huntingtin : Biological Overview | Regulation and Model Systems | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - huntingtin
Cytological map position - 98E2
Function - scaffolding protein
Symbol - huntingtin
FlyBase ID: FBgn0027655
Genetic map position - 3R
Classification - HEAT domain protein
Cellular location - cytoplasmic
|Recent literature||White, J. A., Anderson, E., Zimmerman, K., Zheng, K. H., Rouhani, R. and Gunawardena, S. (2015). Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo. Hum Mol Genet. PubMed ID: 26450517
Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. This study analyzed the in vivo movement of 16 Rab GTPases within Drosophila larval axons and showed that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, these findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease.
|Weiss, K. R. and Littleton, J. T. (2016). Characterization of axonal transport defects in Drosophila huntingtin mutants. J Neurogenet 22:1-10. PubMed ID: 27309588
Polyglutamine expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington's Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. This study characterized a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. The data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt's primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling to motors to regulate their processivity during axonal transport.
|Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2017). Loss of Huntingtin stimulates capture of retrograde dense-core vesicles to increase synaptic neuropeptide stores. Eur J Cell Biol [Epub ahead of print]. PubMed ID: 28129919
The Huntington's disease protein Huntingtin (Htt) regulates axonal transport of dense-core vesicles (DCVs) containing neurotrophins and neuropeptides. DCVs travel down axons to reach nerve terminals where they are either captured in synaptic boutons to support later release or reverse direction to reenter the axon as part of vesicle circulation. Currently, the impact of Htt on DCV dynamics in the terminal is unknown. This study reports that knockout of Drosophila Htt selectively reduces retrograde DCV flux at proximal boutons of motoneuron terminals. However, initiation of retrograde transport at the most distal bouton and transport velocity are unaffected suggesting that synaptic capture rate of these retrograde DCVs could be altered. In fact, tracking DCVs shows that retrograde synaptic capture efficiency is significantly elevated by Htt knockout or knockdown. Furthermore, synaptic boutons contain more neuropeptide in Htt knockout larvae even though bouton size, single DCV fluorescence intensity, neuropeptide release in response to electrical stimulation and subsequent activity-dependent capture are unaffected. Thus, loss of Htt increases synaptic capture as DCVs travel by retrograde transport through boutons resulting in reduced transport toward the axon and increased neuropeptide in the terminal. These results therefore identify native Htt as a regulator of synaptic capture and neuropeptide storage.
Huntingtin is a cytoplasmic protein; its functions are as yet undetermined. In mice, deletion of the huntingtin gene results in early embryonic lethality, whereas later deletion of huntingtin by conditional mutagenesis causes neuronal degeneration (Dragatsis, 2000; Duyao, 1995; Nasir, 1995; Zeitlin, 1995). In rat sciatic nerve axons, huntingtin is transported in both anterograde and retrograde pathways (Block-Galarza, 1997). Immunohistochemical studies in human and rat brains reveal cytoplasmic huntingtin within neurons, and biochemical analysis indicates that huntingtin is enriched in compartments containing vesicle-associated proteins (DiFiglia, 1995; DiFiglia, 1997). Huntingtin interacts with many proteins, including nuclear, transcriptional, and signaling proteins (Cattaneo, 2001; Freiman, 2002). One protein of particular interest is the huntingtin-associated protein 1 (HAP1; Li, 1996). Although its function is currently unknown, HAP1 is also transported in both anterograde and retrograde pathways (Block-Galarza, 1997) and is found associated with vesicle membranes in synaptosomal fractions, indicating that the HAP1 interaction with huntingtin may occur within axons (Engelender, 1997). Additionally, HAP1 strongly associates with p150Glued, a critical component of the dynein-based transport system (Engelender, 1997; Li, 1998). Recent work suggests a role for a Drosophila HAP1-like protein in kinesin-dependent transport of mitochondria (Stowers, 2002). Together, these findings lead to the still untested suggestion that huntingtin has an important function in the axonal transport machinery itself (Gunawardena, 2003 and references therein).
In Huntington's disease (see Drosophila as a Model for Human Diseases: Huntington's disease), effecting the brain, aggregates of mutant huntingtin are observed in nuclear inclusions and in dystrophic neurites (DiFiglia, 1997; Becher, 1998). In HD transgenic mice, N-terminal huntingtin fragments and their aggregates initially accumulate in striatal neurons, and later these neurons form aggregates in axonal processes and terminals (Li, 2000). Neuropil aggregates have been observed in the striatum in the lateral globus pallidus (LGP), a region into which medium spiny neurons project. How a function that may normally be associated with cytoplasmic vesicles can contribute to nuclear dysfunction and whether this reflects a normal nuclear signaling role of huntingtin is unknown. A testable possibility is that (1) normal huntingtin has a role in axonal transport and (2) mutant huntingtin causes neuronal dysfunction by poisoning vesicular transport within neurons, which can ultimately contribute to neurodegeneration. This hypothesis has now been tested in vivo in Drosophila (Gunawardena, 2003 and references therein).
Huntington's disease (HD) is one of nine neurodegenerative diseases that result from the expansion of CAG repeats leading to proteins containing abnormally long polyQ tracts. Although little is known about the mechanism by which polyQ expansion leads to pathogenesis, it has been proposed that misfolding of the mutant protein triggers a cascade of events, ultimately causing disease. The misfolded protein may undergo proteolytic cleavage, interact with other proteins, self-aggregate, and in many cases, translocate into the nucleus. Indeed, a common characteristic of all polyQ diseases is the formation of nuclear or cytoplasmic and axonal or dendritic inclusions of the disease protein (Li, 2000; Li, 2001; Piccioni, 2002; Paulson, 1997; Becher, 1998; Ishikawa, 1999). In the nucleus, aggregated polyQ proteins have been suggested to recruit transcription factors, caspases, and molecular chaperones and other proteins, which may stimulate apoptosis (Gunawardena, 2003 and references therein).
This study finds that wild-type version of Drosophila huntingtin (htt) is needed for normal axonal transport. In addition, pathogenic polyQ proteins alone, or in the context of human htt exon 1 or in the context of another polyQ disease protein Machado-Joseph disease (MJD), also known as spinocerebellar ataxia 3 (SCA3), can interfere with the axonal transport machinery and cause neuronal apoptosis and organismal death. Thus, these findings demonstrate that pathogenic polyQ proteins can poison the axonal transport system and support the proposal that defects in axonal transport may contribute to neuronal failure in HD and other polyQ expansion diseases (Gunawardena, 2003).
An important concern in interpreting these data is whether the transport failures observed are caused by direct poisoning of the transport machinery by pathogenic polyQ proteins, or if it is an indirect consequence in neurons that have become sick or are dying from other causes. There are four strong arguments that support the interpretation that pathogenic polyQ proteins themselves poison the transport machinery leading to neuronal failure. (1) Two different screens carried out for axonal transport mutants suggest that this phenotype is relatively rare. In one screen, only 4 out of 12,000 mutagenized chromosomes exhibited a phenotype diagnostic of transport abnormalities, namely abnormal larval motility combined with organelle accumulations in axons (Bowman, 1999; Bowman, 2000). In another screen, 446 out of 13,000 mutants exhibited the larval motility phenotype. 114 of these were tested for organelle accumulations in axons, but only 3 were found to exhibit this phenotype. In this context, among mutations where the gene product is known, only mutations in genes encoding motor protein subunits, or genes encoding proteins where there is strong evidence to support a role in the transport machinery, cause this phenotype. (2) An overexpression screen using EP elements yielded 36 lines that had the larval motility phenotype out of 2300 tested when driven with 179Y-GAL4; only 3 of these had organelle accumulations within their axons. (3) Overexpression of GFP, or of any protein lacking the C terminus of APP relatives (that is thought to interact with the transport machinery), did not cause a transport phenotype (Gunawardena, 2001). In the current analysis of proteins implicated in polyQ expansion diseases, similar phenotypic selectivity was observed. Proteins with short polyQ regions do not cause transport failures unless motor protein dosage is reduced. (4) Induction of neuronal apoptosis by excess expression of the cell death gene reaper failed to cause axonal transport problems. Expression of MJD-65QNLS, which primarily targets to the nucleus and induces apoptosis, also did not cause transport failures. Thus, cell death or sick cells do not generally cause axonal transport defects (Gunawardena, 2003).
