating axons, whereas secreted APP is correlated with synaptogenesis. In Drosophila, APPL is enriched in growing axons and areas of synapse formation (Torroja, 1999b).

The effects of APP on neuronal development and function have been extensively studied because of their implication in Alzheimer's disease. APP exhibits neurite outgrowth-promoting activities in vitro through interactions with the extracellular matrix and changes in intracellular calcium. Experiments with neuronal cultures suggest that secreted APP modulates excitability via Ca2+-dependent K+ channels and NMDA receptor activity (Torroja, 1999b).

In vivo evidence supports a role for APP in synapse formation, maintenance, and plasticity. APP administration increases synaptic density and memory retention, and exposure of hippocampal slices to secreted APP enhances long-term potentiation and modifies the induction of long-term depression. APP knock-out mice show impaired learning and memory. However, experiments with APP overexpression are contradictory, demonstrating enhancement of synaptic density or Alzheimer's-like pathology and lack of synaptotrophic effects. Much of this controversial evidence may arise from the inability, in these systems, to analyze APP function at the level of single synapses (Torroja, 1999b).

To investigate the role of APP at single, identified synapses, the Drosophila neuromuscular junction (NMJ) was used. APPL overexpression in motoneurons results in a dramatic increase in the number of synaptic boutons. Conversely, NMJs of Applnull larvae exhibit a significant decrease in synaptic bouton number. The synapse-promoting function required a conserved internalization sequence and a putative Go binding site at the cytoplasmic domain. This function was partially suppressed by mutations with increased excitability, which by themselves regulate synapse growth and APPL expression at the NMJ (Torroja, 1999b).

An overexpression approach was used to determine whether APPL plays a role in the regulation of synapse formation. Overexpression of APPL in motoneurons resulted in morphologically distinct NMJs, characterized by an increase in both parent and satellite boutons. To define the APPL domains essential for these effects, an analysis was performed with Appl deletion proteins that were expressed in motoneurons. This demonstrated that a cytoplasmic internalization sequence is essential for satellite bouton formation, whereas a cytoplasmic Go-protein binding sequence and extracellular domains are essential in promoting excess parent bouton formation. Furthermore, it was observed that, in eag Sh mutants (elevated neuronal activity), APPL overexpression-associated effects are partially suppressed, and the endogenous pattern of APPL expression at the NMJ is altered. These results indicate that APPL localization and trafficking at the synaptic bouton membrane can be regulated in an activity-dependent manner and may influence synaptic growth (Torroja, 1999b).

The normal appearance of NMJs in larvae lacking APPL in the loss-of-function mutation demonstrates that APPL is not required for synaptogenesis and/or synapse maintenance. However, bouton numbers are reduced, indicating a possible regulatory role. This notion is bolstered by the striking increase in the number of parent and satellite boutons observed when APPL is elevated. Similar to APPL, human neuronal APP also elicited an increase in satellite and parent bouton number. This provides further support for the notion that APP695 (an alternatively spliced form of App) and APPL are structurally and functionally conserved (Torroja, 1999b).

An increase in bouton number has been observed in several Drosophila mutants that affect synaptic activity, such as eag Sh and dunce. In eag Sh, the increase in bouton number appears to derive from longer and more branched neuronal processes containing normal synaptic boutons. In contrast, APPL overexpression results in satellite boutons that protrude from a larger parent bouton, although parent bouton number is also increased (Torroja, 1999b).

During the postembryonic stage, there is tremendous growth of the NMJ, including elongation and formation of branches, as well as increases in bouton number, number of active zones, and bouton area. These studies suggest that NMJ expansion involves sprouting and elongation of a process, followed by the differentiation of new terminal boutons. Satellite boutons resulting from APPL overexpression may be caused by increased sprouting and abnormal bouton differentiation (Torroja, 1999b).

The distribution of different APPL forms in larval brains suggests that the transmembrane and soluble forms play specific roles in Drosophila neurons (Torroja, 1996). Indeed, it was found that the secretion-deficient APPL form is as effective as wild-type APPL in promoting satellite bouton formation, whereas constitutively secreted APPL has no effect when expressed in one dose and has an effect similar to the Appl null mutant with two doses of the transgene. The observation that two doses of APPLs result in a reduction in the percentage of satellites may indicate an inhibitory role on the satellite bouton-promoting activities (Torroja, 1999b).

Deletion of the APPL cytoplasmic domain abolished both the satellite bouton and parent bouton-promoting activity of APPL. However, these activities can be dissected by smaller deletions of the cytoplasmic and extracellular domains. Deletion of the internalization sequence prevented the formation of satellites but did not prevent the increase in parent boutons. In contrast, deletion of the putative Go-protein binding site prevented the increase in the number of parents, but the satellite bouton-promoting activity remained intact. Similar results were found when portions of the extracellular domain (E1 and E2), which may serve to bind a ligand to regulate Go function (Okamoto, 1995), were deleted (Torroja, 1999b).

In vertebrates, the internalization signal is crucial for regulating APP processing and trafficking. It interacts with several proteins, including X11 and Fe65, which may participate in the regulation of APP metabolism. Thus, APPL-dependent satellite bouton formation is likely to involve APPL internalization. Alternatively, or in addition, APPL may signal by interacting with cytoplasmic proteins, leading to the formation of satellite boutons. The low levels of APPL detected in wild-type NMJs suggests that APPL turnover may be high at the plasma membrane, similar to APP turnover in vertebrates (Torroja, 1999b).

Internalization of synaptic proteins plays an important role in the regulation of synaptic growth. For example, the Aplysia cell adhesion molecule ApCaM and its Drosophila homolog Fasciclin II are internalized at synapses in an activity-dependent manner. This promotes synapse growth, presumably by decreasing adhesion between presynaptic and postsynaptic membranes (Torroja, 1999b).

Studies in vertebrates show that APP695 behaves as a Go-linked receptor; its cytoplasmic region binds to Go-protein. However, whether APP stimulates or downregulates Go is unclear. The observation that deletion of the putative APPL Go-binding site abolishes the ability of APPL to increase parent bouton number indicates that Go activity may be involved. This is consistent with observations implicating Go in growth cone motility and synapse plasticity and with high levels of Go-protein in developing insect neurites (Torroja, 1999b).

Processing and trafficking of APP are known to be affected by neuronal activity. For example, in cortical neurons, the axonal pool of APP holoprotein at the surface was found to be increased after Ca2+ entry. In eag Sh larvae, endogenous APPL signal was consistently increased, especially at the distal-most boutons. Furthermore, when APPL was overexpressed in the hyperexcitable mutant, there was a reduction in satellite bouton number and an increase in parent boutons. This phenotype is similar to the phenotype associated with overexpression of APPL lacking the internalization signal. Thus, deletion of the internalization signal, or high activity, are both likely to increase surface APPL (presumably through a decrease in internalization) and have similar effects on synaptic growth (Torroja, 1999b).

It is suggested that APPL is involved in synaptic plasticity, because APPL is nonessential for formation and maintenance of synapses but can promote synapse formation and appears to be affected by neuronal activity. NMJ phenotypes resulting from the expression of APPL proteins lacking specific domains suggest that APPL trafficking and APPL-dependent signal transduction are two processes that regulate APPL-induced synaptic growth. The evidence indicates that (1) APP can be rapidly internalized; (2) processing and trafficking of APPL and APP is affected by activity; and (3) plasma membrane APP, and possibly APPL, behave as Go-protein linked receptors (Torroja, 1999b).

It is proposed that NMJ expansion occurs by two consecutive steps: the formation of sprouts and the consolidation of some of these sprouts into differentiated boutons. Bouton differentiation entails the proper arrangement of presynaptic and postsynaptic components, as well as the enlargement of the sprout to accommodate all the elements required for synaptic transmission. Both sprouting and differentiation are modulated by APPL. It is suggested that plasma membrane APPL induces sprouting and that this response is independent of APPL signal transduction. In contrast, bouton differentiation is regulated by APPL signal transduction, which may involve Go. Internalization of APPL stops both activities. It is proposed that a satellite bouton is formed when APPL-induced sprouting is initiated, followed by rapid internalization of APPL, thus reducing APPL-dependent signal transduction and, therefore, bouton differentiation. As a result, some degree of differentiation, such as the formation of active zones and transport of vesicles, does occur, but other aspects, such as bouton enlargement, do not (Torroja, 1999b).

Several predictions from this model are in line with these findings. For instance, decreasing APPL internalization (ΔCI) would reduce formation of satellites. A persistent increase in APPL activation, as a result of decreased internalization, is predicted to promote the differentiation of sprouts, thus effectively increasing the number of parent boutons. In contrast, overexpression of APPL variants that are unable to undergo ligand-dependent receptor activation (ΔE1, ΔE2, ΔCg) would reduce bouton differentiation, preventing the increase of parents but not of satellites. Full-length APPL (APPL+, APPLsd) would stimulate both sprouting and differentiation and could be rapidly internalized as in wild type, thus promoting both parent and satellite bouton formation (Torroja, 1999b).

Rapid APPL internalization appears to be key to the normally low level of plasma membrane APPL. Increases in plasma membrane-associated APPL result from increased neuronal activity, a factor shown to increase bouton number (Budnik, 1990). Strikingly, overexpression of APPLsd (which has a cleavage site deleted) in eag Sh results in reduction of satellites, with a concomitant increase in the number of parent boutons. This further supports the idea that neuronal activity can drive APPL-mediated bouton differentiation (Torroja, 1999b).

Based on these observations, it is concluded that APPL has functional significance for the regulation of synapse formation. Moreover, this study has shown that this process involves the APPL domains that are likely to affect APPL signal transduction, suggesting a novel mechanism for the regulation of the size of synaptic arbors (Torroja, 1999b).

Presenilin-dependent transcriptional control of the Aß-degrading enzyme Neprilysin by intracellular domains of ßAPP and APLP

Amyloid β-peptide (Aβ), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent γ-secretase cleavage of β-amyloid precursor protein (βAPP). The presenilins (PS1 and PS2) also regulate Aβ degradation. Presenilin-deficient cells fail to degrade Aβ and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Aβ-degrading enzyme. Neprilysin activity and expression are also lowered by γ-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Aβ, during γ-secretase cleavage of βAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Aβ or by the γ-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Aβ levels with varying levels of γ-secretase activity (Pardossi-Piquard, 2005).

If Aβ production and degradation are tightly linked, this raises the question of why Aβ accumulates in AD. The net accumulation of Aβ in AD pathology likely reflects the cumulative effect of multiple events acting on production, fibrillogenesis, and degradation. In many forms of AD, especially the late-onset sporadic forms, it has not been shown that there is increased β- and γ-secretase activity. In fact, some have suggested that these forms may reflect defective degradation of Aβ. Therefore, AICD levels are likely to be unchanged in these late-onset forms of AD, and as a result, the AICD-mediated ability to upregulate neprilysin activity would not be efficiently brought into play to protect the brain. In contrast, in those cases of AD arising from mutations in APP and PS1, which activate γ-secretase and AICD production, the principal effect is to produce longer Aβ isoforms such as Aβ42. However, although Aβ40 is efficiently degraded by NEP, Aβ42 is degraded by NEP both in vitro and in vivo at a 6-fold lower rate. As a result, the upregulation of AICD (and thus, NEP expression), which would be anticipated in subjects with presenilin mutations, would not completely abolish the accumulation of Aβ42 in these cases. It should be noted that, in agreement with the above hypothesis, neprilysin expression and activity were higher only in brain tissues with familial Alzheimer’s disease linked to various presenilin-1 mutations, while sporadic AD cases displayed neprilysin levels similar to those exhibited by normal brain tissues. Interestingly, PS1 mutations selectively affect neprilysin and do not alter insulin-degrading enzyme expression (Pardossi-Piquard, 2005).

The above observations are also of direct practical interest because they indicate the possibility of new avenues for controlling Aβ levels without directly affecting γ-secretase. This latter concept is important because of the various developmental and postnatal side-effects associated with the inhibition of γ-secretase-mediated cleavage of other signaling molecules, including Notch. This work now suggests that Aβ levels might be modulated by directly increasing neprilysin expression, using AICD or small molecule mimics of AICD. Upregulation of neprilysin by transgenic overexpression, at least to modest levels, appears to be sufficient to reduce brain Aβ levels and to pose few toxic side effects. This strategy would also circumvent the other side effects of γ-secretase inhibitors, including the potentially self-defeating effect of reducing AICD and thus preventing NEP-mediated degradation of Aβ (Pardossi-Piquard, 2005).

Fasciclin II signals new synapse formation through amyloid precursor protein and the scaffolding protein dX11/Mint

Cell adhesion molecules (CAMs) have been universally recognized for their essential roles during synapse remodeling. However, the downstream pathways activated by CAMs have remained mostly unknown. This study used the Drosophila larval neuromuscular junction to investigate the pathways activated by Fasciclin II (FasII), a transmembrane CAM of the Ig superfamily, during synapse remodeling. The ability of FasII to stimulate or to prevent synapse formation was shown to depend on the symmetry of transmembrane FasII levels in the presynaptic and postsynaptic cell and requires the presence of the fly homolog of amyloid precursor protein (APPL). In turn, APPL is regulated by direct interactions with the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-containing protein dX11/Mint/Lin-10, which also regulates synapse expansion downstream of FasII. These results provide a novel mechanism by which cell adhesion molecules are regulated and provide fresh insights into the normal operation of APP during synapse development (Ashley, 2005).

This study demonstrates that the ability of FasII to function either as a permissive or a restrictive influence on synapse growth depends on a balance of FasII levels between the presynaptic and postsynaptic cells. Furthermore, this study shows that FasII and APPL form a biochemical complex in vivo and that the ability of FasII to promote new synapse formation requires an APPL-dependent transduction cascade. Finally, this study shows that dX11 interacts with APPL in vivo and is involved in APPL/FasII-dependent new synaptic bouton formation (Ashley, 2005).

During the development of the larval NMJ, as muscles continuously increase in size, synaptic efficacy is maintained in part by the formation of new synaptic boutons. In this process, FasII plays two fundamental roles: one of maintenance, as exemplified in the absence of FasII, when synaptic boutons begin to form but later retract, and a role in bouton proliferation. Although the role of FasII in synaptic maintenance might be related to its ability to mediate cell adhesion between the presynaptic and postsynaptic membranes, its ability to regulate budding mostly depends on genetic interactions with APPL: in the absence of APPL, a symmetric increase or decrease in FasII levels has no influence or even decreases bouton number. Although APPL is not absolutely required for synaptic growth, elimination of APPL results in significantly smaller arbors (Ashley, 2005).

This study found that APPL was required for both FasII-dependent synaptic growth and for many physiological abnormalities accompanying NMJ structural defects. Although the bouton number decrease in Appl null mutants was correlated with a decreased amplitude of evoked synaptic responses, the bouton number increase in fasIIe76/+ was correlated with increased EJP amplitude. This increase was suppressed (to Appld levels) by eliminating APPL in fasIIe76/+ mutants. In [FasII]-pre-post, however, the dramatic increase in buds was correlated with an EJP amplitude decrease, possibly because these boutons were buds or immature boutons. Similarly, in [APPL]-pre, there was both an increase in buds and a decrease in EJP amplitude. Remarkably, as seen in the fasIIe76/+ mutants, the EJP phenotype is suppressed in the Appld background (again, reaching Appld levels). Thus, as in the morphological studies, many physiological abnormalities elicited by changing FasII levels depended on APPL. Interestingly, Appld does mimic many electrophysiological phenotypes reported previously in fasIIe76 homozygotes, in that both show increased mEJP frequency, increased mEJP amplitude, and decreased bouton number (Ashley, 2005).

Activation of the synapse-promoting activity of APPL by FasII depended on simultaneous changes in the presynaptic and postsynaptic cell, whereas a unilateral change in FasII in either cell alone interfered with synapse formation. This may relate to the ability of FasII to establish cis- and trans-homophilic interactions and to the exclusive presynaptic expression of APPL (Ashley, 2005).

A genetic interaction between Appl and fasII was clearly demonstrated in these studies. It was also demonstrated that both proteins form an endogenous complex at the NMJ and that this complex includes the APPL-binding protein dX11. In these interactions, it was found that FasII could independently interact with both APPL and dX11. In the absence of APPL, an interaction between dX11 and FasII was maintained, whereas in the absence of dX11, interactions between APPL and FasII were preserved. Precisely how APPL and FasII proteins interact physically remains unclear. However, it was found that, as was the case for APPL, the FasII intracellular domain was essential for the budding phenotype, suggesting that they may interact through their intracellular domains. In the case of FasII and dX11, FasII contains a PDZ-binding motif, which interacts with PDZ1-PDZ2 domains of DLG. It is possible that PDZ domains of dX11 are alternative FasII-interacting domains (Ashley, 2005).

These studies have identified a third member of the transduction cascade that promotes synaptic growth, dX11. First, dX11 was found in the same complex with APPL at the body-wall muscles, and the lack of the GYENPTY sequence of APPL or the PTB domain of dX11 suppressed or dramatically reduced this interaction, respectively. Second, a presynaptic increase of dX11 expression mimicked the effects of upregulating APPL. This effect was suppressed by deleting the PTB domain. Third, deleting the dX11-APPL interaction sequence in dX11 (dX11ΔPTB) mimicked the effect of deleting the APPL-dX11 interaction sequence (APPLΔCi) at the NMJ. Fourth, the effects of FasII gain-of-function in both the presynaptic and the postsynaptic cell were suppressed by expressing dX11ΔPTB, suggesting that the APPL and dX11 interaction is required for the effect of FasII. Finally, a hypomorphic dX11 mutant mimicked the effects of eliminating APPL during NMJ expansion (Ashley, 2005).

A variety of proteins that bind to the GYENPTY region of APP either increase APP translocation to the cell surface or alter the stabilization or cleavage of APP at the cell surface. In particular, mammalian X11/Mint is highly expressed in neurons and interacts with the APP GYENPTY sequence through its single PTB domain. Neuronal X11 also associates directly with the exocytotic protein Munc-18, which in turn interacts with syntaxin 1A. In the current studies, it was found that deleting the APPL interaction domain in dX11 resulted in large accumulations of APPL within boutons, suggesting that dX11 may be involved in transporting or facilitating the insertion of APPL into the presynaptic membrane (Ashley, 2005).

Several studies suggest that APPL behaves as a Go-protein-coupled receptor. Go has been shown to be involved in microtubule polymerization, suggesting that one of the actions of APPL during NMJ expansion might be to regulate the cytoskeleton. Recent studies show that microtubule dynamics at the Drosophila NMJ are essential for bud maturation and extension. Based on these known interactions, the following model for APPL and dX11 function can be proposed. Trans-homophilic interactions between FasII molecules localized at the presynaptic and postsynaptic cell activate the binding between the dX11 complex (containing exocytic molecules) and APPL. dX11 then transports its partners to sites of FasII-mediated cell adhesion. This results, on one hand, in the transport of the exocytic machinery and perhaps the addition of new membrane to sites of budding. In contrast, the insertion of APPL into the presynaptic membrane and its interactions with FasII activate Go, resulting in the stimulation of microtubule polymerization, which is required for bud extension (Ashley, 2005).

The notion that FasII might function not only as a cell adhesion molecule but also as a signaling molecule is not without precedence. Indeed, the mammalian FasII homolog NCAM has been shown to initiate a signal transduction cascade after activation of both nonreceptor tyrosine kinases (nRTKs) and RTKs that may influence neurite outgrowth. Interestingly, a genetic interaction between fasII and the nRTK Abelson tyrosine kinase gene (Abl) has been reported previously in flies, and, in mammals, activated Abl interacts directly with and phosphorylates the APP intracellular GYENPTY sequence. A variety of kinases are able to phosphorylate the APP cytoplasmic domain, resulting in regulation of APP metabolism and function. Phosphorylation of Thr668 of APP695 serves as a molecular switch that appears to regulate X11 binding to APP. Thus, phosphorylation of APPL could be the switch that leads to dX11 binding and subsequent translocation of APPL to the presynaptic membrane in the Drosophila NMJ (Ashley, 2005).

These studies show that an asymmetric increase in FasII at the presynaptic cell interferes with normal synaptic bouton formation. This is characterized by formation of grossly abnormal boutons containing internal membrane structures with unusual APPL deposits and microtubule tangles surrounding these deposits. These internal APPL accumulations within the boutons seem reminiscent of intraneuronal amyloid-β accumulation, which may precede extracellular amyloid plaque formation in Alzheimer's disease. This phenomenon may provide additional clues toward a mechanism by which interference with normal APP function could lead to pathological events and subsequent symptoms of Alzheimer's disease (Ashley, 2005).

In conclusion, this study demonstrated via genetic analysis that APPL, FasII, and dX11 are involved in the same pathway that regulates synaptic expansion at the Drosophila NMJ. Altogether, these results suggest that beyond a role in cell adhesion, FasII-mediated signaling depends on a precise balance of its levels of expression at the presynaptic and postsynaptic cell and is likely to activate intracellular transduction pathways that control synapse structure (Ashley, 2005).

The full-length form of the Drosophila amyloid precursor protein is involved in memory formation

The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, APPL, the fly APP ortholog, is required for associative memory. This study aimed to characterize which form of APPL is involved in this process. Expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, was able to rescue the memory deficit caused by APPL partial loss of function. The study next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. Further, in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, transient expression of a constitutive membrane-bound mutant APPL form was sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. This data demonstrates that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes (Bourdet, 2015).

The majority of studies into APP biology have focused on pathogenic mechanisms. However, it remains crucial to understand the normal physiological function of APP, especially as it is possible that APP loss of function elicits early cognitive impairment in AD patients. This study shows that overexpression of secreted APPL rescues the short-term memory deficit caused by a reduction of APPL level. In sharp contrast, overexpression of the α-secretase, KUZ, which produces sAPPL, exacerbates the memory impairment, a phenotype that is likely due to a deficit in full-length APPL protein level. Supporting this hypothesis, it was further demonstrated that expression of a nonprocessed APPL mutant form is able to restore wild-type memory in an APPL partial loss of function background (Bourdet, 2015).

In the past, two main strategies have been considered as therapeutic approaches for AD. First, inhibition of the β- or γ-secretase has been used to achieve an inhibition of Aβ toxic production. However, reduction of Aβ production is not only an ineffective approach for AD, it also can actually promote further pathology, as these enzymes have numerous substrates. A second proposed approach has been to inhibit the amyloidogenic pathway by activating the α-processing of APP. In addition to the potential beneficial inhibition of the amyloidogenic pathway, the advantage of this type of approach is to also increase the production of sAPPα. Indeed, decreased CSF sAPPα levels were found in familial and sporadic AD patients, and correlated with poor memory performance in patients with AD. Thus, in vitro and in vivo studies indicate that sAPPα is downregulated during AD. Numerous analyses have shown that sAPPα ectodomain has neurotrophic and neuroprotective effects in different models of neuronal stress. In addition, sAPPα exhibits memory-enhancing properties. Intracerebroventricular infusion of anti-sAPPα serum was deleterious for memory, while that of sAPPα was beneficial. However, these studies relied on an exogenous excess of sAPPα and mechanisms of action and potential targets remained to be elucidated. With knock-in mice experiments, showed that sAPPα was sufficient to correct the impairments in spatial learning and long-term potentiation that are present in APP KO mice. This study shows in Drosophila that sAPPL is able to fully rescue the STM deficit caused by a reduction in endogenous APPL level, thus establishing that an APPL soluble form plays a role in memory, and giving further support for a role of secreted forms in memory in mammal systems (Bourdet, 2015).

When the fly α-secretase, KUZ, was overexpressed in the adult MB, no STM-enhancing effect was seen and, unexpectedly, KUZ overexpression in the MB of flies with an APPL partial loss of function exacerbated their memory impairment. Thus, KUZ overexpression was actually deleterious for memory, rather than beneficial. These results contrast with a previous study showing that overexpression of the mammalian α-secretase ADAM10 in an AD mice model led to an increase in sAPPα, and was able to overcome APP-related learning deficits. However, these studies showed that α-secretase activation has a positive impact on memory exclusively under conditions where human APP is overexpressed. In wild-type mice, results were not clear because overexpression of either the wild-type or an inactive form of the bovine ADAM10 altered learning and memory. Furthermore, ADAM10 has many substrates, and no evidence was brought to link the memory deficit to APP (Bourdet, 2015).

Interestingly, this study observed that KUZ overexpression decreases membrane nonproteolyzed APPL level, suggesting that its negative impact on memory in APPL LOF flies is linked to a reduction of nonproteolyzed APPL level. Therefore, strategies aimed at increasing APP α-cleavage may not be appropriate as this could provoke a decrease of fl-APP levels that might be deleterious to APP function (Bourdet, 2015).

Transient expression of a constitutive membrane-bound mutant APPL has the capacity to fully rescue the STM deficit caused by APPL partial loss of function. Thus, both sAPPL and fl-APPL appear to be involved in memory processes. This is in apparent contradiction with the observation that mammalian sAPPα was sufficient to correct spatial learning deficit of APP KO mice. However, in this study APP-like proteins APLP1 and ALPL2 were preserved, and as it is known from double KO analyses that the three APP homologs exert functional redundancy, they may have compensated for the loss of essential fl-APP functions. In consequence, one cannot attribute the memory function exclusively to sAPPα (Bourdet, 2015).

If both fl-APPL and sAPPL carry the capacity to restore wild-type STM in APPL partial LOF flies, it is puzzling to observe that KUZ overexpression in this genetic context is deleterious for memory. Indeed, in addition to causing a decrease in fl-APPL, KUZ overexpression leads to a concomitant increase in sAPPL that should be able to complement fl-APPL deficiency. It is suggested that in this context, fl-APPL level is below threshold so that even high levels of sAPPL cannot restore a wild-type memory. This hypothesis is supported by protein quantification experiments showing a 30% decrease in fl-APPL level. Because APPL was extracted from the whole brain, whereas KUZ overexpression was only driven in a subset of neurons, the effective fl-APPL decrease in the MB must be much higher than 30%. In mammalian cells under steady-state levels, ~10% of APP is located at the plasma membrane. APP has long been suggested to act as a cell-surface receptor; however, such a function has not been unequivocally established. Several reports have shown that APP exists as homodimers. Cis-dimerization of APP would represent a potential mechanism for a negative regulation of APP functions and a concomitant impact on Aβ generation via an increase in β-processing. Interestingly, it has been suggested that APP is a receptor for sAPPα as its binding could disrupt APP dimers (Bourdet, 2015).

In Drosophila, it has been reported that the secreted N-terminal ectodomain of APPL acts as a soluble ligand for neuroprotective functions. Furthermore, coimmunoprecipitation experiments from transfected Drosophila MB intrinsic cells revealed a physical interaction between fl-APPL and sAPPL, suggesting that sAPPL could be a ligand for fl-APPL. The current data showing the involvement of both membrane fl-APPL and sAPPL in memory are consistent with the hypothesis that sAPPL could be a ligand for its own fl-APPL precursor (Bourdet, 2015).

In conclusion, these data reveal for the first time a role for membrane fl-APPL in memory, opening new questions about APP nonpathological functions and relations between secreted and full-length forms in memory processes (Bourdet, 2015).

Drosophila full-length Amyloid precursor protein is required for visual working memory and prevents age-related memory impairment

The β-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). APP is cleaved by various secretases whereby sequential processing by the β- and γ-secretases produces the β-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPβ fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPalpha fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPalpha fragments have been suggested to have a neurotrophic and neuroprotective role.Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment. This study shows that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, these results show that specific memory processes require distinct APPL products (Rieche, 2018).

Age-related memory impairment (AMI) affects all animals, and cognitive decline is one of the devastating features of Alzheimer's disease (AD). Although APP, and more specifically the β-amyloid peptide, has been connected with memory deficits in AD, the role of full-length APP and its various other fragments in AMI is unknown. Wild-type Drosophila flies display AMI at middle age (30-40 days) when tested for middle-term or long-term olfactory memory. Furthermore, Drosophila not only encodes an ortholog for APP, called amyloid precursor protein-like (APPL), but also homologs for all three types of secretases; kuzbanian (kuz) corresponds to ADAM10 considered to be an α-secretase, dBace, the fly β-secretase, and Presenilin (Psn), the catalytic subunit of γ-secretase. APPL is processed in a similar way as human APP; however, the cleavage sites of the α- and β-secretase are reversed. Therefore, cleavage by KUZ produces a shorter secreted N-terminal fragment (NTF) than processing by dBACE. Nevertheless, subsequent γ-processing of the β-cleaved C-terminal fragment (βCTF) results in a neurotoxic dAβ peptide, whereas cleavage by KUZ does not. This study asked whether the very short-term (~4 s) visual working memory in flies is also affected by AMI and whether it requires APPL or one of its proteolytic fragments. Therefore, wild-type flies and heterozygous mutants for the three secretases were aged, and their visual orientation memory was assessed (Rieche, 2018).

This working memory is tested in the detour paradigm where walking flies navigate between two inaccessible landmarks. During an approach, the targeted landmark disappears and the fly is lured toward a novel distracting landmark. This distracter disappears one second after reorientation so that the fly is now left without any landmarks. Nevertheless, wild-type Canton-S (CS) flies can recall the position of the initial landmark and try to approach it although still invisible ('positive choices'). Whereas young CS males make about 80% positive choices, aged flies showed a reduced memory when tested at 4 weeks of age and a complete memory loss when 6 weeks old. Interestingly, heterozygosity for any of the three secretases prevented AMI, with 4- and 6-week-old Psn143/+ and kuze29-4/+ flies being indistinguishable from young CS flies. When using heterozygous dBace5243 flies, the improvement compared to age-matched CS controls did not reach significance; however, they made significantly more positive choices than chance level at 6 weeks, whereas CS did not. These findings show that visual working memory is deteriorating with age, and they suggest that reducing APPL processing can suppress AMI (Rieche, 2018).

To address whether increased processing of APPL disrupts this memory, the secretases were overexpressed in the R3 ring neurons of the ellipsoid body (using 189Y-GAL4) the seat of visual working memory. Expression of any of the secretases reduced the performance already in 3-day-old flies compared to controls, supporting a requirement of full-length flAPPL for this type of memory. On the other hand, western blot analyses using an antiserum directed against the NTFs of APPL (Ab952M) established that heterozygous secretase mutants have an overall increase especially in flAPPL which supported the hypothesis on the role of secretases and APPL processing in AMI. Furthermore, a quantitative analysis of the levels of APPL in head extracts from different ages revealed that flAPPL declines with age, whereas the NTF/flAPPL ratio increases, also suggesting that reduced levels of flAPPL are involved in AMI. To verify that APPL is indeed required for visual working memory, homozygous Appld-null mutants and transheterozygous combinations of hypomorphic Appl alleles were tested. All these mutants performed at chance level already when young, confirming that APPL is necessary for this short-termed memory. This function is dose sensitive because even young heterozygous Appld/+ showed a reduced memory that declined faster with age than in CS females (Rieche, 2018).

Next, an RNAi-mediated knockdown of Appl was introduced in the R3 neurons, which resulted in severe memory deficits already in 3- to 5-day-old flies, showing that APPL is required in these neurons for visual working memory. To identify domains in APPL that mediate this function, rescue experiments were performed expressing different APPL constructs via 189Y-GAL4 in young Appld mutant flies. This included full-length flAPPL, secretion-defective sdAPPL, specific deletion constructs, and secreted fragments. Because the exact cleavage sites in APPL are unknown, the secreted fragments are referred to as sAPPLLong (L), which comprises the N-terminal 788 amino acids and should represent the β-cleaved fragment, whereas the 758-amino-acid (aa)-long sAPPLShort (S) should represent the α-cleaved form of Drosophila APPL. In contrast to full-length wild-type APPL, neither of the secreted forms could rescue the memory deficit of Appld when induced in R3 neurons. Notably, sdAPPL very effectively rescued the memory phenotype, confirming that unprocessed flAPPL is crucial for visual working memory (Rieche, 2018).

Next, whether APPL functions as a receptor or ligand in R3 neurons was investigaged. Expression of sdAPPL that in addition lacks the intracellular C terminus (sdAPPL-ΔC) did rescue the Appld memory phenotype, which suggests that intracellular signaling is not required and that APPL does not act as a receptor. Rescue experiments with sdAPPL forms that, in addition, lack one of the two ectodomains (sdAPPL-ΔE1 and sdAPPL-ΔE2) revealed a requirement for E2 for the rescue but not for E1. To confirm this, the rescue experiments were repeated with the hypomorphic Appl4460 allele and APPL, sdAPPL, and sdAPPL-ΔE1 rescued to full extent, whereas sdAPPL-ΔE2 did not. These results suggest that membrane-bound APPL functions as a ligand in the ring neurons. This function was conserved in human APP because expression of APP695 via 189Y-GAL4 in Appld also resulted in a rescue. Using conditional expression of APPL (by combining 189Y-GAL4 with the temperature-sensitive GAL4 repressor Tub>GAL80ts) resulted in the same rescue as constitutive expression, revealing that expression in adult R3 neurons is sufficient to restore the memory in 3- to 5-day-old flies. Notably, using the same expression system to induce moderate overexpression of sdAPPL (at 25°C), it was possible to rescue the AMI in a wild-type background, demonstrating that the secretion-deficient unprocessed APPL can prevent the decline of visual working memory of aged flies. Together with the finding that the levels of endogenous flAPPL decrease with age, this suggests that a loss of flAPPL underlies the visual working memory deficits that occur during normal aging. Interestingly, an age-related increase in BACE1 activity has been described in vertebrates that could reduce levels of full-length APP (Rieche, 2018).

Most of APPL functions in synaptogenesis, neurite outgrowth, and guidance described so far required signaling via the C-terminal domain. Analyzing heterozygous Appld/+ mutant flies or inducing an adult-specific knockdown of Appl in the relevant mushroom body neurons, Preat and Goguel (2016) showed that APPL function is not required for olfactory learning, but for a 2-hr associative memory and long-term memory formation. Similar to findings in mice, overexpression of secreted sAPPLL (as well as APPL and sdAPPL) could restore the 2-hr memory in heterozygous Appld/+ flies, whereas only wild-type APPL could rescue the long-term memory deficit. Because endogenous APPL was still expressed in this rescue experiments, sAPPLL and sdAPPL might act as ligands that bind to flAPPL in mushroom body neurons. The authors therefore suggested that distinct memory phases require different forms of APPL and maybe different intracellular signaling pathways. Notably, overexpression of KUZ in the mushroom body did not affect the 2-hr olfactory memory, whereas KUZ in this study significantly reduced visual working memory. It should also be noted that unprocessed APPL can be deleterious, because expression of sdAPPL in photoreceptor cells caused cell death of lamina glia via an unknown receptor, further emphasizing that individual neuronal networks may require different APPL fragments and signaling pathways (Rieche, 2018).

Due to recent studies suggesting that output from the R3 neurons into the ellipsoid body is instrumental for visual working memory, it was hypothesized that full-length APPL is present at the axonal terminals of R3 neurons in the ellipsoid body. To analyze the sub-cellular localization of APPL and its fragments, a double-tagged version of APPL (dtAPPL) was used that carries an EGFP tag near the N terminus and RFP tag at the C terminus, resulting in yellow fluorescence of full-length dtAPPL (or co-localized fragments that have not been separated yet). Using 189Y-GAL4 to induce dtAPPL, it was observed that dtAPPL is processed differently in individual R3 neurons, whereby full-length as well as fragments of dtAPPL are found in the cell bodies and axonal/dendritic projections. That significant amounts of unprocessed APPL can be found in the R3 axons in the ellipsoid body supports the model, that full-length APPL is needed at the R3 output sites. Note that dtAPPL was able to rescue the memory deficit, as did a dtAPP695, which showed a similar distribution pattern as dtAPPL (Rieche, 2018).

Having established that full-length APPL can be found at the relevant output sides, it was asked whether proteolytic processing of APPL also changes in the R3 neurons with age. Comparing the pattern of dtAPP695 expressed with the 189Y-GAL4 driver in 3-day-old and 6-week-old flies indicated less full-length dtAPP695 in aged flies, but, when quantifying this, it did not reach significance. Therefore, another R3-neuron-specific GAL4 line (VT42759) was used that results in reduced levels of dtAPP695 with increasing age but could nevertheless be used to rescue the memory phenotype of Appld. Compared to 3-day-old flies, there was little co-localization of GFP-tagged N termini and RFP-tagged C termini in 6-week-old VT42759>dtAPP flies, revealing enhanced proteolytic processing of APP. This suggests that AMI of the visual orientation memory is caused by increased ectodomain shedding of APPL and western blot analysis of aged flies supports this notion because during aging the ratio of NTFs to flAPPL increases (Rieche, 2018).

To identify a possible receptor for APPL in visual working memory, focus was placed on the neural cell adhesion molecule Fasciclin 2 (FASII). FASII is enriched in most, if not all types of ring neurons in the ellipsoid body, and it has been shown to interact with APPL in synaptic bouton formation at the neuromuscular junction (NMJ) (Ashley, 2005). Moreover, FASII is the insect homolog of neural cell adhesion molecule (NCAM)-140, which has been demonstrated to bind APP in an E2-depending fashion. When young hemizygous mutants were tested for the strong hypomorphic FasIIe76 allele in the detour paradigm, they showed no working memory, and the same phenotype was observed when an RNAi against FasII was introduced in R3 neurons of 3- to 5-day-old flies. This phenotype could be rescued by expression of FASII in R3 neurons, which reveals that FASII is required in the same ring neuron subtype as APPL, providing a possible binding partner for APPL. At the NMJ, loss of APPL suppressed the increase in bouton number in heterozygous i>FasIIe76/+ larvae. This study therefore investigated whether removing one copy of Appl could rescue the memory deficits of homozygous i>FasIIe76 mutants. This resulted in a significant improvement in performance, as did one copy of the i>FasIIe76 mutant allele in homozygous Appld-null mutant flies. Moreover, reducing FASII expression in R3 neurons by RNAi also ameliorated the memory deficit of Appld-null mutants. Together, this suggests that APPL negatively regulates FASII in R3 neurons and that FASII acts downstream of APPL. That this negative interaction is essential to prevent AMI is demonstrated by the observation that heterozygosity for v suppresses the memory loss of 4-week-old heterozygous Appld/+ flies (Rieche, 2018).

To further investigate interactions between APPL and FASII, overexpression studies were performed using young flies, and elevated levels of flAPPL or sdAPPL were found to ameliorate the memory deficit induced by FASII overexpression. However, only wild-type APPL induced memory deficits when overexpressed without FASII. This suggested that a secreted form of APPL can induce a gain-of-function phenotype, and this study therefore overexpressed sAPPLL and sAPPLS in R3 neurons. Whereas sAPPLL had no effect, sAPPLS caused a severe impairment of visual working memory. Moreover, overexpression of sAPPLS suppressed the effects of elevated FASII levels, whereas sAPPLL did not. This shows that sAPPLS, which corresponds to the α-cleaved (KUZ) fragment, has deleterious effects when overexpressed and that these are also mediated by an interaction with FASII. Whether the lack of an effect of sAPPLL overexpression is due to a less efficient interaction with FASII or an inability to induce downstream pathways causing this gain of function remains to be determined (Rieche, 2018).

Interestingly, overexpression of the dAICD in R3 neurons also disrupted visual working memory of young flies, suggesting that α- and γ-cleavage of APPL has deleterious effects on visual working memory. This is in agreement with the finding that heterozygosity for kuz and Psn ameliorated the age-related decline in visual working memory, whereas heterozygosity for dBACE had only a modest effect. This could be explained by assuming that most of the ectodomain shedding in flies is done by KUZ activity. Therefore, reducing dBACE levels might result in a small increase of flAPPL. In addition, competitive KUZ cleavage in dBace/+ flies could result in more detrimental sAPPLS. Whereas in the case of kuz/+, the effects on memory are mediated by the interaction with FASII, in the case of Psn/+ this may be mediated by a transcriptional function of the AICD, affecting a so far unknown target (Rieche, 2018).

In summary, the results show that full-length APPL acts as a membrane-bound ligand that inhibits the FASII receptor (both acting in R3 neurons), thereby promoting visual working memory. Increased proteolytic APPL processing and therefore reduced suppression of FASII signaling then seems to cause AMI in flies. A similar interaction might also be required for working memories in vertebrates. Aging mice show reduced expression of NCAM-140 in the medial prefrontal cortex and a conditional knockout of NCAM in the forebrain promotes AMI in a delayed matching-to-place test in the Morris water maze and in a delayed reinforced alternation test in the T-maze (Rieche, 2018).

Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, previous work has shown that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). The present study shows that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the alpha'/beta' Kenyon cells. Using a biochemical approach, this study identified the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, this study shows that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the alpha'/beta' neurons. Finally, using a double RNAi approach, it was demonstrated that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, these results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species (Silva, 2020).

AD is the principal neurodegenerative disorder affecting the elderly, and it is characterized by amyloid β (Aβ) deposition derived from proteolytic processing of amyloid precursor protein (APP). A pathological hallmark of AD is a progressive memory decline that correlates intimately with synaptic loss. One of the main hypotheses for the cognitive deficits observed in AD is thus a dysfunction of synapses leading ultimately to synaptic loss and alteration of neural network activity. Therefore, it is essential to understand the physiological role of APP at the synapse. APP is a transmembrane protein expressed on both sides of the synapse (Wang, 2009). The APP extracellular domain can mediate dimerization across the synapse or interact with extracellular matrix components, growth factors and receptor-like proteins. These interactions are involved in synapse stabilization during development and also in regulating synapse plasticity in mature neuronal networks (Montagna, 2017). APP can undergo two types of proteolytic processing, including the non-amyloidogenic pathway, which is initiated by α-secretase and produces a secreted form of APP (sAPPα), and the amyloidogenic pathway, which successively involves β- and then γ-secretase to release Aβ peptide and an APP intracellular C-terminal domain (AICD). Although the manner in which proteolytic processing of APP and its derivatives interferes with neuronal physiology has been extensively studied, little is known about the function of APP intracellular domain at the synapse or its synaptic partners (Silva, 2020).

Studies in mammals suggest that APP can interact via its intracellular domain with synaptic MAGUK proteins such as X11, CASK, or PSD-95. MAGUK proteins are involved in the assembly, maintenance and remodeling of the scaffolding in synaptic compartments mainly via regulation of the targeting of receptors and ion channels to the synapse. Therefore, understanding the interactions between APP and MAGUKs should help decipher the synaptic function of APP (Silva, 2020).

The three mammalian orthologs APP, APLP1 and APLP2 are partially functionally redundant, whereas Drosophila expresses a single APP homolog named APP-like (APPL) that has been implicated in olfactory memory (Bourdet, 2015; Goguel, 2011) and visual memory (Rieche, 2018). APPL is strongly expressed in the adult mushroom body (MB), the main olfactory memory center in insects. Previous work has investigated the function of APPL in Drosophila aversive olfactory memory (Bourdet, 2015; Goguel, 2011; Preat, 2016). However, whether the APP synaptic partners and their interactions might contribute to memory is still unexplored. Several MAGUK homologs in Drosophila have been identified such as dX11, CASK/Caki, and Discs-large (Dlg). Similar to its mammalian counterpart, dX11 binds APPL, and both are necessary for synaptic remodeling at the Drosophila neuromuscular junction (Ashley, 2005). Drosophila CASK regulates CaMKII activity, interacts with dX11. Mammalian Dlg1/SAP97, Dlg2/PSD-93, Dlg3/SAP102 and Dlg4/PSD-95 share similarities with the fly Dlg proteins DlgA and DlgS97), which are encoded by a single dlg gene. Both CASK and Dlg play key roles in neurotransmission, synaptogenesis and plasticity (Silva, 2020).

This study has aimed to decipher the role of APPL and its synaptic partners in appetitive olfactory memory. Additionally, to investigate the AICD interactome, this study used a proteo-liposome recruitment method and found that AICD interacts with the MAGUK synaptic proteins X11, CASK and Dlg. It was then found in flies that APPL, CASK and Dlg are required specifically in the same neuronal subpopulation (the α'/β' KCs) for appetitive LTM. Finally, a double RNAi strategy was used to demonstrate that genetic interaction between APPL and MAGUKs is critical for appetitive LTM. To determine whether this memory deficit could be due to a major disorganization of the synaptic structure, both the pre-synaptic and the post-synaptic sites of MB α'/β' neurons were investigated using confocal immuno-labeling of synaptic proteins (Silva, 2020).

Previous work has showed that APPL is required in the α/β and γ KCs for aversive LTM. Additional work demonstrated that APPL is required for aversive LTM and MTM in the DPM neurons (Turrel, 2017), a pair of serotonergic and GABAergic neurons that project to each of the MB lobes, where they connect both pre- and post-synaptically to the KCs. This study has found that APPL is also required in another form of long-lasting protein synthesis-dependent memory, appetitive LTM, albeit surprisingly in a different subpopulation of KCs, the α'/β' neurons. The requirement of APPL in α'/β' but not in the other KCs for appetitive LTM suggests that it has a specific role in appetitive LTM consolidation, as consolidation of appetitive LTM requires synaptic neurotransmission from α'/β'. Interestingly, recurrent activity of the α'/β'-DPM loop has been described as necessary to consolidate appetitive memories, with LTM eventually being stored in the α/β neurons. An involvement of APPL in memory consolidation may rely on transsynaptic APPL interactions and may also contribute to the molecular support of the α'/β'-DPM loop. Eventually, such a role of APPL in memory consolidation through transsynaptic interaction would be consistent with published research in mammals. Indeed, at the cellular level APP is expressed in pre- and postsynaptic compartments and can form trans-dimers that have been suggested as necessary for synaptic function (Wang, 2009). In addition, perturbation of APP function by intraventricular infusion of an antibody against APP induced memory impairments only when it was performed during the memory consolidation phase of a passive avoidance task. As shown for other synaptic cell adhesion molecules, the regulation of APP expression at the neuronal membrane is critical for hippocampal-dependent memory consolidation in the dentate gyrus, suggesting a potential involvement of APP in synaptic remodeling. Altogether, the present findings combined with research on mammalian models suggest that APP might have a conserved function across species in memory consolidation processes via transsynaptic interactions (Silva, 2020).

