The InteractiveFly: Drosophila as a Model for Human Diseases


Alzheimer's disease
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Drosophila genes associated with
Alzheimer's disease
β amyloid protein precursor-like
forkhead box, sub-group O
Neurotrophin 1
NMNAT
Presenilin
refractory to sigma P
tau
Related terms


Autophagy
Axonogenesis
Circadian clock
Relevant studies of Alzheimer's disease

Fernandez-Funez, P., Zhang, Y., Sanchez-Garcia, J., de Mena, L., Khare, S., Golde, T.E., Levites, Y. and Rincon-Limas, D.E. (2015). Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease. Hum Mol Genet 24: 6093-6105. PubMed ID: 26253732

Abstract
Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. This study shows that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, the study demonstrates the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities (Fernandez-Funez, 2015).

Highlights

  • Two anti-Aß scFvs independently and synergistically suppress Aß42 neurotoxicity in the eye.
  • Anti-Aß scFvs suppress APP neurotoxicity in the eye.
  • Anti-Aß scFvs are secreted in Drosophila.
  • Anti-Aß scFvs co-localize with Aß42 in Drosophila brain neurons.
  • Anti-Aß scFvs protect neuronal architecture against Aß42 neurotoxicity.
  • Anti-Aß scFvs delay the progressive locomotor dysfunction induced by Aß42.
  • Anti-Aβ scFvs suppress neuronal loss in the brain but do not reduce Aß42 accumulation.
  • Anti-Aß scFvs do not reduce steady-state Aß42 levels.

Discussion
ScFvs are a class of engineered antibodies with promising applications for AD immunotherapy. Many anti-Aß scFvs prevent Aß42 aggregation and reduce its neurotoxicity in vitro. Some anti-Aß scFvs also show benefits in mouse models of AD either by direct administration in the brain, nose or muscle or by AAV-mediated expression in brain or muscle. These approaches report reduction of Aß deposition in the brain, supporting the amyloid attenuating effects observed with complete antibodies. Although anti-Aß scFvs have shown promising results in mouse models of AD, these are expensive and time-consuming experiments that mainly focus on Aß burden one scFv at a time. Furthermore, the physiological benefits of the anti-Aß scFvs are unclear because most AD mouse models overexpressing wild-type or mutant APP show no overt neurodegeneration. Thus, a new platform enabling efficient in vivo preliminary screening of the neuroprotective activity of new scFvs may be needed. This study demonstrates the feasibility of testing the neuroprotective activity of anti-Aß scFvs in Drosophila. As proof-of-principle, two scFvs known to reduce Aß42 burden in CRND8 mice were used and compelling evidence of their neuroprotective activity was obtained in several assays. ScFvs have been expressed in flies previously, although modified for intracellular retention against Huntingtin (intrabodies). A camelid heavy-chain antibody against oligomeric Aß has also been tested recently in flies with the surprising result that it promotes the toxicity of Aß40. This study reports for the first time that scFvs are expressed in flies with their original signal peptide, allowing them to interact with secreted Aß42. ScFv9 and scFv42.2 are properly secreted and folded in Drosophila based on their ability to suppress Aß42 and APP neurotoxicity. Overall, the results presented in this study support the use of Drosophila as a platform for screening multiple anti-Aß scFvs in the same experimental conditions to find the most effective in neurotoxicity assays (Fernandez-Funez, 2015).

In the original report, expression of scFv9 and scFv42.2 in CRND8 mice by somatic brain transgenesis shows significant reduction of insoluble Aß42 in brain homogenates and fewer amyloid plaques by histological analysis. But, the neuroprotective activity of the anti-Aß scFvs could not be determined at the time because APP mice do not show overt neurodegeneration. This study demonstrates that both scFv9 and scFv42.2 prevent neuronal loss, protect the structural integrity of the eye and the dendritic fields of mushroom body neurons and increase the activity of motor neurons. Although the protective activity of scFv42.2 was stronger than scFv9 in both the mushroom bodies and the locomotor assay, this difference may be attributed to its higher expression level. In fact, two copies of scFv9 show robust rescue of the eye phenotype, supporting the superior performance of N-terminal antibodies described in other models. Future efforts will take advantage of a common docking site (attP) to ensure similar expression levels, enabling the direct comparison of the relative effectiveness of anti-Aß scFv (Fernandez-Funez, 2015).

The flexibility of Drosophila genetics allows combining both anti-Aß scFvs and to determine that they exert synergistic neuroprotective activities. The combined anti-Aß scFvs drastically improve eye organization and protect axonal and dendritic terminals in the mushroom body neurons. This is the first time that two scFvs have been used in combination in vivo, revealing a novel application for full antibodies or antibody fragments. N-terminal antibodies (e.g. scFv9) are known to bind soluble and insoluble forms of Aß42 because this domain is always exposed. Central and C-terminal antibodies (e.g. scFv42.2) can mostly bind monomeric Aß42, but their mechanism of action is not well understood. It is possible that N- and C-terminal anti-Aß antibodies act by different mechanisms, thus explaining the advantages of combining them. These observations suggest that treatments based on combinations of antibodies against different epitopes may provide significant benefits by exploiting their advantages and minimizing their limitations. As the challenges for delivering AD immunotherapy and reducing negative events have been known for the last 15 years, introducing two antibodies represents a minor modification to the current procedures with potential advantages (Fernandez-Funez, 2015).

The benefits of anti-Aß antibodies are explained by at least three non-exclusive hypotheses. The ‘peripheral sink’ hypothesis poses that antibodies sequester circulating Aß42 in the serum, thus increasing efflux from the brain and reducing Aß42 load in the brain, but subsequent studies have largely excluded this as a primarily mechanism of action. A second hypothesis suggests that Aß42 clearance is mediated by the activation of the immune response, particularly activated microglia and Fc-mediated phagocytosis. The third hypothesis proposes that anti-Aß scFvs directly prevent Aß42 aggregation and/or promote Aß42 disaggregation. Experiments in Drosophila demonstrate the neuroprotective activity of two anti-Aß scFvs in animals lacking an adaptive immune response. This simplified experimental set-up indicates that anti-Aß scFvs are neuroprotective without implicating the first two mechanisms. Therefore, the direct binding of anti-Aß scFvs to Aß42 is the only mechanism that can explain their protective activity in Drosophila. According to some reports, optimal anti-Aß antibodies should remove soluble and insoluble Aß42 without increasing the levels of highly toxic, oligomeric Aß42. But results from this study suggest that anti-Aß scFvs can be protective without clearing or disaggregating Aß42. The study, thus, proposes an alternative mechanism, ‘Aß42 masking’, similar to the action of chaperones on misfolded substrates: binding of anti-Aß scFvs to Aß42 masks neurotoxic epitopes and prevents the interaction of Aß42 with cellular substrates, including neuronal membranes and synaptic proteins. According to this mechanism, the most effective anti-Aß antibodies would be those that stably bind Aß42 monomers, oligomers and other assemblies and mask their neurotoxicity (Fernandez-Funez, 2015).

Although the beneficial effect of the immune system on Aß42 clearance in rodent models and humans could not be discounted, there seems to be serious risks associated with the stimulation of the cellular response. Hence, engineered antibodies that avoid the immune response such as scFvs seem promising alternatives to full antibodies. New anti-Aß scFvs against linear or conformational epitopes can be efficiently selected in flies based on their neuroprotective activity and, then, modified to increase their brain penetration in mice or humans by adding targeting sequences. More complex approaches can include the dual targeting of Aß and tau, the two triggers of neurotoxicity in AD, following the same principle of binding and masking. Thus, changing the way we think about the role of aggregates in the toxicity of amyloids may contribute to the development of more efficient therapies for AD and other incurable proteinopathies (Fernandez-Funez, 2015).

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Helmfors, L., Boman, A., Civitelli, L., Nath, S., Sandin, L., Janefjord, C., McCann, H., Zetterberg, H., Blennow, K., Halliday, G., Brorsson, A.C. and Kågedal, K. (2015). Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease. Neurobiol Dis [Epub ahead of print] PubMed ID: 26334479

Abstract
The hallmarks of Alzheimer disease are amyloid-β plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-β1-40in vitro. This study found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localizes with amyloid-β in plaques. In Drosophila, neuronal co-expression of lysozyme and amyloid-β1-42 reduces the formation of soluble and insoluble amyloid-β species, prolongs survival and improves the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays reveal that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease (Helmfors, 2015).

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Galasso, A., Cameron, C. S., Frenguelli, B. G. and Moffat, K. G. (2017). An AMPK-dependent regulatory pathway in tau-mediated toxicity. Biol Open [Epub ahead of print]. PubMed ID: 28808138

Abstract

Neurodegenerative tauopathies are characterized by accumulation of hyperphosphorylated tau aggregates primarily degraded by autophagy. The 5'AMP-activated protein kinase (AMPK) is expressed in most cells, including neurons. Alongside its metabolic functions, it is also known to be activated in Alzheimer's brains, phosphorylate tau, and be a critical autophagy activator. While stress conditions can result in AMPK activation enhancing tau-mediated toxicity, AMPK activation is not always concomitant with autophagic induction. Using a Drosophila in vivo quantitative approach, this study has analysed the impact of AMPK and autophagy on tau-mediated toxicity, recapitulating the AMPK-mediated tauopathy condition: increased tau phosphorylation, without corresponding autophagy activation. It was demonstrated that AMPK, binding to and phosphorylating tau at Ser-262, a site reported to facilitate soluble tau accumulation, affects its degradation. This phosphorylation results in exacerbation of tau toxicity and is ameliorated via rapamycin-induced autophagy stimulation. These findings support the development of combinatorial therapies effective at reducing tau toxicity targeting tau phosphorylation and AMPK-independent autophagic induction. The proposed in vivo tool represents an ideal readout to perform preliminary screening for drugs promoting this process (Galasso, 2017).

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Chiku, T., Hayashishita, M., Saito, T., Oka, M., Shinno, K., Ohtake, Y., Shimizu, S., Asada, A., Hisanaga, S. I., Iijima, K. M. and Ando, K. (2018). S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy. Neurobiol Aging 71: 255-264. PubMed ID: 30172839

Abstract

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. This study sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. These results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration (Chiku, 2018).

Stapper, Z. A. and Jahn, T. R. (2018) (2018). Changes in glutathione redox potential are linked to Abeta42-induced neurotoxicity. Cell Rep 24(7): 1696-1703. PubMed ID: 30110626

Abstract

Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. This study combined genetically encoded redox sensors with a Drosophila models of amyloid-beta (Abeta) aggregation. Changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. This redox imbalance was found to be specifically in neurons, but not in glia cells. EGSH changes and Abeta42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Abeta42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology (Strapper, 2018).

Furotani, K., Kamimura, K., Yajima, T., Nakayama, M., Enomoto, R., Tamura, T., Okazawa, H. and Sone, M. (2018). Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model. PLoS One 13(9): e0204048. PubMed ID: 30226901

Abstract

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 show 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).

Sun, W, Samimi, H., Gamez, M., Zare, H. and Frost, B. (2018). Pathogenic tau-induced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies. Nat Neurosci 21(8): 1038-1048. PubMed ID: 30038280

Abstract

Transposable elements, known colloquially as 'jumping genes', constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. This study utilize Drosophila melanogaster and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, it was found that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. A significant increase is reported in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer's disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, these data identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy (Sun, 2018).

Younan, N. D., Chen, K. F., Rose, R. S., Crowther, D. C. and Viles, J. H. (2018). Prion protein stabilizes amyloid-beta (Abeta) oligomers and enhances Abeta neurotoxicity in a Drosophila model of Alzheimer disease. J Biol Chem. PubMed ID: 29887525

Abstract

The cellular prion protein (PrPC) can act as a cell-surface receptor for amyloid-beta (Abeta) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. This study has expressed a range of Abeta isoforms and PrPC in the Drosophila brain. Co-expression of Abeta and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Abeta deposition in the fly brain. In contrast, under the same conditions, expression of Abeta or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Abeta as oligomeric assemblies and fragment-preformed Abeta fibers. The ability of membrane-anchored PrPC to trap Abeta as cytotoxic oligomers at the membrane surface and fragment inert Abeta fibers, suggests a mechanism by which PrPC exacerbates Abeta deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Abeta toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Abeta and PrPC represents a potential therapeutic strategy (Younan, 2018).

Martin-Pena, A., Rincon-Limas, D. E. and Fernandez-Funez, P. (2018). Engineered Hsp70 chaperones prevent Abeta42-induced memory impairments in a Drosophila model of Alzheimer's disease. Sci Rep 8(1): 9915. PubMed ID: 29967544

Abstract

Proteinopathies constitute a group of diseases in which certain proteins are abnormally folded leading to aggregation and eventual cell failure. Most neurodegenerative diseases belong to protein misfolding disorders and, among them, Alzheimer's disease (AD) is the most prevalent. AD is characterized by accumulation of the amyloid-beta42 (Abeta42) peptide in the extracellular space. Hence, this study genetically engineered a molecular chaperone that was selectively delivered to this cellular location. It has been reported that the heat shock protein 70 (Hsp70) binds Abeta42 (see Drosophila Appl) preventing self-aggregation. This study employed two isoforms of the Hsp70, cytosolic and extracellular, to evaluate their potential protective effect against the memory decline triggered by extracellular deposition of Abeta42. Both Hsp70 isoforms significantly improved memory performance of flies expressing Abeta42, irrespective of their age or the level of Abeta42 load. Using olfactory classical conditioning, a Drosophila model of AD was established based on Abeta42 neurotoxicity and memory decline was monitored through aging. The onset of the memory impairment observed was proportional to the cumulative level of Abeta42 in the Drosophila brain. These data support the use of this Drosophila model of AD to further investigate molecules with a protective activity against Abeta42-induced memory loss, contributing to the development of palliative therapies for AD (Martin-Pena, 2018).