If expression of pathogenic polyQ proteins directly causes transport failures, what might be the mechanism? One possibility is that aggregation of pathogenic polyQ proteins in narrow axons can physically impair transport of large organelles or vesicles. While attractive, this possibility on its own does not easily account for the observation that polyQ proteins with repeat lengths that are not pathogenic can cause transport failures when combined with reductions in motor protein gene dose (e.g., MJD-27Q). In addition, recent work argues that aggregation per se may not be required for neuronal toxicity (Klement, 1998; Yoo, 2003). A second possibility is that titration of motor proteins from other critical pathways induces vesicle stalling during transport in narrow axons, which can nucleate organelle accumulations that block subsequent transport. While this possibility accounts for the observed ability of pathogenic polyQ proteins to significantly reduce the soluble pool of motor proteins, it does not easily account for the ability of short polyQ repeats to titrate motor proteins without causing transport failure. The third possibility, which is favored, is that motor protein titration and a propensity to aggregate and physically block transport in narrow axons act in concert to poison axonal transport. This mechanism, while more complex than the others, best accounts for a number of important observations including those that are not easily explained by the simplest models. For example, this mechanism accounts for the observed ability of short polyQ repeats to titrate soluble motor proteins but to poison the transport machinery only when motor protein gene dosage is further reduced. It is also consistent with the observation that motor protein gene dosage generally needs to be reduced by more than 50% to cause significant transport phenotypes. This mechanism also accounts for observations (Piccioni, 2002; Li, 2000; Li, 2001) that expression of polyQ proteins in neurons causes axonal inclusions that contain the polyQ proteins themselves. EM examination of the ultrastructure of these inclusions in a mouse model of Huntington's disease reveals a morphology of accumulated vesicles, organelles, and distended axons that is virtually identical to what was observe in axonal blockages formed in the Drosophila system. Similarly, axonal abnormalities, perinuclear and nuclear accumulations, with severe dysfunction in mechanosensory neurons were observed in C. elegans expressing pathogenic polyQ repeats in the context of htt (Parker, 2001). In addition, axonopathies are prominent in a number of polyQ expansion diseases (Li, 2000), and this view also accounts for the beneficial effects of chaperone increases upon transport phenotypes. Live analysis also shows the accumulation of YFP-tagged vesicles into nonmotile aggregates in axons of larvae expressing expanded polyQ repeats. This mechanism would also suggest that there is a phenotypic continuum caused by motor protein reductions or physical aggregation where the burden of either one alone, if substantial enough, can cause phenotypes. However, the relative extent to which axonal blockages contribute to transport failure as compared to motor protein depletion needs to be investigated further. It is also noted that this proposal has the virtue of providing a plausible explanation for the otherwise puzzling ability of broadly expressed proteins to cause neuron-specific toxicity in human disease. Thus, it is conceivable that defects in axonal pathways may contribute to early disease neuropathology (Gunawardena, 2003).
This study provides direct evidence that Htt is required for normal axonal transport. Neuronal depletion of Drosophila htt using RNAi caused an axonal blockage phenotype, which is characteristic of mutations not only in cytoskeletal motor proteins that are required for axonal transport, but also in proteins that function as receptors for motors (Bowman, 2000; Gunawardena, 2001). These axonal blockages were enhanced by a 50% reduction in kinesin. Loss of htt in the eye caused a distinct degenerative phenotype similar to what has been observed in weak mutations of DLC (Bowman, 1999), DIC, and heterozygous dominant mutations of p150Glued (Boylan, 2000). Until now, previous work has only hinted at a possible transport function for Htt but provided no direct evidence in support of this important proposal. For example, mouse models of HD and conditional Htt knockout mice all exhibited degeneration of axon fibers, compatible with, but not establishing, a function for Htt in axonal transport (Dragatsis, 2000; DiFiglia, 1997; Hodgson, 1999; Li, 2000). Thus, the current data together with Htt localization data strongly support a functional role for htt in fast axonal transport. Although how Htt associates with the axonal transport machinery is still unclear, it is proposed that a subclass of vesicles containing Htt may associate with motor proteins via HAP1, or a similar protein, which establishes the link between Htt and p150Glued, thereby enabling transport. Although a true Drosophila homolog of HAP1 has yet to be identified, Drosophila Milton is related to HAP1 and has been suggested to be required for kinesin-dependent transport of mitochrondria (Stowers, 2002). In addition, many coiled-coil linker proteins exist that could facilitate this connection (Gunawardena, 2003).
The proposal that Htt is required for axonal transport explains why both reduction and gain of function cause similar phenotypes, since both can lead to failures of vesicle transport that might physically and biochemically cause organelle blockages in axons. For example, in mouse models, both loss (Dragatsis, 2000) and gain of function of htt causes neurodegeneration (Mangiarini, 1996; DiFiglia, 1997; Li, 2000) and axonal pathology (Sapp, 1999; Li, 2000; Li, 2001). It is striking that in the Drosophila system too, both loss of htt function and polyQ-induced gain of function cause similar axonal blockage phenotypes including neurodegeneration in the adult eye, which may result due to disruption of a specialized neuronal pathway. Both processes could contribute to the observed reduced trafficking of the neurotrophic factor BDNF in mouse HD brains (Zuccato, 2001). Indeed, HD is a dominantly inherited disease with both homozygous and heterozygous individuals affected similarly by a gained toxic function (Gunawardena, 2003).
An important point of controversy is whether neuronal toxicity in HD and other polyQ diseases results from nuclear or cytoplasmic events. PolyQ-induced disease pathogenesis can occur via two mechanisms -- one that induces apoptosis by nuclear accumulation, and the other that induces neuronal dysfunction by disrupting axonal transport -- although these two pathways may not be mutually exclusive. PolyQ-induced neuronal death did not result upon expression of a pathogenic polyQ protein restricted to the cytoplasm by the addition of a nuclear export signal (NES), although axonal blockages formed and organismal death resulted. In contrast, expression of a nuclear-targeted polyQ protein (NLS) caused nuclear accumulations, apoptosis, and lethality. These findings suggest that translocation of polyQ protein into the nucleus is required for cell death and that cytoplasmic polyQ proteins can cause axonal blockage. In fact, similar to the situation of polyQ proteins without an NES, a 50% reduction in kinesin-enhanced organelle blockages and a 50% reduction in dynein caused early organismal lethality of polyQ-NES expressing animals. Taken together, these data support two pathways for pathogenesis by polyQ proteins. In the first, polyQ-induced cytoplasmic perturbations in axonal transport pathways could directly instigate neuronal failure and organismal death. In the second, accumulation of pathogenic polyQ proteins within the nucleus (perhaps enhanced by axonal blockages) and events triggered by the nuclear presence of pathogenic polyQ protein could trigger neuronal death, neuronal degeneration, and finally organismal death. These ideas are consistent with recent findings on the androgen receptor, which, when expanded by a polyQ stretch in the N-terminal A/B domain, causes spinal and bulbar muscular atrophy (SBMA), an X-linked, adult-onset neurodegenerative disorder. While expression of expanded polyQ repeats in the context of the androgen receptor also causes neuropil aggregates and alters the distribution of kinesin (Piccioni, 2002), abnormal binding of the ligand, androgen, to polyQ-expanded human androgen receptor causes neurodegeneration due to ligand-dependent structural alteration that promotes nuclear translocation (Takeyama, 2002). Thus, it is possible that pathogenic polyQ proteins cause polyQ-induced cytoplasmic accumulations and these accumulations may promote abnormal protein-protein interactions that could trigger a cascade of toxic events, ultimately leading to neurodegeneration and organismal death. Indeed, a recent study (Kayed, 2003) suggests that soluble oligomers, which are common to most aggregate forming diseases, may be cytotoxic (Gunawardena, 2003).
Both polyQ toxicity (Warrick, 1999; Kazemi-Esfarjani, 2000) and alpha-synuclein toxicity (Auluck, 2002) observed in the Drosophila adult eye and brain are dramatically modulated by excess chaperones. It is conceivable that polyQ toxicity within axons is also modulated by chaperones. Indeed, axonal blockages and neuronal cell death are completely suppressed by excess HSC70 together in transgenic lines expressing expanded polyQ repeats, although organismal lethality still persists. The chaperone interaction with misfolded mutant polyQ protein may prevent abnormal interactions with motor proteins and other proteins, thereby preventing organelle blockages within axons and neuronal death. Consistently, cytoplasmic axonal aggregations caused by excess polyQ repeats with NES are also suppressed by chaperone expression, although organismal lethality still occurs. Lethality, however, may result due to the fact that although chaperones are modulating abnormal or misfolded proteins, they may be unable to completely prevent the toxic activity of abnormal aggregations of disease protein in time for normal development to proceed (Gunawardena, 2003).
Protein aggregation appears to be a common manifestation in many neurodegenerative diseases, and increasing evidence suggests that such accumulations can be a major trigger of cellular stress and neuronal death (Wyss-Coray, 2002). In Alzheimer's disease, accumulation of the 4 kDa Aß fragment in amyloid plaques and aggregation of phosphorylated Tau in neurofibillary tangles is observed surrounded by degenerating neurites; deposits of aggregated prion proteins with amyloid-like structures are observed in mad cow or Creutzfeld-Jacobs disease; in Parkinson's disease, abnormal alpha-synuclein accumulations known as Lewy bodies are seen; as well, in HD and other expanded polyQ diseases, abnormal accumulations of mutant protein are observed as nuclear and sometimes axonal inclusions. The widespread occurrence of axonal (or dendritic) inclusions leads to the proposal that perturbations in transport could be a common pathway in neurodegenerative disease. In support of this idea, recent findings indicate that dynein (Hafezparast, 2003) and dynactin (Puls, 2003) mutations can induce motor neuron degeneration in mice and humans (Gunawardena, 2003).
In this context, the strongest evidence comes from HD, which is characterized by the preferential loss of striatal neurons. Strikingly, htt accumulations are found in axons of striatal projection neurons (Li, 2001), and it has been argued that these striatal axonal inclusions are better correlated with striatal neuron loss than the presence of nuclear inclusions. Expression of expanded polyQ repeats in the context of the androgen receptor also forms neuropil aggregates and alters the distribution of kinesin (Piccioni, 2002), further supporting the idea that early pathology can occur within axonal processes together with axonal inclusions. It is possible that wild-type htt is required for efficient vesicle trafficking of cortical BDNF, since mutant htt interfers with its anterograde transport, contributing to BDNF depletion in the striatum (Cattaneo, 2001). The importance in transport of neurotrophic factors is also evident in Alzheimer's disease, where one of the earliest detectable signs of disease is the loss of synapses and retrograde degeneration of neurons, accompanied by the decay of intracellular traffic (Terry, 2000). In addition, excess of APP proteins containing the toxic Aß region perturbs axonal transport pathways and causes neuronal cell death (Gunawardena, 2001). Thus, it is proposed that perturbation in axonal transport can contribute to early disease pathology owing to disruption in proper transport of essential neuronal components, triggering a cascade of events leading to neuronal failure and death (Gunawardena, 2003).