This study has shown that in addition to the previously known X11 proteins, the intracellular domain of APP interacts with other scaffolding proteins (CASK, Dlg2/PSD-93 and Dlg4/PSD-95). However, it is not clear whether these proteins interact directly with APP or if their interactions are mediated by the X11 adaptor proteins, as it has been described for CASK. This study demonstrates that the Drosophila homologs of these proteins, i.e. CASK and Dlg, are required in the same neuronal subpopulation as APPL for proper long-term memory. Several arguments in favor of an APPL/X11/CASK/Dlg macromolecular complex can be found in previous studies on mammals or Drosophila. In the Drosophila visual system, the MAGUK complex Lin-7/Dlg/CASK is involved in synaptic stabilization and the interaction between CASK and Dlg proteins has been described as direct (Soukup, 2013). The role of CASK in the recruitment of Dlg1 to the membrane in various cell types has been confirmed in a recent study showing that the N-terminal domain of Dlg1 is critical for its interaction with CASK (Porter, 2019). In mammals, the existence of the APP/X11/CASK ternary complex has also been documented, and the regulation of neuronal excitability through potassium Kir2 channels involves a macromolecular complex consisting of Lin-7/SAP97/CASK/X11. Altogether these studies demonstrate that the interaction between the Dlg, CASK and X11 proteins is both possible and functionally relevant for neuronal physiology. Finally, CASK binds to the Dlg protein SAP97 in mammalian hippocampal neurons to regulate its conformation state and thus its role in glutamate receptor trafficking and insertion at the synapse. Thus, the existence of the APPL/X11/CASK/Dlg macromolecular complex is consistent with previously published reports, and even if this study only demonstrate the genetic interaction between APPL, CASK and Dlg for appetitive LTM, it is likely that such a complex exists in Drosophila α'/β' MB neurons. Complementary studies using genetic tools to impair the interactions between these proteins such as overexpression of an interfering peptide corresponding to the N-terminus of Dlg as in Porter (2019) could bring the confirmation that protein-protein interactions between APPL/X11/CASK/Dlg are required for appetitive LTM via synaptic stabilization of the α'/β' neurons. The present study looked at synaptic organization using confocal microscopy and immuno-labeling of either pre-synaptic proteins of the active zone or post-synaptic scaffold proteins. The levels of the analyzed pre- or post-synaptic proteins were not affected by the concomitant knockdown of APPL and MAGUKs in the adult α'/β' neurons, indicating no obvious alterations in MB synaptic structure. It is noted that as expected this study observed a significant decrease in Dlg levels in the MB calyx upon knockdown of APPL;Dlg in α'/β' neurons, demonstrating the efficacy of the knockdown. To determine the requirement of these proteins for pre- or post-synaptic subtle organization, higher-resolution imaging studies would be required. However, such modification might be difficult to observe if, as suggested by the role of CASK and Dlg proteins, the APPL/X11/CASK/Dlg macromolecular complex is involved in synaptic stabilization specifically when synapses are modified during a plastic event, and not as a basal mechanism for synaptic organization and formation as described for APPL and FasII interactions at the NJM (Silva, 2020).

Interestingly, the Dlg protein SAP97 is involved in trafficking the α-secretase ADAM10 to the synapse through direct interaction, consequently regulating APP processing and the production of the neurotrophic and neuroprotective secreted-APPα fragment. Previous work has shown in Drosophila that a secreted fragment of APPL is involved in aversive memory, as well as KUZBANIAN, the Drosophila homolog of ADAM10 (Bourdet, 2015). Therefore, an APPL/X11/CASK/Dlg supramolecular complex could also be involved in recruiting α-secretase at the synaptic site as well as generating sAPPα (Silva, 2020).

The subcellular localization of APP and MAGUK interactions in the KCs is still an open question. In Drosophila, Dlg and CASK are known to be present at both the pre- and postsynaptic compartments. However, APPL has been described mainly in the neuropil of KCs. Furthermore, the APPL binding protein X11 targets APPL to the axonal compartments and excludes it from MB dendrites via endocytosis. These data suggest that the APPL-MAGUKs complexes would be localized in the α'/β' KC axonal compartment (i.e. the α'/β' lobes), which is importantly also the site of the DPM/KC dialog for appetitive LTM consolidation (Silva, 2020).

In conclusion, this work highlights a novel role of APPL and its synaptic partners in appetitive long-term memory in Drosophila. Genetic interactions between APPL and the MAGUKs are critical for appetitive LTM in the α'/β' KCs, a neuronal sub-population known to be involved in the consolidation of appetitive LTM. A model is proposed in which the role of the interactions between APPL, CASK and Dlg might be the synaptic stabilization of the α'/β'-DPM loop through transsynaptic interactions (Silva, 2020).


Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, transgenic Drosophila was used as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch (Merdes, 2004).

These studies show that the ectopic expression of human APP-family members induces Notch gain-of-function phenotypes during the development of the adult PNS. The severity of the induced phenotypes not only depends on the dosage and the particular APP-family member, but also on particular domains of the molecules. This led to the identification of the NPTY motif as the only critical motif within the ICD for the interference with PNS development and for the interaction of APP with Numb/Pon and Dab in vitro and in vivo (Merdes, 2004).

An interaction between APP and Numb has been demonstrated by Roncarati (2002). In mouse brain lysates as well as in cell culture, APP or APP.ICD bind to all four isoforms of Numb and to Numb-like. Surprisingly, in this study, the processing of APP and the release of the ICD of APP resulted in an inhibition of Notch signaling. Numb is a negative regulator of Notch signaling and binds directly via its PTB domain to Notch. Therefore, a direct interaction between APP and the PTB domain of Numb should result in an increase rather than in a decrease of Notch activation. From the known crystal structure of PTB-NPTY interactions, a trimeric complex between Notch, APP and Numb seems unlikely. In this study, the induced Notch gain-of-function phenotypes, the strong genetic interaction, the dependence of the asymmetric localization of APPL on Numb and the direct binding between APP and Numb support a crosstalk between Notch signaling and APP-family members. One explanation for the APP induced Notch gain-of-function phenotypes during mechano-sensory organ (MSO) development would indeed be the sequestration and inactivation of Numb by APP-family members. However, several lines of evidence are provided that (if APP competes with Notch for the binding to Numb) suggest this binding and competition must be highly regulated and requires factors which have not previously been known to be involved in MSO development (Merdes, 2004).

(1) Expression of the human APP-family proteins induces cell fate transformations during MSO development in a dosage- and construct-dependent manner, but the potency in phenotype induction of the different proteins does not correlate with their in vitro and in vivo binding affinity to Numb. Nevertheless, the NPTY motif proves to be essential both for binding to Numb and phenotype induction, suggesting that the binding to Numb might be necessary but not sufficient for phenotype induction. This implies that there is at least one additional factor which plays an important role and which must have different affinities to the APP-family members than Numb, for example, strong binding to APLP2 but weak binding to APP.
(2) Deletion of the ECD of APP results in an inactive molecule, which can no longer induce any phenotypes. This stands in contrast to all in vitro binding studies that have been performed between the NPTY motif of APP and PTB-containing proteins in cell culture. In these assays, the affinity of such a molecule to Fe65, Dab-1/2, X11L, Numb and Numb-like did not change significantly.
(3) APP molecules with a deletion of the NPTY motif could suppress the phenotypes induced by wt APP and induce the loss of macrochaete in wt flies. Such a dominant-negative effect can only be explained if APP-family members have a receptor-like function. In this scenario, APP.DeltaNPTY would compete with wt APP or APPL for ligand binding, but could not relay the 'signal', for example, crosstalk to Notch and/or inactivating Numb. Another possibility would be the necessity of homodimer formation. Such a dimer formation has been postulated, but so far no in vivo data are available. Furthermore, structural data do not provide any evidence for a dimerization of APP molecules prior to the binding of PTB-containing proteins.
(4) Overexpression of Drosophila APPL induces only very weak phenotypes, whereas the overexpression of induces very strong phenotypes. The difference in phenotype induction could not be correlated with significant differences in expression levels, metabolism or processing. This was surprising, since had been generated to impair secretion and therefore processing. As a consequence, it is postulated that the 33 aa deletion in changes the conformation of the ECD, confirming again the important role the ECD plays in determining the potency of the APP-family members for interference with PNS development.
(5) Overexpression of APLP2 results in bold patches, suggesting that presumptive SOPs are transformed into epidermal cells by the induction of a Notch gain-of-function phenotype very early during MSO development. This step during PNS development is known to be independent of Numb and functions via the lateral inhibition mechanism, indicating that APP-family members can also interact with Numb-independent Notch signaling processes. During these processes, so far unknown factors might take over the role of Numb as a negative regulator of Notch to add an additional level of control to the system. From the literature, it seems to be clear that endocytosis is important for inhibition and for the promotion of Notch signaling, but almost nothing is known about the factors directly involved in these events.
(6) Ectopically expressed APPL and as well as APP and APP/APLP2 are asymmetrically localized during MSO development and co-localization and co-immunoprecipitation with Pon has been be demonstrated in vivo. This is an interesting result since APPL and APP induce only weak phenotypes, but and APP/APLP2 induce very strong phenotypes. Nevertheless, both types of proteins are recognized with the same efficiency by the Numb-dependent machinery responsible for the asymmetric distribution of factors during MSO development, thus completely uncoupling this event from phenotype induction. This implies that the phenotype induction occurs after completion of the separation of the SOP siblings and that APP, even if it binds to Numb, does not compete with other binding partners of Numb for asymmetric segregation (Merdes, 2004).

During MSO development, the asymmetric distribution of Numb ensures that the siblings arising from one mother cell show a difference in response to the activation of the Notch receptor. Numb is responsible for the asymmetric segregation of α-adaptin and binds both the ICD of Notch and α-adaptin, suggesting that Numb may regulate Notch by controlled endocytosis. The difference in response to Notch signaling is further amplified by the asymmetric localization of the E3 ubiquitin ligase Neuralized, which upregulates the endocytosis of the Notch ligand Delta. However, one has to take into account that it has also been reported that Numb can (1) bind the ICD of Notch after release, (2) inhibit the ability of this ICD to cause nuclear translocation of Su(H) and (3) can inhibit Notch signaling during wing development by ectopic misexpression. Therefore, even if it is very tempting to suggest that Numb solely regulates Notch by endocytotic mechanisms, there might still be other Numb functions (Merdes, 2004).

Nevertheless, more and more evidence is emerging that regulated endocytosis is an important general feature for the modulation of developmental signals. In this respect, it is especially intriguing that Drosophila Dab has been identified as an essential factor for the interaction of APP with Notch signaling. Whereas the overexpression of Dab enhances the phenotype induced by APP, a reduction of the endogenous protein level by RNAi suppresses the phenotype. Notch gain-of-function phenotypes during MSO development can be induced by expression of high levels of Dab alone. This is remarkable since it has been proposed that the mammalian Dab-2 homologs belong to a family of cargo-specific adaptor proteins, which, like Numb and β-Arrestin, regulate cargo selection and pit formation. Accordingly, APP molecules could induce the observed phenotypes during PNS development, influencing endocytosis and processing of Notch with the help of Dab. A function for APP as endocytotic receptor is supported by the finding that full-length APP is internalized via clathrin-coated vesicles. Furthermore, a direct interaction between Drosophila Dab and Notch has been demonstated previously (Giniger, 1998). These binding studies have been reproduced, but the binding of Dab to Notch in vitro was shown to be very weak in comparison to the binding affinity of Su(H) or Numb. However, additional studies suggest not only the presence of a second Notch-binding motif within the C-terminal domain of Dab, but also reveal the presence in vivo of a complex which contains Notch and Dab in Drosophila embryos (LeGall and Giniger, personal communication to Merdes, 2004). The second binding motif could allow a direct interaction between the Notch receptor and APP mediated by Dab, and it will be of great interest to elucidate the role of Dab with respect to Notch and APP signaling in the future. A crosstalk between the APP family and Notch receptors has also been shown to take place in the mammalian system (Merdes, 2004).

Originally, mutations in the dab gene were isolated by genetic interactions with the Drosophila Abl homolog. It has recently been reported that these mutations have been erroneously attributed and that all mutations isolated as dab alleles in fact affect the nrt locus (Liebl, 2003). Nrt is a single-pass type-II transmembrane protein and belongs to the family of neuronal cell adhesion molecules (N-CAMs). Nrt mutants are viable and fertile, but its function in growth cone guidance can be revealed in combination with other N-CAM mutants. Since the originally described dab alleles were used for the first genetic studies, mutations were identified in nrt as dominant suppressors of the APP-induced phenotype and also the overexpression of Nrt itself induces very strong and very specific Notch gain-of-function phenotypes. However, genetic studies ruled out an involvement of Abl in the APP-induced phenotype. Preliminary genetic data suggest a genetic interaction between appl and nrt mutations resulting in lethality of the otherwise viable alleles. Additional experiments will be necessary both in Drosophila and vertebrates to further explore this interaction. Especially, the isolation of new mutants for Drosophila dab and appl generated in a clearly defined genetic background, and their use for genetic interactions with Notch, numb and nrt, should provide insights into the mechanisms underlying the potential functions of APP-family members in endocytosis, Notch signaling and PNS development. However, the identification of appl as a quantitative trait locus already provides evidence for a function of appl during PNS developmen (Merdes, 2004).

Although it has not been established that the binding interactions between APP, Numb and Dab are functionally important in AD, signaling pathways emanating from aberrant APP function, as it occurs in AD, may influence Dab/Numb and thus Notch activity. Also, the use of drugs to lower APP processing and Aβ production could result in altered APP functions and an interference with Notch signaling in the adult brain. As already mentioned, an interaction between APP and Numb and Numb-like in the mouse brain has been demonstrated and there is accumulating evidence for a role of the Notch signaling pathway not only in early events during cell fate specifications but also in stem cells, in already differentiated neuronal cells and in neurodegeneration in the adult vertebrate nervous system. Furthermore, the view that only the production and deposition of Aβ plays a decisive role in AD has been challenged by recent evidence from different model systems that attribute numerous functions to APP and derivatives thereof. These findings together with the current data make it likely that alterations in the processing of APP either during the onset and progression of AD or by the use of therapeutics may result in loss- as well as in gain-of-function phenotypes contributing to the disease or side effects (Merdes, 2004).

Identification of novel genes that modify phenotypes induced by Alzheimer's beta-amyloid overexpression in Drosophila

Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of beta-amyloid (Abeta) peptides, the complete network of interactions regulating Abeta metabolism and toxicity is still elusive. To address this, genetic interaction screens were conducted using transgenic Drosophila expressing Abeta and mutations were identified that affect Abeta metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Abeta effects. Several of the mutations that were identified have not been linked to Abeta toxicity before and thus constitute novel potential targets for AD intervention. These mutations were additionally tested for interactions with tau and expanded-polyglutamine overexpression and a few candidate mutations were found that may mediate common mechanisms of neurodegeneration. These data offer insight into the toxicity of Abeta and open new areas for further study into AD pathogenesis (Cao, 2008. Full text of article).

Several transcriptional regulators were identified as modifiers of Aβ phenotypes. Although Aβ is a secreted peptide, it has been proposed that intracellular Aβ pools may regulate the p53 gene. Several studies have also reported RNA expression differences in a variety of models that are associated with overexpression of Aβ peptides, ranging from lymphocytes or other cell types from AD patients to brains from APP transgenic mice and Caenorhabditis elegans expressing Aβ. These studies have proposed that Aβ expression causes misregulation of stress-response genes, cytoskeletal components, DNA repair, and transcription. On the basis of the wide range of pathways that are responsive to overexpression of Aβ, it seems reasonable to propose that Aβ may directly or indirectly titer out components of global regulatory networks that act at the chromatin level, such as the Sin3A complex (see Drosophila Sin3A). Further reduction of the function of these complexes caused by mutations in their components would be expected to enhance the Aβ effects, which is what was observed in these experiments (Cao, 2008).

Throughout its production, intracellular trafficking, and turnover, Aβ is associated with parts of the secretory pathway, making this cellular compartment critical for Aβ function and ultimately the development of Alzheimer's disease. In the current studies carboxypeptidase D, which belongs to a family of enzymes that proteolytically process peptides in the secretory pathway, was identified as a modifier of Aβ toxicity. Previous reports have identified carboxypeptidase B as an enzyme that may degrade Aβ intracellularly by processing its carboxy terminus. The C terminus of Aβ plays an important role in the formation of Aβ fibrils, suggesting that the processing by carboxypeptidases may have an important function in the toxicity of Aβ. In support of this, it was found that in the presence of the loss-of-function mutants, the levels of soluble Aβ were higher and in one of them, (svrKG02090), the difference was statistically significant. Thus, a possible cellular mechanism that is recruited to compensate for Aβ toxicity is carboxy-terminal proteolytic processing that reduces the concentration of fibril formation-competent Aβ species (Cao, 2008).

The amyloid cascade hypothesis considers that accumulation of Aβ is the primary event in triggering Alzheimer's pathology. Consequently, approaches toward the development of disease-modifying therapies have focused on halting the production of Aβ or lowering its levels in the brain. This can be achieved either by manipulating the activity of Aβ-specific proteases or by administering passive or active immunization against Aβ. Great effort is invested in exploring these approaches but their implementation is still at the experimental stage (Cao, 2008).

Although high levels of Aβ are responsible for its neurotoxic properties, a few recent studies have indicated that it is possible to ameliorate its toxic effects by pharmacological approaches that do not change the overall levels of the peptide. Some of these treatments address the inflammatory response that is known to be elicited by Aβ overexpression. Administration of minocycline, an anti-inflammatory drug, did not change the levels of total, soluble, or insoluble Aβ, while ameliorating behavioral deficits in transgenic mice. Similarly, treatment with dithiocarbamates, which can inhibit the preinflammatory agent NFkB, also improved AD pathology without affecting Aβ burden. In addition to anti-inflammatory treatments, the cholesterol-lowering drug simvastatin induced cognitive improvement without changing levels of Aβ in transgenic mice. All of these studies indicate that it is possible to change the toxic effects of Aβ without lowering the levels of the peptide. Supported by these findings, the modifiers of Aβ phenotypes that were identified in this genetic interaction analyses either left total levels of Aβ unchanged or caused a 20%-30% change. Since this small change in levels of total Aβ did not always correlate with the type of phenotypic modification that the mutation caused, it is hypothesized that these changes of total Aβ do not account for the modification of the Aβ phenotype. In contrast, four modifiers caused significant changes in the levels of soluble Aβ. Of those, the mutants in the silver and Snfγ genes increased the levels of soluble Aβ while causing an enhancement of the Aβ phenotype. These results suggest that an increase of the levels of soluble Aβ, possibly inside the cell, could account for the more severe Aβ phenotype. Similarly, the mutation EP(3)3348, which is likely to affect the SAP130 transcriptional coregulator, reduced levels of soluble Aβ, while suppressing the phenotype. These results indicate that soluble intracellular Aβ may contribute to Aβ toxicity. The effects of two additional modifiers, EP(X)0514g and EP(3)3603, are more difficult to explain, since the change in the levels of soluble Aβ does not correlate with the modification of the Aβ phenotype. Histological analyses in these flies might help in the understanding of the mechanism of these particular interactions. In conclusion, it is proposed that small changes (20-30%) in levels of Aβ may not be causative in the modification of Aβ phenotypes. Rather, changes in solubility or subcellular sequestering of Aβ, or interactions with downstream effectors of Aβ toxicity, are possible mechanisms of action of the modifiers that were identified (Cao, 2008).

Although significant advances have been made toward understanding the production of Aβ peptides, less is known about the mechanisms that regulate its turnover or how it becomes toxic to the cells. Analogous mechanisms regulating toxicity of other neurodegenerative agents such as tau- and polyGlu-containing proteins are also less well understood. Neurodegeneration is in general considered an 'aggregation disease' and is associated with elevated levels of peptides or proteins. The induction of proteolytic pathways is a consequence of such accumulation and is considered a natural protective response of the cell. It can be envisioned that a common 'aggregation sensor' operates within cells, identifying the abnormal accumulation of proteins or peptides and activating the ubiquitination machinery. Specific reverse genetic screens using ubiquitination pathway components will help discover on the existence of common ubiquitination responses in neurodegenerative conditions (Cao, 2008).

Despite the common phenotype of protein aggregation, the accumulating proteins and peptides in different neurodegenerative conditions are occupying distinct subcellular localizations. Biogenesis and metabolism of Aβ peptides is closely linked to the various compartments of the secretory pathway, whereas tau is a cytoplasmic protein that associates with the cytoskeleton and was recently also shown to interact with actin. Proteins that contain extended polyglutamine stretches are found normally in the cytoplasm but upon polyglutamine expansion they are translocated to the cell nucleus where they interfere with transcriptional functions. Given the diverse localization of these aggregates, it is challenging to propose common mechanisms of toxicity. This study suggests that several processes, including ubiquitination, vesicular transport, and chromatin regulation, may be common in either mediating or confining toxicity in neurodegenerative conditions. It can be envisioned that such involvement can result either as a primary or as a secondary response. Accumulation of extra Aβ peptides in secretory compartments might affect vesicular trafficking, which might subsequently turn on ubiquitination. In the case of cytoplasmically localized toxic agents, ubiquitination might be induced first, followed by disruption of the secretory pathway and possibly of neurotransmission. Interfering with ubiquitination might result in misregulation of chromatin structure (Cao, 2008).

Although Drosophila models of neurodegeneration are faithfully reproducing several aspects of the human condition, they do not fully reflect the complexity and cell-type specificity of the human brain. This extra level of diversity can account for the differences in symptoms, which correspond to the biochemical pathways that are affected in each case. However, as shown in this study, Drosophila is instrumental in guiding the identification, through unbiased genetic screens, of pathways involved in neurodegeneration and in comparing these pathways among models of different diseases (Cao, 2008).

Activation of JNK signaling mediates amyloid-β-dependent cell death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aβ42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood. This study employed a Drosophila eye model of AD to study how Aβ42 causes cell death. Misexpression of higher levels of Aβ42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. Blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aβ42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aβ42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aβ42 retina as compared to the wild-type retina. When both caspases and JNK signaling were blocked simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone. These data suggests that (1) accumulation of Aβ42 plaques induces JNK signaling in neurons and (2) induction of JNK contributes to Aβ42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies (Tare, 2011).

One of the characteristic features of neurodegenerative disorders like AD and Parkinson disease (PD) is the late onset of neuropathology due to aberrant cellular homeostasis probably due to misregulation of several signaling pathways involved in growth, patterning and survival. Thus, it is apparent that these neurodegenerative disorders are not due to a single gene mutation but a cumulative outcome of impairment of a large spectrum of signaling pathways. Therefore, in order to understand the complexity of the human disorders and to develop therapeutic approaches, it is important to discern the role of various signaling pathways in the neuropathology caused by Aβ42-plaques. This evident complexity is one of the reasons why neurodegenerative diseases are so difficult to understand and treat. The goal of this study was to tease out the role of the cell death pathways in Aβ42 neurotoxicity. It has been known for some time that high levels of Aβ42 result in small and disorganized phenotypes of eyes that contain thin retinas with poorly differentiated photoreceptors. This small eye suggests that Aβ42 induces extensive cell death in the developing eye. To understand when the cell death occurs, how the maturation of photorecepotors is affected by the presence of Aβ42 was studied (Tare, 2011).

The highly versatile model of Drosophila eye was employed to understand the role of signaling pathways involved in cell death in Aβ42-plaque mediated neuropathology. Since the eye is dispensible for the survival of fly, the transgenic Drosophila eye model is ideal for these studies since it is possible to assay the effects throughout eye development without killing the fly. The data suggest that neurodegeneration in the fly retina can be triggered as early as third instar eye imaginal disc using GMR-Gal4 driver mediated misexpression of Aβ42 (GMR>Aβ42), which is only a few hours after Aβ42 expression starts in the developing eye field. It was also found that even though cell death is induced as early as the third instar eye imaginal disc, the morphology of the developing eye field does not dramatically differ between the wild type eye versus the GMR>Aβ42. At this time the toxicity of Aβ42 is only apparent at the level of cell membranes, which shows minor effects on cell arrangement. However, the number of the dying cells shows dramatic increase in GMR>Aβ42 eye imaginal disc as compared to the wild-type eye imaginal disc. Thus, genetic programming that triggers the onset of Aβ42-plaque mediated neurodegeneration is activated soon after the onset of misexpression of Aβ42 in the developing retina. Therefore, the experiments to demonstrate rescue of neurodegeneration phenotype should take this time window into consideration (Tare, 2011).

The larval eye imaginal disc metamorphose into the prepupal retina, which shows clumping of photoreceptor clusters, an indication that photoreceptor specification and signaling are aberrant. The clumping phenotype is caused by fusion of photopreceptor neurons and results in loss of ommatidial cluster integrity. Despite these changes at the photoreceptor neurons level, the outline of the pupal retina shows subtle effects. In the late pupal retina, the size of the retina begins to reduce as the severity of the phenotypes increases at this stage. In the late pupal stage, the retina contains holes due to loss of photoreceptors. The outcome of this cellular aberrations in the eye leads to a small adult eye with glazed appearance and fused ommatidia. Thus, extensive cell death is responsible for some of the phenotypes observed in the adult eye expressing Aβ42. Not surprisingly, the neurodegenerative phenotypes exhibited by Aβ42-plaque are age and dose dependent. Since the Gal4-UAS system is temperature sensitive, it serves as an excellent source to test the dose dependence. The cultures reared at 25°C showed less severe phenotypes as compared to the ones reared at 29°C. Furthermore, the severity of phenotypes increased with the age (Tare, 2011).

It was asked which pathways mediate the extensive cell death induced by Aβ42. The idea was to test the caspase-dependent pathway since the majority of cell death is triggered by activation of caspase-dependent cell death in tissues. To demonstrate the role of caspases in Aβ42-mediated cell death, it was shown that the misexpression of baculovirus P35 protein, significantly reduce the number of TUNEL-positive cells in the larval eye disc. Interestingly, unlike the larval eye disc, the adult eyes did not show comparable strong rescues. It seems there is block in cell death mainly during the larval eye imaginal disc development but the adult eye exhibits a weaker rescue of GMR>Aβ42 neurodegenerative phenotype. This reduction in cell death supports the possible role of caspase-mediated cell death in the small eye induced by Aβ42. However, the eye of GMR>Aβ42+P35 is reduced and disorganized (partial rescue), suggesting that other pathways contribute to Aβ42 neurotoxicity in the eye (Tare, 2011).

JNK-mediated caspase-independent cell death also plays an important role in tissue homeostasis during development. JNK signaling, a family of multifunctional signaling molecules, is activated in response to a range of cellular stress signals and is a potent inducer of cell death. Consistent with this, Aβ42 activates JNK signaling in the eye imaginal disc as indicated by the transcriptional regulation of puc and Jun phosphorylation. Moreover, JNK signaling upregulation increases cell death, supporting the role of JNK in Aβ42 neurotoxicity. Conversely, blocking JNK signaling dramatically reduces cell death in larval eye imaginal disc and the resulting flies from blocking JNK signaling exhibit large and well organized eyes. Thus, it was possible to identify the JNK signaling pathway as a major contributor to cell death observed in the Aβ42 eyes. These studies also highlight that cell death response to misexpression of Aβ42-plaques is way earlier before its affect can be discernible at the morphological level. Since neurons are post-mitotic cells, they can not be replaced. Therefore, early detection of the onset of neurodegeneration is crucial. If the disease is detected later, it may only be possible to block the further loss of healthy neurons. However, the neurons lost prior to block of cell death will not be replaced. It is possible that JNK signaling activation may serve as an early bio-marker for Aβ42 plaque mediated neuropathology. Thus, members of JNK signaling pathway can serve as excellent biomarkers or targets for the therapeutic approaches (Tare, 2011).

Blocking JNK signaling significantly rescued the neurodegenerative phenotypes but the eyes still show subtle signs of Aβ42 in the disorganization of the lattice. Therefore, both caspase dependent cell death and JNK signaling were blocked in fly retina misexpressing Aβ42. Blocking both caspase and JNK pathways simultaneously produced the protection against Aβ42, suggesting that Aβ42 induces cell death by several mechanisms. The results suggest that blocking multiple pathways may result in significant protection against Aβ42 neurotoxicity, an important consideration for potential AD therapies (Tare, 2011).

JNK signaling pathway has been known to be involved in different processes of ageing and development, including tissue homeostasis, cell proliferation, cell survival and innate immune response. Interestingly, evidence collected in several models of AD supports the involvement of JNK signaling in AD. Consistent with the observations of this study, Aβ42 induces JNK activation in primary cultures of rat cortical neurons. Also, the kinase activity of JNK phosphorylates Tau in vitro, thus contributing to the production of hyperphosphorylated Tau, one of the key toxic molecules in AD. Moreover, inhibition of JNK with peptides prevented cell loss in an Tg2576; PS1M146L brain slice model. Additionally, it has been shown that the neuroprotective effect of the diabetes drug rosiglitazone inhibits JNK and results in reduced Tau phosphorylation in rats and mice. The current results support these findings in mammalian models of AD, and provide the first evidence that direct manipulation of JNK activity modulates Aβ42 neurotoxicity in vivo. Despite this evidence, JNK is currently not a major pathway in AD research. These results suggest that more attention should be paid to the role of JNK in AD pathogenesis and its potential as a therapeutic target and biomarker. In fact, the protective activity of JNK may not be limited to AD, as JNK inhibition may show beneficial effects in other diseases, including PD, stroke and others (Tare, 2011).

beta amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons

beta amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease, was found in a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development. Appl expression was found in the eye imaginal disc and it is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, Appl null mutants were analyzed and reduced preference for UV light was found, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, whether Ras1/MAPK is responsible for the high Appl expression was examined. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. Appl was shown to be directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism (Mora, 2013).

Two main conclusions can be drawn from this work. First, Drosophila Appl is involved in R7 axonal targeting. Moreover, the finding that the Appl loss-of-function defects are enhanced when combined with Nrt heterozygous mutant suggest that Appl acts at the membrane of R7, where it interacts with other proteins such as Nrt. Second, Appl activation downstream of the RTK/Ras1 is independent of neural specification, occurs in vivo, and is mediated by direct binding of PntP2 to ETS sequences in the Appl regulatory region (Mora, 2013).

Together, these findings may provide insights into the pathogenesis of neurological disorders such as Alzheimer's disease. The β-amyloid peptides, which accumulate in the amyloid plaques found in the brain of Alzheimer's disease patients, are produced after APP proteolysis. However, Alzheimer's disease has not only been associated to the production of the primary component Aβ by proteolysis of APP, but also by transcriptional regulation. Increased APP transcription underlies the phenotype in some cases of familial Alzheimer's disease. In addition, overexpression of APP appears to be responsible for the early onset of Alzheimer's disease in individuals with Down syndrome. Thus, the current results open the possibility to explore whether in some cases of Alzheimer's disease a burst of RTK/Ras1/MAPK occurs and whether this signaling activity ends with high APP accumulation (Mora, 2013).

Amyloid β peptides are known to be involved in vision dysfunction caused by age-related retinal degeneration in mouse models. Thus, the current in vivo observations could be the basis for further research in mammalian models for neurodegenerative retinal disorders that share several pathological features with Alzheimer's disease (Mora, 2013).

Accumulation of amyloid-like Abeta1-42 in autophagy-endosomal-lysosomal (AEL) vesicles: Potential implications for plaque biogenesis

Abnormal accumulation of Amyloid beta (Abeta) within autophagy-endosomal-lysosomal (AEL) vesicles is a prominent neuropathological feature of Alzheimer's disease (AD) but the mechanism of accumulation within vesicles is not clear. This study expressed secretory forms of human Abeta1-40 or Abeta1-42 in Drosophila neurons and observed preferential localization of Abeta1-42 within AEL vesicles. In young animals, Abeta1-42 appears to associate with plasma membrane while Abeta1-40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Abeta1-40, in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Abeta1-42 accumulation. In samples from older animals, Abeta1-42 is broadly distributed within neurons but only the Abeta1-42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Abeta1-42 containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. It is proposed that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Abeta1-42 expressing Drosophila with potential relevance for AD (Ling, 2014).

Novel neuroprotective function of apical-basal polarity gene crumbs in amyloid β 42 (aβ42) mediated neurodegeneration

Alzheimer's disease (AD), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-β-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). This study has developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. An apical-basal polarity gene crumbs (crb) was identified as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. This study has demonstrated a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration (Steffensmeier, 2013).

Homeotic Gene teashirt (tsh) has a neuroprotective function in amyloid-beta 42 mediated neurodegeneration

Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Abeta42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive. A transgenic Drosophila eye model was generated where high levels of human Abeta42 was misexpressed in the differentiating photoreceptor neurons of the developing eye; this phenocopies Alzheimer's like neuropathology in the neural retina. This model was used for a gain of function screen using members of various signaling pathways involved in the development of the fly eye to identify downstream targets or modifiers of Abeta42 mediated neurodegeneration. The homeotic gene teashirt (tsh) was identified as a suppressor of the Abeta42 mediated neurodegenerative phenotype. Targeted misexpression of tsh with Abeta42 in the differentiating retina can significantly rescue neurodegeneration by blocking cell death. Tsh protein was found to be absent/downregulated in the neural retina at this stage. The structure function analysis revealed that the PLDLS domain of Tsh acts as an inhibitor of the neuroprotective function of tsh in the Drosophila eye model. Lastly, the tsh paralog, tiptop (tio) can also rescue Abeta42 mediated neurodegeneration. This study has identified tsh and tio as new genetic modifiers of Abeta42 mediated neurodegeneration. These studies demonstrate a novel neuroprotective function of tsh and its paralog tio in Abeta42 mediated neurodegeneration. The neuroprotective function of tsh is independent of its role in retinal determination (Moran, 2013).

Presynaptic Aβ40 prevents synapse addition in the adult Drosophila neuromuscular junction

Complexity in the processing of the Amyloid Precursor Protein, which generates a mixture of βamyloid peptides, lies beneath the difficulty in understanding the etiology of Alzheimer's disease. Moreover, whether Aβ peptides have any physiological role in neurons is an unresolved question. By expressing single, defined Aβ peptides in Drosophila, specific effects can be discriminated in vivo. This study shows that in the adult neuromuscular junction (NMJ), presynaptic expression of Aβ40 hinders the synaptic addition that normally occurs in adults, yielding NMJs with an invariable number of active zones at all ages tested. A similar trend is observed for Aβ42 at young ages, but net synaptic loss occurs at older ages in NMJs expressing this amyloid species. In contrast, Aβ42arc produces net synaptic loss at all ages tested, although age-dependent synaptic variations are maintained. Inhibition of the PI3K synaptogenic pathway may mediate some of these effects, because western analyses show that Aβ peptides block activation of this pathway, and Aβ species-specific synaptotoxic effects persists in NMJs overgrown by over-expression of PI3K. Finally, individual Aβ effects are also observed when toxicity is examined by quantifying neurodegeneration and survival. These results suggest a physiological effect of Aβ40 in synaptic plasticity, and imply different toxic mechanisms for each peptide species (Lopez-Arias, 2017).

Synapse dysfunction and loss are key to dementia in Alzheimer's disease (AD). It is currently established that amyloid-β peptides are important contributors to these synaptic alterations, but the precise identity of the Aβ species involved is not well defined. Likewise, it is unclear how aging, the most influential non-genetic risk factor in AD, influences Aβ synaptotoxicity. These are relevant questions to designing suitable therapeutic strategies. It was postulated that the Drosophila adult glutamaergic neuromuscular junction (NMJ) would provide an ideal system in which to address these issues, because its stereotypic morphology would allow uncovering subtle albeit relevant changes. Moreover, this work has revealed a temporal biphasic process of synaptic remodeling at the adult NMJ. An early phase of net synapse addition takes place during the first two weeks of adult life, a critical time period in which several areas of the young fly brain have been shown to undergo experience-dependent structural plasticity. Thenceforth, net synaptic elimination occurs, consistent with the onset of behavioural and synaptic senescence. Thus, this model allows assessing Aβ influence on synaptic dynamics during synaptic maturation and aging. This study shows that: (1) Aβ40 seems to reduce the number of synaptic contacts by preventing synapse addition; (2) Aβ42 synaptotoxicity gradually increases with age; and (3) Aβ42arc produces net synaptic loss from early adulthood, but does not impede synapse addition. In summary, this study demonstrates specific age-dependent synaptic effects for each Aβ peptide, and establish the fly adult NMJ as a suitable model to investigate the mechanisms underlying these peculiarities (Lopez-Arias, 2017).

Studies in Drosophila had shown early memory defects induced by expression of both Aβ40 and Aβ42-derived peptides, but a structural and/or functional synaptic correlate had only been found for Aβ42-derived peptides. This study shows for the first time that Aβ40 reduces the number of synaptic contacts, and it seems to do so by preventing the addition of new synapses that normally occurs in the adult NMJ. At young ages (3-15 days), Aβ42 effect was remarkably similar to Aβ40, which suggests that early in life, Aβ42 may act similarly to Aβ40 by opposing synapse addition. However, Aβ42-expressing brains had the lowest amount of total peptide, which might explain these relatively mild early effects. In contrast, the mutant amyloid peptide Aβ42arc elicited net synaptic loss also at young ages, yet it showed a pattern of age-dependent variation in synapse number similar to controls, indicating that it does not hinder synapse addition. These are significant qualitative differences that point to a physiological role for wild type amyloid species in the process of synaptic plasticity (Lopez-Arias, 2017).

In general, exposure to Aβ is associated with weakening of synapses, which is consistent with a postulated Aβ function in promoting activity-dependent synaptic elimination during mammalian postnatal development. The current data suggest that rather than eliciting synapse removal, Aβ40, and possibly Aβ42, prevents the formation and/or maturation of new synapses in Drosophila. Interestingly, early defects in ocular dominance plasticity in APPswe transgenic mice, which generate elevated levels of wild type amyloid species, are associated with reduced strengthening and expansion of non-deprived eye cortical representations, while deprived eye weakening remains intact. These data argue in favor of a similar effect for Aβ at synapses from juvenile mice and flies. In the Drosophila larval NMJ, the transition from immature to mature synapse involves changes in the relative contribution of specific glutamate receptors in postsynaptic receptor fields opposed to presynaptic active zones. In mammals, synaptic elimination and maturation also involves changes in expression and trafficking of AMPA and NMDA glutamate receptors. The model described in this work represents an invaluable tool for genetically assessing the contribution of Glutamate receptors, and other molecules, in the amyloid-dependent alterations of synaptic refinement (Lopez-Arias, 2017).

Although a mechanistic basis for the specific synaptotoxic activities of each Aβ species cannot be provided, the data on NMJs overexpressing PI3K suggest the direct implication of this pathway. First, despite its strong synaptogenic activity, PI3K over expression was not able to block Aβ-induced synaptic reduction. Second, western quantification showed that the activity of the pathway is reduced by Aβ expression, which is consistent with data from mammalian systems that suggest that amyloid peptides block Akt activation downstream of the PI3K enzyme, and increase GSK3 activity. Moreover, the degree of PI3K pathway inhibition correlated with the extent of synaptic reduction, both being maximum for Aβ42arc. Altogether, evidences point to a direct relationship between Aβ and the PI3K/Akt/GSK3 pathway as central to Aβ-induced synaptic dysfunction, but further studies would be necessary to unambiguously prove it. The importance of this matter is underscored by the relevance of GSK3 in AD, which has been used as a therapeutic target (Lopez-Arias, 2017).

The risk of AD increases with age, but the link between aging and Aβ toxicity is not fully understood. This study has shown that after a stage of synaptic growth, synaptic elimination commences in the fly NMJ sometime between 15 and 20 days after eclosion. This age also represents a point of transition from growth to retraction for the abdominal longitudinal NMJ, and thus defines an age-dependent change in synaptic dynamics. The data suggest that this age also delimits a change in the synaptic action of Aβ42, which would oppose synapse addition in early adulthood, but cause synaptic removal in aged flies. In contrast, Aβ42arc induces net synaptic loss at a similar rate at all ages tested, while Aβ40 maintains an unvarying number of active sites at least until 30 days. These data suggest that the impact of age on Aβ synaptotoxicity differs for each amyloid species. Understanding the mechanisms underlying age-dependent synaptic changes in wild type adult NMJs will be necessary for explaining the observed effects (Lopez-Arias, 2017).

Several in vitro and in vivo studies have demonstrated different aggregation kinetics for the three Aβ species, which result in higher effective concentrations of specific aggregation forms with diverse toxic activities. Specifically, a recent in vivo study expressing tandem dimeric Aβ peptides in Drosophila has shown that the aggregation kinetics of Aβ42 favors a relatively high population of toxic oligomeric species, while this population is undetectable for Aβ40, which seems to more rapidly transit from monomeric to insoluble inert forms. Thus, it is tempting to speculate that Aβ monomeric forms inhibit synapse addition, while oligomeric forms promote synapse removal. Assessing the synaptic effects of the various tandem dimeric Aβ peptide on the Drosophila adult NMJ would provide invaluable data to test this hypothesis (Lopez-Arias, 2017).

Multiple studies in Drosophila, and in other models, suggest that the mechanisms underlying Aβ synaptotoxicity, cytotoxicity, and reduction of life span are different. Even more, in vivo data demonstrate that manipulations that alter aggregation propensity can induce qualitative, rather than quantitative, shifts in the pathology induced. The current data provide further support to this notion. First, Aβ40 was shown to disturb synapses, but not cell survival or life span. Second, neurodegeneration levels were similar in 20 day-old flies expressing either Aβ42 or Aβ42arc, yet both genotypes displayed dramatically different life expectancy. Third, over expression of PI3K reduced early Aβ42 cytotoxicity, measured by degree of neurodegeneration, but it enhanced the deleterious effects of this peptide on life span (Lopez-Arias, 2017).

The differential effect of PI3K hyperactivation on Aβ-related phenotypic outcomes is intriguing. PI3K is an essential signaling pathway with multiple developmental and physiological functions; these include widespread functions such as regulation of cell proliferation and metabolism, or control of cellular remodeling and migration, but also cell-type specific roles such as synapse plasticity in neurons. Moreover, different levels of pathway activation seems to trigger distinct responses. In view of this complexity, it can only be speculated on how PI3K alters Aβ-induced phenotypes. The data show that PI3K overexpression had no influence on Aβ42arc-related phenotypes, a finding that might well be explained by the significant reduction in the activity of the PI3K pathway displayed by Aβ42arc-expressing flies. In contrast, the observed reduced neurodegeneration in flies co-expressing Aβ42 and PI3K could reflect residual hyper-activation of the pro-survival PI3K pathway, as suggested by western quantification. This hypothesis is consistent with studies in mice which suggest that low levels of Akt activity are sufficient to support neuronal survival responses. However, PI3K over-expression had a negative effect on life span in flies expressing Aβ42. Lifespan is an extremely complex trait which may be particularly sensitive to unbalanced conditions, and age-dependent accumulation of toxic amyloid aggregates might further disturb PI3K and related signaling networks, advancing deterioration. These data underscore the complexity of Aβ toxicity and the necessity to test it at different levels when assessing the consequences of therapeutic approaches (Lopez-Arias, 2017).

In summary, using an in vivo model this study demonstrates that (1) different amyloid species disturb synapses by differentially influencing synapse addition or synapse elimination, (2) age-dependent changes in their synaptotoxicity are species-specific, and (3) the toxic actions of each Aβ peptide differ in different contexts. Furthermore, this work demonstrates the value of the Drosophila adult NMJ as an ideal in vivo model for understanding specific effects of amyloid peptides on synaptic plasticity (Lopez-Arias, 2017).

Protein Interactions

Ligand binding to receptors of the LIN-12/Notch family causes at least two proteolytic cleavages: one between the extracellular and transmembrane domains, and the other within the transmembrane domain. The transmembrane cleavage depends on Presenilin, a protein also required for transmembrane cleavage of beta-APP. The substrate requirements for Presenilin-dependent processing of Notch and other type I transmembrane proteins in vivo has been assayed. Presenilin-dependent cleavage does not depend critically on the recognition of particular sequences in these proteins but rather on the size of the extracellular domain: the smaller the size, the greater the efficiency of cleavage. Hence, Notch, beta-APP, and perhaps other proteins may be targeted for Presenilin-mediated transmembrane cleavage by upstream processing events that sever the extracellular domain from the rest of the protein (Struhl, 2000).

Evidence suggests that Presenilin is a component of a general mechanism that cleaves type I transmembrane proteins in the transmembrane domain, provided that they have a relatively small extracellular domain. Little if any processing occurs when the extracellular domain is greater than 200-300 amino acids. However, as the size of the extracellular domain is reduced incrementally, progressive increases in the efficiency of processing is attained, with proteins having very small extracellular domains (<50 amino acids) exhibiting similar, if not higher, levels of processing to those of full-length Notch in response to ligand. These results support the view that ligand activates LIN-12/Notch proteins by inducing a cleavage of the extracellular domain close to the membrane. Consistent with this proposal, recent biochemical studies indicate that mammalian Notch proteins undergo just such a cleavage event in response to ligand. Although this cleavage could activate the receptor in a number of different ways, these findings indicate that the resulting reduction in the size of the extracellular domain should suffice to convert the remainder of the protein into a substrate for Presenilin-dependent cleavage. Hence, the hypothesis is favored that ligand activates Notch by severing the extracellular domain from the rest of the receptor, a process described as 'ectodomain shedding' for other transmembrane proteins (Struhl, 2000).