Talmat-Amar, Y., Arribat, Y. and Parmentier, M. L. (2018). Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila. Int J Mol Sci 19(3). PubMed ID: 29509687

Abstract

Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs). This study took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous) Tau functions. Tau depletion was found to lead to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, this study shows that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau (Talmat-Amar, 2018).

Guo, C., Jeong, H. H., Hsieh, Y. C., Klein, H. U., Bennett, D. A., De Jager, P. L., Liu, Z. and Shulman, J. M. (2018). Tau activates transposable elements in Alzheimer's disease. Cell Rep 23(10): 2874-2880. PubMed ID: 29874575

Abstract

Aging and neurodegenerative disease are characterized by genomic instability in neurons, including aberrant activation and mobilization of transposable elements (TEs). Integrating studies of human postmortem brain tissue and Drosophila melanogaster models, TE activation was investigated in association with Tau pathology in Alzheimer's disease (AD). Leveraging RNA sequencing from 636 human brains, differential expression was discovered for several retrotransposons in association with neurofibrillary tangle burden and highlight evidence for global TE transcriptional activation among the long interspersed nuclear element 1 and endogenous retrovirus clades. In addition, Tau-associated, active chromatin signatures were detected at multiple HERV-Fc1 genomic loci. To determine whether Tau is sufficient to induce TE activation, retrotransposons were profiled in Drosophila expressing human wild-type or mutant Tau throughout the brain. Heterogeneous response profiles were discovered, including both age- and genotype-dependent activation of TE expression by Tau. The results implicate TE activation and associated genomic instability in Tau-mediated AD mechanisms (Guo, 2018).

Panikker, P., Xu, S. J., Zhang, H., Sarthi, J., Beaver, M., Sheth, A., Akhter, S. and Elefant, F. (2018). Restoring Tip60 HAT/HDAC2 balance in the neurodegenerative brain relieves epigenetic transcriptional repression and reinstates cognition. J Neurosci. PubMed ID: 29654189

Abstract

Cognitive decline is a debilitating hallmark during pre-clinical stages of AD yet causes remain unclear. As histone acetylation homeostasis is critical for mediating epigenetic gene control throughout neuronal development, it was postulated that its misregulation contributes to cognitive impairment preceding AD pathology. This study shows that disruption of Tip60 HAT/HDAC2 homeostasis occurs early in the brain of an AD associated amyloid precursor protein (APP) Drosophila model and triggers epigenetic repression of neuroplasticity genes well before Abeta plaques form in male and female larvae. Repressed genes display enhanced HDAC2 binding and reduced Tip60 and histone acetylation enrichment. Increasing Tip60 in the AD associated APP brain restores Tip60 HAT/HDAC2 balance by decreasing HDAC2 levels, reverses neuroepigenetic alterations to activate synaptic plasticity genes, and reinstates brain morphology and cognition. Such Drosophila neuroplasticity gene epigenetic signatures are conserved in male and female mouse hippocampus and their expression and Tip60 function is compromised in hippocampus from AD patients. It is advocated that Tip60 HAT/HDAC2 mediated epigenetic gene disruption is a critical initial step in AD that is reversed by restoring Tip60 in the brain (Panikker, 2018).

Jonson, M., Nystrom, S., Sandberg, A., Carlback, M., Michno, W., Hanrieder, J., Starkenberg, A., Nilsson, K. P. R., Thor, S. and Hammarstrom, P. (2018). Aggregated Abeta1-42 is selectively toxic for neurons, whereas glial cells produce mature fibrils with low toxicity in Drosophila. Cell Chem Biol 25(5):595-610. PubMed ID: 29657084

Abstract

The basis for selective vulnerability of certain cell types for misfolded proteins (MPs) in neurodegenerative diseases is largely unknown. This knowledge is crucial for understanding disease progression in relation to MPs spreading in the CNS. This issue was addressed in Drosophila by cell-specific expression of human Abeta1-42 associated with Alzheimer's disease. Expression of Abeta1-42 in various neurons resulted in concentration-dependent severe neurodegenerative phenotypes, and intraneuronal ring-tangle-like aggregates with immature fibril properties when analyzed by aggregate-specific ligands. Unexpectedly, expression of Abeta1-42 from a pan-glial driver produced a mild phenotype despite massive brain load of Abeta1-42 aggregates, even higher than in the strongest neuronal driver. Glial cells formed more mature fibrous aggregates, morphologically distinct from aggregates found in neurons, and was mainly extracellular. Theses findings implicate that Abeta1-42 cytotoxicity is both cell and aggregate morphotype dependent (Jonson, 2018).

Martin-Pena, A., Rincon-Limas, D. E. and Fernandez-Funez, P. (2017). Anti-Abeta single-chain variable fragment antibodies restore memory acquisition in a Drosophila model of Alzheimer's disease. Sci Rep 7(1): 11268. PubMed ID: 28900185

Abstract

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder triggered by the accumulation of soluble assemblies of the amyloid-beta42 (Abeta42) peptide. Despite remarkable advances in understanding the pathogenesis of AD, the development of palliative therapies is still lacking. Engineered anti-Abeta42 antibodies are a promising strategy to stall the progression of the disease. Single-chain variable fragment (scFv) antibodies increase brain penetration and offer flexible options for delivery while maintaining the epitope targeting of full antibodies. This study examined the ability of two anti-Abeta scFv antibodies targeting the N-terminal (scFv9) and C-terminal (scFv42.2) regions of Abeta42 to suppress the progressive memory decline induced by extracellular deposition of Abeta42 in Drosophila. Using olfactory classical conditioning, both scFv antibodies were observed to significantly improve memory performance in flies expressing Abeta42 in the mushroom body neurons, which are intimately involved in the coding and storage of olfactory memories. The scFvs effectively restore memory at all ages, from one-day post-eclosion to thirty-day-old flies, proving their ability to prevent the toxicity of different pathogenic assemblies. These data support the application of this paradigm of Abeta42-induced memory loss in Drosophila to investigate the protective activity of Abeta42-binding agents in an AD-relevant functional assay (Martin-Pena, 2017).

Galasso, A., Cameron, C. S., Frenguelli, B. G. and Moffat, K. G. (2017). An AMPK-dependent regulatory pathway in tau-mediated toxicity. Biol Open [Epub ahead of print]. PubMed ID: 28808138

Abstract

Neurodegenerative tauopathies are characterized by accumulation of hyperphosphorylated tau aggregates primarily degraded by autophagy. The 5'AMP-activated protein kinase (AMPK) is expressed in most cells, including neurons. Alongside its metabolic functions, it is also known to be activated in Alzheimer's brains, phosphorylate tau, and be a critical autophagy activator. While stress conditions can result in AMPK activation enhancing tau-mediated toxicity, AMPK activation is not always concomitant with autophagic induction. This study analysed in Drosophila the impact of AMPK and autophagy on tau-mediated toxicity, recapitulating the AMPK-mediated tauopathy condition: increased tau phosphorylation, without corresponding autophagy activation. It was demonstrated that AMPK, binding to and phosphorylating tau at Ser-262, a site reported to facilitate soluble tau accumulation, affects its degradation. This phosphorylation results in exacerbation of tau toxicity and is ameliorated via rapamycin-induced autophagy stimulation. These findings support the development of combinatorial therapies effective at reducing tau toxicity targeting tau phosphorylation and AMPK-independent autophagic induction. The proposed in vivo tool represents an ideal readout to perform preliminary screening for drugs promoting this process (Galasso, 2017).

Zhang, X., Wang, W.A., Jiang, L.X., Liu, H.Y., Zhang, B.Z., Lim, N., Li, Q.Y. and Huang, F.D.(2017). Down-regulation of RBO-PI4KIIIα facilitates Aβ42 secretion and ameliorates neural deficits in Aβ42-expressing Drosophila. J Neurosci [Epub ahead of print]. PubMed ID: 28424219

Abstract

Phosphoinositides and their metabolizing enzymes are involved in Aβ42 metabolism and Alzheimer's disease (AD) pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI4P). This study reports that RBO binds to Drosophila PI4KIIIα, and that in an Aβ42-expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorates synaptic transmission deficit, climbing ability decline, and premature death, and reduces neuronal accumulation of Aβ42. Moreover, RBO-PI4KIIIa downregulation increases neuronal Aβ42 release, and PI4P facilitates the assembly or oligomerization of Aβ42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ42 release and consequently reduces neuronal Aβ42 accumulation likely via decreasing Aβ42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for AD treatment (Zhang, 2017).

Lopez-Arias, B., Turiegano, E., Monedero, I., Canal, I. and Torroja, L. (2017). Presynaptic Abeta40 prevents synapse addition in the adult Drosophila neuromuscular junction. PLoS One 12(5): e0177541. PubMed ID: 28520784

Abstract

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).

Frenkel-Pinter, M., Stempler, S., Tal-Mazaki, S., Losev, Y., Singh-Anand, A., Escobar-Alvarez, D., Lezmy, J., Gazit, E., Ruppin, E. and Segal, D. S.Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila. Neurobiol Aging. PubMed ID: 28552182

Abstract

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., beta-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. In an analysis of available expression data sets this study indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled prediction of the occurrence of AD with remarkable accuracy in a test cohort and identification of a set of key genes whose expression determines this classification. Then the effect of reducing expression of homologs of 6 of these genes in was studied in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. These results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology (Frenkel-Pinter, 2017).

Feng, G., Pang, J., Yi, X., Song, Q., Zhang, J., Li, C., He, G. and Ping, Y. (2017). Down-regulation of KV4 channel in Drosophila mushroom body neurons contributes to Abeta42-induced courtship memory deficits. Neuroscience [Epub ahead of print]. PubMed ID: 28627422

Abstract

Accumulation of amyloid-β (Aβ) is widely believed to be an early event in the pathogenesis of Alzheimer's disease (AD). Kv4 is an A-type K+ channel, and previous work has shown that degradation of Kv4, induced by the β42 accumulation, may be a critical contributor to the hyperexcitability of neurons in a Drosophila AD model. This study used well-established courtship memory assay to investigate the contribution of the Kv4 channel to short-term memory (STM) deficits in the Aβ42-expressing AD model. Aβ42 over-expression in Drosophila leads to age-dependent courtship STM loss, which can be also induced by driving acute Aβ42 expression post-developmentally. Interestingly, mutants with eliminated Kv4-mediated A-type K+ currents (IA) by transgenically expressing dominant-negative subunit (DNKv4) phenocopied Aβ42 flies in defective courtship STM. Kv4 channels in mushroom body (MB) and projection neurons (PNs) were found to be required for courtship STM. Furthermore, the STM phenotypes can be rescued, at least partially, by restoration of Kv4 expression in Aβ42 flies, indicating the STM deficits could be partially caused by Kv4 degradation. In addition, IA is significantly decreased in MB neurons (MBNs) but not in PNs, suggesting Kv4 degradation in MBNs, in particular, plays a critical role in courtship STM loss in Aβ42 flies. These data highlight causal relationship between region-specific Kv4 degradation and age-dependent learning decline in the AD model, and provide a mechanism for the disturbed cognitive function in AD (Feng, 2017).

Wu, S. C., Cao, Z. S., Chang, K. M. and Juang, J. L. (2017). Intestinal microbial dysbiosis aggravates the progression of Alzheimer's disease in Drosophila. Nat Commun 8(1): 24. PubMed ID: 28634323

Abstract

Neuroinflammation caused by local deposits of Abeta42 in the brain is key for the pathogenesis and progression of Alzheimer's disease. However, inflammation in the brain is not always a response to local primary insults. Gut microbiota dysbiosis, which is recently emerging as a risk factor for psychiatric disorders, can also initiate a brain inflammatory response. It still remains unclear however, whether enteric dysbiosis also contributes to Alzheimer's disease. This study shows that in a Drosophila Alzheimer's disease model, enterobacteria infection exacerbated progression of Alzheimer's disease by promoting immune hemocyte recruitment to the brain, thereby provoking TNF-JNK mediated neurodegeneration. Genetic depletion of hemocytes attenuates neuroinflammation and alleviated neurodegeneration. It was further found that enteric infection increases the motility of the hemocytes, making them more readily attracted to the brain with an elevated oxidative stress status. This work highlights the importance of gut-brain crosstalk as a fundamental regulatory system in modulating Alzheimer's disease neurodegeneration. Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. This study offers in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease (Wu, 2017).

Singh, S. K., Srivastav, S., Yadav, A. K. and Srikrishna, S. (2017). Knockdown of APPL mimics transgenic Abeta induced neurodegenerative phenotypes in Drosophila. Neurosci Lett 648: 8-13. PubMed ID: 28336338

Abstract

A variety of Drosophila mutant lines have been established as potential disease-models to study various disease mechanisms including human neurodegenerative diseases like Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD). The evolutionary conservation of APP (Amyloid Precursor Protein) and APPL (Amyloid Precursor Protein-Like) and the comparable detrimental effects caused by their metabolic products strongly implies the conservation of their normal physiological functions. In view of this milieu, a comparative analysis on the pattern of neurodegenerative phenotypes between Drosophila APPL-RNAi line and transgenic Drosophila line expressing eye tissue specific human Aβ (Amyloid β) was undertaken. The results clearly show that Drosophila APPL-RNAi largely mimics transgenic Abeta in various phenotypes which include eye degeneration, reduced longevity and motor neuron deficit functions, etc. The ultra-structural morphological pattern of eye degeneration was confirmed by scanning electron microscopy. Further, a comparative study on longevity and motor behaviour between Abeta expressing and APPL knockdown lines revealed similar kind of behavioural deficit and longevity phenotypes. Therefore, it is suggested that APPL-knockdown can be used as an alternative approach to study neurodegenerative diseases in the fly model. This is the first report showing comparable phenotypes between APPL and Abeta in AD model of Drosophila (Singh, 2017).