The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. This study establishes a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. It was found that glia regulates steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery. Remarkably, some of these engulfed neuronal Htt aggregates effect prion-like conversion of soluble, wild-type Htt in the glial cytoplasm. The study provided genetic evidence that this conversion depends strictly on the Draper signalling pathway, unveiling a previously unanticipated role for phagocytosis in transfer of pathogenic protein aggregates in an intact brain. These results suggest a potential mechanism by which phagocytic glia contribute to both protein aggregate-related neuroprotection and pathogenesis in neurodegenerative disease (Pearce, 2015).
This study establishes a Drosophila model that demonstrates an essential role for phagocytic glia in clearance of Htt aggregates from ORN axons undergoing Wallerian degeneration after antennal axotomy or from non-severed axons. It was shown that HttQ91 aggregate clearance is mediated by a pathway that requires the glial engulfment receptor Draper and downstream genes, and is genetically indistinguishable from the pathway that operates in other glial phagocytic processes, including clearance of axonal or cellular debris following injury and axon pruning during development. Surprisingly, it was found that HttQ91 aggregates taken up by this Draper-dependent process are able to access and initiate a prion-like conversion of normally soluble, cytoplasmic HttQ25 in glia. These two findings have important implications for the potential role of glia in the suppression and/or progression of neurodegenerative diseases (Pearce, 2015).
Glial phagocytosis plays an important neuroprotective role in response to many types of brain injury, including insults associated with the production of neurodegenerative disease-linked insoluble protein aggregates. Secretion of pro-inflammatory cytokines and opsonins by activated glia promotes phagocytic clearance of damaged neurons, neuronal processes and cellular debris. In vitro, mouse astrocytes can bind to and degrade extracellular Aβ aggregates in cell culture and in brain slices. In vivo, pharmacological activation of microglia promotes clearance of Aβ deposits in a transgenic mouse model of Alzheimer disease (AD), and antibodies against Aβ or α-synuclein promote microglial-mediated phagocytic removal of the corresponding extracellular aggregates. Compelling evidence for a neuroprotective role for glial phagocytosis has come from recent studies linking missense mutations in TREM2, which encodes a microglial phagocytic receptor, to several neurodegenerative diseases including AD, Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) (Pearce, 2015).
In this study, an essential role for phagocytic glia in clearance
of HttQ91 aggregates from ORN axons was established, but whether
this phagocytosis is initiated as a specific response to the
presence of aggregates or a collateral result of constitutive axon
turnover and remodelling is unclear. This strict dependence on
phagocytosis contrasts with previous work showing that
extracellular aggregates can enter the cytoplasms of many
different types of cultured cells, and cell surface proteins are
only partially responsible for this entry. It is likely that the
discrepancy between these previous studies and the current one at
least in part reflects differences in the extracellular
environment in cell culture, which is homogeneous and effectively
infinite, and in the intact brain, where extracellular space is
severely restricted in volume and is continuously patrolled by
phagocytic glia. This view is supported by observations that, in
the fly brain, aggregate uptake by glia occured only in close
proximity to ORN axons containing HttQ91 aggregates and that
detergent solubilization was required to immunolabel the
aggregates. This study therefore favors a mechanism in which
HttQ91 aggregates are phagocytosed by glia together with
surrounding axonal membrane, analogous to the process by which
supernumerary synapses are eliminated by Draper in Drosophila
development and by the mammalian Draper orthologue, MEGF10, in the
adult mouse brain (Pearce, 2015).
The observation that phagocytosed neuronal HttQ91 aggregates could nucleate aggregation of cytoplasmic HttQ25 in glia is surprising because phagocytosis normally leads to encapsulation and degradation of internalized debris within the membrane-enclosed phagolysosomal system. However, two independent lines of evidence were provided to support the conclusion that ORN-derived HttQ91 aggregates encounter HttQ25 in the glial cytoplasm. First, because HttQ25 is a highly soluble protein that does not aggregate in cells unless seeded by a pre-existing aggregate, the strict dependence of HttQ25 puncta formation on HttQ91 expression in ORNs indicates that these two Htt species must have physically interacted with one another. In principle, it is possible that, instead of HttQ91 aggregates being internalized by glia, this transfer could occur in the opposite direction, namely by the transfer of soluble HttQ25 into ORNs. However, the finding that HttQ25 aggregation was blocked by glial-specific knockdown of Draper and enhanced by antennal injury indicates an absolute requirement for phagocytic uptake of HttQ91, supporting ORN-to-glia transfer (Pearce, 2015).
Second, the majority of HttQ91 and HttQ25 aggregates co-localized with the cytoplasmic chaperones Hsp70/Hsc70 and Hsp90, indicating that aggregated HttQ25 is in direct contact with the cytoplasm. It is conceivable that merging of the phagocytic and autophaghic pathways could provide an opportunity for HttQ91 aggregates and soluble HttQ25 to encounter one another. However, substantial (<15%) co-localization of either HttQ91 or HttQ25 puncta with the autophagy markers, Atg8 and p62, or with the early endosome marker, Rab5 was not detected. Moreover, the amount of soluble HttQ25 that could be captured within an autophagosome was miniscule compared with the total cytoplasmic pool that would be available to be nucleated by an internalized HttQ91 seed. Altogether, these data strongly support the conclusion that phagocytosed HttQ91 aggregates are able to access the glial cytoplasm, thereby affording the opportunity to effect prion-like spreading of disease pathology (Pearce, 2015).
How do these engulfed HttQ91 aggregates breach the membrane barrier that separates the phagolysosomal lumen from the cytoplasm? The dependence of this process on Draper, shark and the actin-remodelling complex indicates that aggregates must access the glial cytoplasm at a step during or subsequent to phagocytic engulfment. It is possible that cytoplasmic entry can be facilitated by interference of phagocytosed aggregates with membrane fusion events during phagosome maturation. However, this ‘foot-in-the-door’ mechanism would be favoured by larger aggregates, which is in opposition to the finding that smaller HttQ91 aggregates were more strongly associated with cytoplasmic nucleation of glial HttQ25. It is proposed instead that slow or inefficient completion of membrane fusion events could provide a temporary conduit to the cytoplasm for HttQ91 aggregates. Such a mechanism would predict an upper size limit for cytoplasmic entry that could be exploited by small, newly formed aggregates. This prediction was supported by the finding that neither the induced HttQ25 aggregates nor the co-localized nucleating HttQ91 puncta in glia were labelled with antibodies to ubiquitin, a marker previously identified with more mature, larger Htt puncta in both cell culture and transgenic mouse models of HD. (Pearce, 2015).
The findings described in this study have broader implications for how potentially toxic protein aggregates are dispersed throughout the diseased brains. Phagocytic removal of aggregates is neuroprotective, but it is likely that phagocytes become impaired in their ability to clear debris as the disease worsens and that chronic glial activation becomes detrimental to the health of nearby neurons. The finding that phagocytosed neuronal Htt aggregates can enter the glial cytoplasm suggests that this transfer process could generate a reservoir of prion-like species inside glia, possibly facilitating their spread to other cells. A growing body of evidence supports the view that glial dysfunction exacerbates neurodegenerative disease pathogenesis by influencing the survival of neurons, and determining the mechanism(s) by which glia contribute to toxicity will be of great value to the development of therapeutic strategies to combat these devastating disorders (Pearce, 2015).
A key feature of many neurodegenerative diseases is the accumulation and subsequent aggregation of misfolded proteins. Recent studies have highlighted the transcellular propagation of protein aggregates in several major neurodegenerative diseases, although the precise mechanisms underlying this spreading and how it relates to disease pathology remain unclear. This study uses a polyglutamine-expanded form of human huntingtin (Htt) with a fluorescent tag to monitor the spreading of aggregates in the Drosophila brain in a model of Huntington’s disease. Upon expression of this construct in a defined subset of neurons, it was demonstrated that protein aggregates accumulate at synaptic terminals and progressively spread throughout the brain. These aggregates are internalized and accumulate within other neurons. The Htt aggregates cause non–cell-autonomous pathology, including loss of vulnerable neurons that can be prevented by inhibiting endocytosis in these neurons. Finally, the release of aggregates requires N-ethylmalemide–sensitive fusion protein 1, demonstrating that active release and uptake of Htt aggregates are important elements of spreading and disease progression (Babcock, 2015).
The ability of misfolded proteins to aggregate and spread throughout the brain has major implications for neurodegenerative diseases. However, there are still many unanswered questions regarding how spreading occurs and its consequences for disease progression. This study demonstrates that mutant huntingtin aggregates spread throughout the Drosophila brain. Although aggregates initially accumulate at ORN synaptic terminals in the antennal lobe, over time these aggregates are distributed more broadly to the far posterior and lateral regions of the brain. After release from ORN terminals, it was found that Htt aggregates become internalized in other populations of neurons. The most prominent accumulation was in a pair of large, possibly peptidergic neurons in the posterior protocerebrum (Babcock, 2015).