Presenilin-dependent processing of betaAPP provides a second example of a possible link between ectodomain shedding and Presenilin-dependent cleavage. betaAPP initially contains a large extracellular domain of approximately 600 amino acids, and the full-length protein is not believed to be a substrate for Presenilin-dependent cleavage. However, full-length betaAPP is a target for cleavage by beta-secretase, a transmembrane aspartyl protease, which cuts at a site around 25 amino acids amino-terminal to the transmembrane domain. This initial cleavage is thought to be responsible for shedding the extracellular domain and for rendering the transmembrane domain susceptible to the Presenilin-associated gamma-secretase activity (Struhl, 2000).

The finding that proteins with diverse transmembrane domains can all be processed in a Presenilin-dependent fashion provided that the extracellular domain is small raises the possibility that such transmembrane cleavages can be viewed as relatively general and indiscriminate scavenging events that allow a cell to clear residual, truncated proteins from the membrane. Such a role could account for the transmembrane cleavages that generate the beta-amyloid peptides, which have no known function in normal cell physiology. However, in the case of LIN-12/Notch receptors, it appears that this cleavage mechanism has been incorporated as a critical step in signal transduction (Struhl, 2000).

Presenilin-dependent cleavage has been implicated in transduction of the unfolded protein response (UPR), which depends on the release and nuclear import of the cytosolic domain of the UPR receptor. Hence, activation of the UPR receptor, like that of Notch, may depend on processing events that cause ectodomain shedding and thereby target the remainder of the receptor for Presenilin-dependent cleavage. It is suggested that LIN-12/Notch proteins and the UPR receptors may belong to a general class of receptors that are activated by ectodomain shedding and which transduce signals by a mechanism involving Presenilin-dependent release of the intracellular domain from the rest of the receptor. It is possible that beta-APP also belongs to this class of receptors, since there is evidence that the intracellular domain of beta-APP interacts via an adaptor protein with a transcription factor (Struhl, 2000).

The mechanism by which ligand might induce dissociation of the Notch extracellular domain from the rest of the protein remains uncertain. Evidence has been found that the Presenilin-dependent cleavage of full-length Notch does not occur in shibire mutant embryos, which are defective in endocytosis due to reduced activity of Dynamin. In contrast, truncated forms of Notch that lack virtually the entire extracellular domain appear to be cleaved in these embryos. These results indicate that the Presenilin-dependent cleavage is not inherently dependent on endocytosis. Instead, endocytosis may be required for upstream events that are necessary to shed the ectodomain and hence to target the rest of the receptor for transmembrane cleavage. For example, endocytosis of the transmembrane ligand Delta in the signaling cell bound to Notch on the receiving cell might expose the Notch extracellular domain to cleavage. Alternatively, Notch may undergo extracellular processing in response to Delta while both proteins are on the cell suface, but endocytosis by the receiving cell might be required to dissociate the cleaved ectodomain from the rest of the receptor (Struhl, 2000).

In mammals, Notch proteins are cleaved at an extracellular Furin site (termed S1) close to the transmembrane domain during their trafficking to the cell membrane. As a consequence, the mature receptor is a heterodimer composed of two components: (1) a large extracellular domain and (2) the remainder of the receptor consisting of a short extracellular stub, the transmembrane domain, and the intracellular domain. In principle, interactions with ligand could activate the receptor by disrupting the association between these two components, causing the ectodomain to be separated from the rest of the protein by displacement rather than by proteolysis. Alternatively, ligand might induce shedding by triggering cleavage at a second site (S2) between the Furin cleavage and the transmembrane domain, a possibility directly supported by biochemical studies of Notch activation in mammalian cell culture. In the case of Drosophila, there is evidence that the mature Notch protein on the cell surface is not normally processed by Furin to form a heterodimer. If the Furin-mediated S1 cleavage does not occur in Drosophila Notch, ectodomain shedding would presumably depend on a ligand-induced S2 cleavage in order to convert the receptor into a substrate for the transmembrane cleavage (referred to as the S3 cleavage), which requires Presenilin (Struhl, 2000).

Recent biochemical evidence in mammals suggests that the metalloprotease TACE can execute the S2 cleavage of Notch in response to ligand. Genetic data in C. elegans and Drosophila suggest that a related metalloprotease, Kuzbanian/SUP-17, is essential for LIN-12/Notch signaling. However, there are conflicting biochemical data concerning whether Kuzbanian cleaves Notch or its ligands, complicating interpretation of whether it plays a direct role in executing the S2 cleavage. The nature of the event that precipitates the S2 cleavage is not known, but genetic evidence in C. elegans raises the possibility that ligand-induced oligomerization is involved (Struhl, 2000).

One determinant of whether a protein is a substrate for Presenilin-dependent cleavage appears to be the size of the extracellular domain. How might the size of the extracellular domain be assayed by the Presenilin-dependent cleavage mechanism? One possibility is that the cleavage mechanism requires the assembly of an active processing complex in close proximity to the transmembrane domain of the substrate. Although Presenilin has been reported to associate with Notch proteins as they move from the endoplasmic reticulum to the cell surface, the presence of a large extracellular domain may interfere sterically with the assembly of the complete complex or with the proteolytic activity of the complex. Another possibility is that the cleavage mechanism recognizes a free amino terminus in close proximity to the transmembrane domain, a condition that may be more likely when the extracellular domain is small. Both of these possible mechanisms are compatible with the finding that there is a progressive decline in cleavage efficiency as the size of the extracellular domain is increased incrementally (Struhl, 2000).

A second factor appears to be the primary sequence of the transmembrane domain. Although all of the transmembane domains tested can be cleaved in a Presenilin-dependent fashion, the amount of cleavage varies. The transmembrane domains of Notch, beta-APP, and Sevenless all appear to be cleaved efficiently, whereas those of Torso, Delta, and GlycophorinA are less efficiently cleaved. Similarly, substitution or deletion of a conserved valine located immediately downstream of the likely S3 cleavage site reduces, but does not eliminate, cleavage in mammalian tissue culture, and evidence has been found in Drosophila. for a reduction in the efficiency of cleavage of such mutated or deleted forms of Notch. These findings suggest that Presenilin-dependent processing may be limited to some extent by the conformational state of the transmembrane domain, a property that is likely to depend on the primary sequence. Nevertheless, it remains striking that many different transmembrane domains, each with a distinct primary sequence, can be cleaved in a Presenilin-dependent fashion. Hence, the protease activity does not appear to require recognition of specific primary sequences (Struhl, 2000).

A third variable that appears to influence substrate specificity is the potential for oligomerization. The transmembrane domain of Glycophorin A, which dimerizes avidly in the membrane, is a relatively poor substrate, whereas a single amino acid substitution, which is expected to severely reduce dimerization of this transmembrane domain, renders it a better substrate for Presenilin-dependent cleavage. Similarly, the presence of an extracellular dimerization domain, a leucine zipper, severely reduces the efficiency of Presenilin-dependent cleavage compared to a control protein that carries a mutated and inactive zipper. Hence, the Presenilin-dependent cleavage reaction appears to work better on isolated monomeric proteins. It is not clear why oligomerization reduces the efficiency of Presenilin-dependent cleavage. One possibility is that the cleavage mechanism depends on the assembly of a protease complex that wraps around a single, isolated transmembrane domain. Another possibility is that oligomerization effectively increases the size of the extracellular domain. The inhibitory effect of oligomerization on Presenilin-dependent cleavage might also be important for stabilizing single-pass transmembrane proteins that normally have short extracellular domains, such as the zeta and eta chains of the T cell receptor CD3 signaling complex. Ligand may intially activate the receptor by inducing oligomerization, but cleavage of the ectodomain may in turn generate truncated proteins that can no longer oligomerize, helping to convert them into substrates for Presenilin-dependent cleavage (Struhl, 2000).

Finally, the amino-to-carboxyl polarity of the transmembrane domain of a protein may also govern whether it is a substrate for Presenilin-mediated cleavage. Notch, betaAPP, and the other proteins assayed are all type I transmembrane domains with amino-to-carboxyl polarity oriented in the extracellular-to-intracellular direction. In contrast, the first transmembrane domains of sterol regulatory element-binding proteins (SREBPs), which are cleaved in response to changes in sterol abundance, have the opposite polarity and do not appear to be Presenilin dependent. There is evidence that Presenilin-dependent cleavage depends on an aspartyl protease activity, perhaps Presenilin itself, and not the S2P metalloprotease, which appears responsible for the transmembrane cleavage of SREBPs. Perhaps these different proteolytic activities reflect distinct mechanisms involved in cleaving type I and type II transmembrane proteins (Struhl, 2000).

Interaction of APP family proteins with APLIP1 and its mammalian homologs, APLIP1 is a scaffold protein of the JNK signaling cascade

A novel protein has been isolated based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease (Taru, 2002; full text of article).

APPs possess a membrane-associated receptor-like structure, and the amino acid sequence of their short cytoplasmic region is highly conserved among a wide variety of species. Protein interactions between the cytoplasmic domains of APPs and cytoplasmic proteins are thought to be important for regulating the metabolism of APPs and/or for putative physiological function of APP. Drosophila APLIP1 and its putative mammalian homologs JIP1b and JIP2 can interact with the cytoplasmic domain of the APPs. These proteins, APLIP1, JIP1b, and JIP2, resemble each other in their structure, especially in their carboxyl-terminal regions that contain SH3 and PI domains. They also share properties such as interactions with APPs, MAP kinase kinase, and kinesin; an abundant expression in the nervous system; and the formation of homo-oligomers. These similarities suggest that APLIP1, JIP1b, and JIP2 belong to same protein family functionally conserved in various species. In mammals, JIP3/JSAP was reported as another member of the JIP family of proteins, which display scaffold function in the JNK signaling pathway as do JIP1 and JIP2. In Drosophila, a putative homolog of JIP3 has been designed Sunday Driver protein (SYD). However, they differ from APLIP1, JIP1b, and JIP2 in their domain structure; they do not possess the SH3 and PI domains that are important regions for binding several proteins including APPs, and they may have some different roles from APLIP1, JIP1, and JIP2 (Taru, 2002).

In mammal several proteins bind the cytoplasmic domain of APP (APPcyt), whereas the physiological role(s) of these interaction have not been sufficiently revealed. JIP1b and JIP2, mammalian counterparts of APLIP1 interact with APPcyt. Regarding JIP1b, the binding to APPcyt is relatively lower than the binding of other APP-binding proteins, such as mDab1, X11, and Fe65. It was also observed that the binding of JIP1b was slightly lower than that of the other binding protein X11L in vitro but not in the cell. However, the faint differences in the binding activities do not necessarily deny the physiological importance of JIP1b for APP. In fact, a novel function of JIP1 and JIP2 was found to be the modulation of the phosphorylation of APP at Thr-668 residue induced by the activation of JNK. Expression of JIP1b slightly enhances the phosphorylation of APP, whereas the expression of JIP2 or JIP1a suppresses the phosphorylation. From the previous reports that JIP1b, JIP1a, or JIP2 equally facilitate the activation of JNK signaling, it was expected that these proteins similarly regulate the phosphorylation of APP when JNK is activated. Nevertheless, only JIP1b facilitates the phosphorylation, and others decrease the level of the phosphorylation of APP. The interaction of JIP2 and JIP1a with APP is remarkably weaker than that of JIP1b in the cell. Therefore, it is conceivable that the effect of JIP1a or JIP2 to decrease the level of the phosphorylation of APP reflects their weaker binding properties to APP rather than their properties of regulating the JNK signaling cascade. It is assumed that formation of the complex between JIP1a or JIP2 and JNK may suppress the approach of JNK to the phosphorylation site of APP, whereas the complex of JIP1b and JNK can easily approach APPcyt. Indeed ut has been reported that formation of the tripartite complex composed of JIP1b, JNK, and APP can be observed in cultured cells. Phosphorylation of APP at Thr-668 has been implicated in the metabolism and/or putative function(s) of APP, and modulation of the phosphorylation level of APP by JIPs in mammal possibly has physiological importance (Taru, 2002).

In invertebrates only Drosophila APLIP1 and dX11L have been reported to interact with APPs except kinesin interacts genetically with APPL. They may all implicated in evolutionarily conserved roles relative to metabolism and/or function of APPs, besides the role of mammalian JIP in the phosphorylation of APP. In Drosophila, APPL does not have a phosphorylation site corresponding to the Thr-668 residue of mammalian APP695. In addition, there are some differences in the function of APLIP1 on the JNK signaling pathway from that of mammalian JIP1 and JIP2 because APLIP1 could not interact with DJNK, whereas it can interact with Drosophila JNK kinase Hep. Thus APLIP1 cannot form a complex with DJNK and facilitate JNK activation in Drosophila in the same manner as JIPs do in mammals, whereas a possibility of regulating JNK signaling through an interaction with Hep still remains. Therefore, the effect modulating the phosphorylation of APP by JIP1 and JIP2 may be acquired in the evolutionary process. Questions of what the evolutionarily conserved role(s) of the interaction of APLIP1, JIP1b, and JIP2 with APPs are remain to be elucidated. Several physiological roles for the mammalian JIP family proteins have been proposed other than acting as scaffold molecules of JNK cascades: as a transactivator of the GLUT2 gene and as cargo for kinesin to mediate the transportation of several transmembrane proteins. In Drosophila APLIP1 interacts with the kinesin light chain as well as mammalian JIP1 and JIP2 do, but interaction with the molecules of the JNK cascades is only partly conserved. The metabolic scheme of APPs is basically conserved between Drosophila and mammals. Kinesin is involved in intracellular transport and metabolism of APP in mammals and is associated with APPL in Drosophila. Accordingly, it is assumed that Drosophila APLIP1 and mammalian JIP share a role in the intracellular metabolism of APPs (Taru, 2002).

In conclusion, Drosophila APLIP1 and mammalian JIP1b and JIP2 are binding proteins of APPs. APLIP1, JIP1b, and JIP2 comprise an evolutionary conserved protein family and share properties in their domain structure, expression pattern, and interaction profiles with proteins such as APPs, kinesin, and JNK kinase, although a few exceptions are observed. It is proposed that a novel function of mammalian JIP1 and JIP2 is to modulate the phosphorylation of APP. Further analysis of conserved or different roles of APLIP1, JIP1b, and JIP2 may contribute to understanding of the mechanisms of APPs metabolism and the pathogenesis of Alzheimer's disease (Taru, 2002).

Abl, acting downstream of β-amyloid, deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration

Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. β-amyloid (Aβ42) initiates Abl kinase activity and blockade of Abl kinase rescues both Drosophila and mammalian neuronal cells from cell death. Activated Abl kinase is necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 is not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. These genetic studies also showed that abl mutations repress Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induces conversion of p35 to p25 in mammalian cells, it does not sufficiently induce Cdk5 activation when c-Abl kinase activity is suppressed. Therefore, it is proposed that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration (Lin, 2007).

Like Cdk5, cellular Abl functions in neural development and its kinase activity and subcellular localization are tightly regulated. This study shows that Abl appears to be essential for Aβ42-triggered Drosophila neurodegeneration both in vivo and in vitro. It is of interest in this regard that Abl may serve as a putative molecular target to stop the progress of neurodegeneration. Interestingly, the anti-leukemic agent Abl kinase inhibitor, STI571, has been shown to rescue the Aβ42-induced neurodegeneration in both Drosophila and mammalian cells. However, STI571 is probably not an ideal reagent for testing this idea in vivo because of its low penetration capability through the blood-brain barrier. Another previous link between Aβ42 and Abl inhibition by STI571 has been reported. Aβ42 production is reduced by STI571 in neuronal cultures and in guinea-pig brain. Therefore, it is reasonable to speculate that Abl kinases might affect amyloid signaling at various points including Aβ42 production (Lin, 2007).

A novel inhibitor of Aβ peptide aggregation: from high throughput screening to efficacy in an animal model for Alzheimer's disease

Compelling evidence indicates that aggregation of the amyloid beta peptide (Aβ) is a major underlying molecular culprit in Alzheimers disease. Specifically, soluble oligomers of the 42-residue peptide (Aβ42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aβ42 aggregation may be an effective strategy for the prevention and/or treatment of disease. This paper describes the implementation of a high- throughput screen for inhibitors of Aβ42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aβ42 aggregation and reducing Aβ42 induced toxicity in neuronal cells. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aβ42. The ability of D737 to prevent Aβ42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the lifespan and locomotive ability of flies in a Drosophila melanogaster model of Alzheimers disease (McKoy, 2010).

The high-resolution structure of the exact molecular species responsible for Aβ42 toxicity is not known. Therefore structure-based drug design is not possible, and high throughput screening remains the most powerful way to identify inhibitors of Aβ42 aggregation that may lead to therapeutics for Alzheimer’s disease. Given the growing body of evidence suggesting that soluble oligomers play a major role in neurodegeneration, it is important to utilize screens capable of identifying compounds that inhibit the formation of oligomers. This study shows that an inexpensive high throughput screen is effective in identifying compounds that not only inhibit Aβ aggregation, but also reduce toxicity in vitro and in vivo. In particular, compound D737, isolated from this screen, inhibits Aβ42 aggregation, reduces the cytotoxicity of Aβ42, and diminishes the cellular accumulation of ROS. Most importantly, D737 leads to significant improvements in both lifespan and locomotive ability in a transgenic Drosophila melanogaster model of AD (McKoy, 2010).

Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model

Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer's disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, inappropriate complex formation may contribute to AD-linked neurodegeneration by misregulation of target genes involved in neurogenesis; however, a direct and causative epigenetic based role for Tip60 HAT activity in this process during neuronal development in vivo remains unclear. This study demonstrates that nervous system specific loss of Tip60 HAT activity enhances APP mediated lethality and neuronal apoptotic cell death in the central nervous system (CNS) of a transgenic AD fly model while remarkably, overexpression of Tip60 diminishes these defects. Notably, all of these effects are dependent upon the C-terminus of APP that is required for transcriptional regulatory complex formation with Tip60. Importantly, this study shows that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. These results are the first to demonstrate a functional interaction between Tip60 and APP in mediating nervous system development and apoptotic neuronal cell death in the CNS of an AD fly model in vivo, and support a novel neuroprotective role for Tip60 HAT activity in AD neurodegenerative pathology (Pirooznia, 2012a).

This study generated a unique transgenic Drosophila model system suitable for investigating a functional link between Tip60 HAT activity and APP in neuronal development, in vivo. Tip60 and APP were shown to functionally interact in both general and nervous system development in Drosophila, in vivo and that this interaction specifically mediates apoptotic neuronal cell death in the CNS, a process that when misregulated is linked to AD pathology. Remarkably, Tip60 appears to display a neuroprotective function in that Tip60 overexpression can rescue both loss of viability and neuronal apoptosis induction in a Drosophila AD model. While a number of in vitro studies supporting the transcription regulatory role of the Tip60/AICD complex in gene control have been reported, this work is the first to demonstrate a functional interaction between Tip60 HAT activity and APP in nervous system development in vivo (Pirooznia, 2012a).

This study shows that misexpression of Tip60 induces neuronal apoptotic cell death in the Drosophila CNS, and that this process is mediated via a functional interaction between Tip60 and the APP C-terminal domain. Since disruption of Tip60 HAT activity induced neuronal cell death, this study examined whether there was specific misregulation of apoptosis linked genes due to loss of Tip60 HAT activity. Pathway analysis of a previously reported microarray data set of genome wide changes in gene expression induced in the fly in response to Tip60 HAT loss (Lorbeck, 2011) revealed genes functioning in 17 different apoptotic pathways to be enriched, many of which were associated with the p53 apoptotic pathway. These findings are consistent with previous studies demonstrating a role for Tip60 as a p53 co-activator in p53 mediated apoptotic pathways. Recent studies have found Tip60 to be required for activation of proapoptotic genes through acetylation of p53 DNA binding domain. TRAF4, one such p53 regulated pro-apoptotic gene that responds to cellular stress was one of the genes that was found to be significantly upregulated in response to Tip60 HAT loss. The Myc family of transcription factors presents another instance of proteins involved in inducing apoptosis that are directly acetylated and stabilized by Tip60 and accordingly, Drosophila dMyc was found to be significantly upregulated in response to Tip60 HAT loss. Thus it is possible that the pro-apoptotic genes enriched in the dataset may represent both direct targets regulated by Tip60 epigenetic function as well as indirect targets of apoptosis regulators such as p53 that are controlled via their acetylation by Tip60. Misregulation of these pro-apoptotic genes in response to disruption of Tip60 HAT activity is also consistent with the observation that nervous system specific expression of dTip60E431Q induces apoptotic cell death in the CNS of dTip60E431Q larvae. This finding is in contrast to previous studies wherein cells expressing mutated Tip60 lacking HAT activity were reported to be resistant to apoptosis. However, these studies examined a role for Tip60 in DNA damage repair following cellular stress using the H4 neuroglioma cells in vitro. While Tip60 HAT activity is vital for DNA repair competency as well as for the ability to signal the presence of damaged DNA to the apoptotic machinery, how Tip60 HAT activity regulates differential gene expression profiles to prevent unwanted neuronal cell death during organismal development remains unclear. A number of mammalian studies have indicated that Tip60 can function not only as a coactivator, but also as a corepressor and as such, Tip60 has been shown to repress a vast array of developmental genes during ESC differentiation to maintain ESC identity. Consistent with these findings, the majority of pro-apoptotic genes identified that were misregulated in response to disruption of Tip60 HAT activity were upregulated, highlighting the crucial role Tip60 HAT activity plays in repression of apoptotic genes during neurogenesis that, when misregulated, likely contribute to dTip60E431Q induced apoptosis (Pirooznia, 2012a).

Interestingly, this study found that overexpression of wild type Tip60 in the nervous system also induced apoptosis in the CNS. Furthermore, overexpressing Tip60 was found to induce expression of pro-apoptotic genes such as CalpA while downregulating others like Wingless, Frizzled and dMyc that have multiple essential functions during Drosophila development. These bidirectional gene expression changes suggest that increasing Tip60 mediated acetylation can also lead to complex changes in the chromatin landscape resulting in inappropriate activation and/or repression of apoptosis competent genes as well as those crucial for development. Accumulating evidence shows that hyperacetylation can be fatal to neurons. Under normal conditions, increasing hyperacetylation by treating neurons with a general HDAC inhibitor like trichostatin A has been found to induce neuronal apoptosis. Similarly, increasing acetylation levels by overexpressing the HAT CBP in resting neurons has been reported to enhance chromatin condensation and neuronal death. In order to maintain cellular homeostasis, HAT/HDAC equilibrium and therefore histone acetylation is strictly regulated as it is essential to maintain the functional status of neurons. Based on these findings, it is speculated that overexpression of Tip60 disrupts the acetylation balance, thus skewing the neuronal survival pathway towards apoptosis and ultimately cell death. In support of this concept, altered levels of global histone acetylation have been observed in many in vivo models of neurodegenerative diseases (Pirooznia, 2012a).

Another striking feature of the apoptotic microarray gene enrichment search was the identification of apoptosis linked pathways associated with neurodegenerative diseases like Parkinson's, Huntington's and Alzheimer's disease. These diseases are also characterized by neuronal cell death that increases over time and underlies an array of symptoms that depend on the function of the lost neuronal population. It has been proposed that in AD, in addition to the deposition of toxic β-amyloid plaques in the brain, neurodegeneration may also be caused via γ-secretase cleavage of APP that generates AICD carboxy terminal fragments that are toxic to neurons. Accordingly, ectopic expression of AICD in rat pheocytoma cells and cortical neurons has been shown to induce apoptosis upon nuclear translocation. Consistent with these reports, induction of apoptosis was observed when APP is expressed in the nervous system of Drosophila in vivo at physiological temperatures, and this phenotype is dependent upon the C-terminal domain of APP. Interestingly, APP C-terminal domain induced apoptosis has previously been reported to be mediated via Tip60 HAT activity in vitro, such that induction of apoptosis in neuroglioma cells transfected with APP C-terminal domain is enhanced by co-transfection of wild type Tip60 and decreased by a dominant negative version of Tip60 lacking HAT activity. In contrast, this study demonstrated that nervous system specific co-expression of APP and HAT defective mutant Tip60 increases apoptosis while overexpression of wild-type Tip60 with APP counteracts this effect and that these phenotypes are dependent upon the Tip60 interacting C-terminus of APP. Such differences may be accounted for by the fact that these studies were carried out in a developmental model system, in vivo. However, the effects this study has show on neuronal apoptosis are also consistent with the effects observed in the viability assay wherein lethality caused by neuronal overexpression of APP was enhanced by reduction of Tip60 HAT activity and suppressed by additional Tip60 levels. Importantly, this finding, in conjunction with previously published reports supporting a causative role for Tip60 in the control of synaptic plasticity (Sarthi, 2011) and the transcriptional regulation of genes enriched for neuronal function (Lorbeck, 2011), support the concept that misregulation of Tip60 HAT activity can lead to aberrant gene expression within the nervous system that contributes to the AD associated neurodegenerative process (Pirooznia, 2012a).

Tip60 has been implicated in AD via its transcriptional complex formation with AICD. Thus, experiments were carried to determine whether the expression of specific genes that are misregulated by dTip60E431Q or dTip60WT are modified by the presence of APP. Intriguingly, a number of these genes were found to be differentially regulated under APP expressing conditions. Two such genes, Wingless and Frizzled, which are upregulated in dTip60E431Q flies and repressed in dTip60WT flies are particularly interesting. Wingless, the Drosophila segment polarity gene and its membrane receptor Frizzled are known to be required for specification and formation of various neurons in the CNS and belong to the Wnt signaling pathway. In addition to Wingless and Frizzled being important for the disease process, they are also crucial for normal growth and development. Intriguingly, it was found that co-expressing APP with either the Tip60 HAT mutant or in the Tip60 overexpressing background has a repressive effect on these essential genes. Recent evidence supports a neuroprotective role for the Wnt signaling pathway and a sustained loss of Wnt signaling function is thought to be involved in aβ induced neurodegeneration. Drosophila Myc is a regulator of rRNA synthesis and is necessary for ribosome biogenesis during larval development and is another instance of a vital gene that exhibited reduced expression under APP expressing conditions. Thus misregulation of such developmentally required genes in conjunction with the other pro-apoptotic genes in the data set likely contributed to the observed enhanced apoptotic cell death in the CNS of APP;dTip60E431Q larvae. In contrast, this study found the Drosophila homolog of Bcl-2 protein, Buffy to be repressed in the APP; dTip60E431Q flies that displayed an increase in apoptosis. Consistent with the findings, recent studies have reported that Buffy has anti-apoptotic functions in vivo and intriguingly, this study found its expression to be significantly induced in the APP; dTip60WT flies that also exhibited a marked reduction in apoptosis induced cell death when compared to flies expressing dTip60WT alone. These findings suggest that induction of such pro-survival factors could mediate the dTip60 induced rescue of APP mediated defects that were observe in these flies (Pirooznia, 2012a).

Differential regulation of the microarray targets were found between flies that express dTip60E431Q alone and in conjunction with APP, in that the majority of genes tested are repressed in the APP;dTip60E431Q double mutants and activated in dTip60E431Q flies. These results indicate that the presence of APP can modulate the transcriptional regulatory potential of Tip60. The APP intracellular domain was recently shown to lower the sensitivity of neuronal cells to toxic stimuli and transcriptionally activate genes involved in signaling pathways that are not active under basal conditions). APP could mediate such effects either by sequestering Tip60 away from its typical target promoters or by displacing another factor in the complex that is also required for regulating transcription. Additionally, Tip60 has been shown to function as a negative regulator of gene expression. In fact, overexpression of Tip60 but not its HAT deficient mutant has been reported to function as co-repressor for gene repression mediated by transcription factors like STAT3 and FOX3, an effect that is mediated through association with specific histone deacetylases. This could partly account for the repressive effects that were observed due to overexpression of wild type Tip60 either alone or in conjunction with APP. Tip60 can also function as a co-activator of gene transcription via displacement of co-repressors on the promoters of specific genes. For instance, it has been reported that following IL-1 stimulation, recruitment of a wild type Tip60 containing co-activator complex leads to activation of p50 target genes like KAI1/CD82 through displacement of a specific NCoR co-repressor complex. Intriguingly, the Tip60-FE65-AICD containing complex was shown to similarly displace the NCoR complex and derepress such targets, suggesting a potential transcription activation strategy that underlies the gene expression changes observed under APP overexpressing conditions. Since loss of Tip60 HAT activity enhances APP induced lethal effects in the nervous system and overexpression of wild type Tip60 diminishes these defects, it is hypothesized that the Tip60-AICD containing complex may mediate these rescue effects either via regulation of a subset of gene targets different from those targeted by either APP or Tip60 alone or by differentially regulating the same gene pool such as that seen in the case of the anti-apoptotic gene Buffy. Thus, although the repertoire of genes that were tested include both mediators as well as inhibitors of apoptosis, taken together the data support a model by which Tip60 HAT activity plays a neuroprotective role in disease progression by complexing with the AICD region of APP to epigenetically regulate transcription of genes essential for tipping the cell fate control balance from apoptotic cell death towards cell survival under neurodegenerative conditions such as excess APP. Therefore, a neuroprotective role is proposed for Tip60 in AD linked induction of apoptotic cell death. Future investigation into the mechanism by which Tip60 regulates these processes may provide insight into the utility of specific HAT activators as therapeutic strategies for neurodegenerative disorders (Pirooznia, 2012a).

Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model

Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60 mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. This study shows that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed small ventrolateral neurons (sLNvs), and their production of the neuropeptide pigment dispersing factor (PDF) that functions to stabilize Drosophila sleep-wake cycles. Using genetic approaches, loss of Tip60 HAT activity in the presence of the Alzheimer's disease (AD) associated amyloid precursor protein (APP) was shown to affect PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of sleep-wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 on sLNv growth and function under APP induced neurodegenerative conditions. These findings reveal a novel mechanism for Tip60 mediated sleep-wake regulation via control of axonal growth and PDF levels within the sLNv encompassing neural network and provide insight into epigenetic based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer's disease (Pirooznia, 2012b).

Chromatin remodeling through histone-tail acetylation is critical for epigenetic regulation of transcription and has been recently identified as an essential mechanism for normal cognitive function. Altered levels of global histone acetylation have been observed in several in vivo models of neurodegenerative diseases and are thought to be involved in the pathogenesis of various memory related disorders. Chromatin acetylation status can become impaired during the lifetime of neurons through loss of function of specific histone acetyltransferases (HATs) with negative consequences on neuronal function. In this regard, the HAT Tip60 is a multifunctional enzyme involved in a variety of chromatin-mediated processes that include transcriptional regulation, apoptosis and cell-cycle control, with recently reported roles in nervous system function. Previous work has demonstrated that Tip60 HAT activity is required for nervous system development via the transcriptional control of genes enriched for neuronal function. Tip60 HAT activity controls synaptic plasticity and growth as well as apoptosis in the developing Drosophila central nervous system (CNS). Consistent with these findings, studies have implicated Tip60 in pathogenesis associated with different neurodegenerative diseases. The interaction of Tip60 with ataxin 1 protein has been reported to contribute to cerebellar degeneration associated with Spinocerebellar ataxia (SCA1), a neurodegenerative disease caused by polyglutamine tract expansion. Tip60 is also implicated in Alzheimer's disease (AD) via its formation of a transcriptionally active complex with the AD associated amyloid precursor protein (APP) intracellular domain (AICD). This complex increases histone acetylation and co-activates gene promoters linked to apoptosis and neurotoxicity associated with AD. Additionally, misregulation of certain putative target genes of the Tip60/AICD complex has been linked to AD related pathology. These findings support the concept that inappropriate Tip60/AICD complex formation and/or recruitment early in development may contribute or lead to AD pathology via epigenetic misregulation of target genes that have critical neuronal functions. In support of this concept, it has been recently reported that Tip60 HAT activity exhibits neuroprotective functions in a Drosophila model for AD by repressing AD linked pro-apoptotic genes while loss of Tip60 HAT activity exacerbates AD linked neurodegeneration (Pirooznia, 2012a). However, whether misregulation of Tip60 HAT activity directly disrupts selective neuronal processes that are also affected by APP in vivo and the nature of such processes remains to be elucidated (Pirooznia, 2012b and references therein)

In Drosophila, the small and large ventrolateral neurons (henceforth referred to as sLNv and lLNv, respectively) are part of the well characterized fly circadian circuitry. Recent studied have implicated the l-and s-LNvs as part of the 'core' sleep circuitry in the fly, an effect that is predominantly coordinated via the neuropeptide pigment dispersing factor (PDF) that serves as the clock output, mediating coordination of downstream neurons. PDF is thought to be the fly equivalent of the mammalian neurotransmitter orexin/hypocretin because of its role in promoting wakefulness and thus stabilizing sleep-wake cycles in the fly. Within this circuit, the sLNvs are a key subset of clock neurons that exhibit a simple and stereotypical axonal pattern that allows high resolution studies of axonal phenotypes using specific expression of an axonally transported reporter gene controlled by the Pdf-Gal4 driver or by immunostaining for the Pdf neuropeptide that is distributed throughout the sLNv axons. These features make the sLNvs an excellent and highly characterized model neural circuit to study as they are amenable to cell type specific manipulation of gene activity to gain molecular insight into factors and mechanisms involved in CNS axonal regeneration as well as those that mediate behavioral outputs like sleep-wake cycle. Importantly, the Drosophila ventrolateral neurons (LNvs) have been previously used as a well characterized axonal growth model system to demonstrate that the AD linked amyloid precursor protein (APP) functions in mediating the axonal arborization outgrowth pattern of the sLNv. Based on these results, and previous studies reporting that Tip60 HAT activity itself is required for neural function and mediates APP induced lethality and CNS neurodegeneration in an AD fly model , It is hypothesized that APP and Tip60 are both required to mediate selective neuronal processes such as sLNv morphology and function that when misregulated, are linked to AD pathology. In the present study, this hypothesis was tested by utilizing the sLNvs as a model system to examine whether Tip60 mediated epigenetic dysregulation under neurodegenerative conditions such as that induced by APP overexpression leads to axonal outgrowth defects and if there is a corresponding effect on sLNv function in sleep regulation, a process that is also affected in neurodegenerative diseases like AD (Pirooznia, 2012b).

This report shows that Tip60 is endogenously expressed in both the sLNv and lLNvs. Specific loss of Tip60 or its HAT activity causes reduction of PDF expression selectively in the sLNvs and not the lLNv and shortening of the sLNv distal synaptic arbors which are essential for the pre-synaptic release of PDF from these cells. The functional consequence of these effects is evidenced by the disruption of the normal sleep-wake cycle in these flies, possibly through disruption of PDF mediated signaling to downstream neurons. By using transgenic fly lines that co-express full length APP or APP lacking the Tip60 interacting C-terminus with a dominant negative HAT defective version of Tip60, it was demonstrated that the APP C-terminus enhances the susceptibility of the sLNvs and exacerbates the deleterious effects that the loss of Tip60 HAT activity has on axon outgrowth and PDF expression. Importantly, these studies identify the neuropeptide PDF as a novel target of Tip60 and APP, that when misregulated results in sleep disturbances reminiscent to those observed in AD. Remarkably, overexpression of wild type Tip60 with APP rescues these sleep defects by increasing PDF expression and inducing overelaboration of the sLNv synaptic arbor area. Taken together, these findings support a neuroprotective role for Tip60 on sLNv growth and function under APP induced neurodegenerative conditions. The data also reveal a novel mechanism for PDF control via Tip60 and APP that provide insight into understanding aspects of sleep dependent mechanisms that contribute to early pathophysiology of AD (Pirooznia, 2012b).

Selective vulnerability of specific neuronal populations to degeneration even before disease symptoms are seen is a characteristic feature of many neurodegenerative diseases. Consistent with these studies, this study shows that when induction of the dTip60 RNAi response or expression of the dTip60 HAT mutant was directed to both the small and large LNvs, only the sLNvs were susceptible to the mutant effects induced under these conditions while the lLNvs were spared. The lack of any morphological effect on the lLNvs in the dTip60E431Q flies could stem from the fact that compared to the sLNvs, these neurons express higher levels of endogenous Tip60 that counteracts the mutant dTip60E431Q protein. However, induction of the RNAi response causes complete loss of Tip60 expression in both the lLNv and sLNv, and yet only the sLNvs are affected while the lLNv are spared, similar to the findings with dTip60E431Q expression. This suggests that the sLNvs may be more susceptible to misregulation of Tip60 or its HAT activity. Of note, the dTip60WT flies did not have any marked effect on the lLNv either, likely because these neurons are not susceptible to the moderate increase in Tip60 levels in the lLNvs induced under these conditions compared to the sLNvs. Developmentally, the sLNvs are known to differentiate much earlier than the large cells and this developmental difference may also in part account for the selective vulnerability of the sLNvs. In many neurodegenerative diseases, axon degeneration is known to involve protracted gradual 'dying-back' of distal synapses and axons that can precede neuron cell body loss and contribute to the disease symptoms. Importantly, loss of synapses and dying back of axons are also considered as early events in brain degeneration in AD. While APP overexpression in the LNvs did not have any observable effect on the sLNv axon growth at normal physiological temperatures, coexpression of the dTip60 HAT mutant with APP C-terminus appears to cause the sLNv axons in the adult animals to retract. The lack of any effect on the sLNv axon in the third instar larva in this case indicates that the axons grow to their full potential in the larval stage, but undergo degeneration post-mitotically in a process similar to 'dying-back' (Pirooznia, 2012b).

A functional interaction between Tip60 and the amyloid precursor protein (APP) intracellular domain (AICD) has been shown to epigenetically regulate genes essential for neurogenesis. Such an effect is thought to be mediated by recruitment of the Tip60/AICD containing complex to certain gene promoters in the nervous system that are then epigenetically modified by Tip60 via site specific acetylation and accordingly activated or repressed. While the E431Q mutation in dominant negative HAT defective version of Tip60 (dTip60E431Q) reduces Tip60 HAT activity, it should not interfere with its ability to assemble into a protein complex. Thus, dTip60E431Q likely exerts its dominant negative action over endogenous wild-type Tip60 via competition with the endogenous wild-type Tip60 protein for access to the Tip60/AICD complex and/or additional Tip60 complexes, with subsequent negative consequences on chromatin histone acetylation and gene regulation critical for nervous system function. This study shows that co-expression of HAT defective Tip60 (dTip60E431Q) with APP in the APP; dTip60E431Q flies exacerbates the mutant effects that either of these interacting partners has on the sLNv axon growth and Pdf expression when expressed alone. In contrast, co-expression of additional dTip60WT with APP alleviates these effects and this rescue is dependent upon the presence of the AICD region of APP. Thus, Tip60 HAT activity appears to display a neuroprotective effect on axonal outgrowth, Pdf expression, with concomitant alleviation of sleep defects under APP expressing neurodegenerative conditions. It is proposed that Tip60 might exert this neuroprotective function either by itself or by complexing with other peptides such as AICD for its recruitment and site specific acetylation of specific neuronal gene promoters to redirect their expression and function in selective neuronal processes such as sLNv morphology and function. Such a neuroprotective role for Tip60 is consistent with previous work demonstrating that excess dTip60WT production under APP expressing neurodegenerative conditions in the fly rescues APP induced lethality and CNS neurodegeneration and that dTip60 regulation of genes linked to AD is altered in the presence of excess APP (Pirooznia, 2012). It is speculated that the degenerative effects observed in the APP; dTip60E431Q flies may result from formation of Tip60E431Q/AICD complexes that ultimately cause activation or de-repression of factors that promote axonal degeneration while excess Tip60/AICD complex formation in the APP;dTip60WT expressing flies promote gene regulation conducive for sLNv outgrowth and Pdf expression (Pirooznia, 2012b).

Sleep or wake promoting neurons in the hypothalamus or brainstem are known to undergo degeneration in a number of neurodegenerative diseases resulting in sleep dysregulation. In AD, such sleep disturbances are characterized by excessive daytime sleepiness and disruption of sleep during the night. These features resemble the symptoms of narcolepsy, a sleep disorder caused by general loss of the neurotransmitter hypocretin/orexin. Hypocretin is involved in consolidation of both nocturnal sleep and diurnal wake and loss of hypocretin levels have been correlated with sleep disturbances observed in AD. While the neuropathological changes in AD may contribute to hypocretin disturbances, a direct and causative role for APP in regulating hypocretin expression is not yet known. The LNv specific neuropeptide PDF is postulated to be the fly equivalent of hypocretin and has been shown to promote wakefulness in the fly. Consistent with these reports, the current data demonstrating somnolence during the light phase due to knock-down of PDF in the sLNv further supports a wake-promoting role for PDF. Accordingly, it was observed that overexpression of APP in the LNvs results in reduction of sLNv PDF expression as well as sleep disturbances that intriguingly, have been associated with AD pathology. The presence of similar effects on PDF and sleep due to loss of dTip60 HAT activity supports a role for both APP and Tip60 in controlling the PDF mediated sleep-wake regulation pathway. Previous studies have reported that the circadian modulators CLOCK and CYCLE regulate PDF expression in the sLNvs but not in the lLNvs. This study also observed a similar sLNv specific regulation of PDF by dTip60 in the adult flies. However, there was no effect on PDF expression in sLNvs in the larvae when Tip60 levels are undetectable. This is also consistent with the sLNv axonal defects that persist only in the adult flies. This suggests that the sLNvs may be subject to differential regulation during development as well as a temporal requirement for Tip60 in these cells in the adult flies. A recent study reported persistence of morning anticipation and morning startle response in LD in the absence of functional sLNv that were ablated due to expression of the pathogenic Huntington protein with poly glutamine repeats (Q128). Consistent with the Sheeba study, this study did not observe any marked effect on the morning and evening anticipatory behavior in LD in the dTip60E431Q flies that exhibit a partial reduction in sLNv PDF. However, while the Q128 expressing flies were arrhythmic under constant darkness, dTip60E431Q flies maintain rhythmicity in DD indicating that the sLNvs are still functional in these flies. The remarkable cell specificity of PDF regulation indicates the presence of additional as yet unidentified clock relevant elements or developmental events that distinguish between the two cell types (Pirooznia, 2012b).

Recent evidence indicates that LNvs are light responsive and that their activation promotes arousal through release of PDF. Furthermore, PDF signaling to PDF receptor (PDFR) expressing neurons outside the clock, such as those found in the ellipsoid body that directly control activity, is thought to be important in translating such arousal signals into wakefulness. Since PDF is released from the sLNv axon terminals, the retraction of the sLNv axon terminals induced by the Tip60 HAT mutant can interfere with PDF mediated interaction of the sLNvs with downstream circuits. In the case of APP overexpression, while sLNv axon structure is unaffected, PDF expression is reduced; it is speculated that the decrease in PDF under these conditions is responsible for the abnormal sleep phenotype observed. In support of this theory, it was found that expression of APP lacking the C-terminus that also has no observable effect on the sLNv axon growth or PDF expression did not have any effect on sleep behavior. Thus the results indicate that the degenerative effect on the sLNv axons and/or the effect on PDF expression could both contribute to the observed sleep disturbances. Likewise, co-expression of the dTip60 HAT mutant with full length APP or APP lacking the C-terminus affected both the sLNv axon growth and PDF expression and consequently resulted in similar sleep disturbances (Pirooznia, 2012b).

In addition to the wake promoting role, the LNvs also express GABAA receptors and are thus subject to inhibition by sleep promoting GABAergic inputs, analogous to those from the mammalian basal forebrain that regulate hypocretin neurons. The current consensus view is that sleep regulation is mediated by mutually inhibitory interactions between sleep and arousal promoting centers in the brain. The normal release of PDF from LNvs is part of the arousal circuitry in the fly and determines the duration of the morning and evening activity peaks while inhibition of these neurons and thus reduction in PDF release is necessary for normal sleep. Current models of sleep regulation suggest that the drive to sleep has two components, the first component is driven by the circadian clock and the second component is homeostatic in nature and the strength of this drive is based upon the amount of time previously awake. PDF release from sLNvs axon terminals exhibits diurnal variation and its release increases the probability of wakefulness by activating arousal promoting centers. However, the homeostatic drive for sleep that accumulates during the wake period eventually inhibits such arousal centers to promote sleep. Consistent with these reports, the reduction of PDF observed due to either dTip60E431Q expression alone or co-expression of dTip60E431Q with APP that leads to flies sleeping more during the day may also lead to a decrease in their homoeostatic drive for sleep, thus resulting in the less consolidated sleep patterns observed for these flies during the night. Conversely, it was found that overexpression of sLNv PDF due to dTip60 overexpression induces wakefulness and arousal. Additionally, these flies exhibit impaired ability to maintain sleep at night that may be mediated through inappropriate activation of arousal circuits due to PDF overexpression. Similar effects have been reported in a Zebrafish model due to hypocretin overexpression that results in hyperarousal and dramatic reduction in ability to initiate and maintain a sleep-like state at night. Despite the moderate increase in sLNv PDF levels in the dTip60Rescue flies, no marked effect on sleep-wake cycle was observed in these flies. Extracellular levels of PDF and its signaling at synapses is thought to be regulated by neuropeptidases like neprilysin (see Neprilysin 4). In fact, neprilysin mediated cleavage of PDF has been shown to generate metabolites that have greatly reduced receptor mediated signaling. Thus, it is speculated that the lack of any corresponding effect on sleep in the Tip60Rescue flies could be because such small increases in PDF might be regulated by endopeptidases like neprilysin. Based on these studies, a model is proposed by which the overelaborated sLNv synaptic arbors observed in flies co-expressing Tip60WT and APP may provide additional input sites for signals from sleep promoting neurons in the vicinity that counteract the arousing effect of PDF overexpression on nocturnal sleep (Pirooznia, 2012b).