Bergkvist, L., Sandin, L., Kagedal, K. and Brorsson, A. C. (2016). AβPP processing results in greater toxicity per amount of Aβ1-42 than individually expressed and secreted Aβ1-42 in Drosophila melanogaster. Biol Open [Epub ahead of print]. PubMed ID: 27387531

Abstract
The aggregation of the amyloid-β (Aβ; see Drosophila Appl) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimer's disease (AD). Aβ peptides are generated from proteolytic processing of the transmembrane Aβ precursor protein (AβPP) via sequential proteolysis through the β-secretase activity of β-site AβPP-cleaving enzyme (BACE1) and by the intramembranous enzyme γ-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo. In one model, the Aβ peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human AβPP is co-expressed with human BACE1, resulting in production of the Aβ peptide through the processing of AβPP by BACE1 and by endogenous fly γ-secretase. This study consisted of a parallel study of flies that expressed the Aβ1-42 peptide alone or that co-expressed AβPP and beta-secretase 1 (BACE1). Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the Aβ1-42, Aβ1-40 and Aβ1-38 peptides were examined. The data reveal that the toxic effect per amount of detected Aβ1-42 peptide was higher in the flies co-expressing AβPP and BACE1 than in the Aβ1-42-expressing flies, and that the co-existence of Aβ1-42 and Aβ1-40 in the flies co-expressing AβPP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the Aβ1-42 peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research (Bergkvist, 2016).

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Arnes, M., Casas-Tinto, S., Malmendal, A. and Ferrus, A. (2017)(2017). Amyloid beta42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K. Biol Open 6(11): 1664-1671. PubMed ID: 29141953

Abstract

The human Abeta42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, this study shows that Abeta42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Abeta42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR) of adult flies at selected ages post Abeta42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Abeta42 toxicity can be suppressed by the joint overexpression of PI3K (Arnes, 2017).

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Malmanche, N., Dourlen, P., Gistelinck, M., Demiautte, F., Link, N., Dupont, C., Vanden Broeck, L., Werkmeister, E., Amouyel, P., Bongiovanni, A., Bauderlique, H., Moechars, D., Royou, A., Bellen, H. J., Lafont, F., Callaerts, P., Lambert, J. C. and Dermaut, B. (2017). Developmental expression of 4-Repeat-Tau induces neuronal aneuploidy in Drosophila tauopathy models. Sci Rep 7: 40764. PubMed ID: 28112163

Abstract
Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. This study used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. The results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, a mitotic delay was observed in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, a high level of aneuploidy was found in post-mitotic differentiated tissue. Finally, it was shown that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, these results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development (Malmanche, 2017).

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Zhang, B., Li, Q., Chu, X., Sun, S. and Chen, S. (2016).Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer's disease. Transl Neurodegener 5: 21. PubMed ID: 27933142

Abstract
Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe treatment against AD. The plant product Salidroside (Sal) is the main effective component of Rhodiola rosea L., which has several pharmacological activities. The objective of this study was to investigate the efficacy of Sal in the treatment of AD transgenic Drosophila and the associated mechanisms. Microtubule associated protein tau transgenic Drosophila line (TAU) was used in which tau protein is expressed in the central nervous system and eyes by the Gal4/UAS system. After feeding flies with Sal, the lifespan and locomotor activity were recorded. The appearance of vacuoles in the mushroom body was examined using immunohistochemistry, and the levels of total glycogen synthase kinase 3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), t-tau and p-tau was detected in the brain by western blot analysis. The results showed that the longevity was improved in salidroside-fed Drosophila groups as well as the locomotor activity. Less vacuoles in the mushroom body, upregulated level of p-GSK-3β and downregulated p-tau were detected following Sal treatment. These data presented the evidence that Sal was capable of reducing the neurodegeneration in tau transgenic Drosophila and inhibiting neuronal loss. The neuroprotective effects of Sal were associated with its up-regulation of the p-GSK-3β and down-regulation of the p-tau (Zhang, 2016).

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Fernandez-Funez, P., Sanchez-Garcia, J., de Mena, L., Zhang, Y., Levites, Y., Khare, S., Golde, T. E. and Rincon-Limas, D. E. (2016). Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila. Proc Natl Acad Sci U S A 113: E5212-5221. PubMed ID: 27531960

Abstract
Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. This study used Drosophila to explore a strategy to block Aβ42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aβ42, a signal peptide was added to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aβ42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aβ42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies (Fernandez-Funez, 2016).

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Niccoli, T., Cabecinha, M., Tillmann, A., Kerr, F., Wong, C.T., Cardenes, D., Vincent, A.J., Bettedi, L., Li, L., Grönke, S. Dols, J. and Partridge, L. (2016). Increased glucose transport into neurons rescues Aβ toxicity in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27524482

Abstract
Glucose hypometabolism is a prominent feature of the brains of patients with Alzheimer's disease (AD). Disease progression is associated with a reduction in glucose transporters in both neurons and endothelial cells of the blood-brain barrier. However, whether increasing glucose transport into either of these cell types offers therapeutic potential remains unknown. Using an adult-onset Drosophila model of Aβ (amyloid beta) toxicity, this study shows that genetic overexpression of a glucose transporter specifically in neurons, rescues lifespan, behavioral phenotypes, and neuronal morphology. This amelioration of Aβ toxicity is associated with a reduction in the protein levels of the unfolded protein response (UPR) negative master regulator Grp78 and an increase in the UPR. Genetic downregulation of Grp78 activity also protects against Aβ toxicity, confirming a causal effect of its alteration on AD-related pathology. Metformin, a drug that stimulates glucose uptake in cells, mimics these effects, with a concomitant reduction in Grp78 levels and rescue of the shortened lifespan and climbing defects of Aβ-expressing flies. These findings demonstrate a protective effect of increased neuronal uptake of glucose against Aβ toxicity and highlight Grp78 as a novel therapeutic target for the treatment of AD (Niccoli, 2016).

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Ray, A., Speese, S. D. and Logan, M. A. (2017) (2017). Glial Draper rescues Abeta toxicity in a Drosophila model of Alzheimer's Disease. J Neurosci 37(49):11881-11893. PubMed ID: 29109235

Abstract

Pathological hallmarks of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Abeta peptides, but the repertoire of molecules required for glial recognition and destruction of Abeta are still unclear. This study shows that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Abeta42arc in adult flies results in robust Abeta accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, while enhanced expression of glial Draper reverses Abeta accumulation, as well as behavioral phenotypes. Stat92E, c-Jun N-terminal Kinase (JNK)/AP-1 signaling, and expression of matrix metalloproteinase-1 (Mmp1) are activated downstream of Draper in glia in response to Abeta42arc exposure. Furthermore, Abeta42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on these findings, it is proposed that glia clear neurotoxic Abeta peptides in the AD model Drosophila brain through a Draper/STAT92E/JNK cascade that may be coupled to protein degradation pathways such as autophagy or more traditional phagolysosomal destruction methods (Ray, 2017).

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Vivien Chiu, W. Y., Koon, A. C., Ki Ngo, J. C., Edwin Chan, H. Y. and Lau, K. F. (2017) (2017). GULP1/CED-6 ameliorates amyloid-beta toxicity in a Drosophila model of Alzheimer's disease. Oncotarget 8(59): 99274-99283. PubMed ID: 29245900

Abstract

Amyloidogenic processing of APP (see Drosophila Appl) by beta- and gamma-secretases leads to the generation of amyloid-beta peptide (Abeta), and the accumulation of Abeta in senile plaques is a hallmark of Alzheimer's disease (AD). Understanding the mechanisms of APP processing is therefore paramount. Increasing evidence suggests that APP intracellular domain (AICD) interacting proteins influence APP processing. This study characterized the overexpression of AICD interactor GULP1 in a Drosophila AD model expressing human BACE and APP695. Transgenic GULP1 significantly lowered the levels of both Abeta1-40 and Abeta1-42 without decreasing the BACE and APP695 levels. Overexpression of GULP1 also reduced APP/BACE-mediated retinal degeneration, rescued motor dysfunction and extended longevity of the flies. These results indicate that GULP1 regulate APP processing and reduce neurotoxicity in a Drosophila AD model (Vivien Chiu, 2017).

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Oka, M., Fujisaki, N., Maruko-Otake, A., Ohtake, Y., Shimizu, S., Saito, T., Hisanaga, S. I., Iijima, K. M. and Ando, K. (2017). Ca2+/calmodulin-dependent protein kinase II promotes neurodegeneration caused by tau phosphorylated at Ser262/356 in a transgenic Drosophila model of tauopathy. J Biochem 162(5): 335-342. PubMed ID: 28992057

Abstract

Abnormal deposition of the microtubule-associated protein tau is a common pathological feature of multiple neurodegenerative diseases, including Alzheimer's disease (AD), and plays critical roles in their pathogenesis. Disruption of calcium homeostasis and the downstream kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) coincides with pathological phosphorylation of tau in AD brains. However, it remains unclear whether and how dysregulation of CaMKII affects tau toxicity. Using a Drosophila model, it was found that CaMKII promotes neurodegeneration caused by tau phosphorylated at the AD-associated sites Ser262/356. Overexpression of CaMKII promoted, while RNA-mediated knockdown of CaMKII and inhibition of CaMKII activity by expression of an inhibitory peptide suppressed, tau-mediated neurodegeneration. Blocking tau phosphorylation at Ser262/356 by alanine substitutions suppressed promotion of tau toxicity by CaMKII, suggesting that tau phosphorylation at these sites is required for this phenomenon. However, neither knockdown nor overexpression of CaMKII affected tau phosphorylation levels at Ser262/356, suggesting that CaMKII is not directly involved in tau phosphorylation at Ser262/356 in this model. These results suggest that a pathological cascade of events, including elevated levels of tau phosphorylated at Ser262/356 and aberrant activation of CaMKII, work in concert to promote tau-mediated neurodegeneration (OKA, 2017).

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Moore, B. D., Martin, J., de Mena, L., Sanchez, J., Cruz, P. E., Ceballos-Diaz, C., Ladd, T. B., Ran, Y., Levites, Y., Kukar, T. L., Kurian, J. J., McKenna, R., Koo, E. H., Borchelt, D. R., Janus, C., Rincon-Limas, D., Fernandez-Funez, P. and Golde, T. E. (2018). Short Abeta peptides attenuate Abeta42 toxicity in vivo. J Exp Med 215(1): 283-301. PubMed ID: 29208777

Abstract

Processing of amyloid-beta (Abeta) precursor protein (APP) by gamma-secretase produces multiple species of Abeta: Abeta40, short Abeta peptides (Abeta37-39), and longer Abeta peptides (Abeta42-43). gamma-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Abeta42 but increase the relative abundance of short Abeta peptides. To evaluate the pathological relevance of these peptides, this study expressed Abeta36-40 and Abeta42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Abeta42 toxicity. In contrast to Abeta42, the short Abeta peptides were not toxic and, when coexpressed with Abeta42, were protective in a dose-dependent fashion. In parallel, the effects were explored of recombinant adeno-associated virus-mediated expression of Abeta38 and Abeta40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Abeta42 deposition, Abeta38 and Abeta40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Abeta42 by raising the levels of short Abeta peptides could attenuate the toxic effects of Abeta42 (Moore, 2018).

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Lee, B. I., Suh, Y. S., Chung, Y. J., Yu, K. and Park, C. B. (2017). Shedding light on Alzheimer's beta-Amyloidosis: Photosensitized methylene blue inhibits self-assembly of beta-amyloid peptides and disintegrates their aggregates. Sci Rep 7(1): 7523. PubMed ID: 28790398

Abstract

Abnormal aggregation of beta-amyloid (Abeta) peptides is a major hallmark of Alzheimer's disease (AD). In spite of numerous attempts to prevent the beta-amyloidosis, no effective drugs for treating AD have been developed to date. Among many candidate chemicals, methylene blue (MB) has proved its therapeutic potential for AD in a number of in vitro and in vivo studies; but the result of recent clinical trials performed with MB and its derivative was negative. In this study, with the aid of multiple photochemical analyses, it is reported that photoexcited MB molecules can block Abeta42 aggregation in vitro. Furthermore, an in vivo study using Drosophila AD model demonstrates that photoexcited MB is highly effective in suppressing synaptic toxicity, resulting in a reduced damage to the neuromuscular junction (NMJ), an enhanced locomotion, and decreased vacuole in the brain. The hindrance effect is attributed to Abeta42 oxidation by singlet oxygen (1O2) generated from photoexcited MB. Finally, it was shown that photoexcited MB possess a capability to disaggregate the pre-existing Abeta42 aggregates and reduce Abeta-induced cytotoxicity. This work suggests that light illumination can provide an opportunity to boost the efficacies of MB toward photodynamic therapy of AD in future (Lee, 2017). Yang, C. N., Wu, M. F., Liu, C. C., Jung, W. H., Chang, Y. C., Lee, W. P., Shiao, Y. J., Wu, C. L., Liou, H. H., Lin, S. K. and Chan, C. C. (2017). Differential protective effects of connective tissue growth factor against Abeta neurotoxicity on neurons and glia. Hum Mol Genet 26(20): 3909-3921. PubMed ID: 29016849

Abstract

Impaired clearance of amyloid-beta peptide (Abeta; see Drosophila Appl) leads to abnormal extracellular accumulation of this neurotoxic protein that drives neurodegeneration in sporadic Alzheimer's disease (AD). Connective tissue growth factor (CTGF/CCN2) expression is elevated in plaque-surrounding astrocytes in AD patients. However, the role of CTGF in AD pathogenesis remains unclear. This study characterized the neuroprotective activity of CTGF. CTGF facilitates Abeta uptake and subsequent degradation within primary glia and neuroblastoma cells. CTGF enhanced extracellular Abeta degradation via membrane-bound matrix metalloproteinase-14 (MMP14) in glia and extracellular MMP13 in neurons. In the brain of a Drosophila AD model, glial-expression of CTGF reduced Abeta deposits, improved locomotor function, and rescued memory deficits. Neuroprotective potential of CTGF against Abeta42-induced photoreceptor degeneration was disrupted through silencing MMPs. Therefore, CTGF may represent a node for potential AD therapeutics as it intervenes in glia-neuron communication via specific MMPs to alleviate Abeta neurotoxicity in the central nervous system.