Selective vulnerability of particular neurons is a common feature of many neurodegenerative diseases, including HD. In HD there is a lack of correlation between neurons in which aggregates accumulate and neuronal loss. For example, striatal spiny projection neurons are particularly vulnerable in HD, yet these neurons accumulate far fewer aggregates than striatal interneurons. A similar outcome was observed in this study: neurons labeled with the nb169 monoclonal antibody accumulate Htt aggregates but they do not seem vulnerable to cell death. In contrast, neighboring neurons that express the R44H11-LexA driver are lost within 10 d after eclosion. One possible explanation for this discrepancy is that the most vulnerable neurons simply are not viable long enough to accumulate a quantity of Htt aggregates. Therefore, the only neurons where accumulation of aggregates can be seen in abundance are those that are most resistant to the toxic effects of the aggregates. Whereas the underlying cause of this selective vulnerability remains unknown, some leading ideas include differences in the microenvironment, metabolic activity, and translational machinery between neuronal populations (Babcock, 2015).
One striking result was that loss of the R44H11-LexA–expressing GFP+ neurons is prevented by blocking endocytosis in these cells. This suggests that Htt.RFP protein is actively internalized by target neurons. Transmission of α-synuclein between cells in culture also depends on endocytosis, demonstrating that there may be some similarities between various pathogenic proteins in mechanism of transfer. Although large aggregates in R44H11-LexA–expressing cells before loss of these neurons were not observed, it is possible that monomers or oligomers are transmitted, which would be difficult to detect. This possibility is also consistent with previous results, demonstrating that both aggregates and more soluble forms of the protein are likely pathogenic (Babcock, 2015).
Understanding the cellular pathways involved in spreading of pathogenic proteins is an important next step because of its potential impact on therapeutic intervention. Although there is abundant evidence that spreading occurs through synaptic connections, other potential mechanisms include spreading between cells via exosomes or tunneling nanotubes. In the current study, unique patterns of spreading were found when mutant Htt is expressed in different subsets of neurons in the brain. This observation supports the idea that transcellular spreading is more likely to involve neurons in close proximity or within the same circuit as those containing aggregates. However, rapid accumulation of Htt aggregates throughout the brain when expressed in olfactory receptor neurons suggests that synaptic connections are not solely responsible for the observed spreading. In addition to transneuronal spreading, mutant Htt aggregates have also recently been shown to spread to nearby phagocytic glia and are responsible for the prion-like conversion of soluble wild-type Htt. Although these glia provide a neuroprotective role through clearance of extracellular aggregates, they may also contribute to disease pathogenesis by spreading the aggregates themselves (Babcock, 2015).
Release of Htt aggregates was shown to require both NSF1 and dynamin, suggesting that SNARE-mediated fusion events play an important role in the spreading of pathology. This is consistent with previous data revealing that tetanus toxins targeting components of the synaptic vesicle fusion machinery block spreading of aggregates in culture. Although inhibition of NSF1 or dynamin significantly limits the spreading, it is not blocked completely. One possible reason for this is that normal protein function is not completely eliminated by genetic manipulation done in this study. Alternatively, spreading of protein aggregates may also operate via additional mechanisms independent of SNARE-mediated fusion events such as release from dead or damaged cells. By use of a candidate gene approach as well as unbiased genetic screens in Drosophila, it should now be possible to identify additional modifiers that regulate spreading of Htt aggregates in vivo (Babcock, 2015).
It was demonstrated that whereas polyglutamine-expanded huntingtin aggregates can spread throughout the brain in Drosophila, polyglutamine-expanded ataxin-3 lacks this property. Furthermore, there is a distinction between the spreading capacities of both a 588-aa fragment of Htt and an 81-aa fragment containing only exon 1. The lack of spreading seen using the exon 1 fragment suggests that specific regions of the protein are required for transmission throughout the brain. These differences should help to identify properties of aggregate-prone proteins that influence the ability to spread and also highlight the need to consider specific forms of proteins used when modeling these diseases. Differences among various disease-associated, aggregate-prone protein in their ability to spread from cell to cell may depend on the type of aggregates they form or the cell type in which they are first expressed. By taking advantage of Drosophila to characterize spreading of other aggregate-prone proteins, it should now be possible to define the precise cellular and molecular mechanisms that are responsible and to determine why some proteins are more likely to undergo spreading (Babcock, 2015).
Huntington's disease (see Drosophila as a Model for Human Diseases: Huntington's disease) is an autosomal dominant neurodegenerative disorder. Disease alleles contain a trinucleotide repeat expansion of variable length, which encodes polyglutamine tracts near the amino terminus of the HD protein, huntingtin. Polyglutamine-expanded huntingtin, but not normal huntingtin, forms nuclear inclusions. A Drosophila model for HD is described. Amino-terminal fragments of human huntingtin containing tracts of 2, 75, and 120 glutamine residues were expressed in photoreceptor neurons in the compound eye. As in human neurons, polyglutamine-expanded huntingtin induces neuronal degeneration. The age of onset and severity of neuronal degeneration correlates with repeat length, and nuclear localization of huntingtin presages neuronal degeneration. In contrast to other cell death paradigms in Drosophila, coexpression of the viral antiapoptotic protein, P35, did not rescue the cell death phenotype induced by polyglutamine-expanded huntingtin (Jackson, 1998).
At least eight dominant human neurodegenerative diseases are due to the expansion of a polyglutamine within the disease proteins. This confers toxicity on the proteins and is associated with nuclear inclusion formation. Recent findings indicate that molecular chaperones can modulate polyglutamine pathogenesis, but the basis of polyglutamine toxicity and the mechanism by which chaperones suppress neurodegeneration remains unknown. In a Drosophila disease model, it has been demonstrated that chaperones show substrate specificity for polyglutamine protein, as well as synergy in suppression of neurotoxicity. This analysis also reveals that chaperones alter the solubility properties of the protein, indicating that chaperone modulation of neurodegeneration in vivo is associated with altered biochemical properties of the mutant polyglutamine protein. These findings have implications for these and other human neurodegenerative diseases associated with abnormal protein aggregation (Chan, 2000).
Analysis of chaperone suppression was extended to the Huntingtons disease protein, Huntingtin. To do this, Hsp70, dHdj1 or dHdj2 were coexpressed with a truncated form of Huntingtin containing an expanded polyglutamine domain of 120 (Htt-Q120). Flies expressing Htt-Q120 show normal eyes at 1 day, but a severely degenerate eye structure by 10 days. On co-expression of chaperones, Hsp70 was found to strongly suppress neuronal degeneration induced by Htt-Q120, restoring a normal photoreceptor rhabdomere structure. However, dHdj1 showed partial and dHdj2 showed no ability to suppress neurodegeneration induced by mutant Huntingtin. These results demonstrate broad effects of Hsp70 on suppression of polyglutamine toxicity and further emphasize substrate specificity among the Hsp40 class of chaperones (Chan, 2000).
Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship. The potential for a relationship between huntingtin and Dorsal was tested by overexpression experiments in Drosophila S2 cells. Drosophila Huntingtin complexes with dorsal via its carboxyl-terminal region, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, it has been found that the carboxyl-terminal segment of huntingtin that mediates the association with Dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein. It is concluded that like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment (Takano, 2002).
Huntington disease is caused by the expansion of a polyglutamine repeat in the Huntingtin protein (Htt) that leads to degeneration of neurons in the central nervous system and the appearance of visible aggregates within neurons. Suppressor polypeptides, containing two polyglutamine repeats separated by a spacer, were developed and tested that bind mutant Htt and interfere with the process of aggregation in cell culture. In a Drosophila model, the most potent suppressor inhibits both adult lethality and photoreceptor neuron degeneration. The appearance of aggregates in photoreceptor neurons correlates strongly with the occurrence of pathology, and expression of suppressor polypeptides delays and limits the appearance of aggregates and protects photoreceptor neurons. These results suggest that targeting the protein interactions leading to aggregate formation may be beneficial for the design and development of therapeutic agents for Huntington disease (Kazantsev, 2002).
Pathogenic polyQ proteins cause axonal transport defects and neuronal apoptosis:
To address whether axonal transport defects are selective to the pathogenic Htt protein, or whether they are a feature of polyQ proteins in general, the effects were examined on axonal transport of various proteins containing polyQ tracts of different lengths and in different contexts. 179Y-GAL4 and APPL-GAL4 were crossed to lines encoding proteins with either a 'normal' length, nondisease-causing polyQ repeat region or proteins with an expanded, disease-causing polyQ repeat region. These proteins consisted either entirely of polyQ repeats (20Q, 22Q, 108Q, 127Q; Marsh, 2000; Kazemi-Esfarjani, 2000) or polyQ repeats embedded in the C-terminal region of the polyQ disease protein Machado-Joseph disease (MJD) protein (MJD-27Q, MJD-78Q; Warrick, 1998). Proteins with normal length polyQ regions (22Q, MJD-27Q) were found to be present within axons, based upon the smooth staining seen with either an antibody against polyQ or an antibody against the HA tag, and these proteins were found to accumulate at neuromuscular junctions, suggesting that they are normally transported within larval axons. In contrast, proteins with expanded polyQ repeats (MJD-78Q, 127Q) exhibited prominent polyQ staining within organelle blockages, while reduced staining was observed at the neuromuscular junctions, suggesting impaired transport of pathogenic polyQ proteins (Gunawardena, 2003).