Light mediated release of PDF from the lLNvs has been reported to modulate arousal and wakeful behavior as well as sleep stability. Thus, it has been suggested that the lLNvs may be part of an arousal circuit that is physiologically activated by light and borders with, but is distinct from the sLNvs and downstream sleep circuits. However, other studies have suggested that both LNv sub-groups promote wakeful behavior and that the lLNv act upstream of the sLNv. The observation of sLNv directed effects on PDF expression and the persistence of sleep-wake disturbances suggest that the sLNvs may be part of the neural circuitry that regulates sleep downstream of the lLNvs via a PDF dependent mechanism. In this regard, the sLNvs may participate in the communication between the lLNvs and other brain regions to promote light mediated arousal. It has been proposed by that the lLNvs may promote neural activity of the Ellipsoid body (EB) in the central complex (CC), a higher center for locomotor behavior that expresses the PDF receptor. However, disruption of sleep-wake cycles was observed even in the absence of any marked effect on the lLNv morphology or PDF expression. While no direct projections from the lLNvs to the EB have been detected, the sLNv axonal projections are relatively closer to the CC and thus may promote PDF receptor mediated signaling in such regions that control activity. Sleep disturbances, while prominent in many neurodegenerative diseases are also thought to further exacerbate the effects of a fundamental process leading to neurodegeneration. For these reasons, optimization of sleep-wake pattern could help alleviate the disease symptoms and slow the disease progression. In this regard, the modulatory effects that Tip60 HAT activity (dTip60E431Q versus dTip60WT) has on the sLNvs, the fly counterpart of the mammalian pacemaker cells, under APP overexpressing conditions, may provide novel mechanistic insights into epigenetic regulation of neural circuits that underlie behavioral symptoms like the 'sundowners syndrome' in AD. Future investigation into the downstream mechanism by which Tip60 regulates the sleep-wake cycle may further provide insight into the utility of specific HAT activators as therapeutic strategies for sleep disturbances observed in AD (Pirooznia, 2012b).

Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, the Drosophila homolog, CG2789/dTSPO, was identified, and the effects of its inactivation was studied by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to gamma-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target (Lin, 2014).

The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling

Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. This study therefore investigated the role of Drosophila APPL during brain development. APPL was found to be involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, APPL was found to be a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover,APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together these data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth (Soldano, 2013).

AD is a neurodegenerative disorder characterized by progressive loss of neurons in specific regions of the brain that correlates with progressive impairment of higher cognitive functions. A growing body of evidence identifies the APP and its metabolite the Aβ peptide as main players in the pathogenesis of AD. In particular, the accumulation of Aβ peptides in the brain seems to be the trigger of the pathological cascade that eventually results in neuronal loss and degeneration. Despite efforts to characterize the molecular mechanisms underlying Aβ's toxic function, it is still not clear what triggers the accumulation of the peptide and how this is correlated with the pathogenesis of the disease and the dementia. In fact, most of the work done to unveil the pathogenesis of the disease has focused on the analysis of Aβ-peptide and the search for its receptors and downstream effectors. Even though the numerous in vitro studies performed in cell culture identified several molecules that interact with Aβ peptide, the in vivo biological relevance of these interactions remains to be clarified. The amyloid cascade hypothesis has also dominated the search for AD treatments, but the promising molecular candidates developed to modulate the Aβ peptide and reached clinical trials failed. Finally, over the last few years many studies indicated that there is no linear correlation between the accumulation of the peptide and the cognitive decline, leading to a revision of the amyloidogenic hypothesis. Taken together, these observations suggest that the accumulation of the peptide is not the only cause of the pathology and that other factors are involved. Interestingly, under physiological conditions APP is mainly found in its uncleaved or α-cleaved form, suggesting that the shift towards amyloidogenic processing not only increases the production of Aβ peptide but also depletes the pool of APP that undergoes non-amyloidogenic processing, with hitherto unknown consequences. It is therefore of paramount importance to understand the physiological role of APP and how perturbing this role could contribute to the pathogenesis of the disease. An important contribution to the study of the function of a protein comes from the analysis of the knock-out (KO) animals. In the case of APP, several KO models have been generated and analyzed in detail both from the morphological and behavioral point of view. Despite these efforts, the normal physiological function of APP in vivo in the nervous system remains largely elusive and highly controversial. This is due to the lack of consensus over the neuronal phenotypes in null mutant animals and the mechanism of action in vivo. The data collected by different labs confirmed the involvement of APPs in development and function of the nervous system, but these studies do not provide an in-depth analysis of the development of the brain during the pre-natal stages or the molecular mechanism underlying APPs' putative functions. Therefore this study took advantage of Drosophila melanogaster to further analyze the consequence of loss of APP Like (APPL) during brain development (Soldano, 2013).

The present study demonstrates that APPL is involved in brain development of Drosophila melanogaster, particularly in the Mushroom Body (MB) neurons. APPL is required for the development of αβ neurons. In Appl-/- flies, MB neurons fail to project the α lobe in 14% of the cases and the β-lobe in 12% of the cases. Further analysis of the phenotype reveals that APPL is required cell-autonomously for the development of the β lobe and non-cell autonomously for the development of the α lobe. In fact, single cell Appl-/- clones display only β-lobe loss and no α loss. The re-introduction of a full-length, membrane-tethered form of APPL, but not a soluble form, rescues β-lobe los. This is of particular interest because it confirms that, similar to mammalian APPs, the physiological role of APPL is mediated both by its full-length form, required in the neurons to achieve the correct β-lobe pattern, and by its soluble form (sAPPL) that regulates the extension of the α lobe. Moreover, the rescue data indicate that, at least in this context, the function of sAPPL is mediated not by homo-dimerization with the full-length form but by some other receptor, hitherto unknown. Further experiments are required to clarify the sAPPL non-cell autonomous effect, but it is hypothesized that it might be involved in modulating signaling mediated by the cells that surround the MB axons. Taken together, the analysis of the Appl-/- animals confirmed the important role of APPs during brain development but reinforced the idea that the phenotypes are present with incomplete penetrance and might be subtle. It would therefore be of interest to analyze the phenotype of the KO mice in greater detail and, in particular, to better characterize the APP's downstream pathway leading to these defects (Soldano, 2013).

Moreover, the results described clearly support a model of APPL as a novel, neuronal-specific positive modulator of the Wnt-PCP pathway. The PCP pathway was initially described because of its role in tissue polarity establishment and, in particular, of its regulation of cell orientation in plane of an epithelium. Among the different processes regulated by PCP signaling, axon growth and guidance is of particular interest. Mice null for Fzd3/Ceslr3-/- genes show severe defects in several major axon tracts like thalamocortical, corticothalamic, and nigrostriatal tracts, defects of the anterior commissure, and similarly to APP KO mice, the variable loss of the corpus callosum (Soldano, 2013).

The molecular mechanism underlying the function of PCP-signaling in regulating tissue polarity has been broadly studied. The current model suggests that, upon polarized expression of the different core proteins, Dsh is recruited to the membrane via Fz and leads to the activation of a cascade of small GTPases finally resulting in cytoskeleton rearrangements. In the case of regulation of axon growth and guidance, it is less clear how the signaling is regulated and transmitted to the cytoskeleton. A recent publication suggested that during axon growth the transmembrane PCP receptor-like Vang and Fzd are localized at the growth cone area on the tip of the fillopodia, thus suggesting that in this context the asymmetric localization is not needed (Soldano, 2013).

Furthermore, Dsh needs to relocalize from the cytoplasm to the membrane to ensure the proper activation of PCP signaling, and this is dependent on its phosphorylation status. Abelson has been shown to be one kinase responsible for this modification, but the receptor upstream of the kinase was not identified (Singh, 2010). Based on current evidence, it is proposed that APPL is a novel regulator of Wnt-PCP pathway involved in axon growth and guidance. This is of interest because while the PCP core proteins are ubiquitously expressed, APPL is restricted to the nervous system, suggesting that it could be the first described tissue-specific modulator of the pathway (Soldano, 2013).

Mechanistically, it is proposed that APPL-Abl complex modulates Dsh via dual protein-protein interactions. First, Abl might have an intrinsic affinity for its substrate Dsh (Singh, 2010). Secondly, this interaction is strengthened or stabilized by the inclusion of APPL in a PCP receptor complex. This dual affinity complex leads to increased PCP signaling efficiency at the developing growth cone. Both biochemical and physiological data show that this function is highly conserved in mammalian APP, suggesting that it may play a similar role in the mammalian brain. The canonical-Wnt signaling pathway has already been connected to AD pathogenesis because of its link to the tau-kinase GSK-3β. Interestingly, no clear link between the Wnt-PCP pathway and this neurodegenerative disorder has been made. Previous reports have show that, in flies and mice, Jun N-terminal Kinase (JNK) is the final effector of PCP in axon outgrowth and JNK was shown to be required for the effect of APP overexpression in the fly. Interestingly, JNK signaling has also been linked to the neuronal loss observed in AD. It is therefore worth investigating whether the physiological function of APP as a neuronal PCP modulator explains the JNK-AD connection (Soldano, 2013).

Endogenous GSK-3/shaggy regulates bidirectional axonal transport of the amyloid precursor protein

Neurons rely on microtubule (MT) motor proteins such as kinesin-1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK-3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin-1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, this study found that endogenous GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK-3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, this study found that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK-3 and APP, and demonstrate that endogenous GSK-3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK-3 controls the number of active motors that are moving a cargo (Weaver, 2013).

Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3beta signaling

Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, this study explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. The Drosophila homolog of DSCR1, Nebula, was found to delay neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, Nebula upregulation was shown to protect against axonal transport defects by restoring calcineurin and GSK-3beta signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, these findings imply that correcting calcineurin and GSK-3beta signaling can prevent APP-induced pathologies. The data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD (Shaw, 2013).

Although upregulation of APP had been shown to negatively influence axonal transport in mouse and fly models, mechanisms by which APP upregulation induces transport defects are poorly understood. Several hypotheses have been proposed, including titration of motor/adaptor by APP, impairments in mitochondrial bioenergetics, altered microtubule tracks, or aberrant activation of signaling pathways. The motor/adaptor titration theory suggests that excessive APP-cargos titrates the available motors away from other organelles, thus resulting in defective transport of pre-synaptic vesicles. The finding that Nebula co-upregulation enhanced the movement and delivery of both synaptotagmin and APP to the synaptic terminal argues against this hypothesis. In addition, earlier findings suggest that Nebula upregulation alone impaired mitochondrial function and elevated ROS level, thus implying that Nebula is not likely to rescue APP-dependent phenotypes by selectively restoring mitochondrial bioenergetics. Furthermore, consistent with a recent report showing normal microtubule integrity in flies overexpressing either APP-YFP or activated GSK-3βM (Weaver, 2013), the data revealed normal gross microtubule structure in flies with APP overexpression. Together, these results suggest that changes in gross microtubule structure and stability is not a likely cause of APP-induced transport defects (Shaw, 2013).

Instead, the current results support the idea that Nebula facilitates axonal transport defects by correcting APP-mediated changes in phosphatase and kinase signaling pathways. First, APP upregulation was found to elevate intracellular calcium level and calcineurin activity, and restoring calcineurin activity to normal suppresses the synaptotagmin aggregate accumulation in axons. The observed increase in calcium and calcineurin activity is consistent with reports of calcium dyshomeostasis and elevated calcineurin phosphatase activity found in AD brains, as well as reports demonstrating elevated neuronal calcium level due to APP overexpression and increased calcineurin activation in Tg2576 transgenic mice carrying the APPswe mutant allele. Second, APP upregulation resulted in calcineurin dependent dephosphorylation of GSK-3β at Ser9 site, a process thought to activate GSK-3β kinase. APP upregulation also triggered calcineurin-independent phosphorylation at Tyr216 site, which has been shown to enhance GSK-3β activity. The kinase(s) that phosphorylates APP at Tyr216 is currently not well understood, it will be important to study how APP leads to Tyr216 phosphorylation in the future. Based on the current results, it is envisioned that APP overexpression ultimately leads to excessive calcineurin and GSK-3β activity, whereas nebula overexpression inhibits calcineurin to prevent activation of GSK-3β. The findings that nebula co-overexpression prevents GSK-3β activation and enhances the transport of APP-YFP vesicles are consistent with a recent report by Weaver (2013), in which it was found decreasing GSK-3β in fly increases the speed of APP-YFP movement. Furthermore, consistent with the current result that APP upregulation triggers GSK-3β enhancement and severe axonal transport defect, Weaver did not detect changes in GFP-synaptotagmin movement in the absence of APP upregulation (Shaw, 2013).

Active GSK-3β has been shown to influence the transport of mitochondria and synaptic proteins including APP, although the exact mechanism may differ between different cargos and motors. One mechanism proposed for GSK-3β-mediated regulation of axonal transport is through phosphorylation of KLC1, thereby disrupting axonal transport by decreasing the association of the anterograde molecular motor with its cargos. Accordingly, this study found that APP reduces KLC-synaptotagmin interaction while Nebula upregulation preserves it. Synaptotagmin transport in both the anterograde and retrograde directions are affected, consistent with previous reports showing that altering either the anterograde kinesin or retrograde dynein is sufficient affected transport in both directions. The results also support work suggesting that synaptotagmin can be transported by the kinesin 1 motor complex in addition to the kinesin 3/imac motor. As kinesin 1 is known to mediate the movement of both APP and mitochondria and phosphorylation of KLC had been shown to inhibit mitochondrial transport, detachment of cargo-motor caused by GSK-3β mediated phosphorylation of KLC may lead to general axonal transport problems as reported in this study. However, GSK-3β activation may also perturb general axonal transport by influencing motor activity or binding of motors to the microtubule tract. Interestingly, increased levels of active GSK-3β and phosphorylated KLC and dynein intermediate chain (DIC), a component of the dynein retrograde complex, have been observed in the frontal complex of AD patients. Genetic variability for KLC1 is thought to be a risk factor for early-onset of Alzheimer's disease. There is also increasing evidence implicating GSK-3β in regulating transport by modulating kinesin activity and exacerbating neurodegeneration in AD through tau hyperphosphorylation. It will be interesting to investigate if Nebula also modulates these processes in the future (Shaw, 2013).

SAlthough calcineurin had been shown to regulate many important cellular pathways, the link between altered calcineurin and axonal transport, especially in the context of AD, had not been established before. This study shows that calcineurin can regulate axonal transport through both GSK-3β independent and dependent pathways. This is supported by observation that the severity of the aggregate phenotype was worse for flies expressing APP and active calcineurin than it was for flies expressing APP and active GSK-3β. These findings point to a role for calcineurin in influencing axonal transport directly, perhaps through dephosphorylation of motor or adaptor proteins. The data also indicate that calcineurin in part modulates axonal transport through dephosphorylation of GSK-3β as discussed above; however, upregulation of APP is necessary for the induction of severe axonal transport problems, mainly by causing additional enhancement of GSK-3β signaling. GSK3 inhibition is widely discussed as a potential therapeutic intervention for AD; results suggest that perhaps calcineurin is a more effective target for delaying degeneration by preserving axonal transport (Shaw, 2013).

DSCR1 and APP are both located on chromosome 21 and upregulated in DS. Overexpression of DSCR1 alone had been contradictorily implicated in both conferring resistance to oxidative stress and in promoting apoptosis. Upregulation of Nebula/DSCR1 had also been shown to negatively impact learning and memory in fly and mouse models through altered calcineurin pathways. How could upregulation of DSCR1 be beneficial? It is proposed that DSCR1 upregulation in the presence of APP upregulation compensates for the altered calcineurin and GSK-3β signaling, shifting the delicate balance of kinase/phosphatase signaling pathways close to normal, therefore preserving axonal transport and delaying neurodegeneration. It is also proposed that axonal transport defects and synapse dysfunction caused by APP upregulation in the Drosophila model system occur prior to accumulation of amyloid plaques and severe neurodegeneration, similar to that described for a mouse model (Shaw, 2013).

DS is characterized by the presence of AD neuropathologies early in life, but most DS individuals do not exhibit signs of dementia until decades later, indicating that there is a delayed progression of cognitive declin. The upregulation of DSCR1 may in fact activate compensatory cell signaling mechanisms that provide protection against APP-mediated oxidative stress, aberrant calcium, and altered calcineurin and GSK3-β activity (Shaw, 2013).

Bidirectional regulation of Amyloid precursor protein-induced memory defects by Nebula/DSCR1: A protein upregulated in Alzheimer's disease and Down syndrome

Aging individuals with Down syndrome (DS) have an increased risk of developing Alzheimer's disease (AD), a neurodegenerative disorder characterized by impaired memory. Memory problems in both DS and AD individuals usually develop slowly and progressively get worse with age, but the cause of this age-dependent memory impairment is not well understood. This study examines the functional interactions between Down syndrome critical region 1 (DSCR1) and Amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. Using Drosophila as a model, this study found that overexpression of nebula (fly homolog of DSCR1) initially protects against APP-induced memory defects by correcting calcineurin and cAMP signaling pathways but accelerates the rate of memory loss and exacerbates mitochondrial dysfunction in older animals. Transient upregulation of Nebula/DSCR1 or acute pharmacological inhibition of calcineurin in aged flies protected against APP-induced memory loss. These data suggest that calcineurin dyshomeostasis underlies age-dependent memory impairments and further imply that chronic Nebula/DSCR1 upregulation may contribute to age-dependent memory impairments in AD in DS (Shaw, 2015).

Down syndrome (DS), due to full or partial triplication of chromosome 21, greatly increases the risk of Alzheimer's disease (AD). By age 65, ~75% of DS individuals will develop dementia as compared to 13% of age-matched controls. Despite an early presence of the neurochemical changes seen in AD brains, dementia is delayed in most DS individuals until after mid-life, suggesting both a genetic risk for dementia and the existence of a neuroprotective period before the onset of memory impairments. The mechanism underlying this age-dependent memory decline is poorly understood, but the well known connection between DS and AD provides a unique opportunity to identify common genetic factors contributing to AD and age-associated dementia (Shaw, 2015).

To uncover mechanisms underlying age-dependent memory decline in AD and DS, this study examined the functional interactions between two genes encoded by chromosome 21 and upregulated in both DS and AD. The amyloid precursor protein (App), encoded by chromosome 21, is a known risk gene for AD because either mutations or duplication of App is associated with familial AD. Studies have shown that overexpression of the wild-type human APP in both mouse and Drosophila causes cognitive deficits before β-amyloid accumulation, suggesting that APP perturbation could contribute to dementia independent of β-amyloid plaques. Another gene encoded by chromosome 21 that is likely to play a crucial role in AD is the Down syndrome critical region 1 (Dscr1; also known as Rcan-1) gene. Postmortem brains from both DS and AD patients show an upregulation of DSCR1 mRNA and protein levels. Oxidative stress, APP upregulation, and β-amyloid exposure have also been shown to induce DSCR1 upregulation. DSCR1 encodes an evolutionarily conserved inhibitor of calcineurin, a serine/threonine calcium/calmodulin phosphatase important for numerous physiological pathways, including memory, cell death, and immunity. Studies have shown that altering levels of DSCR1 in mouse and Nebula (fly homolog of DSCR1) in Drosophila severely impaired memory. However, upregulation of Nebula/DSCR1 has been shown to both promote and inhibit cell survival after oxidative stress, as well as protect against APP-induced neurodegeneration and axonal transport defects. Thus, it remains unknown how Nebula/DSCR1 upregulation will affect APP-induced memory defects (Shaw, 2015).

Drosophila and humans share conserved cell signaling components and pathways essential for learning and memory formation, thus providing an effective model system for studying mechanisms contributing to age-dependent memory impairments and neurological disorders. Drosophila has also been used successfully as a model system to investigate mechanisms underlying various neurological disorders. Using Drosophila, this study shows that overexpression of nebula rescued memory impairments induced by APP upregulation through inhibition of calcineurin. These protective effects did not persist during aging, and Nebula co-upregulation instead accelerated age-dependent memory impairments, increased reactive oxygen species (ROS), and enhanced mitochondrial dysfunctions in aged flies. Furthermore, transient upregulation of Nebula or acute pharmacological inhibition of calcineurin in aged flies was sufficient to restore APP-induced memory loss. These findings suggest that Nebula/DSCR1 upregulation may contribute to progressive dementia by initially rescuing APP-induced memory loss but accelerating the rate of memory impairment in older animals (Shaw, 2015).

These findings reveal a complex and novel role for Nebula/DSCR1 upregulation in regulating APP-induced memory loss during aging. First, it was shown that upregulation of Nebula initially protects against APP-induced memory impairments by restoring calcineurin-mediated signaling in young flies. Second, persistent upregulation of Nebula was found to contribute to the poor memory performance of APP and Nebula flies during aging. Third, aging is accompanied by elevations in calcineurin activity, and acute inhibition of calcineurin can improve the memory performance of older control and APP overexpressing flies. Together, these results suggest that Nebula/DSCR1 upregulation may delay the onset of memory loss but contribute to progressive dementia in older individuals with DS. Therefore, this study has wide implications for memory loss during natural aging and in AD and DS and shines light on restoring calcineurin or regulating Nebula/DSCR1 levels as potential therapeutic strategies for age-dependent memory loss (Shaw, 2015).

Nebula/DSCR1 is a multifunctional protein that inhibits calcineurin and modulates mitochondria function and oxidative stress response. Previous reports have indicated that upregulation of either Nebula/DSCR1 or APP alone impaired memory. Therefore, it is unexpected that co-upregulation of Nebula and APP restored both STM and LTM of young flies. Such results were confirmed using two different mushroom body drivers: C739-Gal4 and MB-GeneSwitch-Gal4. The use of mushroom body drivers is advantageous because it circumvents the problem of locomotor defects associated with pan-neuronal APP overexpression, and the Drosophila mushroom bodies has been shown to be structures important for memory retrieval, a process disrupted in AD-related memory loss. The current biochemical and behavioral data indicate that Nebula rescues memory loss by correcting APP-induced calcineurin hyperactivation, as well as deficits in PKA activity and CREB phosphorylation. These results are consistent with the finding that a fine balance in calcineurin and PKA signaling are crucial for normal memory. However, genetics and behavioral data indicate that restoring GSK-3β hyperactivation in APP overexpressing flies, shown previously to rescue axonal transport defects, is not sufficient to rescue the memory deficits. Furthermore, because APP and Nebula overexpressing flies restored STM despite the presence of mitochondrial dysfunction, the data highlight that correcting calcineurin disturbances in younger flies is more beneficial for memory than restoring mitochondrial dysfunction (Shaw, 2015).

Age-associated memory impairment occurs in many species ranging from Drosophila to humans; understanding mechanisms contributing to this process may provide useful insights into changes responsible for dementia in age-related neurological disorders such as AD. The current data provide two important revelations concerning cellular changes contributing to age-dependent memory decline. First, the finding highlight that elevation in calcineurin activity is a previously unidentified mechanism contributing to memory decline during natural aging in Drosophila. This is supported by biochemical data showing increases in calcineurin activity during aging, as well as behavioral data illustrating that transient pharmacological inhibition of calcineurin can significantly improve the memory performance of old wild-type flies. Second, chronic upregulation of Nebula also triggers severe mitochondrial dysfunction that can override the protective effect of calcineurin inhibition by Nebula in flies overexpressing APP, implying that long-term Nebula upregulation may contribute to memory loss in APP overexpressing flies during aging. By measuring ATP content and ROS levels within the fly brain, this study showed that chronic Nebula overexpression both on its own or in the presence of APP significantly exacerbated mitochondrial dysfunction and elevated ROS. Conversely, short-term upregulation of APP and Nebula in aged flies or transient pharmacological inhibition of calcineurin in older flies with chronic APP overexpression both resulted in normal STM performance compared with age-matched control. These results support the notion that chronic Nebula upregulation during aging enhances age-dependent memory impairments in flies with APP overexpression and further suggest that proper mitochondrial function plays an important role in memory preservation in older flies. This interpretation is supported by a report that STM of older flies is particularly sensitive to mutations that elevate ROS, whereas the STM of younger flies is not affected by ROS elevation (Shaw, 2015).

It is proposed that Nebula/DSCR1 upregulation plays a two-pronged role in regulating APP-induced phenotypes in DS. Nebula/DSCR1 upregulation initially protects against APP-induced memory loss by correcting calcineurin-mediated signaling, but chronic Nebula/DSCR1 overexpression triggers severe mitochondrial dysfunction and ROS elevation that potentially leads to rapid decline in memory during aging in DS. Interestingly, β-amyloid has been shown to trigger upregulation of DSCR1, and DSCR1 upregulation is also associated with tau hyperphosphorylation. It will be particularly interesting in the future to study the effects of Nebula/DSCR1 in modifying β-amyloid and tau-associated memory impairments and to test whether preventing mitochondrial dysfunction and ROS elevations in older animals while correcting calcineurin signaling could alleviate memory problems associated with Nebula/DSCR1 and APP overexpression as seen in some cases of DS and AD (Shaw, 2015).

Htt is a repressor of Abl activity required for APP-induced axonal growth

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. This study asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. htt mutations were found suppress axon outgrowth defects of alphabeta neurons in Appl mutant MB by derepressing the activity of Abl. Abl is required in MB alphabeta neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth (Marquilly, 2021).

Neurodegenerative disease (ND) encompasses a large and heterogeneous group of maladies, including many that are associated with accumulation of specific misfolded proteins. Despite their variety, however, these diseases share a number of cellular pathologies, raising the question of whether different ND-associated genes might function in shared genetic pathways. Several of these disease genes, moreover, have been implicated in neurodevelopmental processes, suggesting that studies of development may be an effective strategy to reveal initially cryptic connections among genes implicated in ND (Marquilly, 2021).

Huntington's disease (HD) is a progressive, autosomal dominant, neurodegenerative disorder. It is a monogenic disease caused by the expansion of a polyglutamine (polyQ) tract at the N-terminus of a large cytoplasmic protein (3144 a.a.), Huntingtin (Htt). Several studies indicate that an alteration of wild-type Htt function might also contribute to disease progression. Consistent with this, numerous biochemical and in vitro studies have suggested that Htt functions in mammalian neuronal development, synaptic function and axonal trafficking. While HD has been characterized as a neurodegenerative disease, a recent study indicates it is also required for normal human brain development (Marquilly, 2021).

Drosophila has been useful as a model to examine the effects of polyQ-expanded human huntingtin transgenes on neuronal form and function. The fly Huntingtin protein (Htt, 3583 a.a.), although lacking a polyQ tract, is similar to the human Htt protein, with four regions of high sequence homology clustered along the protein in the N-, central- and C-terminal regions. Fly Htt is expressed ubiquitously at low level in embryos, larval and adult tissues, with no specific pattern of expression. Fly Htt is found predominantly in the cytoplasm, even when overexpressed. Despite htt being highly conserved across all Drosophila species, indicating an essential role for biological fitness, null htt mutants display no gross developmental defects, although brains from Drosophila Htt mutants have reduced axon complexity. This suggests that it could be necessary to alter the expression of another gene (or genes) during development to be able to detect a htt loss of function phenotype. Therefore, it was asked whether mutant htt modifies a brain axon growth defect present when another neurodegeneration-related protein is lacking, i.e., in a sensitized genetic background (Marquilly, 2021).

In contrast to HD, Alzheimer's Disease (AD) is highly genetically complex. Like HD, AD is also viewed as a proteinopathy, since it is associated with accumulation of amyloid fibrils derived from the Amyloid Precursor Protein (APP). APPs have therefore been investigated intensely, however their normal function in the brain remains unclear and controversial. Drosophila encodes a single APP homologue, called Appl, that is expressed in all neurons throughout development. It has been shown that Appl is a conserved neuronal modulator of a Wnt planar cell polarity (Wnt/PCP) pathway. This signaling pathway is essential for proper axon outgrowth in the learning and memory center of the fly, a bilaterally symmetric pair of structures called the Mushroom Bodies (MB). In this context, it has been proposed that Appl is part of the membrane complex formed by the core PCP receptors, and further that it promotes phosphorylation of the Dishevelled (Dsh) cytoplasmic adaptor protein. Dsh is a core component required for all known Wnt pathways, including the Wnt/PCP pathway. Specifically, Appl recruits a non-receptor protein tyrosine kinase, called Abl, to the PCP receptor complex and positively modulates its phosphorylation of Dsh (Soldano, 2013). Consistent with this view, it has been shown that a 50% reduction of Abl leads to enhancement of the Appl mutant phenotype. Conversely, overexpression of wild-type Abl+, but not a kinase-dead version, in the MB neurons led to a strong rescue of the Appl mutant phenotype. Together with accompanying biochemical experiments, these data suggested that Appl promotes the phosphorylation of Dsh by Abl kinase, and further showed that this mechanism is conserved in mammals (Soldano, 2013). Thus, Abl is a key downstream effector of Appl required for it to stimulate MB axon outgrowth (Marquilly, 2021).

The Abl family of non-receptor tyrosine kinases includes human ABL1 and ABL2 as well as Drosophila Abl. Each Abl protein shares a conserved domain structure consisting of a SH3-SH2-TK (Src homology 3-Src homology 2-tyrosine kinase) domain cassette which confers autoregulated kinase activity. A carboxy-terminal F (F-actin-binding) domain ties Abl-dependent phosphoregulation to actin filament reorganization. ABL1 has been implicated in a range of cellular processes including actin dynamics and cell migration. Abl was discovered as a cellular proto-oncogene that is constitutively active in human chronic myelogenous leukemia and acute lymphocytic leukemia. Kinase activity of Abl in vivo is limited both by intramolecular interactions and by cellular inhibitors, such as Pag/Msp23. After removal of inhibition, Abl acquires substantial catalytic activity that is further enhanced by primary and secondary (auto)phosphorylation. The Abl kinases have also been shown to play a crucial role in the development of the nervous system. Overexpression of active Abl in adult mouse neurons results in neurodegeneration and neuroinflammation and activation of Abl has been shown to occur in human neurodegenerative disease. In contrast to mammalian Abl, Drosophila Abl has not been shown to directly cause tissue hyperplasia or cell fate transformation in vivo, but rather is essential for cell adhesion and morphogenetic processes such as axonogenesis and growth cone motility. Several studies have shown that the precise level of Abl activity is critical to its axonal function, with loss- and gain-of-function both leading to severe defects in neural patterning. Recent experiments revealed the cell biological and biophysical basis of this relationship, showing that either increased or decreased levels of Abl activity induce disorder in growth cone actin and thereby greatly augment the frequency of stochastic errors in growth and guidance. Of particular relevance to this study, Abl has been implicated in axonal arborization and growth in the Drosophila brain, including the MBs though the precise role of Abl in normal MB development has not been documented (Marquilly, 2021).

The MBs, together with the central complex, form the core of the adult central brain of Drosophila. Due to extensive study, they offer an exceptionally powerful system for analyses of genetic and molecular mechanisms of development and function. The MBs are two bilaterally symmetric structures that are required for learning and memory. Each MB is comprised of 2000 neurons that arise from 4 identified neuroblasts. Three types of neurons appear sequentially during development: the embryonic/early larval β', the larval α'β' and the late larval/pupal αβ. Each αβ neuron projects an axon that branches to send an α branch dorsally, which contributes to the formation of the α lobe, and a β branch medially, which contributes to the formation of the β lobe. Both lobes require the PCP mechanism for efficient axon extension. The PCP genes, however, do not act cell-autonomously to promote MB axon growth. Rather, PCP produces a 'community effect' that coordinates the growth decisions of large groups of MB axons, and that overrides the effect of mutations in single PCP components in any single axon or cluster of axons. Appl is required for this PCP-dependent 'community effect'. In β-branches, Appl is evidently also required for some other mechanism that acts cell-autonomously, in addition to its contribution to the non-cell-autonomous PCP mechanism. The molecular nature of this second autonomous Appl function remains unknown (Marquilly, 2021).

This study investigated the genetic and functional interactions of Htt, Appl, Abl and the core PCP gene dsh in Drosophila. Whtt mutations suppress the MB axonal outgrowth defects observed in Appl mutants. Since Abl is known to act downstream of Appl it seemed a potential target for htt mutant-induced suppression of the Appl phenotype. Therefore this study characterized the role of Abl in normal MB development. Using analysis of Abl loss-of function (LOF) alleles in single-cell MARCM clones it was shown that Abl is required for axonal growth in the developing αβ neurons of the MBs and that it is expressed in these neurons. Importantly, the overexpression of Abl in these neurons also leads to axonal growth defects, suggesting the possible existence of cellular proteins that negatively control neuronal Abl activity. Finally, this study demonstrates that Htt acts as a cellular inhibitor of Abl activity, both genetically, as it functions antagonistically to Abl in MB axon growth, and biochemically, as FRET measurement of Abl kinase activity in vivo reveals that is derepressed by reducing Htt expression. These results indicate that Appl and htt, whose human homologs are central players in neurodegeneration, regulate Abl kinase in opposite directions to maintain its activity in the narrow range necessary for normal axon outgrowth in MB αβ neurons (Marquilly, 2021).

Tumorigenesis and neurodegeneration may be two sides of the same coin. Indeed, defining the overlap of molecular pathways implicated in cancer and neurodegeneration may open the door to novel therapeutic approaches for both groups of disorders. Correlative studies have highlighted a decreased cancer incidence in the population with the neurodegenerative disorder Huntington's disease and both wild-type and mutant huntingtin (Htt) have been implicated in tumor progression. Interestingly, it has been proposed that, in the normal physiological situation, the neurodegeneration-related Amyloid precursor protein (APP) recruits the oncogenic Abelson (Abl) kinase in order to promote axonal outgrowth (Soldano, 2013). It is, therefore, tempting to propose that different neurodegenerative diseases (ND) may share components and mechanisms and that Abl may also have a role in ND (Marquilly, 2021).

This study has shown that Abl is required for axonal growth in the MBs, a brain structure that is involved in memory. Furthermore, both Abl overexpression and lack of expression in the MBs result in similar phenotypes, indicating the need to tightly regulate Abl activity during MB axon outgrowth. This raises the question of how Abl activity is normally negatively regulated during MB axon outgrowth. This study confirmed the previous observation that overexpression of Abl rescues the Appld MB phenotypes (Soldano, 2013). Furthermore, Abl overexpression also rescues the dsh1 MB phenotype. These two results support the model that Appl activates Abl, which in turn phosphorylates Dsh. At the genetic level, it was expected that an increase of Abl activity in an individual bearing a loss-of-function mutation of a putative Abl repressor. It was therefore hypothesized that reducing the levels of an Abl repressor would result in suppression of the Appl and dsh mutant MB phenotype. Htt is such an inhibitor of Abl activity in the MBs. The loss of one dose of htt increased the activity of the wild-type Abl still present in Abl2/+ individuals and therefore prevented the enhancement of the MB mutant phenotype. While a number of studies have concluded that Htt deficiency results in significant alterations to kinase signaling pathways, this study is the first to implicate the crucial tyrosine kinase, Abl. Together, these data demonstrate the power that neurodevelopmental studies have to reveal close functional relationships between genes implicated in different forms of neurodegenerative disease (Marquilly, 2021).

It was a surprise that null htt mutants show no obvious developmental defects in Drosophila although strong defects could have been expected from the lack of such a conserved protein. One possible hypothesis is that, due to its fundamental importance, some functional redundancy has been selected to buffer against variation in the production of the Htt protein. It was reasoned that altering the levels of another protein, particularly another protein known to be implicated in neurodegeneration, could reveal cryptic phenotypes of htt mutation during brain development. Following this reasoning, a null Appl mutant was combined with heterozygous null htt mutations in double mutant individuals. Unexpectedly, it was found that mutant htt suppressed the Appl MB axonal outgrowth defect (Marquilly, 2021).

Abl is a key component of the Appl signaling pathway required for axonal arborization and growth in the fly brain and the functional relationship between these two proteins is likely conserved in mammals. While the role of APP-mediated signaling has been shown most clearly in Drosophila, a number of lines of evidence suggest that mammalian APP also fulfills a signaling role. Importantly, and in line with its proto-oncogenic role, Abl tyrosine kinase activity is tightly regulated by intramolecular inhibition. Although Abl is clearly required as a downstream effector of Appl in the MB axon growth, its precise role and regulation in the MBs has not been described previously (Marquilly, 2021).

The three Abl alleles used in this study have all been shown to result in truncated proteins. While Abl1 retains the SH3, SH2 and TK domains of Abl, Abl2 is mutated within the TK domain and only retains the SH3 and SH2 domains, while Abl4 is mutated in the SH2 domain, and only retains an intact SH3 domain. It therefore seems likely that very little or no residual Abl function remains in Abl2/Abl4 and Abl4/Abl1 individuals and may explain why no rescue was observed when one dose of htt was removed in these genetic backgrounds. In contrast, the Abl1/Abl2 allelic combination is likely less severe than the other two genotypes and Abl1/Abl2 animals do accumulate truncated Abl proteins. Indeed, while significant amounts of truncated Abl1 and Abl2 mutant proteins are detectable, only faint protein bands are observed in Abl4 pupae. Therefore, some functionally significant kinase activity could remain in Abl2/Abl1 individuals and the loss of one dose of htt might increase the activity of the remaining kinase activity. Finally, removing one dose of htt in animals overexpressing Abl would result in even more Abl activity, which in turn would exacerbate the mutant phenotype. Contrarily, over-expressing Htt, as in the UAS-Abl, UAS-htt doubly overexpressing individuals, would inhibit Abl function when compared to the UAS-Abl overexpression alone which thus might explain the observed rescue (Marquilly, 2021).

There are three different levels at which Htt might act to modulate Abl activity. First, Htt could either directly or indirectly affect Abl mRNA levels. Although fly Htt has been described as a cytoplasmic protein, htt has been shown to be a suppressor of position-effect variegation, suggesting a possible role in chromatin organization. This hypothesis is unlikely for the MB phenotype described in this study since qRT-PCR analysis of third instar brains did not reveal a significant differences in Abl mRNA levels between httint/+ and control individuals. Second, Htt could play a role regulating Abl protein level in the MBs. This is also considered unlikely since the quantity of the endogenous Abl is unchanged in httint/+ relative to control individuals. Third, Htt could influence the kinase activity of Abl itself. Taking advantage of a FRET biosensor enabling Abl kinase activity to be assayed directly in the MBs, a significant increase in active Abl was seen in httint/+ versus control individuals. Therefore, a model is favored of Htt acting as an inhibitor of Abl kinase activity during normal MB axonal growth. Abl activity in axons needs to be maintained within rather narrow limits. These two recent studies show that either increase or decrease of Abl activity cause disorganization of actin structure in the growth cone and prevent the orderly oscillation of growth cone actin that is the motor for growth cone advance and thus axon extension. Those papers also explain why Abl gain and loss can result in superficially similar mutant axon patterning phenotypes even though the molecular effects of Abl increase versus decrease are opposite (Marquilly, 2021).

The HEAT repeat domains of Htt are thought to function as a solenoid-like structure that acts as a scaffold and mediates inter- and intra-molecular interactions. It is therefore tempting to propose that a scaffolding role of Htt could elicit repression of Abl kinase activity. At least in the MBs, a balance seems to exist in the activity of Abl, positively regulated by the membrane complex formed by the core PCP proteins and Appl, and suppressed by Htt and possibly other proteins, as well. In one case, if Appl is absent, Abl is not optimally activated leading to defects in MB axon growth. Conversely, decrease of Htt to 50% of wild type levels leads to de-repression of Abl kinase activity, which in turn compensates for its sub-optimal level of activation in the absence of Appl. This unexpected apparent balance of activation and inhibition of Abl by Appl and htt, whose mutant orthologs are central players in human neurological disease, may define a conserved functional interaction to maintain Abl activity in the relatively narrow window to appropriately effect axon outgrowth (Marquilly, 2021).


In situ RNA localization has demonstrated that the Appl transcript is found in post-mitotic neurons in all developmental stages in the central and peripheral nervous systems. Within the nervous system, transcripts are observed in neuroblasts, newly generated neurons and at least one class of presumed glial cells. The temporal and spatial specificity of Appl expression suggests that the gene product has a function that is common to most neurons. Appl cDNA predicts an 886-amino acid polypeptide that exhibits strong sequence similarity to the human beta-amyloid protein precursor (APP). It has been suggested that during evolution, a neural-specific function encoded by the APP gene has been selectively maintained (Martin-Morris, 1990).

The recently identified Drosophila gene amyloid protein precursor-like (appl) has a predicted amino acid sequence that shares extensive homology with the beta-amyloid protein precursor (APP) associated with Alzheimer's disease. Characterization of proteins encoded by the appl gene was initiated with the expectation that this simple model system might help elucidate the basic function provided by Appl and APP proteins. Two forms of the Appl protein have been identified in embryonic extracts, primary cultures, and transfected cells. Appl is synthesized as a 145-kDa membrane-associated precursor that is converted to a 130-kDa secreted form that lacks the cytoplasmic domain. Both forms are N-glycosylated. Pulse-chase and subcellular localization studies suggest that the conversion is very rapid. The similarities of biogenesis between APP and Appl provide further evidence that Appl and APP might be functionally homologous, and that the secretion event is of physiological significance. Immunocytochemical studies show that the Appl proteins are first detected in developing neurons concomitant with axonogenesis, and remain associated with differentiated neurons. Appl immunoreactivity is observed in neuronal cell bodies, axonal tracts, and neuropil regions. In the embryo, Appl proteins are expressed exclusively in the CNS and PNS neurons, consistent with the APPL transcript localization. The expression pattern of Appl proteins suggests an ancestral function for this protein in the nervous system (Luo, 1990).

Appl protein is present as a 145 kDa transmembrane protein and a 130 kDa soluble protein (Luo, 1990). The 145 kDa holoprotein is converted to the 130 kDa secreted form by proteolytic cleavage. To gain insights into the function and intracellular trafficking and secretion properties of Appl, the localizations of Appl protein within the different functional regions of the nervous system and at a subcellular level were examined. The insect nervous system can be subdivided operationally into three compartments: the cortical layer where neuronal somata reside; the neuropil region where processes from the overlaying cortex and incoming fibers from other centers ramify and make synaptic contacts, and the fiber pathways that connect cortical areas to the neuropil or connect adjacent neuropil regions. The relative distribution of Appl protein in neuronal cell bodies, neuronal processes, and the extracellular matrix can be revealed by analyzing the presence of the protein in these three compartments (Torroja, 1996).

To reveal Appl proteins, an affinity-purified polyclonal antibody generated against the amino terminal ectodomain common to both Appl forms (Ab952M) was used. In the eye disk only cells posterior to the morphogenetic furrow, where photoreceptors are differentiating, are Appl-immunoreactive. In the CNS, Appl is not detected in regions containing neuroblasts, such as the optic proliferation centers. These observations are consistent with the presence of APPL transcripts in postmitotic neurons (Martin-Morris, 1990). Most neurons in the ventral ganglion (VG) and the brain lobes (BL) show similar levels of Appl immunoreactivity. Under the same conditions, no immunoreactive signal is detected in brains from Appld larvae (Torroja, 1996).

Trafficking and processing of Appl protein is regulated precisely. Differences in trafficking and processing may be dependent on the physiological or developmental stage of a specific neuronal population. To gain insights into the biological significance of the regulation of Appl metabolism, an examination was made of Appl protein distribution during metamorphosis in two regions: the ventral ganglion and the optic lobes. Metamorphosis is a time when the nervous system undergoes dramatic change. For this study both whole-mount preparations and paraffin or cryostat sections stained with anti-APPL antibody Ab952M were used (Torroja, 1996).

The ventral ganglion was chosen as a model to study developmental changes during metamorphosis in Appl distribution because of the simplicity of the pattern of Appl immunoreactivity observed and because the process of remodeling that occurs during metamorphosis has been well characterized in this structure. Appl is observed along the axon tracks in the longitudinal and transverse commissures of the ventral cord in the embryo. In the ventral ganglion of third-instar larval CNS, Appl is no longer discerned along the longitudinal commissures but is concentrated in certain areas of the neuropil. During the first 24 hr of metamorphosis, axons and dendrites of larval neurons prune back, and new arborizations are formed. Axonal growth from new adult-specific neurons and from preexisting larval neurons starts at ~24 hr after pupariation and is completed by 72 hr. Appl immunoreactivity changes during the metamorphosis of the ventral ganglion. Immediately after pupariation, Appl distribution in the ventral ganglion resembles that described for the third-instar larval CNS. The most noticeable changes occur during the early stages of metamorphosis. Six hours after pupariation, the neuropil of the thoracic segments show reduced Appl immunoreactivity compared with the third-instar larval CNS. By 12 hr after pupariation, APPL-immunoreactive signal in the thoracic neuropil is lower and comparable to that in the abdominal segments. By 48 hr, levels of Appl protein in the neuropil are very low, and isolated immunoreactive varicosities and processes are clearly distinguishable. This pattern of Appl immunoreactivity remains unchanged until adult stages (Torroja, 1996).

Most of the neurons that form the optic lobes are new adult-specific neurons. Differentiation of these neurons progresses during late larval and pupal stages and has been well characterized. The first half of metamorphosis is characterized by axonal growth, whereas in the second half, synapses are formed. The process of synaptogenesis seems to continue into the adult. Thus, the optic lobe provides a well-studied structure for correlating Appl metabolism and neuronal differentiation. At 0 hr after pupariation, Appl protein is concentrated in photoreceptor axons within the eye stalk and in the three neuropils of the optic lobe: lamina, medulla, and lobula complex. During development, the optic lobe rotates dorsally, and the relative position of the optic neuropils changes. By 25 hr, Appl signal in the optic neuropils remains intense. Sixty hours after pupariation, Appl immunoreactivity in the medulla is arranged in a modular distribution in three layers that are reminiscent of the synaptic layers described in the adult medulla. Appl staining in the lamina becomes more intense, and axons are distinguished clearly. In the adult, intense Appl immunoreactivity remains in the lamina neuropil. Appl signal in the medulla and lobula neuropils is very low and comparable with the rest of the brain neuropil, except for some isolated axonal processes observed in the medulla (Torroja, 1996).