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Ando, K., Maruko-Otake, A., Ohtake, Y., Hayashishita, M., Sekiya, M. and Iijima, K. M. (2016). Stabilization of microtubule-unbound Tau via Tau phosphorylation at Ser262/356 by Par-1/MARK contributes to augmentation of AD-related phosphorylation and Aβ42-induced Tau toxicity. PLoS Genet 12: e1005917. PubMed ID: 27023670

Abstract
To prevent the cascade of events leading to neurodegeneration in Alzheimer's disease (AD), it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. In this study, using transgenic Drosophila co-expressing human tau and Aβ, tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau was found in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD (Ando, 2016).

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Gerstner, J. R., Lenz, O., Vanderheyden, W. M., Chan, M. T., Pfeiffenberger, C. and Pack, A. I. (2016). Amyloid-β induces sleep fragmentation that is rescued by fatty acid binding proteins in Drosophila. J Neurosci Res [Epub ahead of print]. PubMed ID: 27320125

Abstract
Disruption of sleep/wake activity in Alzheimer's disease (AD) patients significantly affects their quality of life and that of their caretakers and is a major contributing factor for institutionalization. Levels of amyloid-β (Aβ; see Drosophila Appl) have been shown to be regulated by neuronal activity and to correlate with the sleep/wake cycle. Whether consolidated sleep can be disrupted by Aβ alone is not well understood. It was hypothesized that Aβ42 can increase wakefulness and disrupt consolidated sleep. This study shows that flies expressing the human Aβ42 transgene in neurons have significantly reduced consolidated sleep compared with control flies. Fatty acid binding proteins (Fabp) are small hydrophobic ligand carriers that have been clinically implicated in AD. Aβ42 flies that carry a transgene of either the Drosophila Fabp or the mammalian brain-type Fabp show a significant increase in nighttime sleep and long consolidated sleep bouts, rescuing the Aβ42-induced sleep disruption. These studies suggest that alterations in Fabp levels and/or activity may be associated with sleep disturbances in AD. Future work to determine the molecular mechanisms that contribute to Fabp-mediated rescue of Aβ42-induced sleep loss will be important for the development of therapeutics in the treatment of AD (Gerstner, 2016).

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Burnouf, S., Grönke, S., Augustin, H., Dols, J., Gorsky, M.K., Werner, J., Kerr, F., Alic, N., Martinez, P. and Partridge, L. (2016). Deletion of endogenous Tau proteins is not detrimental in Drosophila. Sci Rep 6: 23102. PubMed ID: 26976084

Abstract
Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer's disease (AD), constituting, together with accumulated β-amyloid (Aβ) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, this study aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. It was observed that the specific removal of endogenous dTau proteins does not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function are unchanged in tau KO flies. In addition, any overt positive or negative effect of dTau removal on human Aβ-induced toxicity were not found. Altogether, these results indicate that the absence of dTau proteins has no major functional impact on flies, and suggest that the tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions (Burnouf, 2016).

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Farca Luna, A. J., Perier, M. and Seugnet, L. (2017). Amyloid precursor protein in Drosophila glia regulates sleep and genes Involved in glutamate recycling. J Neurosci 37(16): 4289-4300. PubMed ID: 28314820

Abstract

Amyloid precursor protein (App) plays a crucial role in Alzheimer's disease via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons, the focus of the vast majority of studies investigating its function. Meanwhile, almost nothing is known about App's function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. This report investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in glutamine synthetase (GS) in astrocyte-like glia and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results, the expression of GS and innexin2 are increased following sleep deprivation, indicating that GS and innexin2 genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep, and a glial function for the App family proteins (Farca Luna, 2017).

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Gorsky, M. K., Burnouf, S., Dols, J., Mandelkow, E. and Partridge, L. (2016). Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo. Sci Rep 6: 22685. PubMed ID: 26940749

Abstract
Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer's disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. This study generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. It was observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, these results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD (Gorsky, 2016).

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Sofola-Adesakin, O., Khericha, M., Snoeren, I., Tsuda, L. and Partridge, L. (2016). pGluAβ increases accumulation of Aβ in vivo and exacerbates its toxicity. Acta Neuropathol Commun 4: 109. PubMed ID: 27717375

Abstract
Several species of β-amyloid peptides (&Abeta;; see Drosophila Appl) exist as a result of differential cleavage from amyloid precursor protein (APP) to yield various C-terminal Aβ peptides. Several N-terminal modified Aβ peptides have also been identified in Alzheimer's disease (AD) brains, the most common of which is pyroglutamate-modified Aβ (AβpE3-42). AβpE3-42 peptide has an increased propensity to aggregate, appears to accumulate in the brain before the appearance of clinical symptoms of AD, and precedes Aβ1-42 deposition. Moreover, in vitro studies have shown that AβpE3-42 can act as a seed for full length Aβ1-42. This study characterized the Drosophila model of AβpE3-42 toxicity by expressing the peptide in specific sets of neurons using the GAL4-UAS system, and measuring different phenotypic outcomes. AβpE3-42 peptide was found to have an increased propensity to aggregate. Expression of AβpE3-42 in the neurons of adult flies led to behavioural dysfunction and shortened lifespan. Expression of AβpE3-42 constitutively in the eyes led to disorganised ommatidia, and activation of the c-Jun N-terminal kinase (JNK) signaling pathway. The eye disruption was almost completely rescued by co-expressing a candidate Aβ degrading enzyme, neprilysin2 (see Neprilysin 4). Furthermore, neprilysin2 was capable of degrading AβpE3-42. Also, the seeding hypothesis was tested for AβpE3-42 in vivo, and its effect on Aβ1-42 levels were measured. Aβ1-42 levels were significantly increased when Aβ1-42 and AβpE3-42 peptides were co-expressed. Furthermore, AβpE3-42 enhanced Aβ1-42 toxicity in vivo. These findings implicate AβpE3-42 as an important source of toxicity in AD, and suggest that its specific degradation could be therapeutic (Sofola-Adesakin, 2016).

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Xu, W., et al. (2016). Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration. J Clin Invest [Epub ahead of print]. PubMed ID: 27064279

Abstract
The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. This study explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. It was determined that full-length amyloid precursor protein (APP; see Drosophila Appl) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. The full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. These data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS (Xu, 2016).

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Ping, Y., Hahm, E.T., Waro, G., Song, Q., Vo-Ba, D.A., Licursi, A., Bao, H., Ganoe, L., Finch, K. and Tsunoda, S. (2015). Linking Aβ42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model. PLoS Genet 11: e1005025. PubMed ID: 25774758

Abstract
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).

Highlights

  • Aβ42 induces increased neuronal excitability in 9 days old neuronal cultures.
  • Kv4 current is selectively decreased by Aβ42 expression.
  • Aβ42 induces degradation of Kv4 protein via a pathway dependent on both the proteasome and lysosome.
  • Transgenic restoration of Kv4 rescues hyperexcitability.
  • Loss of Kv4 plays a critical role in Aβ42-induced learning defects.
  • Restoration of Kv4 levels rescues locomotor defects.
  • Restoration of Kv4 slows Aβ42-induced neurodegeneration.
  • Aβ42-induced loss of Kv4 contributes to premature death.

Discussion
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).

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Blake, M.R., Holbrook, S.D., Kotwica-Rolinska, J., Chow, E.S., Kretzschmar, D. and Giebultowicz, J.M. (2015). Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila. Neurobiol Dis 77: 117-126. PubMed ID: 25766673

Abstract
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).

Highlights

  • Over-expression of dBACE in clock cells accelerates aging phenotypes and disrupts rest-activity rhythms.
  • Neuronal over-expression of dBACE disrupts behavioral rest-activity rhythms.
  • Over-expression of dBACE dampens the cycling of PER in central pacemaker neurons.
  • Rest-activity rhythms are disrupted by KUZ over-expression.
  • Expression of AICD disrupts rest-activity rhythms.
  • dAICD is capable of entering the nucleus, but is not toxic to central pacemaker neurons.

Discussion
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).

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Long, D.M., Blake, M.R., Dutta, S., Holbrook, S.D., Kotwica-Rolinska, J., Kretzschmar, D. and Giebultowicz, J.M. (2014). Relationships between the circadian system and Alzheimer's disease-like symptoms in Drosophila. PLoS One 9: e106068. PubMed ID: 25171136

Abstract
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 utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. The study compared development of AD-like symptoms in adult flies expressing Aβ 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 Aβ peptides in flies. However, AD-like pathologies were found to affect the circadian system in aging flies. It was found that rest/activity rhythms are impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide show a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remains intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism (Long, 2014).

Highlights

  • Lifespan reduction caused by Aβ peptides is not exacerbated by the by loss of the clock gene period.
  • Flies expressing Aβ42arc show similar motor decline and neurodegeneration in clock-positive and clock-disrupted backgrounds.
  • Daily locomotor activity rhythms are impaired in aging flies expressing different Aβ peptides.
  • Rhythms in PER cycling continue in lateral neurons of elav>Aβ42arc flies.

Discussion
Associations between AD and impaired daily rhythms are well documented in humans, yet the causes and consequences of AD-related loss of circadian sleep/activity rhythms have not been teased apart. One of the unanswered questions is whether age-related decline of the circadian system contributes to AD progression. This study tested directly whether total arrhythmia caused by mutation in the core clock gene per would exacerbate AD-like phenotypes observed in an AD fly model. It was shown that premature death, progressive locomotor deficits, and vacuolization in the brain occurs with similar timing and intensity in flies with genetically disrupted clock mechanism as in control flies. Consistent with previous reports, the severity of symptoms is proportional to the pathogenicity of the expressed human Aβ fragments. However, within each genotype, symptoms in clock-deficient flies are similar to those in clock-competent flies. While this study's data show that disruption of the clock via removal of the core clock repressor PER does not exacerbate AD symptoms, it cannot be ruled out that disabling the positive clock arm could be more detrimental. A recent report showed that loss of the positive element BMAL1 causes brain neurodegeneration in mice. It was previously demonstrated that the loss of per accelerates death, locomotor impairments, and brain vacuolization in neurodegeneration-prone sniffer and swiss cheese fly mutants. However, the underlying molecular mechanism that mediates this effect is not known. The AD model used in this study is based on the expression of human Aβ peptides, which have been reported to accumulate into insoluble forms in aging flies. Because the disruption of the circadian clock does not affect the pathogenicity of these peptides, the study assumes that it has no effect on Aβ aggregation or clearance. In sum, this study's data show that the molecular and behavioral arrhythmia characteristic for per-null flies is not detrimental in this AD fly model (Long, 2014).

However, the study shows that associations between AD and altered behavioral rhythms, observed in humans and AD model mice, also extend to fly AD models. Pan-neuronal expression of Aβ42 causes age-dependent impairment of circadian rest/activity rhythms, such that a reduced fraction of 50-days old elav>Aβ42 flies remain rhythmic in constant darkness compared to controls. A more dramatic disruption of circadian rhythms is observed in elav>Aβ42arc. In LD, 5-day old flies of this genotype show bimodal activity with an attenuated morning activity peak, while no activity peaks are detected in 15-day old flies, rather they are active around the clock, including nighttime when control flies have prolonged rest. In another related study, a loss of locomotor activity rhythms in elav>Aβ42arc flies even at young age was shown, similar to findings in this study. Together, these results demonstrate that AD model flies have rest/activity rhythm degradation reminiscent of the behavioral degradation observed in humans with AD (Long, 2014).

Loss of rest/activity rhythms in elav>Aβ42arc flies formed the basis of investigation of the functional status of central pacemaker neurons, which are necessary and sufficient for the activity rhythms, at least in young flies. Immunocytochemistry of PDF-positive pacemaker neurons sLNv and lLNv shows the correct number and arborization pattern in elav>Aβ42arc flies. Moreover these neurons show nuclear peak and trough of the core clock protein PER indistinguishable from wild type flies. Similar observations have been published earlier, and it was additionally shown that even expression of the more pathogenic tandem Aβ42 construct leaves molecular oscillations in pacemaker neurons intact. Together, these data show dissociation between functioning molecular pacemaker and disrupted circadian coordination of rest/activity rhythms. This suggests that behavioral rhythm degradation observed in humans and mouse AD models may occur despite the presence of a functional central clock. Importantly, strong body temperature rhythms have been reported in AD patients again suggesting that the central clock may be intact in AD. This is reminiscent of the situation in very old flies and mammals, which show degradation of rest/activity rhythms while their central pacemaker neurons continue to show molecular oscillations (Long, 2014).

While AD-related degradation of behavioral rhythms is not caused by malfunction of the central clock, other contributing factors remain to be investigated. Aβ related arrhythmicity might be due to non-cell-autonomous toxicity as focused expression of toxic peptides in clock containing cells does not affect behavioral rhythmicity, but expression outside of the pacemaker neurons may affect their synaptic connections. Additionally, downstream neuronal or humoral output pathways leading from the central pacemaker network to the motor centers could be adversely affected by Aβ aggregates. For example, recent studies reporting a direct measurement of neuronal activity in elav>Aβ42arc flies reveal increased latency and decreased response stability of the pathways leading from the giant fiber system in the brain into motor neurons of the thoracic ganglia. It is possible that neuronal deficits of this kind can disable output pathways from the central clock leading to fragmented rather than consolidated sleep. This may lead to a vicious cycle as sleep deprivation increases amyloid peptides in mice and Aβ aggregation disrupts the sleep/wake cycle. As flies provide a powerful toolkit to study both AD and circadian rhythms, studies at the intersection of chronobiology and AD should help to provide insights into the mechanisms underlying AD-related pathologies (Long, 2014).