The extent of axonal accumulations induced by polyQ repeats was length dependent, since a correlation was observed between the number of polyQ repeats and the amount of axonal accumulations. Larvae expressing 20Q, 22Q, or MJD-27Q were similar to wild-type in that they exhibited no axonal accumulations. Larvae expressing the pathogenic proteins MJD-78Q, 108Q, or 127Q exhibited a severe sluggish larval movement phenotype, with prominent axonal accumulations observed in all instances. The expression of MJD-78Q and 127Q at 29°C was also observed to be very toxic such that larvae expressing these proteins never survived to adulthood and died at second or third instar larval stage. Western blot analysis ruled out a general expression difference between proteins with normal length polyQ repeats and proteins with expanded polyQ repeats since, if anything, more MJD-27Q expression was observed compared to MJD-78Q. To reduce the level of toxicity, the amount of MJD-78Q and 127Q made was reduced by growing animals at 25°C (GAL4 activity is temperature dependent). Organelle accumulations were still reduced although these larvae now survived much longer, dying at early pupal stages (Gunawardena, 2003).
To confirm the results seen by immunofluorescent staining, EM analysis was conducted on larvae expressing 127Q and on severe genotypes in which expression of MJD-78Q or 127Q was combined with a 50% reduction in KHC gene dose. Prominent axonal blockages were observed characteristic of those observed in homozygous mutations of motor protein genes. Mutant larval nerves also contained enlarged axons, some almost four or five times the diameter of those observed in wild-type. Sometimes 'holes' were observed lacking organelles within the nerve, perhaps indicative of degeneration (Gunawardena, 2003).
To test directly whether pathogenic polyQ proteins block transport by inducing nonmoving blockages in axonal processes, live analysis was performed of vesicular movement within whole-mount larval axons. YFP-tagged human amyloid precursor protein (APP-YFP) was expressed either in the presence or absence of MJD-78Q, using the GAL4 driver pGAL4-62B SG26-1, which is expressed in only a small population of motor neurons. Neurons expressing only APP-YFP contained many actively motile vesicles moving at velocities of approximately 1 microm/s; large bright nonmotile accumulations such as those seen in the presence of MJD-78Q were never observed. Neurons coexpressing MJD-78Q and APP-YFP revealed nonmoving large, bright aggregates of APP-YFP. Thus, polyQ expression can interfere with transport of APP-YFP vesicles (Gunawardena, 2003).
TUNEL analysis was used to test whether proteins with expanded polyQ repeats can induce neuronal apoptosis. A large increase in neuronal apoptosis was observed in lines expressing MJD-78Q and 127Q, but not in lines expressing the nonpathogenic control proteins 22Q or MJD-27Q. Anti-polyQ staining revealed obvious nuclear inclusions within larval brain cells. Many stained nuclei were obviously enlarged and may be undergoing apoptosis. Expression during embryonic cycle 14 revealed smooth cytoplasmic staining for MJD-78Q protein, while staining for 127Q revealed obvious punctate cytoplasmic aggregates with some nuclear aggregates. At later cycles, both MJD-78Q and 127Q were observed as punctate cytoplasmic and nuclear aggregates. In addition, embryos expressing both MJD-78Q and 127Q died soon after they hatched into larvae, indicating substantial polyQ toxicity on normal development. Taken together, these results confirm that proteins with expanded polyQ repeats cause axonal transport defects, perhaps by blocking axonal processes by polyQ accumulations, neuronal cell death, and neurodegeneration (Gunawardena, 2003).
It is possible that polyQ proteins bind and deplete critical components of the molecular motor machinery, perhaps via a Drosophila version of HAP1. This hypothesis makes two predictions: (1) genetic reduction of motor protein dosage should worsen the phenotypes caused by proteins with polyQ expansions by further depleting the motor protein supply, and (2) motor protein depletion should be observable with biochemical methods. To test this hypothesis, proteins with expanded polyQ repeats were expressed and levels of dynein and kinesin were reduced. While a 50% reduction in the dose of KHC has no significant phenotype on its own, when combined with pathogenic polyQ repeats, it dramatically enhances the axonal organelle accumulation phenotype. Similarly, while a 50% reduction in the dose of DLC or components of the dynactin complex (p150Glued, Arp1, and dynamitin) also normally have no significant phenotypes on their own, when combined with pathogenic polyQ proteins, these reductions substantially enhance the organismal phenotype leading to early larval lethality. This finding is consistent with the observation that the neuronal APPL-GAL4 driver turns on during embryonic stage 15 as observed by the expression pattern of UAS-GFP. The enhanced lethality precluded analysis of axonal transport in these genotypes. Interestingly, organelle accumulations now appeared in transgenic lines expressing normally nonpathogenic poly Q repeats (22Q and MJD-27Q) with a 50% reduced dose of dynein, consistent with the hypothesis that all of these proteins may titrate motor proteins, but to varying extents. To test directly for motor protein depletion mediated by expression of proteins with expanded polyQ regions, early embryos expressing httex1-20Q, MJD-27Q, MJD-78Q, httex1-93Q, and 127Q, were examined using the early embryonic GAL4 driver da-GAL4, which turns on at the blastoderm stage based on its UAS-GFP expression pattern (Gunawardena, 2003).
Two considerations led to an evaluation of the effects of polyQ proteins on available motor protein pools by assessing soluble levels of motor proteins: (1) if motor proteins are titrated from normal cargoes by binding to large aggregates, it may be difficult to distinguish motor proteins bound to sedimentable cargoes from sedimentable aggregates; (2) it is not possible to measure the amount of each motor protein associated with normal cargoes owing to the lack of information about such cargoes and how such cargoes fractionate relative to polyQ aggregates. Thus, soluble levels of motor proteins were assessed under the hypothesis that aggregated polyQ proteins may bind motor proteins and deplete both soluble and cargo bound pools in parallel (Gunawardena, 2003).
At 6 hr of development, no significant change in the amount of total motor protein present in these embryos was observed. In contrast to the normal amounts of total motor proteins, an obvious reduction was observed in the amount of soluble motor proteins in embryos expressing MJD-27Q, MJD-78Q, and 127Q compared to wild-type embryos (i.e., da-GAL4 alone and yw). The amount of soluble DHC, DIC, p150Glued, KHC, and KLC were reduced, with no change observed in tubulin, actin, HDAC3, and Rab8. However, for reasons that are not clear, syntaxin was upregulated. Similar observations were evident from 12 and 16 hr embryo collections. The effect of httex1-93Q expression on levels of soluble motor proteins was not obvious at 6 hr of development. However, at 18 hr of development, there is an obvious reduction in soluble p150Glued and KLC in embryos expressing httex1-93Q but not httex1-20Q or wild-type. Expression of polyQ proteins in embryos was obvious as detected by anti-HA antibody and confirmed by anti-polyQ antibody. The level of 127Q was difficult to evaluate, perhaps due to the formation of aggregates, and was convincingly observed only after immunoprecipitation with anti-HA antibody. These observations indicate that expanded polyQ proteins can deplete or sequester available soluble motor proteins, perhaps into polyQ aggregates. The high expression level of MJD-27Q in embryos and the observed depletion of soluble motor proteins in these embryos are consistent with the finding that axonal blockages can be observed in larvae expressing MJD-27Q when motor protein gene dose is reduced by 50%. This finding is also consistent with the proposal that motor titration and aggregation may act in concert to poison axonal transport (Gunawardena, 2003).
Enhanced expression of chaperones restores neuronal transport and suppresses cell death caused by pathogenic polyQ proteins
The neurodegenerative adult eye phenotype caused by polyQ expansion proteins in Drosophila is suppressed by excess chaperone proteins (Warrick, 1999). This suppression has been proposed to occur by modulating soluble properties of pathogenic polyQ proteins, by preventing abnormal interactions with other proteins, or by rescuing chaperone depletion (Bonini, 2002). Whether expression of excess HSC70 protein would suppress axonal blockages and neuronal cell death was tested. Expression of UAS-HSC70 using APPL-GAL4 in the presence of MJD-78Q and 127Q restores axonal transport within larval nerves and suppresses neuronal death. PolyQ accumulations were absent within larval nerves, while cytoplasmic and punctate polyQ staining was present within larval brains. However, while these larvae were now able to pupate (expression of MJD-78Q or 127Q alone causes death at second or third instar larval stages), they still failed to eclose, suggesting that polyQ toxicity was still sufficient to cause lethality. Expression of HSC70 by itself did not cause axonal blockages or neuronal cell death. These results suggest that chaperones could 'clear' larval axons of blockages caused by polyQ proteins and suppress cell death within the larval brain, although organismal toxicity was not completely suppressed (Gunawardena, 2003).
Do axonal defects instigate neuronal dysfunction?
The pathogenic polyQ proteins accumulate in both axonal and nuclear inclusions. To dissect the relative contributions of nuclear and axoplasmic inclusions to the phenotype, transgenes were used that expressed proteins that had different subcellular localizations. One transgene encoded a protein with an expanded polyQ repeat with a nuclear localization sequence (MJD-65QNLS). While expression of MJD-65QNLS within the larval brain caused neuronal apoptosis as observed by TUNEL staining, organelle accumulations within larval axons were absent. These larvae pupated but failed to eclose. While reduction in dynein dose by 50% with excess MJD-65QNLS had no effect, reduction in kinesin dose by 50% with excess MJD-65QNLS caused a small number of accumulations, perhaps due to continued motor titration by these proteins even when targeted to nuclei (Gunawardena, 2003).