Thus during metamorphosis, the pattern of Appl immunoreactivity in the neuropil of the nervous system displays dynamic changes. In the ventral ganglion, these changes coincide with the period of axon retraction and outgrowth. During the major period of synaptogenesis in the optic lobes, the Appl immunoreactivity pattern in the neuropil resembles the distribution of the synaptic layers (Torroja, 1996).

The cell bodies of adult neurons display punctate Appl immunoreactivity. Some isolated cells in the brain and thoracic ganglion show higher levels of Appl expression. The lamina is highly stained, and separate processes are detected in the medulla. Interestingly, the highest levels of Appl protein are localized in the neuropil of the mushroom bodies. Mushroom bodies are a principal site of olfactory information processing and are involved in associative olfactory learning and memory in Drosophila. They consist of two complex bilaterally symmetrical groups of neurons (Kenyon cells) in the dorsal-posterior brain that receive input predominantly from the antennal lobes. Kenyon cell dendrites form the calyces, whereas their axons extend through the peduncle to the anterior of the brain. There, these axons form three different neuropils: the alpha lobe, which extends dorsally, and the beta and gamma lobes, which extend medially. All of the mushroom body axonal neuropils stain intensely, including the peduncle, alpha lobe, and beta/gamma lobes. The cell bodies of the Kenyon cells show slightly higher signal than the rest of the cortex. Their dendrites, however, do not appear enriched in Appl protein. Appl protein is also detected in the central complex, the major structure of Drosophila brain controlling locomotor behavior that has been shown to play an important role in learning in Drosophila. In the rest of the brain neuropil, some isolated processes are highly stained (Torroja, 1996).

Appl is found in the neuropil of the larval mushroom bodies, although the relative amount of protein in these structures, differing from what is observed in the adult brain, is comparable to or even lower than the levels of Appl protein detected in other brain and ventral ganglion neuropil areas. Interestingly, the mutant protein secretion-deficient APPLsd concentrates in the mushroom bodies at much higher levels than the endogenous Appl protein, whereas induced wild-type Appl and mutant secreted APPLs protein are detected at very low levels in this structure. The distribution of Appl protein expressed under a heat-shock promoter in Appld;hsp:Appl+/+ adult brains was examined. Four hr after the heat shock, induced wild-type Appl protein is especially concentrated in the axonal neuropil of the mushroom bodies and in the lamina, those areas where endogenous Appl is enriched. As is the case with the endogenous protein, the mushroom body calyces do not show enrichment of induced Appl. Similar to what was observed in larvae, mutant secretion-deficient APPLsd protein induced in adult brains is found enriched in the axonal neuropil of the mushroom bodies and along processes going from the lamina into the medulla (Torroja, 1996).

In the adult, Appl is concentrated in regions known to mediate behavioral plasticity. It is interesting that Appl is found enriched in the axons but not in the dendrites of the Kenyon cells. Moreover, the cell-type specificity of Appl processing and trafficking is involved in generating its differential distribution in the adult neuropil. The requirement of Appl function in the mature nervous system could explain the behavioral deficits displayed by Appl-null mutants. The fact that Appl is concentrated in the axon termini, but not in dendritic fields, suggests that the protein is transported to the presynaptic terminals in the Kenyon cells. Experiments with mutant Appl forms indicate that the membrane-bound Appl holoprotein is the most likely form concentrated in the mushroom bodies. In response to stimulation of the Kenyon cells, Appl transport, cleavage, and secretion in the presynaptic site could be regulated, and this regulation might be involved in modification of synaptic contacts between Kenyon cells and their synaptic targets (Torroja, 1996).

Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain

The mechanisms regulating the outgrowth of neurites during development, as well as after injury, are key to the understanding of the wiring and functioning of the brain under normal and pathological conditions. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, its physiological role in the central nervous system is not known. Many physical interactions between APP and intracellular signalling molecules have been described, but their functional relevance remains unclear. This study shows that human APP and Drosophila APP-Like (APPL) can induce postdevelopmental axonal arborization, which depends critically on a conserved motif in the C-terminus and requires interaction with the Abelson (Abl) tyrosine kinase. Brain injury induces APPL upregulation in Drosophila neurons, correlating with increased post-traumatic mortality in appld mutant flies. Finally, this study shows interactions between APP and the JNK stress kinase cascade. These findings suggest a role for APP in axonal outgrowth after traumatic brain injury (Leyssen, 2005).

APP and its Drosophila homologue APPL promote de novo axonal arborization in the Drosophila brain. Interestingly, this axonal arborization phenotype can also be induced when high levels of APP are induced only in fully mature adult neurons, a situation similar to that after traumatic brain injury in adult organisms. In contrast to previous reports in cell culture, the effect of APP depends critically on its intracellular domain. To elucidate the mechanisms used by APP to induce axonal arborization, the importance of different conserved residues in the APP molecule was investigated and genetic interaction studies were performed. Deletions and point mutations in APP domains known to mediate physical interactions between APP and components of the Abl signalling pathway (Trommsdorff, 1998), including Abl itself (Zambrano, 2001), abolish the APP effect on axonal outgrowth. Furthermore, APP signalling depends critically on the levels and activity of the Abl tyrosine kinase. Consistently, activation of Abl is sufficient to induce axonal arborization of the Drosophila brain small lateral neurons ventral (sLNv), downstream of APPL. The actin-binding protein Profilin, itself known to mediate Abl effects on axons, is required for APP-dependent axonal arborization, suggesting a functional link between APP and the reorganization of the actin cytoskeleton. It is noteworthy that the Abl adaptor protein Dab is an important factor in neuronal migration. As such, it would be interesting to investigate whether the focal dysplasia seen in the triple APP knockout mice is also linked to the Abl tyrosine kinase cascade (Leyssen, 2005).

Since no axonal outgrowth defects were found in the CNS of appl mutant Drosophila and because APP is able to induce axonal outgrowth phenotypes in fully mature CNS neurons, it was reasoned that APPL might have a role in specific postdevelopmental contexts like brain injury. A model was therefore developed for the induction of brain damage in adult Drosophila. Expression of APPL is increased for several days, specifically in neurons surrounding damaged brain areas after injury, and flies mutant for appl show increased post-traumatic mortality, suggesting that APPL has an important physiological role under these conditions (Leyssen, 2005).

Many neurons in the injured brain areas also show activation of the JNK-signalling cascade. JNK signalling is essential for correct regeneration of axons after injury in mammals. This study found that APP-induced axonal arborization depends on intact JNK signalling. This functional link can be explained by the presence of a molecular link between the pathways provided by the physical binding of JIP/APLIP1 to APP, as well as JNK-signalling components. In contrast, downstream effectors like Profilin may be functionally controlled by APP/Abl, while being under the transcriptional control of JNK signalling (Leyssen, 2005).

Theses data combined suggest a model which brings together several of the proposed APP-binding partners in the context of axonal arborization and trauma response in vivo. In this model, it is proposed that acute trauma results in increased APPL expression and independent, but simultaneous, JNK signalling activation. JNK activity provides, via transcriptional activation of Profilin and other cytoskeletal regulators, a permissive environment for remodelling of the actin cytoskeleton. Regulation of Abl signalling by APP ensures that these components are controlled functionally, resulting in an appropriate neuronal response to axonal damage (Leyssen, 2005).

These findings thus provide the first in vivo evidence for a role of APP in axonal arborization in the central nervous system and propose an integrated explanation for a number of intriguing, but thus far separate, observations from biochemical and cell culture studies in various contexts. Interestingly, while expression of the membrane-bound C-terminal fragment of APP (APP-CTF) is sufficient to induce axonal arborization, expression of the APP intracellular domain (AICD), which results from the γ-secretase-mediated cleavage of APP-CTF, does not. It is therefore speculated that the cleavage of APP-CTF by γ-secretase may terminate APP activity and therefore regulate APP-Abl signalling (Leyssen, 2005).

These findings may also have implications on the understanding of some aspects of the pathophysiology of AD. It is hypothesized that during adult life stressful and/or traumatic events in the brain cause APP upregulation. As a side-effect of this, Aβ peptides are generated, which may cause further disruption of neuronal connection. Whether Aβ peptides have any function under trauma conditions in mammals or are just a toxic side product of the cleavage remains to be seen. This hypothesis would, in part, explain the strong epidemiological relationship between brain trauma and AD, as well as the reports of AD-like brain pathology after severe head trauma. Finally, it would be interesting to investigate whether the downstream components of APP signalling identified in this work are AD susceptibility loci, leading to an inefficient neuronal trauma response and thus longer-lasting APP upregulation after brain injury (Leyssen, 2005).


Appl protein associated with neuronal processes might correspond to the membrane-bound holoprotein, whereas Appl that is not associated with processes might be the secreted ectodomain. To analyze how the different forms of Appl protein contribute to Appl immunoreactivity, use was made of transgenic flies that express wild-type Appl or mutant Appl proteins, which mimic the membrane-bound or the secreted forms, in an Appld genetic background. First the heat-shock response of the hsp:Appl+ transgene was studied by heat-shocking adult flies for 30 min and the Appl proteins were examined by immunoblot analysis after specified intervals. Maximum levels of protein are reached 4 hr after the heat shock. Two forms of APPL+ protein are generated that show a precursor-product relationship and correspond to the membrane-bound holoprotein (145 kDa) and the secreted (130 kDa) forms. Therefore, in vivo processing of APPL+ protein produced from the heat-shock transgene is similar to the processing of endogenous Appl protein. On the contrary, mutant Appld flies carrying the secretion-defective hsp:Applsd transgene express a single form of Appl and are unable to generate a secreted protein. The secreted hsp:Appls transgene contains a mutated Appl cDNA, in which codon 789GAA has been changed to generate a translation-stop TAA, so that the encoded protein lacks the transmembrane and cytoplasmic domains. Flies carrying this construct in an Appld background express only a secreted Appl protein. The overall kinetics of expression of APPLsd and APPLs are similar to those of the APPL+ proteins when they are expressed under the influence of a heat-shock promoter (Torroja, 1996).

Appl immunoreactivity was compared in Canton S brains and in brains from Appld larvae carrying hsp:Appl+, hsp:Applsd, or hsp:Appls transgenes. The antibody Ab952M was used, and larval CNSs were fixed and immunoprocessed 2-4 hr after the heat shock. These preparations were analyzed with confocal microscopy, and the Appl immunoreactivity was compared in the cortex, in the thoracic neuropil, and in the optic lobe neuropil (Torroja, 1996).

Because the differential distribution of Appl in the neuropil could be a consequence of cell-specific differences in the levels of Appl transcription, Appl immunoreactivity generated from the hsp:Appl+ transgene was characterized. Under these conditions, Appl protein should be produced at similar levels in all neurons, because the heat-shock response is expected to be the same in nearly all cells. In general, Appl immunoreactivity in heat-shocked transgenic brains is lower than in the wild-type brain. In the cortex, neuronal cell bodies show a punctate Appl immunoreactivity. In the ventral ganglion, Appl signal in the cortex is quite uniform, and although some cells show slightly higher levels of protein, the position of these cells varies from sample to sample and does not correlate with the pattern of immunoreactivity observed in the neuropil. Appl immunoreactivity in the neuropil, however, resembles the pattern observed in wild-type larval CNS, in both the ventral ganglion and brain lobes. As in the wild type, only the neuropil regions of the three thoracic neuromeres and the eighth abdominal neuromere display positive immunoreactivity. Thus, in a situation where all neurons produce similar amounts of Appl protein, Appl immunoreactivity in the neuropil still mimics the normal pattern, suggesting that Appl trafficking varies between different neuronal cell types and results in differential distribution of this protein in the neuropil (Torroja, 1996).

Mutant Appl proteins are detected in cell bodies at levels similar to the induced wild-type protein, and they display the characteristic punctate aspect; however, in the neuropil the two mutant forms behave differently. In the thoracic neuropil, induced APPLs protein shows a distribution similar to the induced wild-type Appl protein. In contrast, secretion-defective APPLsd induction results in a very low signal in the thoracic neuropil, and the residual immunoreactive signal frequently is found along axonal tracts. The situation in the optic lobe is the converse of that in the thoracic and central brain neuropils. Although induced wild-type Appl and secretion-defective APPLsd proteins show enrichment in this structure at levels similar to endogenous APPL, the secreted APPLs form is found at very low levels (Torroja, 1996).

In summary, these results show that those regions where Appl seems to be associated with neuronal processes (optic lobes) are the regions where secretion-defective APPLsd is enriched. Secreted APPLs, however, concentrates in regions that are rich in the Appl form that is not associated with processes (thoracic neuromeres). These data suggest that Appl is secreted in some areas of the neuropil, whereas in others it remains as a transmembrane protein, indicating that the proteolytic processing of Appl is differentially regulated (Torroja, 1996).

To understand the in vivo function of Drosophila Appl protein, flies deleted for the Appl gene were generated. These flies are viable, fertile, and morphologically normal, yet they exhibit subtle behavioral deficits. A fast phototaxis defect in Appl- flies is partially rescued by transgenes expressing the wild-type, but not a mutant, Appl protein. A functional homology between Appl and APP is demonstrated, since transgenes expressing human APP show a similar level of rescue as transgenes expressing fly Appl (Luo, 1992).

The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (betaA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila was used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of betaA4 is observed in both systems. By using SPA4CT, a short APP form in which the signal peptide is fused directly to the betaA4 region, transmembrane domain, and cytoplasmic tail, betaA4 release in flies and fly-tissue culture cells is observed. Consequently, a gamma-secretase activity has been shown to be present in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. Since the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independent of betaA4 generation (Fossgreen, 1998).

Disruption of axonal transport and neuronal viability by Amyloid precursor protein mutations in Drosophila

The hypothesis was tested that amyloid precursor protein (APP) and its relatives function as vesicular receptor proteins for kinesin-I. Deletion of the Drosophila APP-like gene (Appl) or overexpression of human APP695 (an alternatively spliced version of APP) or APPL constructs causes axonal transport phenotypes similar to kinesin and dynein mutants. Genetic reduction of kinesin-I expression enhances while genetic reduction of dynein expression suppresses these phenotypes. Deletion of the C terminus of APP695 or APPL, including the kinesin binding region, disrupts axonal transport of APP695 and APPL and abolishes the organelle accumulation phenotype. Neuronal apoptosis was induced only by overexpression of constructs containing both the C-terminal and Ab regions of APP695. The possibility is discussed that axonal transport disruption may play a role in the neurodegenerative pathology of Alzheimer's disease (Gunawardena, 2001).

Although reducing the amount of kinesin-I to 50% of normal by deleting one of two copies of either the klc or khc gene ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP proteins that contain the cytoplasmic C terminus is predicted to significantly enhance the axonal blockage phenotype. This behavior is expected, because if kinesin-I becomes limiting by virtue of binding excess APP C termini, then further reduction of kinesin-I by deleting one gene copy should dramatically enhance the axonal transport phenotype. To test this prediction, larvae were generated that overexpressed APPL or APP695 and that were also heterozygous for a null mutation, khc8, so that kinesin-I was reduced to 50% of normal. Although larvae overexpressing APPL or APP by themselves or reduced in KHC dosage alone have no striking organismal phenotype, larvae combining these two features exhibit a dramatic new neuromuscular phenotype. These larvae flip their tail and head upwards during crawling, rocking back and forth as they struggled to crawl. Their neurons also contained an enhanced number of organelle accumulations. The extent of accumulations in larvae expressing wild-type APP695 and APPL in the context of reduced KHC dosage was comparable to homozygotes for kinesin-I or dynein mutants and was similarly lethal. Quantitative analysis has revealed a statistically significant difference between siblings with a normal and reduced dose of KHC (Gunawardena, 2001).

To confirm the specificity of the genetic interactions observed between reduction in KHC and overexpression of APP695 or APPL, larvae heterozygous for a null mutation of klc [Df(3L)8ex94, which removes the entire kinesin light chain gene] were generated in combination with constructs expressing APPL and APP695. Neurons from these larvae contain an increased number of organelle jams relative to larvae with a normal dose of KLC. Quantitative analysis reveals a statistically significant difference between siblings containing a normal and reduced dose of KLC, although the extent of enhancement is not as dramatic as when the dosage of KHC is reduced. Although these larvae do not show a larval neuromuscular phenotype as dramatic as was observed when the dosage of KHC was reduced, these larvae show a clear posterior paralysis phenotype. This result is again consistent with a direct functional interaction of the C-terminal region of APP695 and APPL with kinesin-I (Gunawardena, 2001).

Although reducing the amount of cytoplasmic dynein to 50% of normal by deleting one of two copies of either dynein heavy chain (dhc) or dynein light chain (dlc) genes ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP family members that contain the cytoplasmic C terminus is predicted to suppress significantly the severity of the axonal accumulation phenotype. The basis for this prediction is that dynein drives retrograde axonal transport, which is antagonistic to kinesin-I-mediated anterograde axonal transport. In addition, many vesicles or organelles that exhibit net anterograde movement experience periods of retrograde movement owing to the simultaneous presence of kinesins and dyneins on the same vesicle or organelle. Thus, vesicle stalling and axonal accumulations induced by APP are predicted to be ameliorated by dynein reduction by (1) reducing the rate at which vesicles and organelles moved by dynein are transported into regions that have stalled or accumulated vesicles caused by APP expression; or (2) reducing the contribution of dynein-driven movement to a vesicle experiencing stalling because of reduced kinesin-driven activity; this reduction should attenuate vesicle stalling by restoring the balance of movements toward normal (Gunawardena, 2001).

To test this prediction, larvae overexpressing APP695 or APPL were generated that were also heterozygous for either a deficiency of either dhc [Df(3L)GN24] or dlc (roblk). Reduction of dynein suppresses the extent of organelle accumulations in APP695 or APPL transgenic lines. In addition, no significant larval crawling phenotype was observed in these animals. Surprisingly, reduction in dynein dosage also rescues the inviability of males overexpressing APP695. No effect is seen in the lines expressing a C-terminal deletion of APP695 or APPL. Thus, reduction in dosage of a retrograde motor protein appears to be sufficient to decrease organelle accumulations induced by APP or APPL expression (Gunawardena, 2001).

Whether larvae bearing heterozygous deletions of dynein components and the Appl gene in combination would exhibit abnormal axonal transport was investigated. In contrast to the situation with kinesin and APPL, female larvae with one copy (50% dose) of Appl and one copy of dhc or dlc do not have typical axonal accumulations. Intriguingly, male larvae bearing a deletion of the Appl gene, and hence lacking all APPL function, in combination with one copy of dhc or dlc also lack axonal accumulations. Thus, reduction in dynein levels appears to suppress axonal accumulations induced by loss of APPL (Gunawardena, 2001).

Genetic analysis in Drosophila strongly supports the hypothesis that mammalian APP and its homolog, APPL, have kinesin-I receptor functions in vivo. The genetic data and tests complement the in vitro biochemical evidence for a kinesin-I receptor function for APP. In these experiments, APP has been shown to form a complex with conventional kinesin by directly binding to KLC. Transport of APP depends upon kinesin-I and KLC in particular. In addition, the finding that, in Drosophila, APP695 can enter and be transported down axons to neuromuscular junctions and that this transport depends upon the cytoplasmic C terminus containing the proposed KLC binding region supports this view. In toto, these data strongly support the hypothesis that APP functions as a kinesin-I receptor. Perhaps APP bound to kinesin-I may be required for the axonal transport of a subset of cargoes, such as vesicles containing signaling or other molecules used at the synapse. Identifying these vesicles and their cargoes is an important next step (Gunawardena, 2001).

A surprising finding is the suppression of APP and APPL-induced organelle accumulations by genetic reduction of cytoplasmic dynein. Although further work is needed to define the mechanism of this suppression, one simple explanation comes from previous observations about the functionally antagonistic relationship of dynein and kinesin. A general observation is that kinesin and dynein are both present on many of the same axonal vesicles and organelles. Such vesicles and organelles often exhibit alternating anterograde (kinesin) and retrograde (dynein) movements, with net anterograde or retrograde movement resulting from a regulated bias in the balance of opposing movements along the microtubule. Thus, reduction of kinesin-I on non-APP vesicles caused by binding of kinesin-I to excess APP might cause vesicle stalling and organelle accumulations. Stalling of these vesicles and subsequent phenotypes might be rescued by reducing the antagonistic component of movement produced by dynein (Gunawardena, 2001).

An important related issue is whether APP and APPL have functions in addition to their likely roles as kinesin-I receptors on vesicles. Thus, one extreme possibility is that all phenotypic effects caused by genetic manipulation of APPL or APP result from either titration of available kinesin-I or failure to deliver other components of vesicles whose movement depends upon APP or APPL. However, a number of observations support the view that APP and APPL may have additional functions, perhaps mediated by the extracellular domain of the protein. For example, the secreted form of APP has been implicated in the regulation of hemostasis and neuroprotection, while the intact molecule may be involved in cell-extracellular matrix adhesion and in the sequestration of potentially toxic transition metals. There is also evidence that APPL may have a role in synapse differentiation. Thus, APPL and APP may have additional roles at the nerve terminal following their role in the axon as receptors for kinesin-I-dependent transport (Gunawardena, 2001).

It is also striking that several other proteins thought to play a role in Alzheimer's disease have recently been linked to the axonal transport machinery. For example, axonal transport defects are observed in transgenic mice expressing human ApoE4, a gene whose allelic state is associated with increased risk of AD. In the axonal blockages found in these animals, accumulations of synaptophysin, neurofilaments, mitochrondria, and vesicles are seen. Similarly, overexpression of tau protein, a major component of neurofibillary tangles, has been proposed to inhibit kinesin-I-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum in cultured cells and the PNS of transgenic mice. It is also striking that JIP-1/2 proteins, which are scaffolds for components of JNK signaling pathways, may link kinesin-I to ApoER2 and LRP, which are receptor proteins for ApoE. The allelic state of LRP itself has also been reported to be a predisposing factor for AD. Thus, it is conceivable that not only motor proteins but their cargoes and receptors, such as APP, ApoE, and perhaps secretases and other signaling molecules, coaccumulate when axonal transport is impaired in any way. Accumulating these proteins together at the same site, at the same time, may by itself be neurotoxic, may cause induction of cellular suicide signals, may block neurotrophic and other signaling needed for neuronal viability, or may lead to biochemical changes causing excess production of Aß, any or all of which may lead to cell death. Whether these correlations are unrelated or truly indicative of a causative link remains to be tested (Gunawardena, 2001).

The neurodegeneration mutant löchrig interferes with cholesterol homeostasis and Appl processing

The novel Drosophila mutant löchrig (loe) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK: see SNF4/AMP-activated protein kinase gamma subunit), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved. The loe phenotype is modified by the level of HMG-CoA reductase and suppressed by the statin-induced inhibition of this enzyme; statin has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein-like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimer's disease. Western analysis has revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing that could be mediated through HMG-CoA reductase and cholesterol ester (Tschäpe, 2002).

Cholesterol metabolism has been investigated for a long time in peripheral cells, yet relatively little is known about it in brain cells. This is all the more surprising as the brain is the organ richest in cholesterol. Most cells in the body take up the required amount of cholesterol via the LDL or VLDL (low- and very low-density lipoprotein) receptor pathway. After uptake, the lipoproteins are degraded and the cholesterol released within the cell where it can be either used as free cholesterol or stored in the form of cholesterol ester. This transport mechanism is highly conserved in vertebrates and invertebrates. In addition, vertebrate cells can produce cholesterol by de novo synthesis in the endoplasmic reticulum. Due to the blood-brain barrier, brain cells are unable to receive their supply of lipoproteins from the plasma and it has been suggested that only very little is supplied by uptake. At least oligodendrocytes seem to meet their demand for cholesterol by de novo synthesis. Nevertheless, the cerebrospinal fluid contains special lipoproteins, the apolipoproteins apoE and apoAI, and most probably these brain lipoproteins are not involved in the transport of cholesterol to and from the brain but rather in the redistribution of cholesterol within the brain (Tschäpe, 2002 and references therein).

Cholesterol regulates the physical properties of the cell membrane, and its level is therefore tightly controlled. Recent work has shown that cholesterol plays a role in membrane compartmentalization and in the formation of lipid rafts. This important function might be the reason for the connection between cholesterol and neurodegeneration. Studies have shown that the cholesterol level influences the production of the pathogenic Aß peptide, which is produced from the amyloid precursor protein (APP) by cleavage through ß- and gamma-secretase. It has been suggested that Aß processing occurs within rafts, whereas the non-amyloidogenic alpha-processing occurs outside. Cholesterol synthesis in neurons is regulated by hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which again has been connected to Alzheimer's disease. Inhibition of this enzyme by statins not only reduces cholesterol synthesis but also inhibits ß-secretase cleavage of APP. In addition, clinical studies indicate that patients treated with statins have a decreased prevalence of Alzheimer's disease. HMG-CoA reductase activity is negatively regulated via phosphorylation through the AMP-activated protein kinase (AMPK), a heterotrimeric complex, consisting of the catalytic alpha-subunit and ß- and gamma-subunits, found in all eukaryotes (Tschäpe, 2002).

The Drosophila mutant löchrig (loe) disrupts a specific isoform of the AMPK gamma-subunit, which leads to a low level of cholesterol ester together with a strong neurodegenerative phenotype. loe interacts genetically with HMG-CoA reductase and influences processing of the ß-amyloid protein precursor-like (APPL) gene. Although the regulation and most downstream targets of HMG-CoA reductase are conserved, this enzyme is not involved in cholesterol synthesis in insects, because they cannot synthesize cholesterol de novo. The loe mutant now shows that HMG-CoA reductase and its regulator AMPK are also involved in neurodegeneration in insects. The low level of cholesterol ester suggests that the mediator could be cholesterol ester rather than cholesterol, which might be important in the context of Alzheimer's disease because the level of cholesterol ester has been directly correlated with Aß production in cell culture experiments (Tschäpe, 2002).

loe was isolated from a collection of P-element insertion lines. About 800 lines that have a shortened adult life span were aged and screened histologically for signs of neurodegeneration. Two of these lines showed severe vacuolization of the central nervous system (CNS) which increased with aging, and one of them was named löchrig (the German term for 'full of holes'). The vacuolar pathology is most prominent around the central complex and in the central parts of the brain, while the optic lobes are less affected. Developmental studies have suggested that the vacuolization and degeneration in loe are confined to differentiated, probably synaptically active neurons, whereas neuroblasts and developing neurons are unaffected (Tschäpe, 2002).

cDNAs of the loe gene represent at least six alternatively spliced transcripts for the Drosophila gamma-subunit of AMPK. The different mRNAs encode at least three different protein isoforms, all sharing the same C-terminus while varying in their N-terminal part. The C-terminus includes the so-called CBS (cystathionine-ß-synthase) domains that are highly conserved between yeast, mammals and Drosophila. Interestingly, a region in the unique N-terminus of the LoeI isoform shows homology to the X11alpha protein which can bind to the APP protein (Borg, 1998); LoeI and X11 are 28% identical and 41% similar over a stretch of 80 amino acids. The P-element is inserted in the seventh intron of this transcript and 38 bp upstream of the transcription start site of LoeII, suggesting that one or two transcripts are affected by the insertion (all other transcripts are >10 kb downstream of the insertion site and therefore most probably are not affected by the P-element). A small deletion of 1.3 kb was created around the insertion site, removing exon 1 of the LoeII transcript, and these flies do not show a degeneration phenotype. This indicates that LoeII is not required for CNS integrity (Tschäpe, 2002).

To assess whether the loe mutation influences cholesterol metabolism, a role described for AMPK, the lipid composition of fly heads was measured. The analysis of phospholipids, triglycerides and free cholesterol did not reveal any significant differences between 1- to 5-day-old wild-type and mutant flies. The amount of cholesterol ester, however, was reduced by ~40%. Expressing LoeI in neurons restored the wild-type level of cholesterol ester in the mutant, confirming the role of Loe/AMPK in cholesterol homeostasis. The expression of LoeI restores the cholesterol ester level as well as the neurodegenerative phenotype, directly connecting cholesterol ester and neurodegeneration in the loe mutant. These results reveal an involvement of AMPK in cholesterol ester levels in the brain independent of de novo cholesterol synthesis. In peripheral tissues, vertebrate AMPK inhibits the activation of a hormone-sensitive lipase, an enzyme involved in the breakdown of cholesterol ester. A conserved regulatory pathway in the brain could account for the decreased amount of cholesterol ester (Tschäpe, 2002 and references therein).

A functional homology to the mammalian AMPK is supported further by the accumulation of fatty acids in the mutant, another pathway regulated by AMPK (Tschäpe, 2002).

AMPK negatively regulates HMG-CoA reductase, a key enzyme in cholesterol synthesis in vertebrates. In Drosophila, this protein is encoded by the columbus (clb) gene (Van Doren, 1998). To assess whether loe interacts with the clb mutation, flies were created homozygous for loe and heterozygous for two strong, embryonic lethal alleles of clb (which both had the same effect). clb/+; loe mutants show a weak but significant suppression of vacuolization compared with loe mutant flies. To confirm an interaction, lines expressing Clb were used in the loe background. In contrast to the clb mutation, Clb overexpression enhances the phenotype. Control flies, containing only the UAS-Clb construct but no neuronal promoter construct, did not differ from the original loe mutants. The interaction was quantified by counting holes in the different genotypes and measuring their total volume. The enhancement by Clb overexpression and suppression by the clb/+ mutant suggests that HMG-CoA reductase is negatively regulated by AMPK as in other organisms. In addition, an influence on the cholesterol ester level of loe was investigated. Overexpression of Clb slightly reduced, and introduction of one mutant copy of clb slightly increased, the cholesterol ester level in loe; however, the differences are not significant. Nevertheless, they are in agreement with the results on the neurodegenerative phenotype because the clb mutation suppresses and additional Clb enhances the phenotype. Interestingly, the function of HMG-CoA reductase in cholesterol synthesis is not conserved because insects cannot synthesize cholesterol de novo. However, many other downstream genes and regulatory feedback mechanisms are conserved, and one of them might connect HMG-CoA reductase and cholesterol ester (Tschäpe, 2002).

HMG-CoA reductase can be inhibited pharmacologically by a class of drugs called statins, which have also been shown to decrease the prevalence of Alzheimer's disease. To assess whether treatment with statins influences the neurodegeneration in loe, flies fed on glucose were compared with or without the drug lovastatin. Flies kept on lovastatin showed a suppression of the vacuolization compared with control animals. Treatment of wild-type flies with lovastatin revealed no adverse effects. These results show that the progressive neurodegeneration in loe can be slowed successfully by treatment with statins. The level of cholesterol ester was tested in loe flies treated with statins, but no significant difference could be found (Tschäpe, 2002).

Cholesterol homeostasis has been implicated in the processing of Aß from APP, as has statin treatment, which can dramatically decrease Aß production. Therefore, whether loe influences APPL, the fly homolog of human APP, was investigated. Appld mutants, which carry a deletion in the Appl gene, do not reveal any signs of neurodegeneration. However, crossing Appld with loe flies shows an enhancement of the loe vacuolization. The effect is weaker in loe flies carrying one copy of Appld (loe/loe; Appld/+) compared with homozygous double mutants (loe/loe; Appld/Appld) and can be detected in the central brain as well as the optic system (Tschäpe, 2002).

To determine whether loe might influence the APPL protein, Western blot analysis of brain extracts was performed. Using an anti-APPL polyclonal antibody, two bands were detected in w1118 flies, representing the genetic background used to induce the loe mutation. The bands correspond to the membrane-associated 145 kDa precursor and the 130 kDa secreted form, which are absent in Appld. In the loe mutant, similar amounts of APPL precursor protein are found; however, the level of the processed secreted form is reduced. Conversely, more of the secreted form is found when additional LoeI is expressed in neurons. This reveals a role for loe in APPL processing or stabilization of the processed form. To assess whether this effect is specific for APPL, the processing of Notch, which is cleaved by a mechanism similar to that of APP, was investigated. No differences were detected in the processing of Notch, suggesting a specific function of loe in APPL processing, possibly mediated by the X11alpha similarity domain. In addition, whether Columbus or statin treatment influences APPL processing in loe was investigated. Additional expression of Clb, which enhanced the neurodegenerative phenotype of loe, also enhances the processing effect, causing a slight further reduction of APPL processing. On the contrary, one copy of mutant clb or statin treatment slightly increased processing. This suggests that the neurodegenerative phenotype is correlated with the processing of APPL (Tschäpe, 2002).

Thus, a mutation in the AMPK gamma-subunit causes progressive neurodegeneration in Drosophila. AMPK is a central component of a protein kinase cascade conserved in eukaryotes that acts as a metabolic sensor to monitor the cellular AMP and ATP levels. In cases of ATP depletion, the major ATP function described to date is to activate energy-providing mechanisms while inactivating energy-consuming processes. AMPK is a heterotrimer, consisting of the catalytic alpha-subunit and the ß- and gamma-subunits, which are required for stabilization of the complex and kinase activity. The activity of the complex is regulated by phosphorylation through an upstream kinase, and both phosphorylation and dephosphorylation are sensitive to AMP levels. For all three subunits, different isoforms have been identified that assemble into specific AMPK complexes with distinguishable tissue distribution in peripheral tissues in vertebrates. Whereas most tissues predominantly express one gamma isoform, the human brain expresses three different isoforms. Interestingly, two of them have extended N-termini with no significant homology to each other, LoeI or any other protein. The loe mutation shows, for the first time, that such a brain-specific isoform has a unique function in brain maintenance, which cannot be substituted by other isoforms. This function probably goes beyond the basic role in energy regulation because all isoforms share the C-terminus, which is sufficient for a functional gamma-subunit and, therefore, a functional AMPK complex. It will be interesting to discover whether one of the human isoforms is also required specifically for neuronal survival (Tschäpe, 2002).

AMPK has a central role in cholesterol metabolism by regulating HMG-CoA reductase and hormone-sensitive lipase, which is involved in the breakdown of cholesterol ester in vertebrates. Although hormone-sensitive lipase has not been found in the brain, a cholesterol ester hydrolase activity is described for the brain; however, nothing is known about the potential regulation of this enzyme by AMPK. An inhibitory function of AMPK in the brain would lead to an overactivity of this hydrolase and, therefore, to a reduced level of cholesterol ester. A Drosophila protein with homology to hormone-sensitive lipase can be found in the Drosophila Sequencing Project, but unfortunately no mutant has been described so far. However, a deficiency deleting this enzyme was tested for genetic interactions with loe. Because this deficiency had no influence on the loe phenotype, it is assumed that it is not involved in the neurodegenerative phenotype. In contrast, this study shows a genetic as well as a pharmacologically induced interaction of loe with HMG-CoA reductase (clb). The interaction reveals that, as in vertebrates, AMPK acts upstream of HMG-CoA reductase. Because a mutation in clb suppresses and overexpression enhances the neurodegenerative loe phenotype, the inhibitory function of AMPK on HMG-CoA reductase seems to be conserved. Interestingly, the function of HMG-CoA reductase is not completely conserved between vertebrates and insects, because arthropods cannot synthesize cholesterol de novo. Rather, HMG-CoA reductase is involved in the production of non-sterol isoprenoids from mevalonate. The effect of HMG-CoA reductase on neurodegeneration cannot, therefore, be mediated through cholesterol synthesis and, as measurements show, the cholesterol level is unaltered in loe. However, the amount of cholesterol ester is lowered in loe and adding or removing Clb has a slight influence on it, and APPL processing in loe is influenced by Clb. In this context, it is worth mentioning that statins dramatically decrease Aß production before a reduction in cholesterol can be detected. This suggests that other members of the cholesterol pathway might regulate APP processing, possibly cholesterol ester (Tschäpe, 2002).

The loe mutation reveals a connection between cholesterol ester and progressive neurodegeneration in the model system Drosophila. In vertebrates, such a link has been established by the finding that accumulation of Aß can decrease cholesterol esterification in neurons (Koudinova, 1996; Liu, 1998). found that The level of cholesterol ester directly correlates with Aß production (Puglielli. 2001), and that elevated concentrations of cholesterol ester but not free cholesterol increase the generation of Aß. In contrast, it has been shown that lowering the cholesterol concentration inhibits APP cleavage by secretases and interferes with the localization of APP in membrane rafts (Simons, 1998; Frears, 1999). These are membrane microdomains consisting of lipids, proteins and cholesterol, and their correct composition seems to be required for normal APP processing. These results strengthen the likelihood of a role for cholesterol ester because the loe mutant links a reduced level of cholesterol ester, leaving free cholesterol unaltered, with decreased processing of APPL (Tschäpe, 2002).

These results clearly reveal a function of AMPK in APPL processing. However, the Appl mutant enhances the neurodegenerative phenotype of loe. Like knock-outs of APP in mice, the Appld null mutation of Drosophila displays only subtle neurological deficits. In the loe mutant background, however, Appl can be connected to progressive neurodegeneration, which might help to understand the function of APP proteins. Because the lack of APPL enhances the phenotype, this hints at a neuroprotective function, perhaps specifically of the soluble form of APPL -- this was also suggested by cell culture studies of APP. In this model, neurons would be more vulnerable to the effect of the loe mutation when APPL and its soluble form are missing. The Appl mutant itself might not show degeneration because the damaging event is absent (Tschäpe, 2002).

With the isolation of the loe mutant, a connection has been made between AMPK, a second enzyme (in addition to HMG-CoA reductase involved in cholesterol homeostasis) and neurodegeneration and APPL processing. This underlines the importance of the cholesterol biosynthesis pathway for the maintenance of the nervous system and for understanding of neurodegenerative diseases such as Alzheimer's. With the Drosophila loe mutant available, the role of this pathway in neurodegeneration can now be studied in an easily accessible model organism (Tschäpe, 2002).

Mouse disabled 1 regulates the nuclear position of neurons in a Drosophila eye model

Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Disabled 1, which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhances the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduces the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling that regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons (Pramatarova, 2006).

Aβ42 mutants with different aggregation profiles induce distinct pathologies in Drosophila

Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. This study compared the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. The aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro were compared. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. These results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo (Iijima, 2008).

In summary these results lead to the prediction of two issues. First, the partial prevention of Aβ42 amyloidgenesis by aggregation inhibitors may result in qualitative shifts in the pathogenic effects of Aβ42. Second, the tendency of Aβ42, a natively unfolded polypeptide consist primarily of random-coil structure in their native and soluble states, to aggregate may be affected by a combination of genetic, environmental, and aging factors, and the resultant Aβ42 conformers or species may contribute to the heterogeneous pathogenesis of AD. The existence of different 'Aβ species' has been verified both in vitro (Petkova1, 2005) and in vivo (Meyer-Luehmann, 2006; Iijima, 2008 and references therein).

Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model

APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. It has been shown that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. This study examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, synaptic transmission accuracy was impaired in this model, and this phenotype was improved by yata knockdown. Thus, these data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP (Furotani, 2018).

This study examined whether the inhibition of the synaptic localization of APP affects the phenotypes of the Drosophila model of Alzheimer's disease. For this purpose, the yata gene. The yata gene is required for the intracellular transport of the APPL protein, which is the Drosophila orthologue of mammalian APP. This study induced the expression of the human Swedish mutant APP in larval motor neurons. Knockdown and null mutation of yata resulted in decreased synaptic localization of APP. In this study, immunostaining was performed using the anti-APP antibody MAb 4G8 that recognizes amino acid residues 17–24 of the amyloid β region. Therefore, the observed localization was the expression for full-length APP or the processed fragment that contains the amyloid β region. Notably, yata mutation resulted in the differential suppression of the synaptic localization of a subset of proteins including APP without uniformly affecting all of the synaptic molecules. The data showed that synaptic localization of APP was impaired whereas localization of Fasciclin II was not significantly affected by yata knockdown. Localization of Synaptotagmin and Cysteine string protein was also not significantly affected even in yata null mutants. Such selectivity is a desired property of a tool that therapeutically targets APP. On the other hand, localization of Fasciclin II was significantly decreased in the yata null mutants. yata has a mammalian orthologue, SCYL1. Although both yata and SCYL1 are suggested to be involved in the trafficking of the coated secretory vesicles, the data suggested that the impact of yata loss-of-function is differential among proteins that are transported by vesicular trafficking. The data suggested that APP and Fasciclin II are affected by yata mutation. As a possibility, specific secretory vesicles that contain a subset of proteins such as APP and Fasciclin II as cargos are relatively severely affected by yata mutation. Another molecule whose synaptic localization was found to be affected in yata mutants was the Bruchpilot protein, which is a cytoplasmic protein and is a component of the electron-dense T-bar structures of the presynaptic active zone. This finding was unexpected, because yata/SCYL1 is suggested to play a role in the trafficking of secretory vesicles. Therefore, synaptic dysfunction caused by impaired synaptic development may affect the assembly of presynaptic components such as Bruchpilot. Alternatively, yata may also be involved in the expression or trafficking of synaptic cytoplasmic proteins (Furotani, 2018).

Knockdown of yata partially ameliorated abnormalities in the Alzheimer's disease model, such as increased number of satellite boutons, lethality during development, lethality in the first 10 days after eclosion and impaired synaptic transmission accuracy. In addition, heterozygous introduction of the yata null allele also partially rescued the developmental lethality and lethality within 10 days after eclosion, although the effect was weaker and developmental lethality was not rescued in males. yata knockdown and heterozygosity of the yata null allele themselves caused no apparent abnormalities. The only phenotype observed to be caused by yata knockdown was the elevated variance in the amplitudes of eEJPs. While homozygotes of the yata null allele show phenotypes including developmental abnormalities, progressive brain volume reduction and shortened lifespan, heterozygotes of the yata null allele are viable and fertile and show no apparent abnormalities like many other recessive mutations. The reason why the knockdown of yata causes no apparent abnormalities may be because the effect of knockdown is partial and decreased expression of yata is still sufficient for most of molecular functions of yata. On the other hand, most of the observed rescuing effects were only partial. This finding may possibly be because the synaptic localization of induced APP is still sufficient for the expression of some phenotypes, although the synaptic localization was decreased. Alternatively, synapse-independent mechanisms controlled by induced APP may exist (Furotani, 2018).

Lifespan data showed that the lethality caused by the expression of APP is restricted to the developmental stage from the embryo to the the first 10 days of the adult stage after eclosion. The animals that survived past this stage showed an almost normal lifespan, suggesting that the cause of death is a defect in neural development. Because lethality can be suppressed by yata knockdown and possibly by the decreased synaptic localization of a subset of proteins including APP, this finding suggests that the cause of death is a defect in synapses. In addition, electrophysiological analysis revealed that the expression of APP resulted in impaired synaptic transmission accuracy, because expression of APP caused a significantly elevated variance in the amplitudes of eEJPs, while the amplitudes and frequencies of mEJPs were not significantly affected. The phenotype of the variance of eEJPs was suppressed by yata knockdown and possibly by the decreased synaptic localization of a subset of proteins including APP. Such instability of synaptic transmission may be a result of impaired synaptic development. Notably, knockdown of yata also caused a impairment in the accuracy of synaptic transmission similar to the phenotype caused by the expression of APP, potentially reflecting the physiological function of yata in synaptic physiology. While synaptic localization of ectopically induced APP is decreased by yata knockdown, synaptic localization of other endogenous synaptic proteins may also be affected, and this might contribute to the elevated variance in eEJPs by yata knockdown. Knockdown of yata caused elevated eEJP variance but did not cause premature lethality of animals. These data are against the possibility that the instability of synaptic transmission directly causes the death of animals. The identity of synapses that contribute significantly to the death of animals is also unknown. In this model, the excessive formation of satellite boutons was observed in the neuromuscular synapses on the body wall muscles of larvae. This means that the expression of APP resulted in the loss of proper control of synapse formation. Such a loss of control may cause fatal results in the synapses that are essential for the survival of animals (Furotani, 2018).

In this study, the expression of human Swedish mutant APP associated with familial Alzheimer's disease was induced. Among the phenotypes observed in this study, excessive formation of satellite boutons is known to be caused by the overexpression of human wild-type APP and Drosophila Appl. Therefore, these phenotypes are suggested to be caused by the elevated gene dosage of Appl/APP. It is necessary to examine the contribution of APP mutation to other phenotypes including observed electrophysiological phenotypes, especially because it has been previously shown that the overexpression of Drosophila Appl caused different electrophysiological phenotypes including a decrease in eEJP amplitude, an increase in mEJP amplitude and a decrease in quantal content, although experimental conditions including the selection of Gal4 were different (Furotani, 2018).

Synaptic loss is observed in patients with Alzheimer's disease. On the other hand, previous studies have shown that mammalian APP-family genes are involved in synaptic development. Double knock-out mice of several combinations of APP family genes show embryonic lethality, possibly caused by defects in synaptic morphology and function. Although the APP protein is transported and localized in synapses, whether the role of APP in synaptic morphology and function is attributed to the function of the APP protein localized in the synapses is still unclear. In this study, the data suggested that the suppression of synaptic localization of a subset of proteins including APP partially rescued the synaptic phenotypes caused by APP, suggesting the importance of the function of APP localized in synapses. On the other hand, while Alzheimer's disease is a late-onset disease, this model and double APP knock-out mice show developmental defects in synapses. In fact, loss-of-function mutants of the Drosophila Appl gene show not only defects in synaptic development but also late-onset shortened lifespan. The shortened lifespan of the Appl mutants may reflect the physiological function of Appl in the maintenance of the nervous system against aging. Alternatively, developmental defects may also cause late-onset phenotypes. It remains to be elucidated if the molecular function of Appl in synapses is related to the late-onset premature death of Appl mutant flies, although the relevance of the physiological functions of APP in the pathogenesis of Alzheimer's disease is still unclear and the current model did not show the late-onset premature death of animals (Furotani, 2018).