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Wang, X., Ma, Y., Zhao, Y., Chen, Y., Hu, Y., Chen, C., Shao, Y. and Xue, L. (2015). APLP1 promotes dFoxO-dependent cell death in Drosophila. Apoptosis 20: 778-786. PubMed ID: 25740230

Abstract
The amyloid precursor like protein-1 (APLP1), an engineered human gene into the Drosophila genome, belongs to the amyloid precursor protein family that also includes the Drosophila amyloid precursor protein (APP) and the amyloid precursor like protein-2 (APLP2). Though the three proteins share similar structures and undergo the same cleavage processing by α-, β- and γ-secretases, APLP1 shows divergent subcellular localization from that of APP and APLP2, and thus, may perform distinct roles in vivo. While extensive studies have been focused on APP, which is implicated in the pathogenesis of Alzheimer’s disease, the functions of APLP1 remain largely elusive. This study reports that the expression of APLP1 in Drosophila induces cell death and produces developmental defects in wing and thorax. This function of APLP1 depends on the transcription factor dFoxO, as the depletion of dFoxO abrogates APLP1-induced cell death and adult defects. Consistently, APLP1 up-regulates the transcription of dFoxO target hid and reaper-two well known pro-apoptotic genes. Thus, the present study provides the first in vivo evidence that APLP1 is able to induce cell death, and that FoxO is a crucial downstream mediator of APLP1’s activity (Wang, 2015).

Highlights

  • APLP1 induces caspase-dependent cell death in Drosophila.
  • APLP1 up-regulates dFoxO target gene expression.

Discussion
Amyloid pecursor like protein-1(APLP1) is a mammalian paralog of amyloid precursor protein (APP). While APP has been extensively studied for its involvement in the Alzheimer’s disease, few studies have been directed to APLP1 and its in vivo functions remain largely unknown. This study investigated the in vivo functions of APLP1 using Drosophila as a model organism. It was found that ectopic expression of APLP1 induces cell death and developmental defects in the nervous and non-nervous system. Genetic studies characterize the transcription factor dFoxO as a critical downstream factor that mediates APLP1’s activity, for the depletion of dFoxO significantly suppresses APLP1-induced cell death in larval discs and associated phenotypes in adults. Further, it was shown that APLP1 is able to up-regulate the transcription of dFoxO target genes hid and reaper (Wang, 2015).

APLP1 has been reported to function mainly in the nervous system, as high expression level of APLP1 is detected in the developing central and peripheral nervous systems, yet a weak expression signal of APLP1 is also observed in organs like heart, lung, liver and kidney in mouse embryos, implying a role of APLP1 in the development of non-neuronal tissues. Consistent with this explanation, RNAi mediated knockdown of APLP1 in WI-38 and MCF7 cells dramatically reduced the proliferation of these cells. This study showed that expression of APLP1 could induce cell death and developmental defects in both neuronal and non-neuronal systems in Drosophila, and thus, provides further evidence for the function of APLP1 in non-neuronal cells (Wang, 2015).

Earlier studies have also shown that loss of APLP1 diminishes stress induced apoptosis in neuroblastoma cells, whereas ectopic expression of APLP1 moderately enhances cell death upon stress stimulation. However, expression of APLP1 alone is not sufficient to induce neuroblastoma cell death , suggesting APLP1 induces cell death in a context dependent manner. Data from this study demonstrates that APLP1 by itself is sufficient to induce cell death. APLP1 has been reported to be a direct transcriptional target of the p53 tumor suppressor, which suggests a possible involvement of APLP1 in p53-induced cell death. p53 is known to interact with the transcriptional factor FoxO, and MDM2 is known to act downstream of p53 to promote FoxO ubiquitination and degradation. In the present study, it was shown that FoxO mediates APLP1-induced cell death. The exact relationship between APLP1, FoxO and p53 in cell death will require further investigation. Overall, this study highlights a novel function of APLP1 in promoting FoxO-mediated cell death in vivo, which will shed light on the role of APLP1 in mammalian cells (Wang, 2015).

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Omata, Y., Lim, Y.M., Akao, Y. and Tsuda, L. (2014). Age-induced reduction of autophagy-related gene expression is associated with onset of Alzheimer's disease. Am J Neurodegener Dis 3: 134-142. PubMed ID: 25628964

Abstract
Aging is a major risk factor for Alzheimer's disease (AD). Aggregation of amyloid beta (Aβ) in cerebral cortex and hippocampus is a hallmark of AD. Many factors have been identified as causative elements for onset and progression of AD; for instance, tau seems to mediate the neuronal toxicity of Aβ, and downregulation of macroautophagy (autophagy) is thought to be a causative element of AD pathology. Expression of autophagy-related genes is reduced with age, which leads to increases in oxidative stress and aberrant protein accumulation. This study found that expression of the autophagy-related genes atg1, atg8a, and atg18 in Drosophila melanogaster is regulated with aging as well as their own activities. In addition, the level of atg18 is maintained by dfoxo (foxo) and dsir2 (sir2) activities in concert with aging. These results indicate that some autophagy-related gene expression is regulated by foxo/sir2-mediated aging processes. It was further found that reduced autophagy activity correlates with late-onset neuronal dysfunction caused by neuronal induction of Aβ. These data support the idea that age-related dysfunction of autophagy is a causative element in onset and progression of AD (Omata, 2014).

Highlights

  • Expression of autophagy-related genes is reduced with aging.
  • Expression of atg18 is reduced in dfoxo and dsir2 mutants.
  • Initiation factor 4E-binding protein (4E-BP) negatively regulates autophagy-related gene expression.
  • Aβ transgenic flies produce Aβ42 and exhibit reduced locomotor activity.
  • Expression of autophagy-related genes affects development of AD.
  • Expression of autophagy-related genes correlates with neuronal toxicity caused by Aβ42.

Discussion
This study shows that expression of autophagy-related genes is regulated by age-related signaling. dsir2 (a Drosophila SIRT1 homolog) and dfoxo are required to maintain atg18 expression during aging, suggesting that, among autophagy-related genes, this gene specifically is regulated by foxo/sir2 activity. Interestingly, aging seems to affect expression of all autophagy-related genes tested, suggesting that aging and foxo/sir2 may act at different levels to regulate autophagy-related gene expression (Omata, 2014).

Previous studies show that sir2, foxo and 4E-BP are involved in regulating the Drosophila lifespan. Data from this study, however, indicate that 4E-BP antagonizes expression of autophagy-related genes. 4E-BP is believed to be controlled by TOR signaling. Therefore, the negative effect of 4E-BP on autophagy-related gene expression may be mediated through the effect of TOR signaling pathway, which also seems to antagonize autophagy-related gene expression (Omata, 2014). 

Autophagy is highly correlated with lysosomal activity, and the autophagy-lysosome pathway is thought to be involved in many cellular processes. Earlier studies indicate that lysosomal activity affects expression of autophagy-related genes. The lysosome nutrient sensing (LYNUS) machinery is responsible for sensing whether there are sufficient nutrients. Under a sufficient nutrient status, the mammalian target of rapamycin complex 1 (mTORC1, a member of the LYNUS machinery) phosphorylates transcription factor EB (TFEB) on the lysosomal surface and inhibits its nuclear localization. In this way, TFEB is unable to induce expression of lysosomal and autophagy-related genes under nutrient sufficient conditions. These results suggest that the level of autophagy-related genes might be regulated by the state of lysosome formation and autophagy itself. Here, expression of autophagy-related genes is affected by the activity of other autophagy-related genes as well as their own activity, suggesting that auto-feedback regulation is part of the mechanism used to maintain expression of autophagy-related genes in Drosophila (Omata, 2014). 

It was observed that reducing the expression of autophagy-related genes strongly enhances the neuronal toxicity caused by Aβ expression. Furthermore, reducing atg1 expression using the Df(atg1)/+ heterozygote shows a more severe enhancement of Aβ-dependent neuronal toxicity than reducing atg18 expression using the Df(atg18)/+ heterozygote. Interestingly, atg1 also demonstrates strong auto-feedback regulation, as reducing expression of atg1 results in further defects in expression of atg genes. Therefore, it is possible that a drastic reduction in expression of many atg genes may contribute to the neuronal toxicity of Aβ42, and that aging and autophagy may be determinants of AD onset (Omata, 2014).

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Saitoh, Y., Fujikake, N., Okamoto, Y., Popiel, H.A., Hatanaka, Y., Ueyama, M., Suzuki, M., Gaumer, S., Murata, M., Wada, K. and Nagai, Y. (2015). p62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies. J Biol Chem 290: 1442-1453. PubMed ID: 25480790

Abstract
Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. This study used Drosophila models of neurodegenerative diseases to elucidate the role of p62 in such pathogenic conditions in vivo. It was found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function results in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. It was also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. The study therefore concludes that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases (Saitoh, 2015).

Highlights

  • p62 co-localizes with cytoplasmic polyQ protein aggregates.
  • Loss of p62 function causes exacerbation of eye degeneration in polyQ disease model flies.
  • Loss of p62 function results in an increase in cytoplasmic polyQ protein aggregates.
  • Loss of autophagic function causes the exacerbation of eye degeneration accompanied by the enhanced accumulation of cytoplasmic polyQ protein aggregates.
  • Protective role of p62 against polyQ protein toxicity is dependent on autophagy.
  • Loss of p62 function delays the degradation of the polyQ protein in vivo.
  • p62 plays a protective role in various neurodegenerative disease model flies.

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Lim, J.Y., Reighard, C.P. and Crowther, D.C. (2015). The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aβ. Hum Mol Genet 24: 3929-3938. PubMed ID: 25954034

Abstract
Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. This study identified a synergistic toxic interaction between the amyloid-β peptide (Aβ1–42) and the pro-domains of both DNT1 and BDNF in flies and mammalian cells. Specifically, it was shown that DNT1 pro-domain acquires a neurotoxic activity in the presence of Aβ1–42. In contrast, DNT1 mature-domain is protective against Aβ1–42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aβ1–42. Western blots indicate that this synergistic interaction likely results from the Aβ1–42-induced upregulation of the BDNF pro-domain receptor p75NTR. The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention (Lim, 2015).

Highlights

  • Drosophila DNT1 mRNA levels are high during development but suppressed in adulthood.
  • The subdomains of DNT1 have differential effects on neurotoxic phenotypes in the fly.
  • Co-expression of various domains of DNT1 does not change the abundance or conformation of Aβ.
  • The pro-domain of BDNF and Aβ1–42 exhibit synergistic toxicity in SH-SY5Y human neuroblastoma cell cultures.
  • An elevated ratio of pro- to mature-domain of BDNF correlates with disease status in elderly individuals.

Discussion
NTs are a major class of molecules promoting neuronal survival in vertebrates. They are synthesized as larger precursor forms that are proteolytically processed to yield a mature, biologically active ligand. Among the NTs, BDNF has emerged as a major regulator of synaptic plasticity, neuronal survival and differentiation, and also as a potential molecular target for the treatment of neurological disease. Several studies indicate that the cortex and hippocampus, areas of the brain associated with learning and memory, not only exhibit extensive amyloid pathology but also show decreased levels of BDNF in AD. Interestingly, precursor and mature forms of BDNF are significantly decreased in preclinical and early stages of AD, and this reduction correlates with clinical neuropsychological scores. Low levels of BDNF may favor AD pathogenesis by failing to adequately support neurones and allowing them to succumb to other toxic insults. Furthermore, several studies have linked polymorphisms, specifically Val66Met and Cys270Thr, in the pro-domain of BDNF to an increased risk for AD (Lim, 2015).

BDNF exerts many of its neuroprotective effects by binding to the TrkB receptor, a member of the tumor necrosis factor receptor family. Indeed, some reports indicate that Aβ may have a negative effect on neuronal survival by down-regulating the TrkB receptor. For example, levels of TrkB are reduced in the temporal and frontal cortex of AD brain. In addition, BDNF-induced TrkB autophosphorylation and the activation of the downstream enzymes AKT and ERK are all suppressed in the hippocampus of APP/PS1 mice (Lim, 2015).

The consequences of BDNF binding to its alternative receptor p75NTR and sortilin are less well understood, although they are thought to include promotion of myelination and neuronal migration but also neuronal process retraction and neuronal apoptosis. It has been proposed that the balance between cell death and survival is determined by the relative activity of the precursor versus mature forms of BDNF; indeed, in dorsal root ganglion lesion models in neonatal rats, the signaling appears to be affected by the relative levels of the relevant receptors, namely TrkB, p75NTR and sortilin. Notably, BDNF promotes the death of cultured neurons in vitro when p75NTR is upregulated and TrkB downregulated. Precursor BDNF can also cause cell death in both in vitro and in vivo model systems and the prevailing view is that the apoptotic signal is generated by the pro-domain. Similar domain-specific activities have also been observed for NGF; specifically, the precursor form of this related NT preferentially activates p75NTR resulting in apoptosis, while mature NGF preferentially activates TrkA receptor with neurotrophic effects (Lim, 2015).