To test further if dying neuronal cells induce axonal transport defects, the axonal transport phenotype was analyzed of the cell death gene reaper, which also induces neuronal apoptosis. Transport following reaper expression in these genotypes appeared to be normal based on immunostaining with synaptic vesicle markers even though high levels of neuronal apoptosis were induced. In addition, a 50% reduction in KHC combined with excess reaper expression had no effect on axonal transport. These findings emphasize that not all neuronal death is associated with axonal accumulations, and that the axonal transport defects induced by pathogenic polyQ proteins may be specific to cytoplasmic aggregations of expanded polyQ proteins (Gunawardena, 2003).
To test for cytoplasmic or axoplasmic toxicity, a protein with an expanded polyQ repeat with a nuclear export sequence (MJD-77QNES) was studied. Expression of MJD-77QNES within larval neurons caused large numbers of synaptotagmin-containing organelle accumulations within larval nerves. Consistent with primarily cytoplasmic localization of this protein, polyQ/HA staining was absent from cell nuclei within the larval brain, with bright anterior staining (just distal to the brain) present within larval nerves. Neuronal apoptosis as determined by TUNEL staining was completely absent and these larvae died at second or third instar, similar to MJD-78Q. Quantitative analysis indicates that the extent of organelle accumulations within MJD-77QNES is comparable to accumulations observed in mutations of motor proteins, suggesting that perhaps the extent of accumulations causes lethality. Similar to MJD78Q, a 50% reduction in the dose of KHC with MJD-77QNES enhanced organelle blockages, while 50% reduction in DLC combined with MJD-77QNES caused early larval lethality. Additionally, expression of MJD-77QNES in the adult eye using GMR-GAL4 caused a severe degenerative eye phenotype, indicating that cytoplasmic polyQ protein can cause degeneration of adult neurons (Gunawardena, 2003).
To directly test if excess MJD-77QNES sequestered motor proteins, embryos expressing MJD-27Q, MJD-78Q, and MJD-77QNES were compared with wild-type (da-GAL4). While embryos expressing both MJD-78Q and MJD-77QNES showed normal levels of total motor proteins, they exhibited a striking reduction in the amount of soluble motor proteins. MJD-77QNES also exhibited high molecular weight aggregates, which could be immunoprecipitated with the HA antibody. These high molecular weight aggregates also contained sequestered DHC. Although phenotypically normal when expressed in larvae, MJD-27Q appears to titrate more DHC into high molecular weight aggregates than do MJD-78Q, MJD-77QNES, or 127Q. It is conceivable that pathogenic MJD-78Q, MJD-77QNES, and 127Q form high molecular weight aggregates that were not possible trap or to solubilize using current protocols. Indeed, dramatic phenotypes are only observed in MJD-78Q, MJD-77QNES, and 127Q. Additionally, similar to embryos expressing MJD-78Q or 127Q, embryos expressing MJD-77QNES died soon after they hatch into larvae, indicating significant polyQ toxicity on normal development. It is possible that the 'soluble' polyQ aggregates observed in embryos expressing MJD-77QNES represent a subclass of misfolded proteins, while the class of insoluble aggregates, which were not possible to observe directly on SDS-PAGE, may be responsible for polyQ toxicity (Gunawardena, 2003).
To distinguish if MJD-78Q blockages and MJD-77QNES blockages are comparable, whether blockages caused by MJD-77QNES expression can be suppressed by excess HSC70 was tested. Expression of MJD-77QNES with excess HSC70 completely suppressed axonal accumulations, polyQ aggregates, and rescued larval lethality to pupae, suggesting that axonal blockages caused by either MJD-78Q or MJD-77QNES were comparable. PolyQ-containing accumulations were also absent in larval nerves. However, excess HSC70 was not sufficient to suppress organismal lethality (Gunawardena, 2003).
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, Drosophila transgenic strains were generated expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and leads to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. These findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease (Lee, 2004).
To characterize neuronal defects that result from an expanded polyQ tract within the Htt gene, transgenic Drosophila were generated expressing N-terminal fragments of human Htt containing 0 (Htt-Q0) or 128 (Htt-Q128) glutamines. The Htt constructs were engineered to include the first 548 aa of the human Htt protein; this region includes and extends well beyond the 81-aa product encoded by the first exon of the gene. The 548-aa fragment is truncated close to the site of cleavage by caspase-3, thought to be a crucial step in the generation of aggregate-forming Htt fragments (Kim, 2001; Wellington, 2002). This region also encompasses the highest stretch of homology between the Drosophila and human Htt proteins. Htt-Q0 and Htt-Q128 fragments were expressed by using UAS/GAL4 or a heat-shock promoter. To confirm transgene expression, Htt protein was compared between pHS-Htt Drosophila maintained at room temperature and after exposure to a heat-shock paradigm. Western blotting with anti-human Htt antibodies detected no Htt protein in control Canton S or in pHS-Htt lines maintained at room temperature. In contrast, Htt-Q0 and Htt-Q128 lines showed abundant Htt expression after heat shock. Transgene expression was also established in pUAST-Htt strains that were crossed to a neuronal GAL4 driver (Lee, 2004).
To determine the functional consequences of Htt-Q128 expression on neuronal activity and morphology, effects in the visual system were examined. Previous Drosophila models of polyQ diseases have demonstrated that eye-specific expression of expanded polyQ proteins leads to a rough-eye phenotype and photoreceptor degeneration. To determine whether the 548-aa Htt transgene caused similar effects, Htt-Q0 and Htt-Q128 were expressed by using the eye-specific GMR-GAL4 driver, and the resulting eye phenotypes were observed by external morphology and the corneal pseudopupil method. Whereas expression of Htt-Q0 does not perturb external-eye appearance or ommatidial morphology, expression of Htt-Q128 causes a rough-eye phenotype with corresponding photoreceptor degeneration. Thus, polyQ expansion in the context of a larger Htt fragment results in neurodegeneration, as observed in other polyQ disease models (Lee, 2004).
To characterize the physiological effects of mutant Htt expression, electroretinograms were recorded from transgenic animals. A normal electrical response to light was seen in Drosophila expressing the GMR-GAL4 driver alone, Htt-Q0 with GMR-GAL4, or Htt-Q128 without the GMR-GAL4 driver. In contrast, Drosophila expressing Htt-Q128 with the GMR-GAL4 driver showed reduced photoreceptor depolarization and complete abolishment of synaptic transmission in response to light. Similar abnormal electroretinograms were observed in heat-shocked pHS-Htt-Q128 lines after a developmental heat shock paradigm but not with control pHS-Htt-Q0 strains. Synaptic activity was also assayed in the giant fiber flight circuit, a pathway important in escape responses and flight initiation. Wild-type Drosophila display little to no spontaneous activity when the temperature is raised to 38°C. In contrast, robust spontaneous seizure activity was recorded in Htt-Q128 flies at 38°C after a developmental heat-shock paradigm. No seizure activity was recorded in Htt-Q0 flies at 38°C. Together, these results indicate that Htt-Q128 expression results in neurodegeneration, accompanied by widespread defects in membrane excitability and brain activity (Lee, 2004).
To establish whether neuronal Htt-Q128 transgene expression causes defects at earlier stages of Drosophila development, quantitative locomotion assays were performed to examine the function of the motor central pattern generator in third-instar larvae. When Htt transgenes were expressed with the pan-neuronal elav-GAL4 driver C155, Htt-Q128 larvae showed a significant reduction in locomotor speed of >25. Adult transgenic flies also display abnormal motor behavior caused by pan-neuronal expression of the Htt-Q128 protein. Whereas expression of Htt-Q128 with the C155 neuronal GAL4 driver causes pharate adult lethality with no viable adult escapers, Htt-Q128 driven by a weaker second chromosome elav-GAL4 driver results in fully viable adults. Several days after eclosion, flies expressing Htt-Q128, but not Htt-Q0, begin to exhibit uncoordinated movement and abnormal grooming behaviors. The behavioral defects worsen with age, resulting in premature death. To quantify the reduction in viability, lifespan curves were generated for control adults, Htt-Q128 adults without elav-GAL4, or adults expressing Htt-Q0 or Htt-Q128 with elav-GAL4. Compared to controls, Htt-Q128/elav-GAL4 animals showed a dramatic reduction in lifespan, with a decrease in the T50 (age at which 50% of the culture has died) by 70%, indicating a highly significant effect of Htt-Q128 expression on viability in Drosophila (Lee, 2004).
A hallmark of HD is the formation of Htt-immunopositive intracellular aggregates in neurons. To determine whether intracellular aggregates are formed in transgenic Htt Drosophila, both Htt-Q128 and Htt-Q0 strains were crossed to flies containing C155 elav-GAL4 to direct expression of Htt within the nervous system. Htt-immunopositive staining was visualized in both central and peripheral neurons of dissected third-instar larvae. Whereas Htt staining remained diffuse throughout the cytoplasm of neurons in Drosophila expressing Htt-Q0, distinct aggregates were observed in the cytoplasm and processes of neurons in lines expressing Htt-Q128. Contrary to what has been observed in exon 1 HD models, no evidence was found of nuclear aggregate localization. To verify that Htt aggregation is based on the length of the polyQ tract and not on protein concentration, Htt levels were quantitated for several Htt-Q0 and Htt-Q128 transgenic lines crossed to elav-GAL4. Levels of Htt protein were generally higher in Htt-Q128 lines than in Htt-Q0 lines, likely because of sequestration of the mutant protein into stable aggregates. However, low-expressing Htt-Q128 lines that produced transgenic protein at a level comparable with that in Htt-Q0 strains still exhibited aggregates, whereas Htt-Q0 lines did not, indicating that polyQ tract expansion and not protein concentration alone is necessary for formation of aggregates. Aggregate formation was also time-dependent. Although Htt levels were visibly high in the central and peripheral nervous system of Htt-Q128/elav-GAL4 embryos, the protein remained largely diffuse in the cytoplasm with rare occurrence of aggregates. By the third instar larval stage, essentially all Htt was observed in aggregates, with relatively little nonaggregate staining. It is concluded that Htt-Q128 forms cytoplasmic neuronal aggregates in a time-dependent manner (Lee, 2004).