Because Drosophila yata mutants and SCYL1 null mutant mice share neurodegeneration phenotypes, and the human SCYL1 gene has been identified as a causative gene of a genetic disease causing peripheral neuropathy and cerebellar atrophy, the molecular function of these orthologues seems to be evolutionarily conserved. The synaptic pathology of Alzheimer's disease may be able to be modified if one could control the synaptic localization of APP. Although the mammalian orthologue of yata, SCYL1, is a candidate target molecule to affect the synaptic localization of APP, complete ablation of both Drosophila yata and mammalian SCYL1 result in fatal phenotypes including neurodegeneration. Moreover, knockdown of yata itself causes impaired synaptic transmission accuracy. Therefore, it is necessary to examine if there is a way to control the synaptic localization of APP without fatal side-effects, if it is possible to control the functional expression of SCYL1 strictly and if SCYL1 can be used as a target molecule in a therapeutic approach for the treatment of Alzheimer's disease (Furotani, 2018).

Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening

The subcellular localization of membrane and secreted proteins is finely and dynamically regulated through intracellular vesicular trafficking for permitting various biological processes. Drosophila Amyloid precursor protein like (APPL) and Hikaru genki (HIG) are examples of proteins that show differential subcellular localization among several developmental stages. During the study of the localization mechanisms of APPL and HIG, a novel mutant was isolated of the gene, CG1973, which was named yata. This molecule interacted genetically with Appl and is structurally similar to mouse NTKL/SCYL1, whose mutation was reported to cause neurodegeneration. yata null mutants showed phenotypes that included developmental abnormalities, progressive eye vacuolization, brain volume reduction, and lifespan shortening. Exogenous expression of Appl or hig in neurons partially rescued the mutant phenotypes of yata. Conversely, the phenotypes were exacerbated in double null mutants for yata and Appl. The subcellular localization of endogenous APPL and exogenously pulse-induced APPL tagged with FLAG was examined by immunostaining the pupal brain and larval motor neurons in yata mutants. These data revealed that yata mutants showed impaired subcellular localization of APPL. Finally, yata mutant pupal brains occasionally showed aberrant accumulation of Sec23p, a component of the COPII coat of secretory vesicles traveling from the endoplasmic reticulum (ER) to the Golgi. Thus this study identified a novel gene, yata, which is essential for the normal development and survival of tissues. Loss of yata resulted in the progressive deterioration of the nervous system and premature lethality. The genetic data showed a functional relationship between yata and Appl. As a candidate mechanism of the abnormalities, it was found that yata regulates the subcellular localization of APPL and possibly other proteins (Sone, 2009).

Drosophila amyloid precursor protein-like is required for long-term memory

The amyloid precursor protein (APP) plays an important role in Alzheimer's disease (AD), a progressive neurodegenerative pathology that first manifests as a decline of memory. While the main hypothesis for AD pathology centers on the proteolytic processing of APP, very little is known about the physiological function of the APP protein in the adult brain. Likewise, whether APP loss of function contributes to AD remains unclear. Drosophila has been used extensively as a model organism to study neuronal function and pathology. In addition, many of the molecular mechanisms underlying memory are thought to be conserved from flies to mammals, prompting a study of the function of APPL, the fly APP ortholog, during associative memory. It was previously shown that APPL expression is highly enriched in the mushroom bodies (MBs), a specialized brain structure involved in olfactory memory. This study analyzed memory in flies in which APPL expression has been silenced specifically and transiently in the adult MBs. The results show that in adult flies, APPL is not required for learning but is specifically involved in long-term memory, a long lasting memory whose formation requires de novo protein synthesis and is thought to require synaptic structural plasticity. These data support the hypothesis that disruption of normal APP function may contribute to early AD cognitive impairment (Goguel, 2011).

This study took advantage of inducible RNAi expression to study the function of endogenous amyloid precursor protein-like in the Drosophila olfactory memory center. Specifically reducing APPL expression in the adult MBs led to LTM disruption, showing that the observed memory defect is not caused by a subtle alteration in brain development. Furthermore, although this study did not search for amyloid deposits in knock-down flies, it seems unlikely that decreasing APPL expression for 2 d may result in their production. Rather, memory impairment is most likely caused by the loss of APPL function in young flies (Goguel, 2011).

The construction and analysis of flies lacking the APPL gene (Appld) has been reported previously. Significantly lower STM scores were observed after one cycle of aversive conditioning but it could not be concluded that Appl-null mutation led to a memory defect, because Appld flies did not react normally to electric shock. In the current study, flies showed normal reaction to electric shock, odor avoidance, and STM. A possible explanation is that previously observed behavioral deficits may have been caused by a mild defect in brain development or by the complete lack of APPL in the adult or both. Because APPL is highly expressed in the central complex, the major Drosophila brain structure controlling locomotor behavior, it is possible that the complete lack of APPL in this structure could lead to motor defects (Goguel, 2011).

In mice, several loss-of-function analyses have suggested that APP might be involved in learning and memory. These studies have relied upon knock-out (KO) or knock-in techniques or injection of anti-APP antibodies or antisense oligonucleotides. Single APP KO leads to relatively mild behavioral defects, probably caused by functional redundancy, while the adult APP-/-APLP1-/-APLP2-/- triple KO cannot be analyzed because of early postnatal lethality. APP-null mice display neuroanatomical abnormalities and reduced brain weight, suggesting that APP plays a role in development and somatic growth. It is thus difficult to determine in KO studies the relative contributions of defects caused by a role in the adult brain versus those caused by a requirement during the development and maintenance of neuronal networks. Also, injection experiments should be interpreted with caution, as antibodies might not be specific for a single APP species, and in addition, interaction of APP with an antibody might activate intracellular APP-mediated signaling events. Not only does Drosophila present the advantage of a single APP-encoding gene, but the genetic tools available in this organism allow specific silencing in a restricted part of the adult brain (Goguel, 2011).

It has been proposed that AD physiopathology might in part be caused by loss of normal APP function, in particular during the early stages of the disease characterized by memory impairment. APP processing by the amyloid pathway precludes its cleavage by α-secretase, thus leading to a decrease in the production of secreted APPα (sAPPα), a neurotrophic and neuroprotective peptide. Indeed, several studies have shown that sAPPα levels are decreased in both familial and sporadic AD, while low levels have been correlated with poor memory performance in humans. It was thus suggested that decreased amounts of sAPPα in AD brain contributed to the memory deficit characterizing AD, independently of Aβ toxicity. Consistent with these observations, analyses in rodents have shown a role for sAPPα in spatial memory. The current data establish that in the fly a memory deficit is caused by loss of APPL function independently of the toxicity of the amyloid pathway, thus giving further support to the hypothesis that loss of function might contribute to early AD cognitive decline (Goguel, 2011).

The first symptom of AD is a disability to encode new memories while old declarative memory remains unaffected. This study reports that APPL loss of function in the fly affects LTM formation, whereas learning, STM, and ARM remain unaffected. Although it is not straightforward to compare complex behavioral deficits occurring in AD patients to those of invertebrates, at first glance these observations could appear contradictory. In Drosophila, LTM is the only memory form that relies on de novo protein synthesis and is therefore likely to involve long-lasting changes in synaptic plasticity. This leads to the hypothesis that APPL dysfunction causes a failure of synaptic plasticity such as that thought to underlie the earliest cognitive features of AD, namely an impairment in acquiring new information. It is possible that initial AD symptoms might be generated by APP loss of function, while further cognitive decline might result from progressive loss of synapses and neurons due, at least in part, to an additional toxic pathogenic mechanism such as Aβ accumulation. In the future, the power of genetic manipulation in the fly, together with behavioral studies, should allow unraveling of APPL function in memory (Goguel, 2011).

Relationships between the circadian system and Alzheimer's disease-Like symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, this study used transgenic Drosophila expressing various Amyloid-beta (Abeta) peptides, which are prone to form aggregates characteristic of AD pathology in humans. Development of AD-like symptoms were compared in adult flies expressing Abeta peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per01). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Abeta peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. Rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Abeta peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Abeta peptides causes rhythm degradation downstream from the central clock mechanism (Long, 2014; PubMed).

Epigenetic control of learning and memory in Drosophila by Tip60 HAT action

Disruption of epigenetic gene control mechanisms in the brain causes significant cognitive impairment that is a debilitating hallmark of most neurodegenerative disorders including Alzheimer's disease (AD). Histone acetylation is one of the best characterized of these epigenetic mechanisms that is critical for regulating learning and memory associated gene expression profiles, yet the specific histone acetyltransferases (HATs) that mediate these effects have yet to be fully characterized. This study investigated an epigenetic role for the HAT Tip60 in learning and memory formation using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. Tip60 is endogenously expressed in the Kenyon cells, the intrinsic neurons of the MB and in the MB axonal lobes. Targeted loss of Tip60 HAT activity in the MB causes thinner and shorter axonal lobes while increasing Tip60 HAT levels cause no morphological defects. Functional consequences of both loss and gain of Tip60 HAT levels in the MB are evidenced by defects in immediate recall memory. ChIP-Seq analysis reveals that Tip60 target genes are enriched for functions in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, it was found that both learning and immediate recall memory deficits that occur under AD associated amyloid precursor protein (APP) induced neurodegenerative conditions can be effectively rescued by increasing Tip60 HAT levels specifically in the MB. Together, these findings uncover an epigenetic transcriptional regulatory role for Tip60 in cognitive function and highlight the potential of HAT activators as a therapeutic option for neurodegenerative disorders (Xu, 2014).

Gr33a modulates Drosophila male courtship preference

In any gamogenetic species, attraction between individuals of the opposite sex promotes reproductive success that guarantees their thriving. Consequently, mate determination between two sexes is effortless for an animal. However, choosing a spouse from numerous attractive partners of the opposite sex needs deliberation. In Drosophila melanogaster, both younger virgin females and older ones are equally liked options to males; nevertheless, when given options, males prefer younger females to older ones. Non-volatile cuticular hydrocarbons, considered as major pheromones in Drosophila, constitute females' sexual attraction that act through males' gustatory receptors (Grs) to elicit male courtship. To date, only a few putative Grs are known to play roles in male courtship. This study reports that loss of Gr33a function or abrogating the activity of Gr33a neurons does not disrupt male-female courtship, but eliminates males' preference for younger mates. Furthermore, ectopic expression of human amyloid precursor protein in Gr33a neurons abolishes males' preference behavior. Such function of APP is mediated by the transcription factor Forkhead box O (dFoxO). These results not only provide mechanistic insights into Drosophila male courtship preference, but also establish a novel Drosophila model for Alzheimer's disease (AD) (Xue, 2015).

To avoid futile reproductive efforts, an animal must distinguish conspecifics from other species and differentiate the sex of conspecific partners. It must also determine the most suitable mates from large amounts of available partners in order to maximize reproductive efficiency. Evolution endows Drosophila melanogaster males with the instinct to discriminate conspecifics from other Drosophila species1 and to discern females from males. It also bestows on them the ability to select the most favorable mates among masses of desirable virgin females (Xue, 2015).

In Drosophila, non-volatile cuticular hydrocarbons (CHCs) have been recognized as a type of major sex pheromone, which convey information of an individual such as species and sex. Female-specific CHCs are detected by male gustatory receptors (Grs), a chemosensory receptor family mainly responsible for detecting non-volatile chemicals, during tapping and licking steps in stereotypical male courtship behavior. So far, only a few Grs, including Gr32a, Gr33a and Gr39a are reported to be engaged in Drosophila male courtship behavior. While Gr32a acts to assist males to discriminate conspecifics from other species, Gr39a is required for males to distinguish females from males. On the other hand, Gr33a functions to inhibit male-male courtship. Despite these findings, roles of the Grs in males' choices for the most favorable mates have remained largely unknown (Xue, 2015).

A previous study (Hu, 2014) set a paradigm of choice model in which both options (younger virgin females and older ones) are proved to be attractive to Drosophila males, but males still intensely prefer younger mates to older ones. Using this model, this study explored the mechanisms by which males bias their potential mates. Gene loss-of-function, gain-of-function, and cell-inactivation experiments demonstrated that Gr33a and Gr33a neurons are essential for males' preference for younger mates. Since the previous data indicated that pan-neuronal expression of human amyloid precursor protein (APP) ablates males' preference for younger mates, this study sought to investigate whether APP expression in Gr33a neurons would affect this behavior. Indeed, it was found that Gr33a neurons-specific expression of APP abolished males' preference for younger mates, and this function of APP is mediated by the transcription factor forkhead box O (dFoxO) (Xue, 2015).

Drosophila male courtship choice has been frequently applied for studying decision making in animals, yet most of the past studies have focused on male courtship choices between likes and dislikes, such as court towards females vs. males, or virgin vs. non-virgin females. Previous study has characterized a choice behavior between two equally-liked options: mature virgin females, whether younger or older, were similarly attractive to naive males; nevertheless, when given the option, males turn out to be picky and prefer younger virgin females to older ones. This study found that a gustatory receptor, Gr33a, is necessary for males' preference for younger mates. Gr33a is thought to be necessary to inhibit homosexual behavior; its role in heterosexual behavior, however, is rarely pondered. This study has revealed the critical role of Gr33a in males' preference for younger mates. Furthermore, ectopic expression of APP in Gr33a neurons eliminates males' preference behavior, and such function is mediated by dFoxO, a recently reported downstream factor of APP27. Therefore, this work demonstrates the genetic interaction of APP and dFoxO in Gr33a neurons, which modulates males' preference for younger mates. APP is identified as a potential causative protein of AD, a common progressive neurodegenerative disorder, in which cognitive decline is the prime symptom. Although Drosophila has long been utilized for building AD models to investigate the pathogenesis and possible cure for AD, accepted Drosophila AD models are limited to locomotion model and life span model, which have little correlation with cognitive ability. The current findings, however, have offered the possibility for establishing a novel Drosophila AD model that is related to cognitive ability (Xue, 2015).

In all CHCs produced by files, 7, 11-HD and 7, 11-ND have been identified as female specific aphrodisiac pheromones to Drosophila melanogaster males. GC and MS studies suggest that both 7, 11-HD and 7, 11-ND are expressed at lower concentration in younger virgin females than the older ones. Hence, it appears unlikely that 7, 11-HD or 7, 11-ND is the cause that leads males to court younger virgin females more vigorously than the older ones. On the contrary, since Gr33a has been reported as a receptor of aversive odors, it is more likely that older females produce certain aversive odors that can be recognized by males and repel them. Consistent with this explanation, this study found that the concentrations of most detected CHCs are significantly higher on the older virgin females than the younger ones. Nevertheless, at this stage it was not possible to identify the CHC(s) that serves as the aversive pheromone to males. Besides, it cannot be excluded that younger virgin females produce unknown attractive pheromones other than 7, 11-HD or 7, 11-ND. However, all CHCs detected on younger virgin females also present on older ones at a similar or higher lever. Thus, the tentative conclusion is drawn that younger virgin females do not produce more attractive pheromones than the older ones. The results, taken together, unravel the role of bitter sensory Gr33a neurons in males' preference for younger mates and infer that older females might produce certain aversive odors that cause males to turn to younger mates (Xue, 2015).

Linking Aβ42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model

Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. β-amyloid (Aβ) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aβ has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. This study shows that overexpression of human Aβ42 in a Drosophila model indeed induces increased neuronal activity. It was found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, it was found that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aβ42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, Kv4 was transgenically over-expressed to near wild-type levels in Aβ42-expressing animals. It was shown that restoration of Kv4 attenuates age-dependent learning and locomotor deficits, slows the onset of neurodegeneration, and partially rescues premature death seen in Aβ42-expressing animals. The study concludes that Aβ42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aβ42-Drosophila model, and possibly in AD (Ping, 2015).

Aβ-induced hyperexcitability is indeed intriguing, with interesting implications especially for seizure-like activity and epilepsy, which are potentially associated with AD. Little, however, has been done previously to determine whether Aβ-induced hyperactivity contributes to downstream behavioral pathologies. Recent studies, however, suggest that neuronal hyperactivity may precede neurological dysfunction and may be improved by pharmacologically reducing activity. This study shows that Kv4 channels are specifically down-regulated by Aβ42 expression, while other K+ currents (eg. Kv2 and Kv3) remain unaltered in cultured neurons and in the intact brain. The resulting increase in neuronal excitability is present in the adult brain at an age (8 days AE) before the appearance of locomotor (14–15 days AE) and learning defects (14 days AE), and before the onset of neurodegeneration (25 days AE), supporting the hypothesis that hyperactivity precedes and contributes to these downstream pathologies. The study also shows that increasing Kv4 channel levels in Aβ-expressing animals restores normal excitability to neurons, and as a result, completely rescues learning and locomotor defects, slows neurodegeneration, and slightly increases lifespan. It is significant to note that the expression of a UAS-GFP or UAS-CD8-GFP transgene does not rescue any of these pathologies, suggesting that any rescue effects by UAS-Kv4 are not simply due to the introduction of another UAS target for GAL4 that would dilute the expression of Aβ42; indeed, quantification of Aβ42 is not any lower in Aβ42+Kv4 flies. In future studies, it will be interesting to examine the temporal requirement for reducing excitability with Kv4 expression; for example, is early hyperexcitability more “toxic” to the system than later stage hyperexcitability? (Ping, 2015).

Although specificity of rescue by Kv4 varies from one pathology to another, the genetically engineered EKO channel that acts as a general activity inhibitor does not ameliorate any of the cognitive, motor, or survival deficits tested. This suggests that general dampening of excitability is not sufficient to replace Kv4 loss. Kv1, the other A-type K+ channel present in Drosophila, however, is able to rescue Aβ42-induced locomotor dysfunction, but, interestingly, not learning or premature death. These results are consistent with the fact that Kv1 and Kv4 share some, but certainly not all, biophysical properties. For example, Kv4 channels have a much more hyperpolarized voltage-operating range than Kv1 channels, making them much more likely to play roles at subthreshold potentials. Also, while both Kv4 and Kv1 channels display fast inactivation, the inactivation rate is voltage-independent for Kv4 channels and voltage-dependent for Kv1 channels. Finally, the subcellular localization of Kv4 and Kv1 channels are thought to be distinct, with Kv4 channels restricted to dendrites and cell bodies, and Kv1 channels localized in axons and nerve terminals (Ping, 2015).

The study also examined whether the loss of Kv4 function alone is sufficient to lead to cognitive and motor pathologies. Previously, it was shown that expression of a dominant-negative Kv4 subunit, DNKv4, results in the elimination of the Kv4 current. Loss of Kv4 function leads to increased excitability and locomotor deficits. In the present study, it was found that expression of DNKv4 also induces learning defects and a shortened lifespan, consistent with a key role for the Aβ42-induced reduction in Kv4 in these downstream pathologies. In mammalian systems, Kv4.2 has been shown to play a role in the induction of long-term potentiation (LTP), and hippocampal dependent learning/memory defects. Loss of Kv4 function alone, however, does not induce any significant neurodegeneration, suggesting that while Aβ42-induced loss of Kv4 exacerbates degeneration, it is not sufficient to trigger neurodegenerative pathway(s) (Ping, 2015).

Previous reports over the years have shown different effects of Aβ on A-type K+ currents in vitro, with some identifying decreases in A-type K+ currents (IA) and others reporting increases in IA. Differences between these studies are likely to be due to a variety of factors including the species of Aβ tested (eg. Aβ1–40, Aβ1–42, Aβ25–35; some studies finding clear differences with different Aβ species, the cell type examined (eg. HEK cells, hippocampal neurons, or cortical neurons), and the time course of the effect (eg. from seconds to days in different studies). For example, the Aβ species applied, the concentration used, and time incubated with cells all affect the assembly state of Aβ, which has also been proposed to have differential downstream effects on K+ currents and excitability/activity. In the future, it will be interesting to see how effects on Kv4 develop, and possibly change, throughout the assembly of Aβ42 from monomers to oligomers, protofibrils, and mature fibrils in vivo (Ping, 2015).

Much remains to be understood about the mechanism by which Kv4 channels are lost in response to Aβ42 expression. In this study, pharmacological and genetic approaches suggest a degradation pathway for Kv4 that depends on both the proteasome and lysosome, similar to the EGF receptor. This scenario is likely to be complicated since previous studies have shown that Aβ directly inhibits the proteasome, and that clearance of Aβ depends on the proteasome. Further study is needed to understand how Kv4 channels are targeted for turnover by Aβ42, and what other component(s) are involved (Ping, 2015).

Further study is also needed to unravel specific mechanisms by which Kv4 channels function in downstream Aβ42 pathologies. For example, how does the loss of Kv4 exacerbate neurodegeneration? One possibility is that cell death is induced by an “excitotoxic” pathway due to excess Ca2+ entry, and ultimately, necrosis. Interestingly, previous studies have shown that Aβ42 induces an increase in various K+ currents that are linked to cell death in vitro, consistent with evidence that efflux of K+ is required as an early step in apoptosis. While it is not clear how to reconcile these findings with ours, it does seem that proper K+ homeostasis is critical for neuronal survival. The role of Kv4 in lifespan, however, is complex, given that neurodegeneration, learning/memory formation, and locomotor activity all contribute to survival. The partial to full rescue of multiple Aβ42-induced pathologies by Kv4, however, underscores the importance of the loss of Kv4 in vivo and suggests that Kv4 is a critical target of Aβ42 in this model, and perhaps in AD (Ping, 2015).

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. This study examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. It was shown that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. The study's data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, it was shown that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) causes disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintain stronger circadian rhythms during aging. In summary, this study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).

Loss of rest-activity rhythms is a well-established early symptom of AD in humans. Because disruption of circadian rhythms is detrimental to neuronal homeostasis, it is important to understand relationships between AD and circadian rhythms at the cellular and molecular levels. To address this question, this study examined how manipulations of the fly ortholog of APP and its cleaving enzymes affect endogenous rest-activity rhythms and clock mechanism in Drosophila. Over-expression of dBACE was found to disrupt behavioral rest-activity rhythms, and this effect is most severe in aged flies suggesting an age-dependent mechanism. Furthermore, dBACE expression resulted in dampened oscillation of the core clock protein PER in central pacemaker neurons, which are master regulators of rest activity rhythms. Significantly reduced PER levels are observed in the sLNv and lLNv neurons of age 50d flies expressing dBACE in all clock cells (including glia), all neurons, or only in PDF-positive sLNv and lLNv neurons. These data suggest that manipulation of APP-cleavage by dBACE over-expression directly affects the oscillation of PER protein in central pacemaker neurons in a cell-autonomous manner. Since a functional clock mechanism in sLNv is necessary and sufficient to maintain free running activity rhythms, reduced oscillations of PER in these neurons could be responsible for the loss of activity rhythms in age 50d flies. Importantly, the decline in PER levels occurrs only in flies with manipulated dBACE, not in old control flies. This is in agreement with earlier findings that aging does not dampen PER oscillations in pacemaker neurons of wild type flies, while it reduces clock oscillations in peripheral clocks  (Blake, 2015).

While this study reports that the loss of behavioral rhythms after manipulation of dBACE is associated with reduced expression of clock genes in the central pacemaker, other recent work shows that expression of human Aβ peptides leads to disruption of rest activity rhythms without interfering with PER oscillations in the central pacemaker. Even strongly neurotoxic Aβ peptides, such as Aβ42 arctic, do not cause rhythm disruption when expressed in central pacemaker neurons; rather, pan-neuronal expression is required. The fact that even the most neurotoxic Aβ peptides are not capable of dampening PER oscillation in pacemaker neurons suggests that Aβ production does not affect clock oscillations and that it is not Aβ production that causes the phenotype observed in this study upon over-expression of dBACE. This was confirmed by expression of KUZ, whose activity does not increase dAβ production; however, it also leads to disruption of rest-activity rhythms. Similar rhythm disruption by dBACE and KUZ suggests that an excess cleavage product of both pathways might be responsible for the disruption. Like in the mammalian APP cleavage pathway, in Drosophila cleavage of APPL by KUZ or dBACE results in a C-terminal fragment (CTF) that is subsequently cleaved by the ϒ-secretase resulting in the production of dAICD. Indeed, it was shown that expression of dAICD results in an age-dependent decline in rhythmic locomotor activity. As with dBACE and KUZ expression, dAICD expression causes weakening or complete loss of behavioral rhythms while age-matched control flies remain highly rhythmic. In this context, it is worth noting that α-secretase activators are considered for clinical trials to reduce Aβ production in AD patients. However, according to results in this study, this could lead to disruptions of circadian rhythms and sleep patterns thus negatively impacting the lives of patients and their caretakers (Blake, 2015).

This study's data suggest that increased dAICD may be the proximal cause of decay in rest-activity rhythms. The role of AICD in AD is increasingly evident but poorly understood. AICD is able to enter the nucleus and has been implicated in transcriptional regulation that may affect cell death, neurite outgrowth and neuronal excitability. Interestingly, transgenic mice expressing AICD have increased activity of GSK-3, which in flies affects the circadian clock. Over-expression of GSK-3 in Drosophila leads to altered circadian behavior by hyper-phosphorylation of TIMELESS (TIM), a key circadian protein which forms dimers with PER that enter the nucleus and regulate the clock mechanism. Of further interest, increased GSK-3 activity has been implicated in AD, and in Drosophila, increased GSK-3 activity mediates the toxicity of Aβ peptides (Blake, 2015).

Cleavage of APPL likely results in a significant decline in intact APPL, and this could be detrimental as APPL has neuroprotective effects. It was also recently shown that loss of full-length APPL induces cognitive deficits in memory. This study reports that flies over-expressing full-length APPL in central pacemaker neurons maintain stronger behavioral rest-activity rhythms during aging than control flies; however this effect is not observed when APPL is expressed pan-neuronally. This could be caused by negative effects of APPL when expressed in other unspecified neurons, or could be related to driver strength. Overall, the study suggests that the loss of full-length APPL might negatively affect circadian behavior by way of the central pacemaker neurons (Blake, 2015).

Over-expression of dAICD induces a severe phenotype, disrupting rest-activity rhythms as early as age 5d when expressed in central pacemaker neurons and by age 35d with pan-neuronal expression. Taken together these results suggest that while loss of full-length APPL by over-expression of its secretases might negatively impact circadian behavior, the cleavage product dAICD induces the most severe behavioral rest-activity disruption. Interestingly, the observed effect is not likely a product of neurodegeneration as it was previously shown that dAICD has no effect on neurodegeneration, and this study shows that the pacemaker cells appear intact in pdf > dAICD flies. In addition, it was shown that dAICD, like the vertebrate AICD, can be found in the nucleus. Therefore, this study suggests that dAICD may directly or indirectly affect the expression of clock genes. This offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).


The amyloid precursor protein is a conserved Wnt receptor

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. This study shows that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner (Liu, 2021).

This study has identified a previously unknown conserved Wnt receptor function for APP proteins. APP was shown to bind both canonical and non-canonical Wnt ligands via a conserved CRD and that this binding regulates the levels of full-length APP by regulating its intracellular trafficking from early endosomes to the trans Golgi network versus the lysosome. Finally, it was shown that APP through the CRD regulates neurite growth and axon branching complexity in primary mouse cortical neurons (Liu, 2021).

A function for APP as a cell surface receptor has be proposed for quite a long time. The first strong evidence came from the structure of APP which shares similarity with type I transmembrane receptors. For example the growth factor like domain (GFLD) in the E1 region of APP could act as a ligand-binding site, and disulfide bridges within the same E1 area could further facilitate ligand-induced signal transduction by stabilizing the structure of APP ectodomain. In addition, the site-specific proteolytic processing property of APP resembles several membrane receptors and constitute a second line of evidence. For instance, both APP and Notch are processed by γ-secretase and release the intracellular domain AICD and NICD, respectively, to induce transcriptional activity by nuclear transfer of the ICDs. Several putative ligands have been proposed including proteolytic products of APP itself. Interestingly, a conserved CRD within the E1 region of APP makes APP a putative Wnt receptor as the CRD is required for the binding between Wnt and its receptor and co-receptor like Frizzled Ror-2 and Musk, although the crystal structure of the APP CRD is different from that of classic Wnt receptors (Liu, 2021).

Using immunoprecipitation this study has shown that mouse APP binds in a CRD-dependent manner to Wnt3a and Wnt5a the two well-known ligands for triggering canonical or non-canonical Wnt signaling pathways, respectively. Based on these data, it is speculated that APP might act as a conserved Wnt receptor as its CRD is highly conserved across species, and that the binding may not be limited to Wnt3a or Wnt5a, but also apply to all other Wnt family members. As Wnt signaling is critical for development and tissue homeostasis, the role of APP as a component or putative receptor or co-receptor in Wnt signaling should be carefully studied. The difficulty to explore the exact role of APP may not only come from the complexity of Wnt signaling, but also from the structural properties of APP itself. For example, the extracellular domain of APP harbors several binding site for the molecules in the extracellular matrix (ECM), such as the Heparin binding domain (HBD) is not only exist in E1 area a second HBD has been found in the E2 region. A recent study has shown that LRP6, the co-receptor of canonical Wnt signaling, could directly bind to APP. Using peptide Mapping array that study further revealed several binding sites (20-70 amino acids in length) in the ectodomain of APP, and one of the sites (34 amino acids) is located within the APP CRD. Based on IP results indicating a Wnt receptor function for APP, an obvious question arises as to whether LRP6 and APP compete for Wnt. Before trying to address this problem, several issues need to be resolved. First, since in the IP experiments the whole CRD (150 amino acids) was deleted, it is not clear if specific sequences inside the CRD are critical for Wnt binding that may or may not overlap with the reported LRP6-binding site. A second important point is that the results of the peptide mapping array may not reflect the exact binging site for LRP6 as these short synthesized peptides may lack information embedded in the 3D structure like the disulfide bridges formed by Cysteines that may be critical for maintaining the stability of the APP extracellular domain. Significant further biophysical and biochemical analysis is required to understand the details of the interaction and various components of the Wnt receptor complex (Liu, 2021).

APP has been extensively reported to be involved in regulating neurite outgrowth, with conflicting conclusions as to whether APP promotes or inhibits neurite outgrowth. In the current experiments, it was found that while in Drosophila APPL loss reduced axonal growth, the comparison of axonal outgrowth and branching in primary cortical neuron derived from mAPP wild type or mAPP knock out mice at DIV2, DIV3 and DIV7 showed that loss of mAPP significantly accelerated axonal maturation. Specifically, it was found that the initial phase of axonal growth at DIV2 is unaffected, but that APP mutant axons grow longer at DIV3 and then show increased axon complexity at DIV7. It is therefore suggested that the conflicting data in the literature may arise from examining different types of neurons at different time points, where the requirement of APP may differ in a context-specific manner. It is speculated that this context specificity may in part be due to the levels and types of Wnt ligands present in the environment (Liu, 2021).

Finally, the current findings suggest that in addition to the well-described proteolytic processing of APP, the regulation of its recycling by Wnt ligands may be crucial for its function. It is important to note that, like proteolytic processing, Wnt ligands regulate APP stability post-translationally, as no effect was found on App mRNA levels upon Wnt treatment. With regard to the role of APP processing in Alzheimer's disease, recently published work suggests that an imbalance between Wnt3a/canonical signaling pathway and the Wnt5a/PCP signaling pathway at the initial step of amyloid beta production could trigger a vicious cycle favoring the amyloidogenic processing of APP (Sellers, 2018; Elliott, 2018). The findings of this study, that Wnt3a and Wnt5a have opposite effects on amyloid beta production, provide a mechanistic framework for understanding how the normal physiological function of APP directly impacts the generation of a key marker of AD, and thus potentially links the normal activity of APP to its role in neurodegeneration. Whether and how the modulation of APP's role in Wnt signaling may offer future therapeutic avenues for AD is an exciting venue for future research (Liu, 2021).

Amyloid-beta (Abeta) plaques promote seeding and growth in spreading of alpha-Synuclein and Tau in a mouse model of Lewy body disorders with Abeta pathology

Studies have shown an overlap of Abeta plaques, tau tangles, and alpha-synuclein (alpha-syn) pathologies in the brains of Alzheimer's disease (AD) and Parkinson's disease (PD) with dementia (PDD) patients, with increased pathological burden correlating with severity of cognitive and motor symptoms. Despite the observed co-pathology and concomitance of motor and cognitive phenotypes, the consequences of the primary amyloidogenic protein on the secondary pathologies remain poorly understood. To better define the relationship between alpha-syn and Abeta plaques, this study injected alpha-syn preformed fibrils (alpha-syn mpffs) into mice with abundant Abeta plaques. Abeta deposits dramatically accelerated alpha-syn pathogenesis and spread throughout the brain. Remarkably, hyperphosphorylated tau (p-tau) was induced in alpha-syn mpff-injected 5xFAD mice. Finally, alpha-syn mpff-injected 5xFAD mice showed neuron loss that correlated with the progressive decline of cognitive and motor performance. These findings suggest a 'feed-forward' mechanism whereby Abeta plaques enhance endogenous alpha-syn seeding and spreading over time post-injection with mpffs (Bassil, 2020).

Fe65 is the sole member of its family that mediates transcription regulated by the amyloid precursor protein

The amyloid precursor protein (APP), a central molecule in Alzheimer's disease (AD), has physiological roles in cell adhesion and signaling, migration, neurite outgrowth and synaptogenesis. Intracellular adapter proteins mediate the function of transmembrane proteins. Fe65 (also known as APBB1) is a major APP-binding protein. Regulated intramembrane proteolysis (RIP) by gamma-secretase releases the APP intracellular domain (AICD), together with the interacting proteins, from the membrane. The impact was studied of the Fe65 family (Fe65, and its homologs Fe65L1 and Fe65L2, also known as APBB2 and APBB3, respectively) on the nuclear signaling function of the AICD. All Fe65 family members increased amyloidogenic processing of APP, generating higher levels of beta-cleaved APP stubs and AICD. However, Fe65 was the only family member supporting AICD translocation to nuclear spots and its transcriptional activity. Using a recently established transcription assay, this study dissected the transcriptional activity of Fe65 and provides strong evidence that Fe65 represents a transcription factor. Fe65 relies on the lysine acetyltransferase Tip60 (also known as KAT5) for nuclear translocation. Furthermore, inhibition of APP cleavage reduces nuclear Tip60 levels, but this does not occur in Fe65-knockout cells. The rate of APP cleavage therefore regulates the nuclear translocation of AICD-Fe65-Tip60 (AFT) complexes, to promote transcription by Fe65 (Probst, 2020).

APLP2 modulates JNK-dependent cell migration in Drosophila

Amyloid precursor-like protein 2 (APLP2) belongs to the APP family and is widely expressed in human cells. Though previous studies have suggested a role of APLP2 in cancer progression, the exact role of APLP2 in cell migration remains elusive. This report shows that ectopic expression of APLP2 in Drosophila induces cell migration which is mediated by JNK signaling, as loss of JNK suppresses while gain of JNK enhances such phenotype. APLP2 is able to activate JNK signaling by phosphorylation of JNK, which triggers the expression of matrix metalloproteinase MMP1 required for basement membranes degradation to promote cell migration. The data presented in this study unraveled an in vivo role of APLP2 in JNK-mediated cell migration (Wang, 2018).

Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

Abeta peptide that accumulates in Alzheimer's disease brain, derives from proteolytic processing of the amyloid precursor protein (APP) that exists in three main isoforms derived by alternative splicing. The isoform APP695, lacking exons 7 and 8, is predominately expressed in neurons and abnormal neuronal splicing of APP has been observed in the brain of patients with Alzheimer's disease. This study demonstrates that expression of the neuronal members of the ELAVL protein family (nELAVLs) correlate with APP695 levels in vitro and in vivo. Moreover, evidence is provided that nELAVLs regulate the production of APP695; by using a series of reporters it was shown that concurrent binding of nELAVLs to sequences located both upstream and downstream of exon 7 is required for its skipping, whereas nELAVL-binding to a highly conserved U-rich sequence upstream of exon 8, is sufficient for its exclusion. Finally, this study reports that nELAVLs block APP exon 7 or 8 definition by reducing the binding of the essential splicing factor U2AF65, an effect facilitated by the concurrent binding of AUF-1. This study provides new insights into the regulation of APP pre-mRNA processing, supports the role for nELAVLs as neuron-specific splicing regulators and reveals a novel function of AUF1 in alternative splicing (Fragkouli, 2017).

Amyloid-beta Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

Alzheimer's disease (AD) is characterized by excessive production and deposition of amyloid-beta (Abeta) proteins (see Drosophila Appl) as well as synapse dysfunction and loss. While soluble Abeta oligomers (AbetaOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. This study screened synaptic organizer proteins for cell-surface interaction with AbetaOs and identified a novel interaction between neurexins (NRXs; see Drosophila Neurexin) and AbetaOs. AbetaOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of beta-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AbetaOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2; see Drosophila Neuroligin) and the leucine-rich repeat transmembrane protein LRRTM2. Although AbetaOs do not interfere with the binding of NRX1beta to NLG1 or LRRTM2, time-lapse imaging revealed that AbetaO treatment reduces surface expression of NRX1beta on axons and that this reduction depends on the NRX1beta HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of beta-NRXs, but not alpha-NRXs, decreases. Thus these data indicate that AbetaOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal beta-NRXs, providing molecular and mechanistic insights into how AbetaOs lead to synaptic pathology in AD (Naito, 2017).

Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease

The greatest risk factor for Alzheimer's disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid β (Aβ) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. An adult-onset Drosophila model of AD has been developed. Mutant Aβ42 accumulation leads to increased mortality and neuronal dysfunction in the adult flies. Furthermore, lithium was shown to reduce Aβ42 protein, but not mRNA, and was able to rescue Abeta42-induced toxicity. The current study investigated the mechanism/s by which lithium modulates Aβ42 protein levels and Aβ42 induced toxicity in the fly model. Lithium was found to cause a reduction in protein synthesis in Drosophila and hence the level of Aβ42. At both the low and high doses tested, lithium rescued the locomotory defects induced by Aβ42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. These data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis (Sofola-Adesakin, 2014).

Altered synapses in a Drosophila model of Alzheimer's disease

Alzheimer's Disease (AD) is an age related neurodegenerative disease characterized by memory loss and decreased synaptic function. Advances in transgenic animal models of AD have facilitated understanding of this disorder, and have aided in the development, speed, and efficiency of testing potential therapeutics. The characterization of a novel model of AD has been described in the fruit fly, where the human AD associated proteins APP and BACE were expressed in the central nervous system of the fly. This study describes synaptic defects in the larval neuromuscular junction (NMJ) in this model. The results indicate that expression of human APP and BACE at the larval NMJ lead to defective larval locomotion behavior, decreased pre-synaptic connections, altered mitochondrial localization in presynaptic motor neurons, and decreased postsynaptic protein levels. Treating larvae expressing APP and BACE with the γ-secretase inhibitor L-685, 458 suppresses the behavioral defects as well as the pre- and postsynaptic defects. It is suggested that this model will be useful to assess and model the synaptic dysfunction normally associated with AD, and will also serve as a powerful in vivo tool for rapid testing of potential therapeutics for AD (Mhatre, 2014).

The central molecular clock is robust in the face of behavioural arrhythmia in a Drosophila model of Alzheimer's disease
Circadian behavioural deficits, including sleep irregularity and restlessness in the evening, are a distressing early feature of Alzheimer's disease (AD). These phenomena were investigated by studying the circadian behaviour of transgenic Drosophila expressing the amyloid beta peptide (Aβ). Aβ expression was found to result in an age-related loss of circadian behavioural rhythms despite ongoing normal molecular oscillations in the central clock neurones. Even in the absence of any behavioural correlate, the synchronised activity of the central clock remains protective, prolonging lifespan, in Aβ flies just as it does in control flies. Confocal microscopy and bioluminescence measurements of molecular clock function point to the output pathway as the main site of Aβ toxicity. In addition there appears to be significant non-cell autonomous Aβ toxicity resulting in morphological and likely functional signalling deficits in central clock neurones (Chen, 2014).

Though it is widely accepted that amyloid-β (Aβ) is a key factor in Alzheimer's disease (AD) pathology, its underling mechanism remains unclear. In order to study the association between Aβ and neural circuitry dysfunction, a primary culture preparation derived from the nervous system of transgenic Drosophila melanogaster larvae was developed expressing human Aβ1-42 (Aβ42). Cultured neurons undergo a consistent developmental process, culminating in an elaborate neuronal network with distinct functional and morphological characteristics. Throughout this development, a time-dependent increase in intracellular expression levels of Aβ42 was detected, followed by extracellular staining at a later time point. When compared to controls, Aβ42 cultures exhibited enhanced levels of apoptosis, resulting in reduced cell viability. Moreover, as primary culture preparations enable high resolution monitoring of neuronal phenotypes, it was possible to detect subtle morphological changes in neurons expressing Aβ42, namely an enhancement in neurite outgrowth and arborization, which preceded the effect of neurodegeneration. These results establish D. melanogaster primary neuronal cultures as a rapid, accessible and cost-effective platform for AD molecular studies and drug screening, and suggest a possible role for Aβ42 in the organization of neuronal processes (Saad, 2014).

APP homodimers transduce an amyloid-β-mediated increase in release probability at excitatory synapses

Accumulation of amyloid-β peptides, the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. This study shows that Aβ40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aβ40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aβ40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease (Fogel, 2014).

Activity-dependent p25 generation regulates synaptic plasticity and Aβ-induced cognitive impairment

Cyclin-dependent kinase 5 regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. This study shows that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIalpha-dependent manner. Moreover, a knockin mouse model was developed in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Deltap35KI) to prevent p25 generation. The Deltap35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 phosphorylation at Ser845. Finally, crossing the Deltap35KI mice with the 5XFAD mouse model of Alzheimer's disease (AD) resulted in an amelioration of beta-amyloid (Aβ)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Aβ-associated neurotoxicity and AD-like pathology (Seo, 2014).

Human LilrB2 is a β-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model

Soluble β-amyloid (Aβ) oligomers impair synaptic plasticity and cause synaptic loss associated with Alzheimer's disease (AD). Murine PirB (paired immunoglobulin-like receptor B) and its human ortholog LilrB2 (leukocyte immunoglobulin-like receptor B2), present in human brain and acting as known receptors for major histocompatibility complex class I antigen, are receptors for Aβ oligomers, with nanomolar affinity. The first two extracellular immunoglobulin (Ig) domains of PirB and LilrB2 mediate this interaction, leading to enhanced cofilin signaling, also seen in human AD brains. In mice, the deleterious effect of Aβ oligomers on hippocampal long-term potentiation require PirB, and in a transgenic model of AD, PirB not only contribute to memory deficits present in adult mice, but also mediate loss of synaptic plasticity in juvenile visual cortex. These findings imply that LilrB2 contributes to human AD neuropathology and suggest therapeutic uses of blocking LilrB2 function (Kim 2013).

A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-Amyloid production

Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. An RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, was performed complemented with a Rab-GTPase-activating protein screen, and a road map is presented of the membrane-trafficking events regulating Aβ production. Rab11 and Rab3 were identified as key players. Although retromers and retromer-associated proteins control APP recycling, Rab11 controls β-secretase endosomal recycling to the plasma membrane and thus affects Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. These results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD (Udayar, 2013).

The zebrafish amyloid precursor protein-β is required for motor neuron guidance and synapse formation

The amyloid precursor protein is a transmembrane protein mostly recognized for its association with Alzheimer's disease. The physiological function of APP is still not completely understood much because of the redundancy between genes in the APP family. This study used zebrafish to study the physiological function of the zebrafish APP homologue, appb, during development. appb was shown to be expressed in post-mitotic neurons in the spinal cord. Knockdown of appb by 50%-60% results in a behavioral phenotype with increased spontaneous coiling and prolonged touch-induced activity. The spinal cord motor neurons in these embryos show defective formation and axonal outgrowth patterning. Reduction in Appb also results in patterning defects and changed density of pre- and post-synapses in the neuromuscular junctions. Together, these data show that development of functional locomotion in zebrafish depends on a critical role of Appb in the patterning of motor neurons and neuromuscular junctions (Abramsson, 2013).

Cloning of APP homologs

The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. Several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans have been isolated and the corresponding gene has been designated as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP (Daigle, 1993).

Complimentary DNA clones have been isolated from Xenopus larva to delineate a protein highly homologous to the human beta-amyloid precursor protein (APP). Developmental change of Xenopus APP gene expression has been analyzed with molecular probes. From early oogenesis, there is a high accumulation of maternal APP. After fertilization, the mRNA is degraded, reaching a minimum level around the gastrula stage. Then zygotic transcription appears to be initiated, and this continues during the subsequent embryonic and larval stages. Splicing patterns differ between the maternal and zygotic transcripts. The ratio of mRNA including the protease inhibitor domain (PID) sequence is extremely low for the transcript of maternal origin as compared to that for the transcript of zygotic origin. These results suggest some roles for the APP molecule in Xenopus early development (Okado, 1992).

Mouse and human cDNA clones encoding amyloid precursor-like proteins (APLP1 and APLP2) have been identified and exhibit extensive sequence similarity to the Drosophila Appl and mammalian APP genes. To define the potential role of APLP in the mammalian brain, APLP1 localization was examined within the complex cortical synaptic structure. A focused was placed on the postsynaptic density (PSD), which appears to be central to synaptic function. The 90 kDa APLP1, the first known APLP, is localized to the PSD from rat and human cerebral cortex. APLP1 increases during cortical synaptic development, suggesting a role in synaptogenesis or synaptic maturation. In contrast, APP is predominantly expressed in the synaptic membrane fraction, but is barely detectable in the PSD, including the different subcellular locations of APP and APLP1. These observations raise the possibility that APLP1 participates in brain synaptic function in mammals (Kim, 1995).