Mammalian NTs are similar in many ways to their insect orthologues. Sequence analysis has identified Drosophila neurotrophin (DNT1), also called Spatzle 2 (Spz 2), as the closest fly orthologue of human BDNF. Like the NTs, the Spz polypeptides are synthesized with a signal peptide, followed by a pro-domain and then the cysteine knot-containing mature domain. The characteristic NT cysteine knot, formed by antiparallel β-sheets held together by three disulfide bonds, is conserved in the crystal structures of both Spz and NGF. There is also functional conservation between DNT1, Spz and the mammalian NTs in the nervous system. Indeed, during Drosophila embryogenesis DNT1 is expressed in neurons and muscle cells where it promotes neuronal survival and suppresses apoptosis. This study shows that the high levels of endogenous DNT1 mRNA that are present during larval development are rapidly suppressed at the beginning of adult life in both control flies and similarly in those expressing various isoforms of the Aβ peptide (Lim, 2015).

Because of the functional similarities between mammalian and Drosophila NTs, this study analyzed the interaction between Aβ and DNT1 in the fruit fly. It was found that transgenic expression of the DNT1 mature-domain protects flies against Aβ toxicity and that this benefit is not due to spurious reductions in Aβ levels. Similarly, in human SH-SY5Y culture, the mature-domain of BDNF protects cells from Aβ toxicity. In both the fly and the mammalian models, the pro-domains of both DNT1 and BDNF, while being harmless alone, are nevertheless toxic when added in combination with Aβ. In SH-SY5Y cells, this synergistic toxic interaction between Aβ and the BDNF pro-domain is only apparent for the Met66 variant and is absent for the wild-type Val66 isoform. This finding provides mechanistic underpinning for the clinical observation that the Met66 polymorphism is linked to poor prognosis, particularly in individuals with high Aβ burden on PiB photon emission tomography brain scans (Lim, 2015).

The expression of the receptor p75NTR is upregulated in both SH-SY5Y cells following Aβ treatment and also in transgenic mice expressing the human APPswe transgene. Aβ treatment also enhances sortilin expression via p75NTR, which is thought to activate the downstream effectors JNK and Rho, resulting in apoptotic cell death. Furthermore, the accumulation of Aβ in humans is also accompanied by an increased hippocampal membrane-associated p75NTR. However, it seems unlikely that Aβ binds directly to p75NTR, rather this study suggests that Aβ amplifies the pro-apoptotic signaling of pro-domain BDNF by upregulating p75NTR in the human SH-SY5Y cells (Lim, 2015).

Next, analysis of post-mortem hippocampal tissue from age-matched healthy elderly and patients with AD underlines the clinical significance of experimental findings in this study. As expected, the mature-domain of BDNF is reduced in AD when compared with control brain tissue. In analysis of 10 cases and controls, it was found that control subjects fall into two groups—those with levels of BDNF mature-domain that are up to 20 times higher than AD cases; however, an equal number of controls have mature-domain levels that are equivalent to AD patients. The separation between cases and controls is better when BDNF pro-domain levels are measured: cases have elevated levels of pro-domain, spread over almost 2 orders of magnitude while all but one of the controls are closely grouped. The average pro-domain level is ∼16-fold higher in AD cases when compared with controls. These reciprocal changes in the levels of pro- and mature-BDNF are surprising considering that the peptides are generated stoichiometrically. Receptor-mediated clearance is an unlikely explanation because the levels of TrkB are low and p75 high in AD cases when compared with controls. Conceivably, in AD the BDNF pro-domain is being stabilized by binding to another protein; this intriguing possibility requires further investigation (Lim, 2015).

When these relative changes in pro- and mature-domains are combined as a ratio, it was found that cases and controls have an average 30-fold difference and only one of the controls overlaps with the AD range. This particular control individual is interesting because, despite being symptom-free and relatively young (aged 63), she was the only control to have accumulated significant Aβ. In fact, she had the highest level of Aβ amongst all cases and controls. That this control individual's pro:mature domain ratio was in the AD range raises the tantalizing possibility that disease changes can be predicted before the onset of symptoms (Lim, 2015).

In conclusion, this study shows for the first time that the pro- and mature-domains of NTs have opposing effects on Aβ neurotoxicity in vitro and in vivo. The mature-domains of NTs protect against Aβ1–42 toxicity, whereas the pro-domains acquir a toxic role in the presence of Aβ1–42. In case of post-mortem clinical brain samples, the ratio of pro- to mature-domain of BDNF is significantly higher in patients with AD when compared with controls. Taken together, the finding that patients with AD have elevated levels of BDNF pro-domain underlines the importance of its synergistic toxic interaction with Aβ. Conceivably, individuals with an unfavourable pro:mature domain ratio could be targeted with therapy aimed at restoring a more neurotrophic environment in the brain (Lim, 2015).

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Ott, S., Dziadulewicz, N. and Crowther, D.C. (2015). Iron is a specific cofactor for distinct oxidation- and aggregation-dependent Aβ toxicity mechanisms in a Drosophila model. Dis Model Mech 8: 657-667. PubMed ID: 26035384

Abstract
Metals, including iron, are present at high concentrations in amyloid plaques in individuals with Alzheimer's disease, where they are also thought to be cofactors in generating oxidative stress and modulating amyloid formation. This study presents data from several Drosophila models of neurodegenerative proteinopathies indicating that the interaction between iron and amyloid beta peptide (Aβ) is specific and is not seen for other aggregation-prone polypeptides. The interaction with iron is likely to be important in the dimerisation of Aβ and is mediated by three N-terminal histidines. Transgenic fly lines systematically expressing all combinations of His>Ala substitutions in Aβ were generated and used to study the pathological role of these residues. Developmental eye phenotypes, longevity and histological examinations indicate that the N-terminal histidines have distinct position-dependent and -independent mechanisms. The former mediate the toxic effects of metals and Aβ aggregation under non-oxidising conditions and the latter are relevant under oxidising conditions. Understanding how Aβ mediates neurotoxic effects in vivo will help to better target pathological pathways using aggregation blockers and metal-modifying agents (Ott, 2015).

Highlights

  • The iron-binding protein ferritin is a specific suppressor of Aβ toxicity in vivo.
  • Iron modifies Aβ aggregation in vitro and in vivo.
  • Position-dependent effects of His>Ala substitutions on Aβ toxicity in a non-oxidising environment.
  • Position-dependent effects of His>Ala substitutions on the metal-dependent component of Aβ toxicity in a non-oxidising environment.
  • Position-independent effects of His>Ala substitutions on Aβ toxicity in an oxidising environment.

Discussion
A number of neurodegenerative disorders are characterized by the abnormal metabolism of metals, such as copper and iron; however, the relevance of this observation to pathological mechanisms remains unclear. Consequently, this study used several models of protein aggregation disorders to investigate the role of metals. In an earlier study using a Drosophila model of Aβ toxicity, it was found that the iron-binding protein ferritin and other iron-chelators are powerful suppressors of both Aβ toxicity and the associated markers of oxidative damage. Consequently, this study asked whether ferritin expression also rescues disease-related phenotypes in other Drosophila models of common neurodegenerative disorders. Specifically, flies expressing tau, the Q48 peptide and TDP43, in addition to various isoforms of Aβ, were analyzed (Ott, 2015).

The inclusion of these particular polypeptides in the study is justified by varying degrees of evidence linking their pathogenicity to interactions with metal cofactors. Considering tau, its hyperphosphorylation and deposition as neurofibrillary tangles are characteristic of AD and other tauopathies. Some investigators have highlighted the role of metal binding in generating tau deposits and the consequent neuronal microtubule dysfunction. It is thought that this process is induced by the ability of Fe3+ to bind to phosphorylated tau, causing it to aggregate. Furthermore, tau, like Aβ, may form complexes with iron and copper, resulting in the generation of reactive oxygen species, in turn favouring tau phosphorylation (Ott, 2015).

Similarly, in HD there is evidence for abnormal metal metabolism, with clinical MRI scans showing iron accumulation in susceptible brain regions. Iron levels tend to be higher in advanced disease and in patients with longer polyQ repeat lengths. In mouse and fly models of HD there is good evidence that both copper and iron metabolism are disturbed and that chelator therapy may be beneficial. In vitro, metals promote oxidative damage, with a truncated huntingtin polypeptide interacting with both Cu2+ and Fe3+ (Ott, 2015).

Finally, familial amyotrophic lateral sclerosis (ALS) may result from mutations in the genes for TDP43, FUS, C9ORF72 and, classically, SOD1. Although there is considerable evidence that copper plays a role in the pathogenicity of SOD1 variants, the role of metals in ALS as a whole is less clear. For example, mice expressing disease-linked variants of TDP43 show abnormal metabolism of a number of metals, although iron appears to be normal (Ott, 2015).

Considering this evidence that iron is more or less important in the pathological actions of a series of aggregation-prone polypeptides, it is remarkable that, in this study, iron chelation by ferritin was beneficial only for flies expressing Aβ. The finding in this study that ferritin offers Aβ-specific rescue from both longevity and eye phenotypes indicates that an important component of Aβ toxicity in vivo is not generic. Thus, the overall toxicity of an aggregating polypeptide seems to be composed of a core generic component, which is likely to be related to the presence of oligomeric aggregates. To this core are added peptide-specific effects that are modulated by environmental cofactors – in this case the presence of iron or other metals. One way in which metals could enhance Aβ toxicity is by stabilising particular conformers in the aggregation pathway. The formation of an initial dimer is likely to be a fundamental step and this was investigated in vivo by expressing either the normal monomeric peptide or a ‘pre-dimerised’ tandem Aβ peptide. It has been previously shown that both tandem Aβ42 and tandem Aβ40 aggregate rapidly in vivo; however, of these, only the 42 amino acid isoform is able to generate stable oligomeric aggregates and exhibit pronounced toxicity. This study presents tentative evidence in vitro that amyloid generation by partially purified, recombinant Aβ and by tandem Aβ responds differently to the addition of iron. Specifically, the addition of iron to a preparation of partially purified tandem Aβ does not cause the slowing of ThT amyloid signal that is observed with the monomeric peptide preparation. Concordant with these in vitro results, it was found that the toxicity associated with tandem Aβ in the fly is not suppressed by co-expression of ferritin. Furthermore, ferritin has no effect on the number of tandem Aβ deposits in the brain, whereas flies expressing monomeric Aβ consistently have up to 25% fewer deposits in the presence of ferritin. Importantly, this reduced plaque load is not due to decreased Aβ transgene expression or peptide production. In summary, these results suggest that, both in vitro and in vivo, iron is interacting with Aβ to accelerate a step that is redundant in the tandem peptide; this step is likely to be the formation of dimeric aggregates. These results in a Drosophila model are reminiscent of the reduction in plaque load in tg2576 mice treated with the broad-spectrum metal chelator clioquinol and support the relevance of this study for mammalian systems (Ott, 2015).

After evaluating the specificity of iron for Aβ-induced toxicity and its effect on aggregation, potential interactions between Aβ and iron were studied. Such interactions are thought be mediated by the three N-terminal histidine residues at positions 6, 13 and 14. The study systematically assessed how each of the seven possible combinations of His>Ala substitutions modifies the elaboration of three key disease-linked phenotypes, namely: (1) survival in an oxidising environment; (2) survival in a non-oxidising environment; and (3) the proportion of Aβ toxicity that is metal dependent (Ott, 2015).

To assess the response of the flies to oxidative stress, survival after oral challenge with hydrogen peroxide was measured. Whereas control flies are robust when treated in this way, there is a marked reduction in survival after 78 and 96?h when Aβ is expressed. Remarkably, only the number of histidines in a particular Aβ variant determine the susceptibility to oxidative stress. This histidine dose dependency and position independence makes it unlikely that the mechanism involves specific changes in peptide conformation or overall aggregation propensity; indeed, the hydrogen peroxide feeding experiments are performed in 6-day-old flies in which there is no detectable peptide aggregation. Moreover, the predicted aggregation propensities of the various peptide isoforms are essentially identical. A possible molecular scale interpretation of these results is that the formation of redox-active metal-peptide complexes is rather flexible and peptide promiscuous. In this model, histidines from distinct peptides could co-ordinate to a shared metal ion and generate a redox-active complex. An alternative explanation might be that there is an oxidative reaction in the presence of Aβ that uses histidines as a substrate, thereby generating toxic oxygen species. In either case, these results predict that reducing total brain Aβ levels might be most effective in reducing the oxidative damage in AD (Ott, 2015).

By contrast, the remaining two disease-linked phenotypes are sensitive to the positions of each of the His>Ala substitutions. In a non-oxidising environment the survival of flies expressing the various peptides is determined by position-dependent factors. Some in vitro studies have shown that the histidines at positions 13 and 14 interact similarly with copper in models of amyloid fibrils, whereas others suggest that the H14A substitution in synthetic Aβ42 reduces toxicity, possibly by preventing the interaction of the peptide with cell membranes. The longevity effects observed in this study, although relatively modest, are nevertheless both robust and significant, and present with a contrasting observation. Specifically, it was found that H14A enhances toxicity, as evidenced by a reduction in longevity, whereas the H13A substitution has the opposite effect. Interestingly, the H6A substitution has a modulating role in the fly model; in particular, H6A acts to amplify the longevity effects of substitutions at positions 13 and 14. Accordingly, H14A reduces the median survival and H6/14A has the shortest lifespan of all the fly lines. By contrast, Drosophila expressing the H13A variant live longer than wild-type flies and H6/13A live the longest of all the peptide-expressing lines. H6 also modulates the accumulation of peptide deposits in the brain, with H6/14A showing remarkably high levels of Aβ deposition, higher than H14A alone. The use of the φC31 system to target all of the Aβ transgenes to the same 51D genomic insertion site in the same acceptor fly line makes it highly unlikely that the phenotypic differences observed in this study are due to artefactual fluctuations in transgene expression levels. Furthermore, the genetic background of the experimental flies should be essentially identical. It should also be noted that the density of Aβ deposits is comparable between lines despite highly significant differences in median survival (Ott, 2015).