Transcriptional regulation of APP

The role of betaAPP gene transcription and promoter regulation in modifying amyloid beta-peptide (Abeta) levels is not well understood. Increased production of Abeta or changes in Abeta42/Abeta40 ratio by fibroblasts occurs in the presence of mutant presenilin or betaAPP alleles in familial Alzheimer's disease subjects. Both betaAPP mRNA and Abeta levels are increased in trisomy 21. The APP gene promoter is in a class of housekeeping genes and contains two putative consensus sites for the binding of transcription factor AP1. Electrophoretic mobility shift (EMSA) and DNase protection assays using human fibroblast and HeLa nuclear extract have identified specific protein binding with novel Sp1-like properties to both a near-upstream and a downstream domain of the betaAPP promoter. The upstream binding activity is localized to a putative AP1 consensus site and its immediate 5'-adjacent GC-rich element. However, c-Jun antibody and competition experiments have no effect on binding to this domain. A series of 5'-deleted betaAPP promoter-reporter gene transfections in HeLa and fibroblast cells shows that the domain-containing region, n.t. -383 to -348, exerts a 2.9-fold activating influence on basal pbetaAPP-reporter transcription. When subcloned to test enhancer function, the 5'-GC element/'AP1 site' tandem construct confers four-fold greater activity than either element alone and two-fold greater than the more 3'-situated HSE consensus sequence. Phorbol ester treatment has no effect in these reporter assays. This element shares homology and binding properties with a domain immediately 5' to the downstream E-box/USF element. An interaction model involving both domains and looping of interjacent DNA is proposed. It is concluded that this newly described binding protein-enhancer complex is required for full betaAPP promoter activation (Querfurth, 1999).

Proteases that cleave APP and endocytic processing of APP

For information on gamma-secretase see the Presenilin overview and The role of presenilin in beta-amyloid precursor protein processing.

Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increases the amount of beta-secretase cleavage products: these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreases the amount of beta-secretase cleavage products, and purified BACE protein cleaves APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE are consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease (Vassar, 1999).

The release of amyloidogenic amyloid-beta peptide (Abeta) from amyloid-beta precursor protein (APP) requires cleavage by beta- and gamma-secretases. alpha-secretase, in contrast, cleaves APP within the Abeta sequence and precludes amyloidogenesis. Regulated and unregulated alpha-secretase activities have been reported, and the fraction of cellular alpha-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated alpha-secretase cleavage of APP has been shown to occur at the cell surface, attempts have been made to identify the intracellular site of PKC-regulated alpha-secretase APP cleavage. To accomplish this, levels of secreted ectodomains and C-terminal fragments of APP generated by alpha-secretase (sAPPalpha) (C83) versus beta-secretase (sAPPbeta) (C99) were measured and secreted Abeta was measured in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. PKC stimulation increases sAPPalpha but decreases sAPPbeta levels by altering the competition between alpha- versus beta-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for beta-secretase activity prompted the hypothesis that PKC-regulated alpha-secretase(s) also resides in this organelle. To test this hypothesis, studies were performed demonstrating proteolytically mature TACE intracellularly, and regulated alpha-secretase APP cleavage was shown to occur in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated alpha-secretase APP cleavage in the TGN by TACE/ADAM-10, ADAM activity was revealed in a novel location. Finally, the competition between TACE/ADAM-10 and beta-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease (Skovronsky, 2000).

Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate APP cleavage. A new membrane-bound aspartyl protease (Asp2) with beta-secretase activity has been identified. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease (Yan, 1999).

Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). The amyloidogenic processing of APP was examined using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface. The wild-type sequence of CD3gamma was used to create an APP chimera predicted to remain in the ER [gammaAPP(ER)]. Deletion of the RKK motif at the C-terminus directs the protein directly to the lysosomes [gammaAPP(LYS)]. A third chimera was created by removing both lysosomal targeting signals in addition to RKK [gammaAPP(DeltaDelta)]. This last construct does not contain known targeting signals and consequently accumulates at the cell surface. All three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, evidence is provided that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42) (Soriano, 1999).

APP1, a transmembrane protein with homology to glycosylated cell surface receptors, can reside at the cell surface and is reinternalized via clathrin-coated pits. Some internalized APP remains intact to be recycled to the cell surface plasma membrane. However, internalized APP can also be proteolytically processed into several distinct secreted fragments, which include the large secreted N-terminal APP ectodomain (APPs), and Abeta, the major protein component of senile plaques in Alzheimer's disease (AD). Because Abeta deposition may be central to AD pathogenesis, the mechanism by which Abeta is generated from the precursor is an important focus of AD research. At least two species of Abeta, differing by two amino acids at the C terminus (Abeta40 and Abeta42), are released from cells during normal cellular metabolism. Abeta42, which readily aggregates in vitro appears to be more pathogenic and may serve as a seed for plaque formation in individuals with AD, hereditary cerebral hemorrhage with amyloidosis Dutch type, and Down's syndrome. The source of Abeta deposited in brain tissues is still uncertain. However, cell lines expressing wild type APP can produce and release Abeta primarily after internalization of APP from the cell surface. Although familial mutations in APP can enhance Abeta secretion, almost all humans express wild type APP. Therefore, the major pathway for Abeta production appears to involve endocytic recycling of APP from the cell surface. To date, the specific contribution of APP endocytic processing to Abeta42 production in particular has not been established (Perez, 1999).

APP endocytosis relies on signals in the cytoplasmic C-terminal domain. An NPXY sequence similar to that found in the C terminus of the low density lipoprotein receptor and the LDLR-related protein is also found in APP. Because the tyrosine in NPXY is crucial for LDLR endocytosis, it has long been assumed that the C-terminal motif, NPXY, is the internalization signal for beta-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr743 by APP751 numbering, Tyr682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743) were mutated. Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impair APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (DeltaC), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr743 regulates rapid APP turnover because half-life increases by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, Abeta, is tightly correlated with APP internalization, such that Abeta secretion is unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Abeta42 isoform is also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic Abeta species (Perez, 1999).

The Alzheimer's beta-amyloid precursor protein (APP) is internalized from the axonal cell surface. Biochemical and cell biological methods have been used to characterize endocytotic compartments that participate in the trafficking of APP in central neurons. APP is present in presynaptic clathrin-coated vesicles purified from bovine brain, together with the recycling synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2. In contrast, APP is largely excluded from synaptic vesicles purified from rat brain. In primary cerebellar macroneurons, cell-surface APP is internalized with recycling synaptic vesicle integral membrane proteins but is subsequently sorted away from synaptic vesicles and transported retrogradely to the neuronal soma. Internalized APP partially co-localizes with rab5a-containing compartments in axons and with V-ATPase-containing compartments in both axons and neuronal soma. These results provide direct biochemical evidence that an obligate sorting compartment participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals. Moreover, APP is now, the first demonstrated example of an axonal cell-surface protein that is internalized with recycling synaptic vesicle membrane proteins but is subsequently sorted away from synaptic vesicles (Marquez-Sterling, 1997).

The pheochromocytoma PC12 cell line has been used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increases neurite outgrowth, APP mRNA expression, and APP secretion in a dose-dependent fashion, with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolishes NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone does not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide (an analog of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase) mimics the effects of NGF on cell morphology and stimulates both APP mRNA levels and APP secretion. In contrast, specific stimulation of TrkA receptors by receptor cross-linking selectively stimulates neurite outgrowth and APP secretion but not APP mRNA levels, which are decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, these data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Specific activation of TrkA receptors, in contrast to p75NTR-associated signaling, stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels (Rossner, 1998).

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite in the brain. The role of neprilysin (see Drosophila Neprilysin4), a candidate Abeta-degrading peptidase, in Abeta metabolism was examined using neprilysin gene-disrupted mice. Neprilysin deficiency results in defects both in the degradation of exogenously administered Abeta and in the metabolic suppression of the endogenous Abeta levels in a gene dose-dependent manner. The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, where hippocampus has the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD. These observations suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Abeta accumulation (Iwata, 2001).

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Aß42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in C. elegans, Drosophila, and mammals. Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupts the HMW complex formation, and abolishes gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescues the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolishes the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn is associated with a drastic overproduction of Aß1-42 from human ßAPP in N2a cells, but not in S2 cells. These data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals (Takasugi, 2002).

The formation of the stabilized HMW complex of mammalian PS, that requires the integrity of the conserved PS C terminus, is essential to the acquisition of gamma-secretase activity, and an aspartate residue within 7th TMD (TMD7) is crucial to the gamma-secretase activity in mammalian PS. To verify the effects of missense mutations in Psn that cause Notch (i.e, loss-of-function) phenotype in Drosophila in vivo, on the metabolism of Psn polypeptides, the two types of amino acid substitutions (i.e., P507L or G516E) were introduced and stably expressed the mutant Psn in N2a NL/N cells. In addition, N2a NL/N cells, stably coexpressing Psn carrying D461A mutation that replaces the highly conserved aspartate residue in the TMD7 with alanine, were established to see if this mutation works as a dominant negative mutant on gamma-cleavage as in mammalian PS. Neither Psn/P507L, Psn/G516E nor Psn/D461A undergo endoproteolysis to give rise to NTF and CTF that normally occurs with wild type Psn. The replacement of endogenous PS1 did not occur in N2a NL/N cells coexpressing Psn/P507L or Psn/G516E. Upon CHX treatment of the N2a cells, the Psn/P507L or Psn/G516E holoproteins were rapidly degraded in a similar manner to wild type Psn holoprotein. In contrast, the overexpression of Psn/D461A results in a complete replacement of endogenous murine PS1 fragments, and a portion of Psn/D461A is stabilized as a holoprotein, as previously described in aspartate mutants of mammalian PS (i.e., PS1/D385A, PS2/D366A). The HMW complex formation of Psn and its derivatives was analyzed. The unstable Psn/P507L or Psn/G516E holoproteins were fractionated exclusively in the LMW range. In contrast, Psn/D461A, which was stabilized but not cleaved, was present as holoproteins broadly within LMW and HMW ranges in a similar manner to that of mammalian PS2/D366A (Takasugi, 2002).

Psn-dependent gamma-secretase activity in Drosophila has been shown to cleave Notch and other transmembrane proteins in vivo. The amino acid sequence of APPL, a Drosophila homolog of ßAPP, is not homologous to that of mammalian ßAPP especially within the TMD, and gamma-cleavage of APPL has not been documented. However, it has been shown that overexpression of the C-terminal 99 amino acid fragment of human ßAPP elicits the cleavage to generate Aß1-40 by a gamma-secretase-like activity in Drosophila SL-2 cells, although Drosophila cells lack ß-secretase activity. To evaluate the gamma-secretase-like activity for proteolytic processing of the TMD sequence of human ßAPP in Drosophila S2 cells, a cDNA encoding SC100, that corresponds to the C-terminal fragment of human ßAPP starting at the 1st residue of Aß preceded by a signal peptide, was transiently transfected and the conditioned media by ELISA was analyzed. Aß secretion was readily detectable in conditioned media of cells expressing SC100; surprisingly, however, percent Aß42 was ~15%, which was in sharp contrast to the robust Aß1-42 overproduction in mouse N2a cells, that is mediated by the same PS species, i.e., wild type Psn. To exclude the possibility that gamma-secretase-like activity in S2 cells is incapable of producing excessive amounts of Aß1-42, a cDNA encoding SC100 was constructed harboring an isoleucine to phenylalanine substitution at residue 716 of ßAPP (SC100/I716F); this substitution has been shown to cause robust increase in Aß1-42 secretion in COS cells. Transfection of SC100/I716F into S2 cells results in a dramatic increase in Aß1-42 secretion and simultaneous decrease in Aß40 secretion, suggesting that the endogenous gamma-secretase-like activity mediated by Psn normally cleaves the TMD sequence of human ßAPP predominantly at Aß40 position, but is capable of cleaving predominantly at position 42 under pathogenic conditions (e.g., ßAPP mutation) in S2 cells. Thus, Psn-dependent gamma-cleavage in S2 cells shows similar characteristics to those in mammalian cells, whereas it may be shifted to position 42 by some unknown mechanism in mouse N2a cells (Takasugi, 2002).

To examine whether Psn plays an essential role in Aß generation by a gamma-secretase-like activity in S2 cells, an S2 cell line was generated stably expressing SC100 (S2-SC100) and the expression of endogenous Psn gene was suppressed by double-stranded RNA (dsRNA)-mediated interference (RNAi). After a 48-h transfection of Psn dsRNA, the expression of Psn polypeptide in the form of fragments was completely and specifically abolished in S2-SC100 cells. After incubation in fresh media for additional 24 h, the cell lysates and conditioned media were analyzed. Immunoblot analysis has revealed an accumulation of SC100 as well as of a ~10-kDa polypeptide comigrating with C83 of mammalian cells. The latter band presumably represents the SC100 derivative cleaved by an alpha-secretase-like activity, that has been reported in Drosophila and SL-2 cells. No Aß secretion was observed in conditioned media, suggesting that the total suppression of the expression of Psn by RNAi results in a complete loss of gamma-secretase activity. Thus, Psn-dependent gamma-secretase activity is required for Aß generation from a human ßAPP derivative (i.e., SC100) in Drosophila S2 cells (Takasugi, 2002).

Alzheimer's disease is associated with increased production and aggregation of amyloid-ß (Aß) peptides. Aß peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent gamma-secretase cleavage. Presenilin interacts with nicastrin (see Drosophila nicastrin) , APH-1 and PEN-2, all of which are required for gamma-secretase function. Presenilins also interact with alpha-catenin, ß-catenin and glycogen synthase kinase-3ß (GSK-3ß), but a functional role for these proteins in gamma-secretase activity has not been established. Therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Aß peptides by interfering with APP cleavage at the gamma-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Aß peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3alpha, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3alpha offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease (Phiel, 2003).

In summary, this study shows that GSK-3alpha facilitates APP processing and that lithium inhibits the generation of Aß peptides through inhibition of GSK-3alpha. In support of this conclusion: (1) lithium reduces Aß production in cultured cells and in the brains of mice that overproduce Aß peptides; (2) kenpaullone, an alternative GSK-3alpha inhibitor, also inhibits Aß production; (3) RNAi-mediated depletion of GSK-3alpha reduces Aß production, and (4) moderate overexpression of GSK-3alpha increases Aß production. Lithium inhibits the GSK-3-mediated phosphorylation of tau, which, in its hyperphosphorylated state, is the main component of neurofibrillary tangles. Thus, GSK-3alpha offers an attractive target for pharmacological agents aimed at reducing the formation of amyloid plaques and neurofibrillary tangles, the pathological hallmarks of Alzheimer's disease. Lithium also protects neurons from proapoptotic stimuli and could therefore reduce neuronal cell death associated with Alzheimer's disease. Lithium has been used for more than 50 years to treat bipolar disorder, but has a narrow therapeutic window and a higher frequency of side effects in older patients. Thus, although lithium might be considered for the prevention of Alzheimer's disease, especially in younger patients with FAD mutations or Down's syndrome, new agents that specifically target GSK-3alpha may prove to be valuable in the treatment of Alzheimer's disease (Phiel, 2003).

The abnormal accumulation of beta-amyloid (Abeta) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Abeta. As with any peptide, however, the degree of Abeta accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models endothelin-converting enzyme-1 (ECE-1) has been characterized as an Abeta-degrading enzyme that appears to act intracellularly, thus limiting the amount of Abeta available for secretion. To determine the physiological significance of this activity, Abeta levels were analyzed in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Abeta40 and Abeta42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Abeta levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Abeta accumulation in the brain and also provides further insight into the factors involved in Abeta clearance in vivo (Echman, 2003).

Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. IDE has been identified as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, mice with homozygous deletions of the IDE gene were characterized. IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE minus mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which is degraded by IDE in vitro. Together with emerging genetic evidence, these in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD (Farris, 2003).

Factors that elevate Abeta peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Abeta degradation based on its hydrolysis of Abeta peptides in vitro. In this study, in vivo levels of brain Abeta40 and Abeta42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the gamma-secretase-generated C-terminal fragment of the Abeta precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased approximately 50%, brain Abeta peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Abeta peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease (Miller, 2003).

Converging evidence suggests that the accumulation of cerebral amyloid ß-protein (Aß) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Aß thus represents a novel therapeutic approach to lowering steady-state Aß levels, but the consequences of sustained upregulation in vivo have not been studied. Transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons has been shown to significantly reduce brain Aß levels, retard or completely prevent amyloid plaque formation and its associated cytopathology, and rescue the premature lethality present in amyloid precursor protein (APP) transgenic mice. These findings demonstrate that chronic upregulation of Aß-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo (Leissring, 2004).

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. This study reports the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147 (see Drosophila Basigin), a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression has no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides (Zhou, 2005; full text of article).

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 2005).

ApoE promotes the proteolytic degradation of Aβ

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Aβ homeostasis. ApoE plays a role in facilitating the proteolytic clearance of soluble Aβ from the brain. The endolytic degradation of Aβ peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Aβ degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Aβ degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Aβ degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Aβ from the brain and suggest that LXR agonists may represent a novel therapy for AD (Jiang, 2008).

An isoform of apolipoprotein E, ApoE4, has been shown to confer dramatically increased risk for late-onset AD (LOAD); however, the basis for this remains one of the major unanswered questions of disease pathogenesis. ApoE plays critical roles in regulating brain Aβ peptide levels, as well as their deposition and clearance. Thus, processes that regulate ApoE expression and functional state could affect its ability to influence brain Aβ homeostasis. ApoE is the predominant apolipoprotein in the brain and is synthesized and secreted mainly by astrocytes (but also by microglia) within unilamellar HDL-like particles. ApoE is lipidated principally through the action of the ATP-binding cassette transporter ABCA1 (and related transporters), which acts in a variety of cell types to transfer both phospholipids and cholesterol to ApoE, and, in this way, ApoE acts to traffic lipids throughout the brain. The lipidation status of ApoE is an important functional parameter, governing its conformation, intrinsic stability, and interactions with membrane receptors. Importantly, ApoE binds to Aβ, and this, too, is influenced by its lipidation status. Studies with APP transgenic mice have demonstrated ApoE isoform-specific effects on the propensity of Aβ to be deposited in the brain (E4 > E3 > E2), the nature of the deposits, and a gene-dosage-related influence on the magnitude of these effects. ApoE lipidation status is also a significant determinant of whether its interaction with Aβ leads to efflux of the peptides from the brain or, alternatively, to the formation of fibrils and their deposition into plaques. Recently, three independent studies have reported that inactivation of the Abca1 gene in APP-expressing transgenic mice resulted in reduced levels of ApoE. Remarkably, these mice exhibited a seemingly paradoxical elevation of brain Aβ peptide levels and a doubling of Aβ plaque burden. The outcomes of these studies strongly suggested that lipidated forms of ApoE act to enhance the clearance of Aβ peptides from the brain. The aim of the present study was to establish the mechanism by which ApoE and its lipidation affect Aβ homeostasis (Jiang, 2008).

Liver X receptors (LXRs) are ligand-activated transcription factors that induce the expression of Apoe, Abca1, and other genes of lipid metabolism. LXRs act physiologically as cellular cholesterol sensors and are activated by oxysterols. There are two LXR isoforms, LXRα and LXRβ (encoded by Nrlh3 and Nrlh2, respectively), both of which are expressed in the brain, and their activation results in the rapid and robust increase in the levels of lipidated forms of ApoE. Thus, regulation of LXR transcriptional activity provides a mechanism to regulate brain ApoE levels and its lipidation status (Jiang, 2008).

The brain possesses robust intrinsic Aβ clearance mechanisms. Aβ peptides are proteolytically degraded within the brain principally by neprilysin (NEP) and insulin-degrading enzyme (IDE, insulysin). Genetic inactivation of these genes or administration of inhibitors of these proteinases into the brain results in substantial elevation of Aβ levels in the brain and induction of plaque deposition. Conversely, overexpression of IDE or neprilysin lowered brain Aβ levels and reduced plaque formation. It has been argued that the predominant mode of Aβ42 clearance from the brain is through its proteolytic degradation because this peptide is not efficiently exported through the vasculature. Microglia, the brain's resident macrophages, play an essential role in Aβ clearance through their ability to take up and degrade soluble and fibrillar forms of Aβ. Moreover, both microglia and astrocytes secrete proteinases, including IDE, that mediate the degradation of Aβ peptides in the extracellular milieu (Jiang, 2008).

Despite considerable effort, the cellular mechanisms through which ApoE influences Aβ clearance remain unresolved. This paper reports that ApoE acts to facilitate the proteolytic degradation of Aβ, a previously unappreciated action of this apolipoprotein. Moreover, the lipidation status of ApoE is a critical determinant of its ability to stimulate Aβ degradation, and this finding provides a mechanistic explanation of the increased Aβ levels and deposition observed in APP-expressing mice lacking the Abca1 gene. Importantly, it was demonstrated that elevation of lipidated forms of ApoE, through activation of LXRs, results in reduced Aβ peptide and plaque levels in an animal model of AD and is associated with improved contextual memory. Therapeutic agents that increase the abundance of highly lipidated forms of ApoE, including LXR agonists, may attenuate disease pathogenesis and represent a promising strategy for the treatment of AD (Jiang, 2008).

Subcellular localization, internalization and transport of APP

Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). The mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives were examined in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail has no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine alters the distribution of cell surface beta APP so that approximately 40%-50% of the molecules are inserted apically. In parallel, A beta is now secreted from both surfaces. Surprisingly, this change in surface beta APP has no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules that give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, intracellular APPs are readily detected in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment results in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it does not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653 (Haass, 1996).

Embryonic cortical neurons in culture contain transmembrane amyloid precursor protein (APP) capable of associating with the detergent-insoluble cytoskeleton through interactions requiring the presence of its C-terminal. These transmembrane APPs are not detectable at the surface of living cells. When neurons are fixed with paraformaldehyde alone, APP is mainly visualized close to the membrane of the axon and cell body of 40% of neurons, with virtually no dendritic staining. Membrane permeabilization with detergent or methanol extends APP immunostaining to 100% of the cells and to all compartments, including the dendrites. Taken together, these results suggest that APP in embryonic neurons is present in two compartments, one more readily detectable in some axons and cell bodies and the other distributed throughout all neurons. The axonal and somatic pool of APP detectable after paraformaldehyde fixation alone is highly and rapidly augmented after exposure to calcium ionophores. It has been proposed that calcium entry increases the amount of axonal APP close to the cell surface, but that the stabilization of the protein at the cell surface and its subsequent secretion require further physiological stimuli (Allinquant, 1994).

Beta-amyloid precursor protein is a transmembrane protein that can be processed to release a large secretory product or processed in the endosomal/lysosomal pathway without secretion. Previous studies have shown that from the cell surface, beta-amyloid precursor protein may be released after cleavage or internalized without cleavage, the latter in a pathway that both produces amyloid beta-protein and also targets some molecules to the lysosomal compartment. Analysis of beta-amyloid precursor protein trafficking is confounded by the concomitant secretion and internalization of molecules from the cell surface. To address this issue, an assay was developed, based on the binding of radioiodinated monoclonal antibody, to measure the release and internalization of cell surface beta-amyloid precursor protein in transfected cells. With this approach, it has been shown that surface beta-amyloid precursor protein is either rapidly released or internalized, such that the duration at the cell surface is very short. Approximately 30% of cell surface beta-amyloid precursor protein molecules are released. Following internalization, a fraction of molecules are recycled while the majority of molecules are rapidly sorted to the lysosomal compartment for degradation. When the C terminus of beta-amyloid precursor protein is deleted, secretion is increased by approximately 2.5-fold as compared to wild-type molecules. There is concomitant decrease in internalization in these mutant molecules as well as prolongation of the resident time on the cell surface. This observation is consistent with recent evidence that signals within the cytoplasmic domain mediate beta-amyloid precursor protein internalization (Koo, 1996).

The principal component of Alzheimer's amyloid plaques, Abeta, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear. APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both alphaAPPs and Abeta secretion. The dramatic (4-fold) FE65-dependent increase in Abeta secretion suggests that agents that inhibit the interaction of FE65 with APP might reduce Abeta secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation (Sabo, 1999).

The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1; see Drosophila APP-like protein interacting protein 1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. This study tested whether APP and JIP-1 are transported together or separately on different vesicles. It was found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP (Muresan, 2005).

Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. This study reports the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the mu4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances gamma-secretase-catalyzed cleavage of APP to the pathogenic amyloid-beta peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein (Burgos, 2010).

Aß deposition and Alzheimer's disease

Considerable circumstantial evidence suggests that Aβ42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Aβ42 for amyloid deposition has never been demonstrated in vivo. This was addressed by developing transgenic models that express Aβ1-40 or Aβ1-42 in the absence of human amyloid β protein precursor (APP) overexpression. Mice expressing high levels of Aβ1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Aβ1-42 accumulate insoluble Aβ1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Aβ deposits. When mice expressing Aβ1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Aβ1-42 is essential for amyloid deposition in the parenchyma and also in vessels (McGowan, 2005).

Much of the data that support a pivotal role for Aβ42 in AD have come from the study of mutations in the APP and presenilin genes that cause early-onset familial forms of AD. The vast majority of these mutations selectively increase the relative levels of Aβ42. However, even in typical late-onset AD there is evidence that Aβ42, a minor Aβ species, usually representing less then 20% of the total Aβ secreted, is both the earliest form and the predominant species deposited in the brain parenchyma. In contrast, Aβ40, the major Aβ peptide secreted by cells, appears to be the predominant species deposited in the amyloid deposits in the cerebral vasculature (congophillic angiopathy, CAA). Transgenic mouse studies using mutant APP and PS transgenes have provided some insights into the effects that altering the ratio of Aβ40 and Aβ42 have on time to onset of deposition, type of deposit (e.g., diffuse versus compact), and extent of CAA. However, such studies have not definitively identified which Aβ species are responsible for seeding amyloid deposition in either the parenchyma or vasculature (McGowan, 2005).

To address this question, transgenic mice were generated that express Aβ1-40 or Aβ1-42 without APP overexpression. For these studies cDNAs were used that express fusion proteins between the BRI protein, involved in amyloid deposition in Familial British (FBD) and Danish Dementia (FDD) and Aβ1-40 (BRI-Aβ40) or Aβ1-42 (BRI-Aβ42). Transfection of BRI-Aβ cDNAs results in high-level expression and secretion of the encoded Aβ peptide through proteolytic cleavage of the fusion protein at a furin cleavage site immediately preceding Aβ. Efficient secretion of Aβ from the BRI fusion protein distinguishes this approach from studies using Aβ minigene constructs that generate high levels of intracellular Aβ and minimal secreted Aβ. The BRI-Aβ transgenic mice generated provide substantial evidence that Aβ1-42 but not Aβ1-40 is sufficient to promote Aβ deposition in mice (McGowan, 2005).

Progressive memory loss and cognitive dysfunction are the hallmark clinical features of Alzheimer's disease (AD). Identifying the molecular triggers for the onset of AD-related cognitive decline presently requires the use of suitable animal models, such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. This study characterizes the onset of learning and memory deficits in this model. Two-month-old, prepathologic mice are cognitively unimpaired. The earliest cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala. Plaque or tangle pathology is not apparent at this age, suggesting that they contribute to cognitive dysfunction at later time points. Clearance of the intraneuronal Aβ pathology by immunotherapy rescues the early cognitive deficits on a hippocampal-dependent task. Reemergence of the Aβ pathology again leads to cognitive deficits. This study strongly implicates intraneuronal Aβ in the onset of cognitive dysfunction (Billings, 2005).

Amyloid beta-peptide interaction with LRP (low-density lipoprotein receptor-related protein)

LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice (see Hsiao, 1996). While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the blood-brain barrier mediate brain accumulation of neurotoxic Abeta (Deane, 2004).

The protease inhibitor function of APP

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP that contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decays in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 h and 5.7 h, respectively. A small fraction (10%) of the total APP is secreted into the culture medium where it is degraded with a half-life of 9 h. Studies using specific protease inhibitors have demonstrated that this extracellular breakdown is due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease is inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease (Caswell, 1999).

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that 'protease nexin 2 (PN2)', the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibits the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Moreover, PTP and PN2 form SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP, as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass, were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP is also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrates the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease (Hook, 1999).

Phosphorylation of APP

The cytosolic domain of the ß-amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of these is Mena, which binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologs of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr682 of APP695 is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling (Zambrano, 2001).

It is worth noting that the Tyr682 of human APP695 and the YENP motif are both conserved among all the known APPs in primates, rodents, Drosophila, and Caenorhabditis and are present also in the APP-related proteins APLP1 and APLP2. Considering that the overall sequence identity between Drosophila APP (Appl) and the mammalian APPs is less than 30%, the 100% conservation of the cytosolic motif containing the phosphorylated tyrosine suggests that it plays a key functional role. This means that the understanding of the molecular basis of the different phenotypes observed in insects bearing mutations of Drosophila Abl (DAbl) and/or disabled and/or enabled should also take into account the involvement of APP. Appl null flies show behavioral defects that are rescued by human APP, and the possible correlation with the defects caused by DAbl, disabled, and enabled gene mutations is not apparent. However, one could gain better insight by the analysis of the phenotypes of insects bearing combined mutations of Appl with the other three genes. For example, the effects of disabled gene mutation on the Abl -/- flies also could be the consequence of the direct interaction of these two proteins with APP, whereas the amelioration observed in Drosophila Abl-/-;disabled-/- following the mutation of the enabled gene could be also based on the competition between the enabled and DAbl gene products for the binding to Appl through Drosophila Fe65 (Zambrano, 2001).

Although the WW domain of Fe65 interacts in vitro with both c-Abl and Abl-PP, only the complexes between Fe65 and the active form of Abl, and not those with the wild type c-Abl, were found in cell extracts. This effect could be due to a lower amount of c-Abl than Abl-PP available for the formation of the in vivo complexes; or it could be due to a low affinity of c-Abl for the WW domain of Fe65 so that, in vivo, it cannot form a significant number of complexes with Fe65 because of the competition of the other ligands of the WW domain of this protein. Furthermore, active Abl probably has a different conformation from that of c-Abl, thus acquiring a higher affinity for the WW domain. On the contrary, the APP-Abl direct interaction probably requires an active Abl, because the binding is based on a pTyr-SH2 interaction (Zambrano, 2001).

It has been hypothesized often that APP could have some role in signaling, and in a recent review article, Bothwell and Giniger (2000) suggested the possibility that intracellular signaling could be involved in the development of AD. Their hypothesis takes into account the numerous reports on various proteins that could be involved in the pathogenesis of AD and suggests a role for c-Abl as a modulator of APP biology. The results presented here support their hypothesis. A point that deserves attention concerns the possible involvement of p73 in the molecular machinery under examination. In fact, this protein is a key regulator of apoptosis that binds to and is activated by Abl as a response to DNA damage. An isoform of p73 functions as an anti-apoptotic protein in developing neurons, and the role of its phosphorylation by Abl has not been addressed. The finding that active Abl binds to APP suggests an examination of the possible regulatory effects of this binding on the p73 phosphorylation by Abl and the consequences on this regulation of the enhanced APP proteolytic processing characteristic of AD (Zambrano, 2001).

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β-peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ and cause the much more common sporadic forms of AD are largely unknown. This study shows that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD (Kumar, 2011).

Cytoplasmic interactions of APP and signaling downstream of APP

Disabled gene products are important for nervous system development in Drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. The amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences has shown that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. It is proposed that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal (Howell, 1999).

APP forms a complex with Go, a major GTP-binding protein in brain. The cytoplasmic APP sequence His 657-Lys 676 shows a specific Go-activating function and is necessary for complex formation. Go protein treated with GTP-gamma S loses the ability to associate with APP. This suggests that APP is a receptor coupled to Go and that abnormal APP-Go signaling is involved in the Alzheimer's disease process (Nishimoto, 1993).

Amyloid precursor protein (APP), a transmembrane precursor of beta-amyloid, possesses a function whereby it associates with Go through its cytoplasmic His657-Lys676. APP has a receptor function. In phospholipid vesicles consisting of baculovirally made APP695 and brain trimeric Go, a monoclonal antibody against the extracellular domain of APP (22C11) increases GTP gamma S binding and the turnover number of GTPase of Go without affecting its intrinsic GTPase activity. This effect of 22C11 is specific among various antibodies and is observed neither in Go vesicles nor in APP695/Gi2 vesicles. In APP695/Go vesicles, synthetic APP66-81, the epitope of 22C11, competitively antagonizes the action of 22C11. Monoclonal antibody against APP657-676, the Go binding domain of APP695, specifically blocks 22C11-dependent activation of Go. Therefore, APP has a potential receptor function whereby it specifically activates Go in a ligand-dependent and ligand-specific manner (Okamoto, 1995).

APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations (V642I/F/G) have been discovered in APP695, which, multiple studies suggest, is a cell surface signaling receptor. Normal APP695 encodes a potential Go-linked receptor with ligand-regulated function; expression of the three FAD mutants (FAD-APPs), not normal APP, induces cellular outputs by Go-dependent mechanisms. This suggests that FAD-APPs are constitutively active Go-linked receptors. Direct evidence for this notion is provided in this study. Reconstitution of either recombinant FAD-APP with Go vesicles induces activation of Go, which is inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD-APP. Consistent with the dominant inheritance of this type of FAD, this function is dominant over normal APP, because little activation is observed in APP695-Go vesicles. Experiments with antibody competition and sequence deletion indicates that His657-Lys676 of FAD-APP, which has been specified as the ligand-dependent Go-coupling domain of normal APP, is responsible for this constitutive activation, confirming that the three FAD-APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP as a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors (Okamoto, 1996).

Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, induces nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation requires the cytoplasmic domain of the mutants and appears to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins) (Yamatsuji, 1996).

In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. An investigation of NK1 cells was carried out to determine which subunit is responsible for the induction of apoptosis by V642I APP. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induces little apoptosis. Apoptosis by V642I APP is antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G betagamma combinations induces DNA fragmentation in a manner sensitive to bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue). These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP (Giambarella, 1997).

Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, proteins were sought that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, the neuronal adaptor proteins FE65 and mammalian Disabled have been shown to bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal (Trommsdorff, 1998).

Amyloid precursor protein requires the insulin signaling pathway for neurotrophic activity. Picomolar concentrations of purified amyloid precursor protein (APP) potentiate the neurotrophic activity of suboptimal concentrations of NGF on PC12 cells. To understand the molecular basis for this potentiation, the signal transduction pathway used by APP for its neurotrophic activity was characterized. APP stimulates the tyrosine phosphorylation of a number of proteins including insulin receptor substrate-1 (IRS-1). Incubation of naive cells with antisense oligonucleotides to IRS-1 mRNA results in a dramatic reduction of IRS-1 levels and inhibition of APP stimulated neurite outgrowth. Phosphotidylinositol 3-kinase become associated with IRS-1 and activated upon APP stimulation. Extracellular signal-regulated kinase (ERK 1 and ERK 2) phosphorylation is detected by both immunoblot analysis and immunocytochemistry using antibodies directed to their phosphorylated (and hence, activated) form. There was also an elevation of ERK kinase activity. The potentiation of NGF activity is reflected in a correspondingly synergistic elevation of tyrosine phosphorylated ERK. The pattern of signal transduction targets indicates that APP potentiates the neurotrophic effects of NGF via the activation of the IRS-1 signaling pathway (Wallace, 1997b).

Using a yeast two-hybrid method, amyloid precursor protein (APP)-interacting molecules were sought by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID), the following were identified as novel APP-interacting molecules: Fe65, Fe65L, Fe65L2, X11, and mDab1, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1 (the established scaffold proteins for JNK). The APP amino acids Tyr682, Asn684, and Tyr687 in the G681YENPTY687 region are all essential for APP/JIP-1b interaction, but neither Tyr653 nor Thr668 are necessary. APP-interacting ability is specific for this additional isoform containing PID and is shared by both human and mouse homologs. JIP-1b expressed by mammalian cells is efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly681-Asn695 domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs are precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitate JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1ß1 forms a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrates that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules (Matsuda, 2001).

JIP-1 was initially characterized as a cytoplasmic inhibitor of JNK family kinases and subsequently found to interact with MKK7, MLK, DLK, and HPK-1 in addition to JNK. Coexpression of JIP-1 and JNK with MKK7 or MLK3 increases JNK activation. These findings have established that JIP-1 scaffolds the kinase components of the JNK signaling pathway. An additional isoform of JIP-1 has been reported in mouse (JIP-1b), rat [islet-brain-1 (IB1)], and human (IB1). This isoform contains a 47-residue insertion that completes the PID region at the C terminus, which was originally identified in Shc interaction with NPXY in the cytoplasmic domain of the epidermal growth factor receptor. Neither the physiological nor the pathological role of the JIP-1 proteins has become totally clear, whereas expression of JIP-1 has been reported to transcriptionally activate the GLUT2 promoter and is implicated in the pathogenesis of a form of type 2 familial diabetes mellitus and in the cytoprotection of insulin-secreting cells. The present study thus provides the first line of evidence that the JNK scaffold protein, abundant in the brain and in islet ß-cells, could be relevant to Alzheimer's disease. Interestingly, it has been reported that in vivo, neurotoxicity by hippocampal administration of Ab1-42 occurs only in diabetic rats (Matsuda, 2001 and references therein).

Analysis of subcellular localization using transfected cells indicates that JIP-1b and APP colocalize in the cytoplasm but both are not detected in the nuclei. Similar cytoplasmic localization of JIP-1b has been reported although other studies have reported that IB1 is a nuclear protein. Nuclear localization of JIP-1 proteins have been demonstrated in cerebellar granule cells, and JIP-1 proteins localize in the cytoplasm in unpolarized NIE115 and PC12 cells but are concentrated at neurites when the cells are polarized. These differences in JIP-1 localization thus may reflect different functions of JIP-1 proteins assigned in different cell environments. Although the present study provides evidence that JIP-1b interacts with APP inside the transfected cells, it would be necessary to investigate whether endogenous APP and JIP-1b interact in nontransfected cells. Yet the notion that JIP-1b/IB1 colocalizes with APP is consistent with earlier studies indicating that the subcellular and brain regional localizations of JIP proteins considerably overlap with those of APP. Because the putative alpha-secretase ADAM10 and the putative ß-secretase BACE are expressed in the same neurons that express APP in the mouse brain, APP cleavage by these putative secretases would lose the interaction of APP with JIP-1b/IB1, causing, in turn, a loss in the ability of JIP-1b/IB1 to specifically colocalize signaling molecules with APP. Although so far coimmunoprecipitate APP with IB1 from rat brain homogenates has not been demonstrated, it remains unclear whether this failure is caused by inappropriate experimental conditions for specific immunoprecipitation of the APP/IB1 complex from solubilized brain homogenates or whether it implies that, with the APP/IB1 complex being a minor fraction, the majority of APP and IB1 in the brain does not complex with each other or form complexes with different partners. The latter notion is consistent, at least in part, with the observed relatively lower maximal binding of JIP-1b to the cytoplasmic domain of APP, as compared with those of the other PID-containing proteins tested (Matsuda, 2001).

By constructing deletion and point mutants, it has been shown that the domains necessary for the APP/JIP-1b interaction are the cytoplasmic G681YENPTY687 region in APP and the PID region in JIP-1b, completed by the insertion specific for this isoform. This accounts for the PID-nonbearing isoform JIP-1 not interacting with APP. As noted above, X11, Fe65, Fe65L, and mDab1 have been shown to interact with the C terminus of APP. The present study indicates that the APP/JIP-1b interaction requires Tyr682, Asn684, and Tyr687 contained in the G681YENPTY687 region. This is different from the mode of APP interaction with Fe65 and X11 and similar to that with mDab1. Interestingly, the JIP-1b isoform, which is interactive with APP, is the major transcript in the brain, and the noninteractive JIP-1 transcript is hardly detected, pointing to certain specific roles of the JIP-1b isoform in neuronal functions (Matsuda, 2001).

The mechanism underlying the observed JIP-1b/IB1 interaction with APP is thus consistent with the established NPXY motif interaction of PID in Shc and IRS-1. Yet in the present GST pull-down experiments, the cytoplasmic domains of APP, APLP1, and APLP2, all of which contain the same NPXY structure GYENPTY, show largely different binding intensities for JIP-1b/IB1, with APP being the strongest among them. These different binding characteristics might reflect the difference in the primary to ternary structures surrounding the NPXY motif, suggesting the presence of an additional structural requirement allowing NPXY to interact efficiently with PID. In support of this idea, the most recent literature, in which PID of JIP-1b is shown to interact with p190 rhoGEF, indicates that the binding region of p190 does not contain the classical NPXY motif (Matsuda, 2001).

Because JIP-1b shows binding similar to full-length APP regardless of the presence of four different FAD mutations, JIP-1b is most likely involved in the basic function of APP. Although the binding of Fe65 or X11 to APP has been shown to affect Aß secretion from APP, so far remarkable changes in Aß42 secretion from NL-APP have not been demonstrated by cotransfection with JIP-1b. The JIP-1 proteins have been shown to serve as scaffold proteins for the organization of active JNK signaling complexes. In fact, APP associates with JNK via JIP-1b. It has also been established that APP interacts with the GTP-binding protein Go through the middle portion in the APP cytoplasmic domain adjacent to the NPXY-containing C terminus. It is likely, therefore, that APP may serve as a membrane-anchoring protein that further scaffolds the JIP-scaffolding complex with other signaling molecules. Taking into account the recently cloned members of the JIP family, JIP2 and JIP3 (PID is contained in JIP2 but not in JIP3), it may be worthwhile to investigate whether APP might regulate the JNK signaling pathway through the binding of these various JIP proteins to the cytoplasmic domain of APP (Matsuda, 2001).

The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. This study shows that membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling: positive when cleaving Notch and generating NICD, and negative when processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).

The APP - cytoskeleton connection

Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. The secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulate the 43-kDa form of MAPK by greater than 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevents the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer's disease, a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation was found. In the ras dominant inhibitory cell line, beta-APP fails to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau (Greenberg, 1994).

This study examined the mechanism of axonal transport of the amyloid precursor protein (APP), which plays a major role in the development of Alzheimer's disease. Coimmunoprecipitation, sucrose gradient, and direct in vitro binding have demonstrated that APP forms a complex with the microtubule motor, conventional kinesin (kinesin-I), by binding directly to the TPR domain of the kinesin light chain (KLC) subunit. The estimated apparent Kd for binding is 15-20 nM, with a binding stoichiometry of two APP per KLC. In addition, association of APP with microtubules and axonal transport of APP is greatly decreased in a gene-targeted mouse mutant of the neuronally enriched KLC1 gene. It is proposed that one of the normal functions of APP may be as a membrane cargo receptor for kinesin-I and that KLC is important for kinesin-I-driven transport of APP into axons (Kamai, 2000).

It is intriguing that overexpression of an APP homolog (APPL) in Drosophila neurons leads to axonal blockage and neuronal dysfunction that is synergistic with overexpression of the microtubule-associated protein tau (Torroja, 1999a). This axonal blockage phenotype is similar to what is observed in Drosophila mutants lacking known kinesin and dynein motor subunits or other membrane proteins that bind kinesin-I. Besides the phenotypic similarity between APPL overexpression and mutations in microtubule motor subunits, a genetic interaction between APPL and the KHC gene has also been demonstrated (Torroja, 1999a), further supporting a direct functional association of APP and kinesin-I (Kamai, 2000).

One of the most poorly understood aspects of microtubule-dependent trafficking is the identity of the membranous cargo that each motor carries. It is thought that motor-cargo recognition may require three players: the motor proteins, a cargo-bound receptor, and accessory components. These results suggest that APP may be a membrane cargo receptor for kinesin-I and might link kinesin-I to a particular subset of axonal transport vesicles. This idea is consistent with the finding that APP, a kinesin-I cargo, is enriched in Rab5-positive vesicles, whereas there is virtually no APP present in synaptophysin positive vesicles that are most likely cargoes for the UNC104/KIF1A kinesin. These data suggest that different motors could interact with different membrane cargo receptors on particular subsets of axonal transport vesicles (Kamai, 2000 and references therein).

A potential receptor for kinesin-I is kinectin, an integral membrane protein that is localized to the endoplasmic reticulum. However, proteins other than kinectin might be important for axonal transport since kinectin has been reported to be absent from axons. In addition, no direct connection between kinectin and either subunit of kinesin-I has been demonstrated, and kinectin is not found in C. elegans or Drosophila (Kamai, 2000).

Analysis of an axonal transport mutant in Drosophila led to the identification of a novel membrane-associated protein, Sunday-driver (SYD), which may also be a membrane receptor for kinesin-I (Bowman, 2000). GFP-tagged mammalian SYD localized to tubular and vesicular elements that costained with kinesin-I and Golgi markers, suggesting that SYD might function as a membrane-associated receptor for the axonal transport of post-Golgi vesicles. Thus, both APP and SYD could be membrane cargo receptors for kinesin-I in axonal transport and post-Golgi transport. Further work is needed to understand the functional relationship of these two proteins (Kamai, 2000).

These studies of APP suggest that KLC interacts with membrane-associated proteins through one or more of its TPR repeat domains. The observed binding stoichiometry of two APP molecules per KLC fits well with the atomic structure of other TPR domains, which suggests that three TPR repeats fold together to bind one ligand. The KLC construct used has six TPR repeats, so the observed binding stoichiometry fits the theoretical binding saturation that is predicted from the atomic structure. It is intriguing that the observation that APP directly binds to the TPR domain of KLC and that APP binding to KLC is inhibited by the KLC-All antibody (which binds specifically to the TPR domain of KLC) is similar to recent work on the SYD protein. The SYD protein directly interacts with the TPR domain of KLC by yeast two-hybrid analyses and the KLC-All antibody also inhibits binding of SYD to KLC in GST pulldown experiments (Bowman, 2000). Strikingly, in an in vitro organelle motility system, the KLC-All antibody inhibits the binding of kinesin-I to membranes and blocks fast axonal transport, while no such effects are seen with the 63-90 antibody, which recognizes the N-terminal domain of KLC. Together, these results demonstrate that the TPR domains of KLC directly interact with membrane-associated proteins of vesicular cargo (Kamai, 2000).

Although the data in isolation are most consistent with the simple suggestion that the function of the KLC subunit of kinesin-I is to directly bind cargo receptor proteins such as APP, previous studies of the relative roles of KLC and KHC in cargo attachment and motor regulation have yielded apparently contradictory results. For example, while antibody inhibition studies suggest that KLC is needed for interaction of kinesin-I with membranes, another study has shown that KHC alone is sufficient to bind membranes. This latter finding is consistent with recent work on a null mouse mutant of KLC1 that found KHC accumulation in the absence of KLC at the Golgi apparatus, a presumed site of cargo transport initiation. This apparent binding of KHC to potential cargoes in vivo, in the absence of KLC, is also consistent with work on fungal kinesin-I, which has no KLC subunit, yet appears to be capable of cargo binding. There has also been conflicting evidence about whether KLC, or the tail of KHC, or both repress kinesin-I motor activity in the absence of membrane or cargo binding. The inconsistencies among these studies could be attributable to the various experimental systems used. However, the following simple and testable unifying hypothesis is proposed that accounts for virtually all of the results described above, including those on APP and SYD. It is suggested that both KLC and the tail of KHC combine to fully repress motor activity; that the tail of KHC binds relatively indiscriminately to membrane cargoes, and that KLC interaction with specific membrane proteins (such as APP or SYD) relieves motor repression and activates transport. Thus, the role of KLC may be to provide specificity for cargo binding and transport, perhaps via an activation function. Clearly, further work is needed to test this model rigorously (Kamai, 2000).

There are numerous suggestions that aberrant trafficking or transport of APP may contribute to the development of AD. The finding of a direct interaction of APP and the microtubule transport machinery leads to the intriguing suggestion that abnormal interactions of APP and kinesin-I could play a role in the pathogenesis of AD, perhaps by blocking or otherwise interfering with normal axonal transport. Future studies using APP transgenic and kinesin-I mutant mice will help elucidate the significance of these findings for the pathology of AD (Kamai, 2000).

Growth factor effects on APP processing

Altered processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease. To investigate whether cellular APP processing is controlled by cell-surface neurotransmitter receptors, human embryonic kidney (293) cell lines were transfected with the genes for human brain muscarinic acetylcholine receptors. Stimulation of m1 and m3 receptor subtypes with carbachol increases the basal release of APP derivatives within minutes of treatment, indicating that preexisting APP is released in response to receptor activation. Receptor-activated APP release is blocked by staurosporine, suggesting that protein kinases mediate neurotransmitter receptor-controlled APP processing (Nitsch, 1992).

Proteolytic processing of the beta-amyloid protein precursor (APP) is regulated by cell-surface receptors. To determine whether neurotransmitter release in response to neuronal activation regulates APP processing in brain, superfused rat hippocampal slices were electrically depolarized and soluble APP derivatives released into the superfusate were measured. Electrical depolarization causes a rapid increase in the release of both neurotransmitters and amino-terminal APP cleavage products. These derivatives lack the APP carboxyl terminus and are similar to those found in both cell culture media and human cerebrospinal fluid. Superfusate proteins including lactate dehydrogenase are not changed by electrical depolarization. The release of amino-terminal APP derivatives increases with increasing stimulation frequencies from 0 to 30 Hz. The increased release is inhibited by the sodium-channel antagonist tetrodotoxin, suggesting that action-potential formation mediates the release of large amino-terminal APP derivatives. These results suggest that neuronal activity regulates APP processing in the mammalian brain (Nitsch, 1993).

Release of large soluble NH2-terminal fragments of the amyloid precursor protein (APP) of Alzheimer's disease was measured in two Swiss 3T3 fibroblast cell lines (designated SF1.4 and SF3.2), overexpressing the alpha subtype of protein kinase C, and in two control cell lines (SC1 and SC2). Basal release of APP is significantly increased in SF1.4 cells, but not in SF3.2 cells, relative to controls. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, elicits a concentration-dependent increase in APP release in all four cell lines. However, the estimated EC50 for this effect is lower in the two cell lines overexpressing protein kinase C-alpha than in control SC1 and SC2 cells. The absolute amount of APP released by maximal concentrations of phorbol ester is not altered by overexpression of protein kinase C alpha. A protein kinase C inhibitor significantly reduces the response to phorbol esters in control cells but not in cells that overexpress protein kinase C alpha. Levels of cell-associated APP are slightly elevated, and rates of APP turnover are unchanged in SF1.4 cells, relative to controls. However, cell-associated APP levels are lower in SF3.2 cells than in controls. The results demonstrate that protein kinase C alpha regulates APP release in Swiss 3T3 fibroblasts, and perhaps in other tissues, including brain, and may be the isozyme that mediates receptor-evoked release of APP (Slack, 1993).

The amyloid protein precursor (APP) can be processed via several alternative processing pathways. Alpha-secretase processing by cleavage within the amyloid beta-peptide domain of APP is highly regulated by several external and internal signals, including G protein-coupled receptors, protein kinase C and phospholipase A2. In order to demonstrate that G protein-coupled neuropeptide receptors for bradykinin and vasopressin can increase alpha-secretase processing of APP, endogenously expressed bradykinin or vasopressin receptors were stimulated in cell culture with the neuropeptides. The secreted ectodomain (APPs) were measured in the conditioned media. Both bradykinin and vasopressin rapidly increase phosphatidylinositol (PI) turnover in PC-12 and in NRK-49F cells, indicating that these cell lines constitutively expressed functional PI-linked receptors for these neuropeptides. Both bradykinin and vasopressin readily stimulate APPs secretion. Increased APPs secretion is concentration-dependent and saturable, and it is blocked by receptor antagonists indicating specific receptor interaction of the peptides. The bradykinin-induced increase in APPs secretion in PC-12 cells is mediated by protein kinase C (PKC), whereas vasopressin receptors in NRK-49F cells are coupled to APP processing by PKC-independent signaling pathways. These data show that neuropeptides can modulate APP processing in cell culture. In as much as increased alpha-secretase processing is associated with decreased formation of A beta(1-40), a major constituent of amyloid plaques, these findings suggest a possible role for modulating neuropeptide receptors as a strategy for altering amyloid metabolism in Alzheimer's disease brain (Nitsch. 1998).

APP and neural activity: APP could keep neuronal hyperactivity in check

A large body of evidence has implicated Aß peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimer's disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Neuronal activity is shown to modulate the formation and secretion of Aß peptides in hippocampal slice neurons that overexpress APP. In turn, Aß selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Aß could contribute to cognitive decline during early AD. In addition, it is proposed that activity-dependent modulation of endogenous Aß production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD (Kamenetz, 2003).

While these results are consistent with the notion that high Aß levels may disrupt synaptic function, the data suggest that Aß may also have a normal negative feedback function. Increased neuronal activity produces more Aß; the enhanced Aß production depresses synaptic function; the depressed synaptic function will decrease neuronal activity. Examples of synaptic homeostasis have recently been reported, as well as intercellular depression following strong tetanic stimulation, although the signaling molecules mediating these processes have not been identified. In support of this model, it has been fond that in addition to human Aß, rodent Aß can also depress synaptic transmission. This is important because rodent Aß is believed not to have amyloidogenic properties. In wild-type rat tissue, multiple tetani lead to greater synaptic potentiation in the presence of the gamma-secretase inhibitor L-685,458. This suggests that multiple tetani drive APP processing, producing a synaptic depression (in addition to LTP) that can be revealed by gamma-secretase inhibition. This phenomenon is seen when multiple tetani are delivered suggesting that the negative feedback system mediated by APP processing may normally only be rapidly recruited under very high activity levels. This may explain the enhanced kainate-induced seizure activity in APP knockout mice. It has also been found that 24 hr application of gamma-secretase inhibitor L-685,458 leads to enhanced synaptic transmission (increased miniature EPSC frequency). The absence of an overt phenotype in mice lacking ß-secretase suggests that other mechanisms can compensate for this in these mice (Kamenetz, 2003).

How could disturbances in this proposed negative feedback loop contribute to AD? One can envision a number of scenarios. For instance, if synapses lose sensitivity to Aß-induced depression, persistently elevated neuronal activity may go unchecked. High levels of neuronal activity could lead to excitotoxicity, as well as higher levels of secreted A peptides, which may in turn form neurotoxic fibrils that eventually kill neurons. Alternatively, Aß production may become constitutive (lose sensitivity to synaptic activity), with resulting synaptic depression and neuronal toxicity (Kamenetz, 2003).

APP acts as a growth factor

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulates phosphorylation mediated by mitogen-activated protein kinase and effectively evokes proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliferative effect of sAPP is of physiological relevance, antisense techniques were used to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. After the reduced APP expression and sAPP secretion, a strong suppression of the TSH-induced cell proliferation down to 35% was observed. Recombinant sAPP but not TSH is able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH: it is released from thyroid epithelial cells during TSH-induced differentiation. It is proposed that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells (Pietrizik, 1998).

beta-Amyloid precursor protein (beta APP) is an integral membrane polypeptide expressed in many neural and non-neural cells. beta APP occurs in part at the cell surface and undergoes proteolytic processing to release the large soluble ectodomain (APPs) and the amyloid beta-peptide (A beta), both of which have apparent trophic activity in vitro. Despite intense interest in beta APP expression and metabolism, there is limited knowledge about the function mediated by beta APP inserted at the cell-surface. A coculture system has been established in which beta APP-transfected CHO cells serve as a substrate for the growth of primary rat hippocampal neurons. Compared to nontransfected CHO cells, the increased surface beta APP of the transfectants stimulate short-term neuronal adhesion and longer-term neurite outgrowth, whereas the increased amount of secreted APPs and A beta in conditioned medium produce no such effects when neurons are grown either on untransfected CHO cells or on a polylysine substrate. Moreover, a peptide that has been shown to block the trophic effects of secreted APPs fails to interrupt the neurite promoting activity mediated by the surface-expressed beta APP. Surface-expressed beta APP751 or beta APP770 isoforms mediate more neurite outgrowth than does the beta APP695 isoform. Antibody blocking and regional deletion experiments indicate that the mid-region of the beta APP ectodomain (residues 361-648) is involved in promoting neurite outgrowth. It is concluded that surface-expressed cellular beta APP has a neurite-promoting function that is distinct from the trophic function of the secreted beta APP derivatives and may have special significance during brain development (Qui, 1995).

Cortical amyloid precursor protein (APP) is induced and secreted in response to subcortical lesions of cholinergic innervation. To understand the physiological role of the induced APP, its neurotrophic activity on PC12 cells was characterized. Highly purified human APP751 (50-1000 pM) induces outgrowth of neurites. The neurotrophic activity is inhibited by an antibody that is directed to the C-terminal portion of the secreted APP but not by an antibody directed to the KPI domain. The neurotrophic activity of APP is independent of the TrkA NGF receptor because neither phospholipase C-gamma1 nor TrkA exhibit tyrosine phosphorylations with APP treatment. Furthermore, APP stimulates neurite outgrowth from PC12 cells lacking TrkA receptors. At lower concentrations (10-50 pM), APP synergistically potentiates the neurotrophic effects of NGF when added with NGF or before NGF as a priming pretreatment. These results implicate APP, a rapidly induced protein in the injured cortex, as a potentiating agent that may render compromised neurons more responsive to low levels of NGF or other neurotrophins (Wallace, 1997a).

Amyloid precursor protein (APP) is known to be widely expressed in neuronal cells, and enriched in the central and peripheral synaptic sites. Although it has been proposed that APP functions in synaptogenesis, no direct evidence has yet been reported. In this study the involvement of APP in functional synapse formation was investigated by monitoring spontaneous oscillations of intracellular Ca2+ concentration ([Ca2+]i) in cultured hippocampal neurons. As more and more neurons form synapses with one another during the culture period, increasing numbers of neuronal cells show synchronized spontaneous oscillations of [Ca2+]i. The number of neurons that show synchronized spontaneous oscillations of [Ca2+]i is significantly lower when cultured in the presence of monoclonal antibody 22C11 against the N-terminal portion of APP. Moreover, incubation with excess amounts of the secretory form of APP or the N-terminal fragment of APP also inhibits the increase in the number of neurons with synchronized spontaneous oscillations of [Ca2+]i. The addition of monoclonal antibody 22C11 or secretory form of APP does not, however, affect MAP-2-positive neurite outgrowth. These findings suggest that APP play a role in functional synapse formation during CNS development (Morimoto, 1998).

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. Since neurogenesis is an ongoing process in the adult mammalian brain, a role for sAPP in adult neurogenesis has been investigated. The subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site; binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system (Caillé, 2004).

A nuclear function for the APP intracellular domain

Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase. gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. The cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60 (see Drosophila Tip60). This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression (Cao, 2001).

Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease. The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma). This processing resembles regulated intramembrane proteolysis of transmembrane proteins such as Notch, where the released cytoplasmic fragments enter the nucleus and modulate gene expression. This study examines whether the analogous Cgamma fragments of APP also exert effects in the nucleus. Ectopically expressed Cgamma is present both in the cytoplasm and in the nucleus. Interestingly, expression of Cgamma59 causes disappearance of PAT1 (a protein that interacts with the APP cytoplasmic domain) from the nucleus and induces its proteosomal degradation. Treatment of cells with lactacystin prevents PAT1 degradation and retains its nuclear localization. By contrast, Cgamma57, a minor product of gamma-cleavage, is only marginally effective in PAT1 degradation. Furthermore, Cgamma59 but not Cgamma57 potently represses retinoic acid-responsive gene expression. Thus, these studies provide the evidence that, as predicted by the regulated intramembrane proteolysis mechanism, Cgamma seems to function in the nucleus (Gao, 2001).

Defining the molecular mechanisms that integrate diverse signaling pathways at the level of gene transcription remains a central issue in biology. Interleukin-1ß (IL-1ß) causes nuclear export of a specific N-CoR corepressor complex, resulting in derepression of a specific subset of NF-kappaB-regulated genes, exemplified by the tetraspanin KAI1 that regulates membrane receptor function. Nuclear export of the N-CoR/TAB2/HDAC3 complex by IL-1ß is temporally linked to selective recruitment of a Tip60 coactivator complex. Surprisingly, KAI1 is also directly activated by a ternary complex, dependent on the acetyltransferase activity of Tip60, consisting of the presenilin-dependent C-terminal cleavage product of the amyloid ß precursor protein (APP), Fe65, and Tip60, identifying a specific in vivo gene target of an APP-dependent transcription complex in the brain (Baek, 2002).

This work defines a molecular mechanism that links inflammation to derepression of a specific subset of NF-kappaB-regulated genes via control of a previously unknown stable N-CoR complex. This N-CoR/TAB2/HDAC3-containing complex binds to p50-regulated target genes and undergoes a nuclear to cytoplasmic translocation in response to IL-1ß signaling. TAB2 (Drosophila homolog: TGF-ß activated kinase 1) itself enhances N-CoR-dependent repression, but the apparently critical function of TAB2 is to regulate IL-1ß-mediated translocation of the N-CoR complex out of the nucleus. TAB2 thus seems to have dual roles upon activation of the NF-kappaB pathway, serving to both derepress p50-dependent transcription units (nuclear function) as well as to activate the p50/p65 targets (cytoplasmic function) (Baek, 2002).

Evidence is provided indicating that the molecular basis of IL-1ß-dependent nuclear export of the nuclear N-CoR likely represents a MEKK1-dependent phosphorylation of TAB2 in the nucleus, putatively causing an allosteric alteration that exposes the TAB2 nuclear export signal. Thus, MEKK1 might also serve to integrate signal transduction pathways, both in the nucleus and in the cytoplasm (Baek, 2002).

These data indicate that KAI1/CD82 is an IL-1ß-induced NF-kappaB target gene based on binding of p50 homodimer. Under unstimulated conditions, p50, but not p65, was detected on the KAI1 promoter, while after IL-1ß stimulation, the level of promoter-associated p50 remained constant, without any binding by p65. Bcl3 occupies the KAI1 promoter in the presence or absence of IL-1ß, and several independent studies have suggested that Bcl3 can act as a bridging factor linking NF-kappaB to nuclear coregulators. Tip60 has been suggested to be a binding partner of Bcl3, enhancing Bcl3/p50-activated transcription through a NF-kappaB binding site (Baek, 2002).

Thus, the recruitment of Tip60 to KAI1/CD82 promoter after IL-1ß treatment, possibly requiring Bcl3, appears to be of functional importance to activation of the gene, which is accompanied by acetylation of histones H3/H4. Tip60 has been identified as a component of a multimeric protein complex containing histone acetylase, ATPase, DNA helicase activity, and structural DNA binding activity, which links it to DNA repair function. In the case of KAI1/CD82 promoter, Tip60 appears to be recruited as a component of a TRRAP-containing complex, likely distinct from the purified repair complex, although the precise complement of corecruited factors remains to be defined. The Tip60 HAT function appears to be required, directly or indirectly, for effective gene activation, because Tip60 HATmut abolishes histone H3 and H4 acetylation and recruitment of Pol II. The observed acetylation of histones H3/H4 during IL-1ß-stimulated KAI1 transcription could reflect direct acetylation by Tip60 in a promoter-specific fashion, or it could reflect recruitment of an as yet unidentified histone acetyltransferase. In contrast to KAI1, examination of two other NF-kappaB-regulated genes that recruit p50/p65 heterodimers revealed no recruitment of the N-CoR/TAB2/HDAC3 complex. The identification of a large number of transcriptional coactivators and corepressors, capable of interacting with distinct DNA bound transcription factors, has raised questions regarding their potential specificity and complementarity in gene regulation events. The NF-kappaB-regulated genes examined appear to recruit distinct coactivator machinery during gene activation events in response to IL-1ß, which is in contrast to the apparently more uniform, ligand-dependent recruitment of many coactivator complexes in estrogen receptor-regulated genes (Baek, 2002).

gamma-Secretase cleavage of APP releases not only Aß from the membrane but also the intracellular fragment AICD, which was identified only very recently because of its instability. A number of investigators have speculated that by analogy to signaling by NICD derived from Notch 1 receptor, the corresponding AICD may also function in signal transduction. In this manuscript, it has been shown that transgenic mice overexpressing APP, which develop age-related amyloid deposits and associated pathologic changes, unexpectedly exhibit increased expression of both Fe65 and Tip60 in the CNS. All three components of the AICD/Fe65/Tip60 complex are unexpectedly induced, forming a complex binding to the KAI1/CD82 promoter. This complex is capable of displacing the N-CoR/TAB2/HDAC3 complex in the absence of an IL-1ß signal and causing target gene activation. KAI1 itself provides a potentially intriguing transcriptional target of APP overexpression. As many cell surface receptors and cell adhesion molecules that regulate cytoskeletal functions are impacted by the tetraspanin KAI1/CD82, it is tempting to speculate that expression of KAI1 might contribute to later pathological events (Baek, 2002).

Remarkably, the acetyltransferase function of Tip60 is required to form the ternary complex that can displace an N-CoR/TAB2/HDAC3 corepressor complex. In addition to autoacetylation of Tip60, increased levels of acetylated Fe65 are found in the trimeric complex, suggesting that acetylation of Fe65 might be a regulatory component of ternary complex formation. These data provide a striking example of acetylation as a critical regulatory aspect of coactivator complex assembly, required for specific gene activation events. Since the transcriptional activation of KAI1 by AICD/Fe65/Tip60 is abolished by both the NSAIDs ibuprofen and naproxen with restoration of the binding of the NCoR/TAB2/HDAC3 complex on the promoter, it is postulated that NSAIDs may act at some step(s) distal to generation of AICD by an as yet unknown mechanism (Baek, 2002).

Together, these data are consistent with a model in which IL-1ß acts physiologically to cause dismissal of a specific N-CoR corepressor complex and recruitment of a Tip60-containing coactivator complex resulting in activation of p50 target genes. The AICD/Fe65/Tip60 trimeric complex can similarly displace the N-CoR complex, derepressing gene targets such as KAI1/CD82, providing a potential transcriptional activation strategy that may underlie specific aspects of APP function, both in normal physiology and in Alzheimer's disease (Baek, 2002).

The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11alpha (MINT1) and the chromatin-associated protein Tip60, their interactions were analyzed by confocal microscopy and co-immunoprecipitations. APP intracellular domain (AICD) corresponding to S3-cleaved APP binds to Fe65 that transports it to nuclei and docks it to Tip60. These proteins form AICD-Fe65-Tip60 (AFT) complexes that are concentrated in spherical nuclear spots. gamma-Secretase inhibitors prevent AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transports AICD to nuclei and docks it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes have different, speckle-like morphology. By contrast, X11alpha traps AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3beta and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling (von Rotz, 2004).

Amyloid-beta precursor protein (APP) forms a transcriptionally active complex with the adaptor protein Fe65 and the histone acetyltransferase Tip60, but the mechanism of transcriptional activation that is mediated by APP and Fe65 remains unclear. APP is cleaved by gamma-secretase similar to Notch, whose intracellular domain activates transcription by interacting with nuclear transcription factors. To test whether the APP intracellular domain (AICD) functions analogously, how APP and Fe65 transactivate a Gal4 fusion protein of Tip60 was investigated. Consistent with the Notch paradigm, it was observed that gamma-cleavage of APP and nuclear translocation of Fe65 are required for transactivation. Surprisingly, however, it was found that nuclear translocation of the AICD may be dispensable and that only membrane-tethered AICD (i.e. AICD coupled to a transmembrane region) and not free AICD (i.e. soluble AICD) is a potent transactivator of transcription. Membrane-tethered AICD recruits Fe65 and mediates the activation of bound Fe65 that is then released for nuclear translocation by gamma-cleavage together with the AICD. These data suggest that transcriptional transactivation by APP and Notch may involve distinct mechanisms; whereas the Notch intracellular domain directly functions in the nucleus, the AICD acts indirectly by activating Fe65 (Cao, 2004).

Suppression of beta-amyloid precursor protein signaling into the nucleus by estrogens mediated through complex formation between the estrogen receptor and Fe65

The C-terminal fragment of the beta-amyloid precursor protein produced after cleavage by gamma-secretase, namely, APPct or AICD, has been shown to form a multimeric complex with the adaptor protein Fe65 and to regulate transcription through the recruitment of the histone acetyltransferase Tip60. The present study shows that 17beta-estradiol inhibits the transcriptional and apoptotic activities of the APPct complex by a process involving the interaction of estrogen receptor alpha (ERalpha) with Fe65. ERalpha-Fe65 complexes were detected both in vitro and in the mouse brain, and recruitment of ERalpha to the promoter of an APPct target gene (KAI1) was demonstrated. These studies reveal a novel mechanism of estrogen action, which may explain the well-known neuroprotective functions of estrogens as well as the complex role of this female hormone in the pathogenesis of neuronal degeneration diseases (Bao, 2006).

APP interaction with neurotrophin receptor

Amyloid beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP), is a major component of the plaques found in the brain of Alzheimer's disease (AD) patients. These plaques are thought to cause the observed loss of cholinergic neurons in the basal forebrain of AD patients. In these neurons, particularly those of the nucleus basalis of Meynert, an up-regulation of 75kD-neurotrophin receptor (p75NTR), a nonselective neurotrophin receptor belonging to the death receptor family, has been reported. p75NTR expression has been described to correlate with beta-amyloid sensitivity in vivo and in vitro, suggesting a possible role for p75NTR as a receptor for Abeta. A human neuroblastoma cell line to investigate the involvement of p75NTR in Abeta-induced cell death. Abeta peptides were found to bind to p75NTR resulting in activation of NFKB in a time- and dose-dependent manner. Blocking the interaction of Abeta with p75NTR using NGF or inhibition of NFKB activation by curcumin or NFKB SN50 attenuates or abolishes Abeta-induced apoptotic cell death. The present results suggest that p75NTR might be a death receptor for Abeta, thus being a possible drug target for treatment of AD (Kuner, 1998).

APP modulation of neuronal excitability and synaptic function

In several pedigrees of early onset familial Alzheimer's disease (FAD), point mutations in the beta-amyloid precursor protein (APP) gene are genetically linked to the disease. This finding implicates APP in the pathogenesis of Alzheimer's disease in these individuals. To understand the in vivo function of APP and its processing, an APP-null mutation has been generated in mice. Homozygous APP-deficient mice are viable and fertile. However, the mutant animals weigh 15%-20% less than age-matched wild-type controls. Neurological evaluation shows that the APP-deficient mice exhibit a decreased locomotor activity and forelimb grip strength, indicating a compromised neuronal or muscular function. In addition, four out of six homozygous mice show reactive gliosis at 14 weeks of age, suggesting an impaired neuronal function as a result of the APP-null mutation (Zheng, 1995).

The Alzheimer's beta-amyloid precursor protein (beta-APP) is widely expressed in neural cells. In neurons, secreted forms of beta-APP (sAPPs) are released from membrane-spanning holo-beta APP in an activity-dependent manner. Secreted APPs can modulate neurite outgrowth, synaptogenesis, synaptic plasticity and cell survival; a signal transduction mechanism of sAPPs may involve modulation of intracellular calcium levels ([Ca2+]i). Whole-cell perforated patch and single-channel patch-clamp analysis of hippocampal neurons was used to demonstrate that sAPPs suppress action potentials and hyperpolarize neurons by activating high-conductance, charybdotoxin-sensitive K+ channels. Activation of K+ channels by sAPPs is mimicked by a cyclic GMP analog and sodium nitroprusside and blocked by an antagonist of cGMP-dependent kinase and a phosphatase inhibitor, suggesting that the effect is mediated by cGMP and protein dephosphorylation. Calcium imaging studies indicate that activation of K+ channels mediates the ability of sAPPs to decrease [Ca2+]i. Modulation of neuronal excitability may be a major mechanism by which beta-APP regulates developmental and synaptic plasticity in the nervous system (Furukawa, 1996).

The secreted form of beta-amyloid precursor protein (sAPP alpha) is released from neurons in an activity-dependent manner: data suggest sAPP alpha may play roles in regulating neuronal excitability, plasticity, and survival. In cultured hippocampal neurons sAPP alpha can suppress elevation of [Ca2+]i induced by glutamate and can protect neurons against excitotoxicity. Whole-cell patch-clamp data from studies of cultured embryonic rat hippocampal neurons demonstrate that sAPP alpha selectively suppresses N-methyl-D-aspartate currents without affecting currents induced by AMPA or kainate. sAPP alpha suppresses N-methyl-D-aspartate current rapidly and reversibly at concentrations of 0.011 nM. Suppression of N-methyl-D-aspartate current by sAPP alpha is apparently mediated by cyclic guanosine monophosphate because 8-bromo-cyclic guanosine monophosphate suppresses N-methyl-D-aspartate current in a manner similar to sAPP alpha, and two different inhibitors of cyclic guanosine monophosphate-dependent protein kinase prevent sAPP alpha-induced suppression of N-methyl-D-aspartate current. In addition, okadaic acid prevents suppression of N-methyl-D-aspartate-induced current, suggesting the involvement of a protein phosphatase in modulation of N-methyl-D-aspartate current by sAPP alpha. These data identify a mechanism whereby sAPP alpha can modulate cellular responses to glutamate, and suggest important roles for sAPP alpha in the various physiological and pathophysiological processes in which N-methyl-D-aspartate receptors participate (Furukawa, 1998).

The secreted form of amyloid precursor protein (sAPPalpha), which is released from neurons in an activity-dependent manner, can modulate neurite outgrowth, synaptic plasticity, and neuron survival. sAPPalpha can enhance glucose and glutamate transport in synaptic compartments. Treatment of cortical synaptosomes with nanomolar concentrations of sAPPalpha results in an attenuation of impairment of glutamate and glucose transport induced by exposure to amyloid beta-peptide and Fe2+. The protective effect of sAPPalpha is mimicked by treatment with 8-bromo-cyclic GMP and blocked by a cyclic GMP-dependent protein kinase inhibitor, suggesting that protective action of sAPPalpha is mediated by cyclic GMP. These data suggest that glucose and glutamate transport can be regulated locally at the level of the synapse and further suggest important roles for sAPPalpha and cyclic GMP in modulating synaptic physiology under normal and pathophysiological conditions (Mattson, 1999).

APP and neural development

The amyloid precursor superfamily is composed of three highly conserved transmembrane glycoproteins, the amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1, APLP2), whose functions are unknown. Proteolytic cleavage of APP yields the betaA4 peptide, the major component of cerebral amyloid in Alzheimer's disease. Five post-translationally modified, full-length species of APP and APLP2 (but not APLP1) arrive at the mature presynaptic terminal in the fastest wave of axonal transport and are subsequently rapidly cleared (mean half-life of 3.5 h). Rapid turnover of presynaptic APP and APLP2 occurs independently of visual activity. Turnover of the most rapidly arriving APP species is accompanied by a delayed accumulation of a 120-kDa, APP fragment lacking the C terminus, consistent with presynaptic APP turnover via constitutive proteolysis. Turnover of APLP2 is not accompanied by detectable APLP2 fragment peptides, suggesting either that APLP2 either is more rapidly degraded than is APP or is retrogradely transported shortly after reaching the terminus. A single 150-kDa APLP2 species containing the Kunitz protease inhibitor domain is the major amyloid precursor superfamily protein transported to the presynapse. Presynaptic APP and APLP2 are sialylated and N- and O-glycosylated, and some also carry chondroitin sulfate glycosaminoglycan and/or dermatan sulfate glycosaminoglycan. The rapid kinetics for turnover of APP and APLP2 predict a sensitive balance of synthesis, transport, and elimination rates that may be critical to normal neuronal functions and metabolic fates of these proteins (Lyekman, 1998).

The beta-amyloid precursor protein (beta APP) gene of the mouse was disrupted by inserting into exon 2 a cassette containing a neomycin resistance gene and a putative transcription termination sequence. Contrary to expectation, brain and other tissues from mice homozygous for the insertion still contain beta APP-specific RNA, albeit at a level 5- to 10-fold lower than wild type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice are severely impaired in spatial learning and exploratory behavior and show increased incidence of agenesis of the corpus callosum (Muller, 1994).

To address the question of the actual function of APP in normal developing neurons, a study aimed at blocking APP expression using antisense oligonucleotides was undertaken. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed for studies with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation results in a marked but transient decrease in APP neosynthesis. Although transient, the decrease in APP neosynthesis is sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease is not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture is sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there are no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture (Allinquant, 1995).

Alzheimer's amyloid precursor protein (APP), the precursor of beta-amyloid (Abeta), is an integral membrane protein with a receptor-like structure. The mature APP (mAPP; N- and O-glycosylated form) is phosphorylated at Thr668 (numbering for APP695 isoform), specifically in neurons. Phosphorylation of mAPP appears to occur during, and after, neuronal differentiation. The phosphorylation of mAPP begins 48-72 hr after treatment of PC12 cells with NGF and this correlates with the timing of neurite outgrowth. The phosphorylated form of APP is distributed in neurites and mostly in the growth cones of differentiating PC12 cells. PC12 cells stably expressing APP with Thr668Glu substitution show remarkably reduced neurite extension after treatment with NGF. These observations suggest that the phosphorylated form of APP may play an important role in neurite outgrowth of differentiating neurons (Ando, 1999).

The amyloid precursor protein (APP) and APP-like (APLP) material, as visualized with the Mab22C11 antibody, have been shown to be associated with radial glia in hypothalamus. Radial glia are known to promote neurite outgrowth. By Northern blot analysis, APP 695 mRNA levels increases steadily over hypothalamic development, APP 770 mRNA is transiently expressed at 12 days postnatally, and APLP mRNA is only weakly expressed in the hypothalamus. The developmental pattern of APP moeities in mouse hypothalamus and in fetal hypothalamic neurons in culture has been compared with a presenilin 2 (PS2) related protein using an antibody developed against the N-terminal part of PS2. By Western blot analysis, APP and PS2-like immunoreactivity are visualized as a 100-130kDa band and a 52 kDa band, respectively. An APP biphasic increase is observed during hypothalamic development in vivo. APP immunoreactivity is equally detected in neuronal and glial cultures, while PS2-like material is more concentrated in neurons. A correlation between APP/APP-like and PS2-like levels was observed during development in vivo. While APP is mostly associated with membrane fractions, a significant portion of PS2-like material is also recovered from cytosolic fractions in vitro. In contrast to native PS2 in COS-transfected cells, the PS2-like material does not aggregate after heating for 90 s at 90 degrees C. These results indicate a close association between APP and PS2-like material during hypothalamic development in vivo, and suggest that neuronal and glial cultures may provide appropriate models to test their interactions (Apert, 1998).

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, [35S]methionine was injected into the rat entorhinal cortex and the trafficking and processing of de novo synthesized APP was examined in the perforant pathway and at presynaptic sites in the hippocampal formation. Both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. At these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta (Buxbaum, 1998).

APP and synaptic plasticity

The secreted form (sAPP) of the Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in the in vitro regulation of fibroblast growth and neurite extension from neuronal cells. The active site of sAPP responsible for these functions is within a small domain just C-terminal to the Kunitz-type protease inhibitor (KPI) insertion site. A 17-mer peptide, containing this active domain of sAPP, can induce cellular and behavioral changes when infused into rat brains. After 2 weeks of APP 17-mer peptide infusion, the animals were tested for reversal learning and memory retention and were sacrificed for morphological examination of brains. Administration of the APP 17-mer peptide results in an 18% increase in the number of presynaptic terminals in the frontoparietal cortex. At the behavioral level, 17-mer-infused animals with nonimpaired learning capability show an increased memory retention that seems to interfere with reversal learning performance. This APP 17-mer effect on memory retention is not observed in animals with impaired initial learning capacity. These results suggest that APP is involved in memory retention through its effect on synaptic structure (Roch, 1994).

The secreted form of beta-amyloid precursor protein (sAPP alpha) is released from neurons in an activity-dependent manner, and has been reported to modulate neuronal excitability in dissociated hippocampal neurons. sAPP alpha shifts the frequency dependence for induction of long-term depression of synaptic transmission (LTD) in hippocampal slices from adult rats. Whereas low frequency stimulation (1 Hz) of Schaffer collateral axons induces LTD of the post-synaptic response of CA1 neurons in control slices, it does not induce LTD in slices pretreated with sAPP alpha. However, while a 10 Hz stimulation normally induces neither LTD or LTP, it does induce LTD in slices pretreated with sAPP alpha. sAPP alpha potentiates LTP induced by high frequency stimulation. sAPP alpha induces cGMP production in hippocampal slices, and pretreatment of slices with 8-bromo-cyclic GMP mimics the effect of sAPP alpha on LTD, suggesting a role for cyclic GMP in modulation of LTD. The data suggest an important role for sAPP alpha in the modulation of synaptic plasticity in the hippocampus (Ishida, 1997).

Abnormal processing of amyloid precursor protein (APP), in particular the generation of beta-amyloid (Abeta) peptides, has been implicated in the pathogenesis of Alzheimer's disease. This study examined the consequences of deleting the APP gene on hippocampal synaptic plasticity, and upon the biophysical properties of morphologically identified neurons in APP-null mice. The hippocampus of APP-null mice has a characteristic increase in gliosis throughout the CA1 region and a disruption of staining for the dendritic marker MAP2 and the presynaptic marker synaptophysin. The disruption of MAP2 staining is associated with a significant reduction in overall dendritic length and projection depth of biocytin labeled CA1 neurons. In two groups of APP-null mice examined at 8-12 months, and 20-24 months of age, there was an impairment in the formation of long-term potentiation (LTP) in the CA1 region, compared to isogenic age matched controls. This LTP deficit is not associated with an alteration in the amplitude of EPSPs at low stimulus frequencies (0.033 Hz) or facilitation during a 100 Hz stimulus train, but is associated with a reduction in post-tetanic potentiation. Paired-pulse depression of GABA-mediated inhibitory post-synaptic currents is also attenuated in APP-null mice. These data demonstrate that the impaired synaptic plasticity in APP deficient mice is associated with abnormal neuronal morphology and synaptic function within the hippocampus (Seabrook, 1999).

Synaptic communication and plasticity were investigated in hippocampal slices from mice overexpressing mutated 695-amino-acid human amyloid precursor protein (APP695SWE). These mice show behavioral and histopathological abnormalities simulating Alzheimer's disease. Although aged APP transgenic mice exhibit normal fast synaptic transmission and short term plasticity, they are severely impaired in in-vitro and in-vivo long-term potentiation (LTP) in both the CA1 and dentate gyrus regions of the hippocampus. The LTP deficit is correlated with impaired performance in a spatial working memory task in aged transgenics. These deficits are accompanied by minimal or no loss of presynaptic or postsynaptic elementary structural elements in the hippocampus, suggesting that impairments in functional synaptic plasticity may underlie some of the cognitive deficits in these mice and, possibly, in Alzheimer's patients (Chapman, 1999).

A presenilin-1 (PS1) conditional knockout mouse (cKO) has been generated in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities in contrast to the pleiotropic phenotypes associated with PS1 deficiency in the embryonic brain. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of ß-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Aß generation and subtle cognitive deficits without affecting expression of Notch downstream genes (Yu, 2001).

The most striking feature of the disrupted APP processing is the 30-fold accumulation of the APP C-terminal fragments (CTFs) in PS1 cKO mice by the age of 6 months. Interestingly, the APP ß-CTFs (C89 and C99) accumulate differentially in the absence of PS1, with the increase in the level of C89 and C99 cleavage fragments measuring approximately 30- and 3-fold, respectively. The levels of the alpha-CTF (C83) are also elevated by as much as 30-fold. Since the CTFs represent the substrates for gamma-secretase cleavage, these results are consistent with a requirement of PS1 for gamma-secretase activity. The differential accumulation of the CTFs, particularly C89 and C99, which are cleavage products of ß-secretase, is likely due to the differences in their half lives. Alternatively, C99 could be converted to C89 by ß-secretase, resulting in much lower levels of C99 relative to C89 in the cKO mice. Furthermore, all APP CTFs are present in phosphorylated and nonphosphorylated forms in the brain of both control and cKO mice. The cytoplasmic domain of full-length APP has been shown to be phosphorylated in cultured neurons and adult rat brain by cdk5 on Thr668, which resides in the C-terminal region common to all APP CTF species. This phosphorylation event may therefore account for the observed phosphorylation of the CTFs. Although the physiological significance of APP phosphorylation is unclear, there is evidence suggesting that it may be associated with the regulation of Aß generation and neurite extension (Yu, 2001).

Although extensive data support a central pathogenic role for amyloid ß protein (Aß) in Alzheimer’s disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Aß and the nature of its effects on synaptic function have not been defined in vivo. Fibrillar (but not monomeric) forms of Aß akin to those present in the amyloid plaques of Alzheimer’s disease are neurotoxic in culture. However, relatively weak correlations between fibrillar plaque density and severity of dementia are found in Alzheimer’s diseased brains, whereas correlations between soluble Aß levels and the extent of synaptic loss and cognitive impairment are stronger. Natural oligomers of human Aß are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Aß monomers but no amyloid fibrils markedly inhibit hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Aß species completely abrogates this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aß monomers but not oligomers, does not prevent the inhibition of LTP. Therefore, Aß oligomers, in the absence of monomers and amyloid fibrils, disrupt synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevents oligomer formation at doses that allow appreciable monomer production, and such medium no longer disrupts LTP, indicating that synaptotoxic Aß oligomers can be targeted therapeutically (Walsh, 2002).

Detrimental effects of normal and mutant APP

Adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) was used to evaluate the role of various h-APPs in causing neuronal cell death. PC12 cells were infected with very high efficiency: approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA show high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons results in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells (Luo, 1999).

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APP delta NL) secrete comparable amounts of soluble forms of APP (sAPP). B103/APP cells produce sAPP cleaved at amyloid beta/A4 (A beta) 16 (the alpha-secretase site). B103/APP delta NL cells produce sAPP beta cleaved at A beta 1 (the beta-secretase site). B103/APP delta NL cells develop fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells are increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APP delta NL cells. Chemically synthesized A beta, at concentration levels higher than 1 nM, reduces numbers of neurites from B103 or B103/APP delta NL cells. However, A beta at 1-100 nM does not reduce the neurite number of B103/APP cells. The protective activity against A beta's deleterious effect of reducing neurite numbers is attributable to sAPP alpha in the CM. Although sAPP alpha can block the effect of A beta, sAPP beta can not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPP alpha. Nevertheless, sAPP alpha's protective activity requires the N-terminal sequence around RERMS, previously identified as the active domain of sAPP beta. The overall effect of APP mutation, which overproduces A beta and sAPP beta and underproduced sAPP alpha, is a marked decline in the neurotrophic effect of APP. It is suggested that the disruption of balance between the detrimental effect of A beta and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation (Li, 1997).

Cholinergic deficits are one of the most consistent neuropathological landmarks in Alzheimer's disease (AD). Two transgenic mouse models, one overexpressing presenilin-1 (PS1) and a second overexpressing a mutant amyloid precursor protein (APP), and a doubly transgenic line overexpressing both genes, were examined to investigate the effect of AD-related gene overexpression and/or amyloidosis on cholinergic parameters. The size of the basal forebrain cholinergic neurons and the pattern of cholinergic synapses in the hippocampus and cerebral cortex were revealed by immunohistochemical staining for choline acetyltransferase and the vesicular acetylcholine transporter, respectively. At the time point studied (8 months), no apparent changes in either the size or density of cholinergic synapses were found in either of the two single overexpressors, relative to the nontransgenic controls. However, the doubly transgenic line showed a significant elevation in the density of cholinergic synapses in the frontal and parietal cortices. Most importantly, the double mutant, which had extensive amyloidosis, demonstrated a prominent diminution in the density of cholinergic synapses in the frontal cortex and a reduction in the size of these synapses in the frontal cortex and hippocampus. Nonetheless, no significant changes in the size of basal forebrain cholinergic neurons were observed in these three mutants. This study shows a novel role for APP and a synergistic effect of APP and PS1 that correlates with amyloid load on the reorganization of the cholinergic network in the cerebral cortex and hippocampus at the time point studied (Wong, 1999).

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimers disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimers disease (Takahashi, 2000).

Most Down's syndrome (DS) patients develop Alzheimer's disease (AD) neuropathology. Astrocyte and neuronal cultures derived from fetal DS brain show alterations in the processing of amyloid ß precursor protein (AßPP), including increased levels of AßPP and C99, reduced levels of secreted AßPP (AßPPs) and C83, and intracellular accumulation of insoluble Aß42. This pattern of AßPP processing is recapitulated in normal astrocytes by inhibition of mitochondrial metabolism, consistent with impaired mitochondrial function in DS astrocytes. Intracellular Aß42 and reduced AßPPs are also detected in DS and AD brains. The survival of DS neurons is markedly increased by recombinant or astrocyte-produced AßPPs, suggesting that AßPPs may be a neuronal survival factor. Thus, mitochondrial dysfunction in DS may lead to intracellular deposition of Aß42, reduced levels of AßPPs, and a chronic state of increased neuronal vulnerability (Busciglio, 2002).

Impaired mitochondrial function could result from direct toxic effects of Aß or from the metabolic cost of clearing aggregated proteins through chaperones and the degradative apparatus, processes that are highly energy dependent. This model may also apply to other chronic neurodegenerative diseases characterized by intracellular protein aggregation, such as polyglutamine repeat disorders, synucleinopathies, and tauopathies. The metabolic cost of chronic protein aggregation in these diseases may lead to a state of impaired mitochondrial function and ATP depletion, giving rise to a pathological feedback loop in which protein aggregation increases and mitochondrial function is impaired further. Furthermore, impaired energy metabolism would increase neuronal vulnerability to exogenous toxic insults, such as reactive oxygen species, giving rise to a stochastic process of neuronal cell death, consistent with recent mathematical models of cell death in neurodegenerative diseases. As such, therapeutic strategies should be targeted to the prevention of protein aggregation and the restoration of normal mitochondrial function (Busciglio, 2002).

Aβ plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks

Alzheimer's disease is characterized by the deposition of senile Aβ plaques and progressive dementia. The molecular mechanisms that couple plaque deposition to neural system failure, however, are unknown. Using transgenic mouse models of AD together with multiphoton imaging, neuronal calcium was measured in individual neurites and spines in vivo using the genetically encoded calcium indicator Yellow Cameleon 3.6. Quantitative imaging revealed elevated [Ca2+]i (calcium overload) in ~20% of neurites in APP mice with cortical plaques, compared to less than 5% in wild-type mice, PS1 mutant mice, or young APP mice (animals without cortical plaques). Calcium overload depended on the existence and proximity to plaques. The downstream consequences included the loss of spinodendritic calcium compartmentalization (critical for synaptic integration) and a distortion of neuritic morphologies mediated, in part, by the phosphatase calcineurin. Together, these data demonstrate that senile plaques impair neuritic calcium homeostasis in vivo and result in the structural and functional disruption of neuronal networks (Kuchibhotla, 2008).

This study has determined at least three specific downstream functional consequences of calcium overload: spinodendritic calcium decompartmentalization, structural neuritic alterations, and CaN activation, and has also highlighted an important intermediary stage that can be targeted for therapeutic intervention -- inhibiting CaN activity leads to amelioration of the structural and functional deficits and may have behavioral implications. A critical factor that remains to be determined is the specific pathway that amyloid-β activates to induce calcium influx. The effect may be mediated by the formation of calcium-conducting pores comprised of the Aβ peptide, modulation of voltage-gated calcium channels, or an Aβ-mediated oxidative stress that results in altered calcium regulation. Nonetheless, this study shows that mutant APP processing and subsequent plaque deposition play a critical role in inducing calcium overload in these AD models and activating calcineurin-dependent neurodegenerative processes (Kuchibhotla, 2008).

The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

Amyloid beta 42 (Abeta42; see Drosophila Appl) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, this study identified the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. It was demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity (Ring, 2022).

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Biological Overview

date revised: 18 February 2024

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