The modulating role of H6 is seen most clearly in the third phenotype, where the degree to which the metal chelator clioquinol prolongs the longevity of the various fly lines was measured. When the percentage increase in median survival upon treatment with clioquinol is calculated, it is apparent that the histidines at positions 13 and 14 each have their position-dependent effects on the clioquinol response. However, there is an overwhelming additive effect of histidine at position 6, such that each corresponding H6A variant shows a 25-30% reduction in clioquinol responsiveness. The implication of these results is that much of the metal-mediated toxicity, in a non-oxidising environment, is dependent on H6A and that, thereafter, there are smaller position-dependent effects of residues 13 and 14. A possible mechanistic interpretation is that metal interactions with H6 initiate metal-mediated Aβ dimerisation and, thereafter, histidines 13 and 14 determine the particular conformations of downstream aggregates and their toxicity. These position-dependent properties of individual histidine residues make it likely that particular peptide conformations are mediating the combined Aβ-iron effects; such structures might be amenable as therapeutic targets (Ott, 2015). 

In summary, this study's analysis of the effects of metals and their specific interactions with Aβ in vivo indicates that dimerisation of the peptide, perhaps mediated by H6, is an early step in generating toxicity. The subsequent toxic consequences of Aβ aggregation are then largely determined by the histidines at positions 13 and 14. In a strongly oxidising environment the importance of peptide aggregation is complemented, if not overwhelmed, by the histidine-dependent oxidative damage mediated by Aβ (Ott, 2015).

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Sofola-Adesakin, O., Castillo-Quan, J.I., Rallis, C., Tain, L.S., Bjedov, I., Rogers, I., Li, L., Martinez, P., Khericha, M., Cabecinha, M., Bähler, J. and Partridge, L. (2014). Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease. Front Aging Neurosci 6: 190. PubMed ID: 25126078

Abstract
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 beta (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 earlier in which mutant Aβ42 accumulation leads to increased mortality and neuronal dysfunction in the adult flies. Furthermore, lithium reduces Aβ42 protein, but not mRNA, and is able to rescue Aβ42-induced toxicity. This study investigated the mechanism/s by which lithium modulates Aβ42 protein levels and Aβ42 induced toxicity in the fly model. It was found that lithium causes a reduction in protein synthesis in Drosophila and hence the level of Aβ42. At both the low and high doses tested, lithium rescues the locomotory defects induced by Aβ42, but it rescues lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulates 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).

Highlights

  • Lithium reduces Aβ load in arctic Aβ42 expressing flies through protein clearance/degradation-independent mechanisms.
  • Lithium down-regulates overall protein synthesis/translation.
  • Lithium also inhibits protein synthesis in fission yeast.
  • Lithium extended lifespan of flies expressing Aβ.

Discussion
Human life expectancy continues to increase at a steady rate in most countries worldwide, and has done so by almost 3 months per year in the last 160 years. Therefore, it is of great importance to tackle ageing-related diseases such as AD, because they are becoming increasingly prevalent. Because age is the biggest risk factor for AD, interventions that promote general increases in health during ageing could also be important and beneficial in AD (Sofola-Adesakin, 2014).

Lithium is becoming increasingly implicated as a drug that can ameliorate ageing and neurodegenerative diseases. Several groups have shown that it extends lifespan in model organisms such as the nematode worm C. elegans and Drosophila. This study shows that lithium also extends lifespan in fission yeast Schizosaccharomyces pombe, highlighting that this effect is conserved over large evolutionary distances. Fission yeast is an ideal organism for genetic screens, and future work should identify the molecular targets of lithium both for control of protein synthesis and of lifespan. Furthermore, slightly higher levels of lithium present in the drinking water have been reported as associated with reduced mortality in a Japanese human population (Sofola-Adesakin, 2014).

A substantial body of work has demonstrated that several neurodegenerative diseases and neurological disorders, including but not confined to stroke, schizophrenia, Fragile X syndrome, Huntington's disease and Parkinson's disease, benefit from the therapeutic properties of lithium. In addition, several studies have investigated whether lithium has a beneficial effect in AD pathogenesis. Clinical trials conducted with lithium have yielded conflicting results; some have found benefits, whilst others have not. Interestingly, a correlative study conducted in patients with bipolar disorder, suggests that patients that have been on chronic lithium treatment show a reduced incidence of AD in comparison to patients that have not been on treatment. And a small-scale clinical trial on mild cognitive impaired (MCI) patients finds that low doses of lithium slow cognitive decline. The investigators suggest that a reason for the previous conflicting data on the efficacy of lithium is probably attributable to the pathological states/stages at which the patients were given lithium. It is becoming increasingly evident that drug trials are most likely to yield positive effects when initiated early, at the MCI stage (Sofola-Adesakin, 2014).

Results from this study add to existing data suggesting that lithium could be beneficial in ameliorating Aβ toxicity, and should be considered for a potential large-scale trial on MCI patients. It has the added advantage of being an already approved drug, used to treat bipolar patients. It does have side-effects, but these are minimal at the low doses used in a recent small-scale clinical study. It was also found that there are limits to the beneficial/therapeutic benefits of lithium in fission yeast in chronological lifespan—lithium is unable to increase chronological lifespan at higher doses as well as in the Drosophila AD model. Previously, it was shown that administering both 30 and 100 mM lithium into the fly food is effective in modulating Aβ neuronal toxicity as evidenced by the improved locomotor function in young flies. These lithium concentrations were initially chosen based on another study in which it was shown that lithium concentrations ranging from 10 to 100 mM lithium in the fly food translates to roughly 0.05–0.4 mM in the fly tissue, so well below the toxic levels in patients and mice. This study shows that both 25 and 100 mM lithium reduce Aβ levels in a dose dependent manner at an early time point. It was also found that lower doses of lithium (10 and 25 mM) rescue the shortened longevity of the Aβ flies, but 100 mM lithium is unable to extend lifespan when given to the flies throughout adulthood. It will be important to determine the therapeutic thresholds for lithium in patients that could offer therapeutic benefits without overt side effects (Sofola-Adesakin, 2014).

Similar to the published data on the role of GSK-3 inhibition in down-regulating translation in HCC1806 cells, it was found that lithium is able to reduce translation in fission yeast and flies, suggesting that perhaps some of the effect of lithium on translation down-regulation is via GSK-3 inhibition. However, this is correlative and future work will involve carrying out epistasis interactions between lithium and GSK-3, and identifying molecular targets of GSK-3 and lithium for control of protein synthesis. Nonetheless, this study highlights the potential benefits of lithium through down-regulation of translation, associated with extension of lifespan in very distantly related organisms. By reducing protein synthesis, lithium may reduce the increased proteostatic burden in ageing, a recognized hallmark of ageing. Lithium is also of specific benefit in AD, because of its ability to down-regulate translation, and hence levels of proteins involved in promoting the presence of toxic Aβ (Sofola-Adesakin, 2014).

The mutant Arctic Aβ42 protein present in the transgenic flies used in this study has a propensity to aggregate faster than wild type Aβ. However, both soluble and insoluble Aβ in the Arctic Aβ42 flies were observed, and the ability of lithium to reduce translation of the Aβ peptide without affecting its clearance may lower the level of soluble Aβ. In a wider context, lithium might be beneficial in ameliorating toxicity of AD by lowering expression of APP and of proteins that are involved in the generation of Aβ from APP. The AD model used in this study does not express full length APP, and may therefore not include other potential/additional benefits of lithium on Aβ toxicity. As well as the increased ratio of Aβ42 to Aβ 40 peptide observed in familial AD cases with APP mutations, increased levels of APP could also contribute to AD pathogenesis. Indeed, patients with Down syndrome have a high risk of developing AD possibly due to trisomy of the APP gene which leads to increased APP expression. Also, several mutations in the APP promoter region have been found to significantly increase APP expression in SH-SY5Y cells, and are associated with risk for AD (Sofola-Adesakin, 2014).

The ability of lithium to down-regulate translation could therefore be beneficial at several stages in AD pathogenesis. Lithium might also have therapeutic benefits for other neurodegenerative disorders that are caused by over-expression of wild type or mutant forms of proteins such as α-synuclein in Parkinson's disease. Lithium could also reduce the production of mis-translated polypeptides, and free proteases or/and chaperones that can then participate in cellular proteostasis. Furthermore, diseases where protein turnover is compromised by loss of function of the degradation machinery could also benefit from lowering the burden of protein production hence reducing cellular stress. This could be particularly important in lysosomal storage diseases, where the intrinsic function of the degradative machinery is compromised. Moreover, induction of autophagy in some cases increases the load of an already dysfunctional lysosome, worsening the cellular proteostatic stress. Hence lowering the production of proteins could again be a viable mechanism to restore proteostasis. Other neurodegenerative models where the role of lithium in lowering protein translation could be beneficial are, for example, the Drosophila models of Pink1 and Parkin, which do not include the over-expression of toxic proteins. Flies lacking either of these proteins accumulate unfolded proteins in mitochondria, resulting in mitochondria impairment. It would be interesting to study whether lithium could ameliorate mitochondrial stress by reducing the production of the proteins accumulating in the mitochondria of the Pink1 or parkin null flies. Lithium could hence be a useful drug with an overall benefit for health during ageing and protection against AD and other neurodegenerative diseases (Sofola-Adesakin, 2014).

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Allan, K., Perez, K.A., Barnham, K.J., Camakaris, J. and Burke, R. (2014). A commonly used Drosophila model of Alzheimer's disease generates an aberrant species of amyloid-β with an additional N-terminal glutamine residue. FEBS Lett 588: 3739-3743. PubMed ID: 25171862

Abstract
Expression of human amyloid-β (Aβ) in Drosophila is frequently used to investigate its toxicity in vivo. This study expressed Aβ1–42 in the fly using a secretion signal derived from the Drosophila necrotic gene, as described in several previous publications. Surface-enhanced laser desorption/ionization TOF MS analysis revealed that the Aβ produced contains an additional glutamine residue at the N-terminus. AβQ+1–42 was found to have increased protein abundance and to cause more severe neurodegenerative effects than wild type Aβ1–42 as assessed by locomotor activity and lifespan assays. These data reveal that a commonly used model of Alzheimer’s disease generates incorrect Aβ peptide (Allan, 2014).

Highlights

  • Amyloid-β (Aβ) peptide produced with the Necrotic signal peptide contains an additional N-terminal glutamine.
  • Aβ produced with the Necrotic signal peptide gives dose-dependent neurodegenerative phenotypes.

Discussion
Alzheimer’s disease (AD) is characterized by the presence of brain extracellular plaques rich in amyloid-β (Aβ), a peptide derived from the proteolytic cleavage of Amyloid-β Precursor Protein (AβPP). Differential processing of AβPP can produce several different Aβ species of varying lengths. Aβ42 is significantly more cytotoxic than the abundant Aβ40 normally found in healthy brains and the Aβ42: Aβ40 ratio is considered to be an important factor in neuronal health and AD progression (Allan, 2014).

When attempting to replicate AD-like neurodegeneration in the vinegar fly Drosophila, the need for AβPP processing has been circumvented by directly expressing Aβ in fly neuronal tissues. A secretory signal sequence at the N-terminus of the Aβ peptide, allowing its secretion from producing cells, is known to be essential for inducing age-dependent phenotypes including reduced adult lifespan, reduced locomotor activity, learning defects, deposition of amyloid plaques and visible brain and retinal degeneration. This model system has since been used to investigate the effect of single amino acid substitutions in the Aβ42 peptide sequence and numerous other aspects of AD including pharmacological and genetic modification of Aβ-mediated toxicity (Allan, 2014).

This study demonstrates that one of the signal peptides frequently used to generate secreted Aβ, derived from the fly Necrotic protein, actually produces an aberrant Aβ1–42 peptide with an additional glutamine residue at the N-terminus, referred to as AβQ+1–42. A similar situation has been seen with a C. elegans Aβ transgenic line that produces a truncated form of Aβ, Aβ3–42, which begins with glutamic acid. Using transgenic lines with equivalent transcription levels, it was found that a wild type Aβ1–42 construct gives dramatically lower peptide levels and no detectable neurodegenerative phenotypes compared to the aberrant AβQ+1–42 form (Allan, 2014).

Since TSpAβ1–42 could not be expressed at sufficient levels for repeating the mass spectrometry analysis performed on NSpAβ1–42, this study cannot confirm that TSpAβ1–42 is being correctly processed. However, Aβ1–42 constructs using the pre-proenkephalin signal sequence (also predicted to cleave correctly) are toxic when expressed at high levels. Therefore the study proposes that the addition of an N-terminal glutamine enhances Aβ abundance and therefore toxicity compared to wild type Aβ, a property revealed when the two species are transcribed at similar levels (Allan, 2014).

The slightly higher NSpAβQ+1–42 transcript levels do not account for the strong difference in peptide levels seen between the NSpAβ1–42 and TSpAβ1–42 constructs. Possibly, NSpAβQ+1–42 is more resistant to degradation and/or clearance. In the human AD brain, N-terminally truncated Aβ3–42 and Aβ11–42 peptides represent ∼50% of total Aβ found in senile plaques. Both peptides begin with a glutamic acid residue which can undergo post-translational cyclization to produce pyroglutamate (pE). pE-modified Aβ displays increased stability due to reduced protease susceptibility. Glutamine can also undergo cyclization. This study therefore hypothesizes that aberrant signal peptide cleavage from the NSpAβ1–42 construct results in a cyclized pQ form of Aβ that displays greater stability and hence increased toxicity. These findings emphasize the importance of determining the species of Aβ being generated experimentally and the transcription levels of each different construct (Allan, 2014).

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Hu, Y., Han, Y., Shao, Y., Wang, X., Ma, Y., Ling, E. and Xue, L. (2015). Gr33a modulates Drosophila male courtship preference. Sci Rep 5: 7777. PubMed ID: 25586066

Abstract
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 (CHCs), 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 (APP) 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) (Hu, 2015).

Highlights

  • The roles of Grs in males' courtship preference behavior.
  • Gr33a is required for males' preference for younger mates.
  • Activity of Gr33a neurons is essential for males' preference for younger mates.
  • Expression of APP in Gr33a neurons results in dFoxO-dependent choice defect.
  • Female CHC profiles change with age.

Discussion
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. The choice behavior between two equally-liked options: mature virgin females, whether younger or older, has been previously shown to be 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 shows 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 reveals 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 APP. Therefore, this study demonstrates the genetic interaction of APP and dFoxO in Gr33a neurons, which modulates males' preference for younger mates (Hu, 2015).

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. Findings in this study, however, offer the possibility for establishing a novel Drosophila AD model that is related to cognitive ability (Hu, 2015).

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Lau, H.C., Lee, I.K., Ko, P.W., Lee, H.W., Huh, J.S., Cho, W.J. and Lim, J.O. (2015). Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor. PLoS One 10: e0117810. PubMed ID: 25714733

Abstract
Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. A previous study reports the presence of excess salivary sugars, in patients with Alzheimer’s disease (AD). This study developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor is built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), results in an increase in the sensitivity and reliability of detection. The biosensor is designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, 20 saliva samples were collected from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson’s disease (PD), and a control group composed of healthy individuals. Then, the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor was studies. The cell-based biosensor significantly distinguishes salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, the study propose that salivary trehalose might be a potential biomarker for AD and could be detected using the cell-based EG-ISFET biosensor developed in this study. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and may be used in the future as a screening method for AD (Lau, 2015).

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Frenkel-Pinter, M., Tal, S., Scherzer-Attali, R., Abu-Hussien, M., Alyagor, I., Eisenbaum, T., Gazit, E. and Segal, D. (2016). Naphthoquinone-tryptophan hybrid inhibits aggregation of the Tau-derived peptide PHF6 and reduces neurotoxicity. J Alzheimers Dis [Epub ahead of print]. PubMed ID: 26836184

Abstract

Tauopathies, such as Alzheimer's disease (AD), are a group of disorders characterized neuropathologically by intracellular toxic accumulations of abnormal protein aggregates formed by misfolding of the microtubule-associated protein tau. Since protein self-assembly appears to be an initial key step in the pathology of this group of diseases, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes. Aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly. Specifically, series of small aromatic naphthoquinone-tryptophan hybrid molecules were designed as candidate aggregation inhibitors of β-sheet based assembly, and their efficacy was demonstrated toward inhibiting aggregation of the amyloid-β peptide, another culprit of AD, as well as of various other aggregative proteins involved in other protein misfolding diseases. This study tested whether a leading naphthoquinone-tryptophan hybrid molecule, namely NQTrp, can be repurposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. The molecule was shown to inhibit the in vitro assembly of PHF6, the aggregation-prone fragment of tau protein, reduces hyperphosphorylated tau deposits and ameliorates tauopathy-related behavioral defect in an established transgenic Drosophila model expressing human tau. It is suggested that NQTrp, or optimized versions of it, could act as novel disease modifying drugs for AD and other tauopathies (Frenkel-Pinter, 2016).

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Bouge, A. L. and Parmentier, M. L. (2016). Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death. Dis Model Mech [Epub ahead of print]. PubMed ID: 26822478

Abstract
In neurodegenerative diseases like Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown in particular with respect to the microtubule-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of patients. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for microtubules, there has been no indication that an excess of this protein could affect mitosis. The effect of an excess of human Tau (hTau) protein on cell mitosis was studied in vivo. Using the Drosophila developing wing disc epithelium as a model, this study shows that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. The mechanism of action of hTau was studied and it was found that the MT-binding domain of hTau is responsible for these defects. hTau effects occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). This deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc) as well as in human Hela cells.By demonstrating that microtubule-bound Tau inhibits the Eg5/KIF11 kinesin and cell mitosis, this work provides a new framework to consider the role of Tau in neurodegenerative diseases (Bouge, 2016).

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Kilian, J.G., Hsu, H.W., Mata, K., Wolf, F.W. and Kitazawa, M. (2017). Astrocyte transport of glutamate and neuronal activity reciprocally modulate tau pathology in Drosophila. Neuroscience [Epub ahead of print]. PubMed ID: 28215745

Abstract
Abnormal buildup of the microtubule associated protein tau is a major pathological hallmark of Alzheimer's disease (AD) and various tauopathies. The mechanisms by which pathological tau accumulates and spreads throughout the brain remain largely unknown. It is known that a restoration of the major astrocytic glutamate transporter, GLT1, ameliorates a buildup of tau pathology and rescues cognition in a mouse model of AD. In this study, it was hypothesized that aberrant extracellular glutamate and abnormal neuronal excitatory activities promote tau pathology. Consequently, the genetic interactions between tau and the GLT1 homolog dEaat1 were investigated in Drosophila melanogaster. Neuronal-specific overexpression of human wildtype tau markedly shortens lifespan and impairs motor behavior. RNAi depletion of dEaat1 in astrocytes worsens these phenotypes, whereas overexpression of dEaat1 improves them. However, the synaptic neuropil appears unaffected, and there is no major neuronal loss with tau overexpression in combination with dEaat1 depletion. To mimic glutamate-induced aberrant excitatory input in neurons, repeated depolarization of neurons via transgenic TrpA1 was applied to the adult Drosophila optic nerves, and the change of tau deposits was examined. Repeated depolarization significantly increases the accumulation of tau in these neurons. The study propose that increased neuronal excitatory activity exacerbates tau-mediated neuronal toxicity and behavioral deficits (Killian, 2017).

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Li, A., Hooli, B., Mullin, K., Tate, R.E., Bubnys, A., Kirchner, R., Chapman, B., Hofmann, O., Hide, W. and Tanzi, R.E. (2017). Silencing of the Drosophila ortholog of Sox5 leads to abnormal neuronal development and behavioral impairment. Hum Mol Genet [Epub ahead of print]. PubMed ID: 28186563

Abstract
SOX5 encodes a transcription factor that is expressed in multiple tissues including heart, lung and brain. Mutations in SOX5 have been previously found in patients with amyotrophic lateral sclerosis (ALS) and developmental delay, intellectual disability and dysmorphic features. To characterize the neuronal role of SOX5, this study silenced the Drosophila ortholog of SOX5, Sox102F, by RNAi in various neuronal subtypes in Drosophila. Silencing of Sox102F leads to misorientated and disorganized michrochaetes, neurons with shorter dendritic arborization (DA) and reduced complexity, diminished larval peristaltic contractions, loss of neuromuscular junction bouton structures, impaired olfactory perception, and severe neurodegeneration in brain. Silencing of SOX5 in human SH-SY5Y neuroblastoma cells results in a significant repression of WNT signaling activity and altered expression of WNT-related genes. Samples of SOX5 variants reveals several variants that show significant association with Alzheimer’s disease disease status. These findings indicate that SOX5 is a novel candidate gene for AD with important role in neuronal function. The genetic findings warrant further studies to identify and characterize SOX5 variants that confer risk for AD, ALS and intellectual disability.

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Cutler, T., Sarkar, A., Moran, M., Steffensmeier, A., Puli, O.R., Mancini, G., Tare, M., Gogia, N. and Singh, A. (2015). Drosophila eye model to study neuroprotective role of CREB Binding Protein (CBP) in Alzheimer's disease. PLoS One 10: e0137691. PubMed ID: 25714733

Abstract
The progressive neurodegenerative disorder Alzheimer's disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. This study utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer's like neuropathology. The histone acetylase, CREB Binding Protein (CBP), was identified as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. It was found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Structure-function analysis using truncated constructs lacking one or more domains of CBP protein in transgenic flies reveals that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. In conclusion, the study identifies CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, BHQ domain of CBP is responsible for its neuroprotective function (Cutler, 2015).

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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: 25714733

Abstract
Brain amyloid plaques are a hallmark of Alzheimer's disease (AD), and primarily consist of aggregated Aβ 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 are quite toxic, with 3-42 similar to 1-42, while 11-42 shows a pronounced but less severe phenotype. N-terminal mutations in 3-42 (E3A) or 11-42 (E11A) result in reduced toxicity for 11-42, and reduced aggregation for both variants. Strikingly, C-terminal truncation of Aβ (1-41, -40, -39, -38, -37) are non-toxic. In contrast, C-terminal extension to 1-43 results in reduced lifespan and activity, but not to the same extent as 1-42. Mutating residue 42 in 1-42 (A42D, A42R and A42W) greatly reduces Aβ accumulation and toxicity. Histological and biochemical analysis reveals 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 (Jonson, 2015).

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Haddadi, M., Nongthomba, U., Jahromi, S. R. and Ramesh, S. R. (2015). Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration. Behav Brain Res [Epub ahead of print]. PubMed ID: 26706888

Abstract
The ε4 isoform of Apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. This study created a transgenic model of neurodegenerative disorder by expressing ε3 and ε4 isoforms of human ApoE in the Drosophila. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity.

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Ali, Y. O., Ruan, K. and Zhai, R. G. (2012). NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy. Hum Mol Genet 21(2): 237-250. PubMed ID: 21965302

Abstract

Tauopathies, including Alzheimer's disease, are a group of neurodegenerative diseases characterized by abnormal tau hyperphosphorylation that leads to formation of neurofibrillary tangles. Drosophila models of tauopathy display prominent features of the human disease including compromised lifespan, impairments of learning, memory and locomotor functions and age-dependent neurodegeneration visible as vacuolization. This study used a Drosophila model of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), in order to study the neuroprotective capacity of a recently identified neuronal maintenance factor, nicotinamide mononucleotide (NAD) adenylyl transferase (NMNAT), a protein that has both NAD synthase and chaperone function. NMNAT is essential for maintaining neuronal integrity under normal conditions and has been shown to protect against several neurodegenerative conditions. However, its protective role in tauopathy has not been examined. This study shows that overexpression of NMNAT significantly suppresses both behavioral and morphological deficits associated with tauopathy by means of reducing the levels of hyperphosphorylated tau oligomers. Importantly, the protective activity of NMNAT protein is independent of its NAD synthesis activity, indicating a role for direct protein-protein interaction. Next, it was shown that NMNAT interacts with phosphorylated tau in vivo and promotes the ubiquitination and clearance of toxic tau species. Consequently, apoptosis activation was significantly reduced in brains overexpressing NMNAT, and neurodegeneration was suppressed. This report on the molecular basis of NMNAT-mediated neuroprotection in tauopathies opens future investigation of this factor in other protein foldopathies (Ali, 2012).

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Kong, Y., Wu, J. and Yuan, L. (2014). MicroRNA expression analysis of adult-onset Drosophila Alzheimer's disease model. Curr Alzheimer Res 11: 882-891. PubMed ID: 25274109

Abstract
Alzheimer's disease (AD) is the most common reason for dementia in elderly population. Its neuropathological features include senile plaques, neurofibril tangles and neuronal death. Many AD animal models have been established including yeast, Caenorhabditis elegans, Drosophila melanogaster, mice, rats and non-human primates. Drosophila AD models are much more efficient for genetic manipulation and screening assay than mammals. microRNAs (miRNAs) are ~22nt small RNA molecules that fine-tune gene expression at posttranscriptional level. The dysregulation of miRNAs could participate in AD progression by influencing targets' expression and functions. However, miRNA expression profile of AD flies has not yet been investigated. Using the latest µParaflo™ miRNA microarray assay, this study found that 17 miRNAs are consistently dysregulated in adult-onset AD Drosophila brains: eight of which are upregulated (miR- 8, miR-13b, miR-277, miR-279, miR-981, miR-995, miR-998, miR-1017) and nine are downregulated (let-7, miR-1, miR-9a, miR-184, miR-193, miR-263b, miR-276a, miR-285, miR-289). KEGG pathway annotations using DIANA miRPath or targets predicted by Targetscan identifies 7 pathways (Valine, leucine and isoleucine degradation; MAPK signaling pathway; Dorso-ventral axis formation; Propanoate metabolism; Sphingolipid metabolism; Lysine degradation; Jak- STAT signaling pathway) which might be influenced by these miRNAs. Integrative miRNA/mRNA regulatory network analysis reveals functional cluster with transaminase activity to be potentially regulated by miRNAs in AD. Taken together, dysregulation of miRNA profile may participate in AD pathogenesis by interrupting the metabolism of amino acids in the brain (Kong, 2014).

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Reviews

Fernandez-Funez, P., de Mena, L. and Rincon-Limas, D.E. (2015). Modeling the complex pathology of Alzheimer's disease in Drosophila. Exp Neurol [Epub ahead of print]. PubMed ID: 26024860

Chen, K.F. and Crowther, D.C. (2015). Insights into amyloid disease from fly models. Essays Biochem 56: 69-83. PubMed ID: 25131587

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More in IF

Role of Nck-associated protein 1 (Nap1) in Alzheimer's disease

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Recent Updates

Khanna, M.R. and Fortini, M.E. (2015). Transcriptomic analysis of Drosophila mushroom body neurons lacking amyloid-β precursor-like protein activity. J Alzheimers Dis 46: 913-928. PubMed ID: 26402626

Shaw, J.L., Zhang, S. and Chang, K.T. (2015). Bidirectional regulation of amyloid precursor protein-induced memory defects by Nebula/DSCR1: A protein upregulated in Alzheimer's disease and Down syndrome. J Neurosci 35: 11374-11383. PubMed ID: 26269644

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

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Back to Drosophila as a Model for Human Diseases


Date revised: 03 Nov 2015

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