Although the causative proteins for many of the polyQ repeat diseases are expressed widely or ubiquitously, aggregate formation and cell death occur in subsets of neurons that differ between the diseases. To examine the effect of cellular context on aggregate formation, the Htt-Q128 protein was expressed in different cell types by using a tubulin GAL4 driver. Transgenic lines were generated containing both the UAS-Htt-Q128 construct and UAS-GFP fused to a nuclear localization signal, allowing for covisualization of Htt-immunopositive aggregates and GFP-stained cell nuclei in expressing cells. Immunocytochemical analysis has demonstrated the formation of cytoplasmic aggregates in both neuronal and nonneuronal tissues, including CNS neurons, gut, salivary glands, and trachea. Interestingly, Htt aggregates were differentially distributed in polarized cells such as the gut, with transport of Htt aggregates to the basolateral domain and exclusion from the apical surface. Similar aggregate transport was found in neurons, indicating that Htt aggregates undergo a cytoskeletal association that allows for directed transport. The Htt-Q128 protein was found in a more diffuse, nonaggregated state in certain cell types, including muscle and epidermis, suggesting that some tissues may be more resistant to Htt aggregation. In cell types in which aggregate formation occurred, only cytoplasmic aggregates (as opposed to nuclear aggregates) were observed, suggesting differences between the larger Htt fragments used in this study compared with exon 1 HD models (Lee, 2004).
Although the polyQ repeat diseases share a similar CAG repeat expansion in the causative gene, the pattern of neurodegeneration and behavioral dysfunction is distinct for each, indicating that protein context for expanded polyQ tracts is critical to disease manifestation. To examine the importance of protein context in the subcellular localization of polyQ-containing proteins, immunocytochemical analysis was performed on larvae expressing an expanded polyQ tract alone (Q127), the mutant polyQ protein responsible for Machado-Joseph disease (SCA3-Q78), or an expanded polyQ tract (Q108) previously engineered into the nonpathogenic dishevelled gene. In contrast to the cytoplasmic localization of Htt aggregates, both Q127 and SCA3-Q78 aggregates localized exclusively to the nucleus. Very few Dishevelled-immunopositive aggregates were observed, and the protein was present diffusely in the cytoplasm. These results demonstrate that the protein context in which the polyQ tract is found exquisitely controls both aggregate localization and aggregate formation (Lee, 2004).
To test whether Htt-Q128 can interact and coaggregate with other polyQ repeat proteins, double transgenic Htt-Q128; Q127 and Htt-Q128; SCA3-Q78 strains were generated. When the Htt-Q128 and Q127 proteins were coexpressed, both central and peripheral neurons showed localization of Htt-Q128 aggregates to the cytoplasm, whereas Q127 aggregates were restricted to the nucleus. Likewise, in strains expressing Htt-Q128 and SCA3-Q78, aggregates were segregated independently in the cytoplasm and nucleus (respectively) of both neuronal and nonneuronal cells. These results suggest that the trafficking and aggregation of nuclear and cytoplasmic aggregates are independently regulated (Lee, 2004).
To determine whether Htt-Q128 might interact with cytoplasmic proteins containing an expanded polyQ tract, double transgenic strains were made containing Htt-Q128 and Dishevelled-Q108 (Dsh-Q108). Dsh-Q108 formed few aggregates when expressed alone; however, when coexpressed with Htt-Q128, the subcellular distribution of Dsh-Q108 shifted from a diffuse cytoplasmic pattern to a complete sequestration into aggregates that colocalized with Htt-Q128. These findings indicate that Htt aggregates are able to trap and sequester Dsh-Q108 into cytoplasmic aggregates. Similar interactions between Htt aggregates and endogenous cytoplasmic polyQ-containing proteins might be predicted to play an important role in disease pathology (Lee, 2004).
Htt-Q128 forms cytoplasmic aggregates that are associated with cytoskeletal transport systems. When Htt-Q128 was expressed with the eye-specific driver GMR-GAL4, Htt aggregates were abundantly transported along axons entering the CNS of the developing visual system and accumulated in pathfinding photoreceptor growth cones. Similarly, when Htt-Q128 was expressed with the C155 driver, aggregates were transported in larval motor axons and accumulated at presynaptic neuromuscular junction terminals. No aggregates were observed in axons from animals expressing Htt-Q0. Axonal transport of aggregates was not observed in transgenic animals that produce exclusively nuclear aggregates (Q127 and SCA3-Q78), suggesting that axonal and synaptic defects that may occur downstream of cytoplasmic aggregate formation are likely specific to HD. Additionally, no Dsh-Q108 aggregates were observed in axon bundles. However, when Dsh-Q108 was coexpressed with Htt-Q128, Dsh-Q108 protein trapped by Htt-Q128 aggregates was also transported along axons. Similar trapping of endogenous cytoplasmic polyQ proteins may sequester them away from their natural cellular locations and contribute to neuronal dysfunction (Lee, 2004).
In observing axonal aggregates in Htt-Q128-expressing animals, it was noted that the diameter of Htt aggregates often exceeds that of normal larval axons. This suggests that large Htt aggregates might physically block axonal transport, as would be manifested by axonal swellings at the sites of blockage. This hypothesis was tested in Htt-Q128-expressing animals by observing the localization of synaptotagmin I, a synaptic vesicle protein localized to synapses in Drosophila. Normal transport of the synaptotagmin protein along axons is below the threshold for immunocytochemical detection. This pattern of trafficking, as observed in Htt-Q0-expressing animals, is abruptly altered in Htt-Q128-expressing Drosophila. Instead of the normal diffuse localization along axonal tracts, synaptotagmin became concentrated at specific points along axons that corresponded to large areas of Htt-immunopositive aggregate accumulation, suggesting sites of axonal blockage. These synaptotagmin-rich areas of Htt aggregate accumulation were quantified in 100-µm segments along peripheral nerves and were observed at a density of 6.1 +/- 2.6 sites per 100 microm. In contrast, synaptotagmin-immunopositive accumulations alone without Htt aggregate colocalization were only observed at a density of 1.3 +/- 0.9 per 100 microm. The average diameter of Htt-Q128 aggregate accumulations at putative axonal blockage sites was 2.4 +/- 0.6 µm. These findings suggest that axonal segments can be obstructed by Htt aggregates. Over time, the cumulative blockage of axons and synaptic terminals in postmitotic neurons is likely to contribute to the progressive physiological defects and neuronal dysfunction that has been documented in Htt-Q128-expressing Drosophila, as well as to late-onset neurodegeneration in HD patients (Lee, 2004).
Many human neurodegenerative diseases have been successfully modeled in Drosophila with replication of key neuropathological features, including late onset and progressive neurodegeneration. Existing Drosophila models of HD target expression of the first exon of the mutant Htt protein to the fly retina, either by using an eye-specific promoter (Jackson, 1998) or the GAL4/UAS system (Steffan, 2001). In both HD models, expression of Htt with a pathogenic number of glutamine repeats results in nuclear accumulation of aggregates and progressive neurodegeneration of photoreceptor cells, suggesting a role for nuclear aggregates in disease pathology. Indeed, nuclear aggregate-mediated impairment of transcription has become a favored hypothesis to explain polyQ-mediated neurodegeneration. The findings using a larger Htt transgene suggest that nonnuclear pathology associated with cytoplasmic and neuritic aggregates is likely to play an essential role in disease progression as well. Given that Drosophila polyQ disease models with either nuclear-restricted (expanded polyQ alone, mutant SCA-3, or Htt exon 1) or cytoplasm-restricted (Htt-Q128) aggregates both exhibit neurodegeneration, it is likely that multiple pathways for polyQ-mediated dysfunction exist. Indeed, it has been possible to rescue adult lethality in Htt-Q128-expressing Drosophila with any of the previously published genetic suppressors of Drosophila transgenic polyQ models. These negative results likely reflect distinct modes of toxicity between nuclear and cytoplasmic aggregates and suggest that preventing polyQ-mediated Htt toxicity may require more research on the role of nonnuclear aggregates. A potential role has been demonstrated for cytoplasmic and neuritic aggregates in the sequestration of cytoplasmic polyQ proteins and in the blockage of axonal transport. Consistent with the hypothesis that Htt-Q128 expression causes neurodegeneration secondary to impairment of axonal transport, recent studies have found that neurodegeneration is a primary consequence of axonal transport defects in non-polyQ diseases as well, including Alzheimer's disease. During review of this manuscript, two reports have been published that document similar axonal transport defects in HD models. Determining the precise mechanisms by which Htt aggregates physically attach to the axonal cytoskeleton will likely provide important insights into the mechanisms of axonal transport blockage in HD. In summary, these results indicate that cytoplasmic aggregate formation in HD sequesters endogenous polyglutamine proteins and blocks axonal transport, contributing to neurophysiological dysfunction and neurodegeneration (Lee, 2004).
Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. This study demonstrates that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress was attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which released ULK1 from negative regulation by mTOR. This data uncovers an important physiological function of Huntingtin and provides a missing link in the activation of selective macroautophagy in metazoans (Rui, 2015).
Homozygous flies lacking the single htt homologue (dhttko) are fully viable with only mild phenotypes. In a genetic screen for the physiological function of Htt, ectopic expression of a truncated form of the microtubule-binding protein Tau (Tau-ΔC; truncated after Val 382) induced a prominent collapse of the thorax in dhttko flies due to severe muscle loss not observed by Tau expression alone, and accelerated decline in mobility and lifespan. These phenotypes were fully rescued by the dhtt genomic rescue transgene (‘dhttRescue’), suggesting that dhtt protects against Tau-induced pathogenic effects (Rui, 2015).
Although heterozygous dhttko/+ flies expressing Tau
(ATau; dhttko/+) seem normal, removing a single copy
of the fly LC3 gene, atg8a
(atg8ad4 mutant), in these flies also induces a collapsed
thorax and muscle loss, which can be phenocopied by expressing Tau
in homozygous atg8ad4−/− flies alone. Four
additional components of the early steps of the autophagy pathway,
atg1 (ULK1), atg7
and an adaptor for the selective recognition of autophagic cargo,
also exhibit strong genetic interactions with dhtt.
Consistent with its pivotal role in autophagy initiation, loss of
atg1 induces the strongest defect, and Tau expression can
induce a mild muscle loss phenotype even in heterozygous null atg1Δ3d.
Collectively, these genetic interaction studies suggest a role for
dhtt in autophagy (Rui, 2015).
By using the mCherry–GFP–Atg8a fusion reporter to directly measure autophagic flux in adult dhttko−/− brains, this study found similar number of red fluorescent punctae (acidic autolysosomes originating from autophagosome/lysosome fusion) in young mutant and control flies, but the number of punctae were reduced in old dhttko−/− brains when compared with age-matched controls. As autophagosome accumulation (co-localized green and red puncta) was not observed, it was concluded that the absence of dhtt in older animals was associated with reduced autophagosome formation. The fact that levels of Ref(2)P are significantly higher in old dhttko−/− brains compared with brains from age-matched wild-type controls suggests a possible preferential compromise in selective autophagy in these animals (Rui, 2015).
Consistent with the role of basal autophagy in quality control in non-dividing cells, it was found that brains from 5-week-old dhttko−/− contained almost double the amount of ubiquitylated proteins, a marker of quality control failure, compared with wild-type flies. As genetic interaction analysis and specific ubiquitin proteasome system (UPS) reporters all failed to reveal a functional link between dhtt and the UPS pathway, the study proposes that the defects in autophagic activity are the main cause of diminished quality control and increased accumulation of ubiquitylated proteins in dhttko−/− mutants (Rui, 2015).
Selective autophagy is induced in response to proteotoxic stress. The truncated Tau-ΔC used in genetic experiments in this study is preferentially degraded through autophagy in cortical neurons, serving as a model of proteotoxicity when ectopically expressed. The lower stability of Tau-ΔC compared with full-length Tau in wild-type flies and in UPS mutants was confirmed, but significantly higher levels of Tau-ΔC when expressed in atg8a and in dhttko−/− mutant flies were found, suggesting that autophagy is essential for the clearance of Tau-ΔC also in flies and that dhtt plays a role in this clearance (Rui, 2015).
In contrast, loss of dhtt does not affect flies’ adaptation to nutrient deprivation, which typically induces robust ‘in bulk’ autophagy. Fat bodies of early third instar larvae expressing mCherry–Atg8, where starvation-induced autophagy can be readily detected, fail to reveal any significant difference between wild-type and dhttko−/− flies; these flies die at the same rate as wild-type flies when tested for starvation resistance. Thus, although dhtt is necessary for selective autophagy of toxic proteins such as Tau-ΔC, it is dispensable for starvation-induced autophagy in flies (Rui, 2015).
Expression of human Htt (hHTT) in dhttko−/− null flies rescues both the mobility and longevity defects of dhttko−/− mutants and partially rescues the Tau-induced morphological and behavioural defects of dhttko−/− flies. hHTT also suppresses almost all of the autophagic defects observed in dhttko−/−, including decreased levels of autolysosomes, increased levels of Ref(2)P and of total ubiquitylated proteins, and accumulation of ectopically expressed Tau-ΔC, suggesting that the involvement of dhtt in autophagy is functionally conserved. In fact, confluent mouse fibroblasts knocked down for Htt (Htt(−)) exhibit significantly lower basal rates of long-lived proteins’ degradation than control cells, which are no longer evident on chemical inhibition of lysosomal proteolysis or of macroautophagy, thus confirming an autophagic origin of the proteolytic defect. Htt(−) fibroblasts also exhibit higher p62 levels and accumulate ubiquitin aggregates even in the absence of a proteotoxic challenge. As in dhttko−/− flies, Htt knockdown in mammalian cells does not affect degradation of CL1–GFP (a UPS reporter), β-catenin (a UPS canonical substrate) or proteasome peptidase activities. Reduced autophagic degradation in Htt(−) cells is not due to a primary lysosomal defect, as depletion of Htt does not reduce lysosomal acidification, endolysosomal number (if anything, an expansion of this compartment was observed) or other lysosomal functions such as endocytosis (for example, transferrin internalization). In fact, analysis of the lysosomal degradation of LC3-II reveals that autophagic flux and autophagosome formation are preserved and even enhanced in Htt(−) fibroblasts at basal conditions (Rui, 2015).
The expression of the Drosophila huntingtin transcript was examined at different developmental stages by using Northern blot analysis. A transcript of the predicted size, ~12 kb in length, was detected in all embryonic stages examined, in third-instar larvae and in adults. A second, fainter band of ~5 kb was also observed when a 5' riboprobe was used to probe the blot, but not when riboprobes from other regions of Drosophila HD were used. The transcript is expressed widely, which is similar to the case in mammalian HD genes. A hybridization signal was detected only in poly(A)+ fractions and not in total RNA from Drosophila tissues. The Drosophila huntingtin transcript is present at very low levels, consistent with the results of embryonic cDNA library screens where only three partial length positive clones were found among 3x106 plaques screened (Li, 1999).
Drosophila larvae with mutations in genes encoding axonal transport proteins show dramatic neuromuscular pathology. Segmental nerves of such mutant animals contain prominently stained accumulations of synaptic vesicle proteins such as synaptotagmin (SYT) and cysteine string protein (CSP) (Hurd, 1996; Bowman, 1999). Recently, deletants of the Drosophila APPL gene (which is proposed to function as a vesicular receptor for kinesin-I) were found to exhibit phenotypes characteristic of axonal transport defects (Gunawardena, 2001). To test if reduction of Drosophila huntingtin (htt) causes axonal transport phenotypes, tissue-specific reduction of Drosophila htt was generated by using a modified RNAi method. Three independent UAS double-strand htt hairpin RNAi transgenic lines (dhtt#1, #9, and #13) were combined with the neuronal APPL-GAL4 driver. Expression of htt RNA in the dhtt-RNAi lines was observed by RT-PCR to be reduced, in some instances to as little as 10%-30% of normal. All three lines exhibited defects characteristic of axonal transport problems (organelles accumulate within larval nerves) as well as a small amount of neuronal cell death in the larval brain. As controls, UAS double-strand RNAi lines of RhoGAP were combined with APPL-GAL4. These combinations did not cause axonal transport defects, although disruption of RhoGAP in the adult eye using GMR-GAL4 or eyeless-GAL4 caused a rough eye phenotype (Gunawardena, 2003).
Ordinarily, deleting one of two copies of motor genes does not produce a significant phenotype, but such a reduction combined with a reduction of a putative cargo binding partner is predicted to enhance an axonal transport phenotype because of the additional reduction in components required for transport. To test this prediction, larvae were generated that had reduced levels of both htt and motor proteins. Axonal blockages in the presence of reduced htt gene function were enhanced by a 50% reduction in kinesin heavy chain (KHC) or kinesin light chain (KLC) gene dosage; a small enhancement of neuronal cell death was also observed. A 50% reduction of dynein heavy chain (DHC) or dynein light chain gene (DLC) dose did not dramatically increase the amount of organelle accumulations or the amount of neuronal cell death (Gunawardena, 2003).
Since adults and pupae mutant for DLC, dynein intermediate chain, and the dominant-negative mutation p150Glued have rough eye phenotypes, it was asked if loss or disruption of htt would also exhibit this phenotype. Reduction of htt in the eye using the GMR-GAL4 driver caused a rough eye phenotype in adults; this is also characteristic of neurodegeneration. This phenotype was progressive over time (10 days), causing severe problems of morphology and pigmentation, leading to patches of dark areas indicative of death. Sections of aged mutant eyes showed loss of cells beneath the external surface of the eye and disruption in the normal ommatidial morphology, suggesting loss of photoreceptor integrity. These results suggest that htt has a function in the axonal transport machinery and that loss of htt can lead to neurodegeneration (Gunawardena, 2003).
The observation that reduction of htt causes an axonal transport phenotype, combined with previous work linking htt to the transport machinery, led to the hypothesis that excess htt, particularly htt containing pathogenic polyQ repeats, should cause axonal blockages by titrating motor proteins away from other cargo. To test this proposal, human huntingtin exon 1 was expressed with either a 'normal' length of polyQ (httex1-20Q) or with a disease-causing length of polyQ (httex1-93Q; Steffan, 2001) in larval neurons. These transgenic lines were crossed to two neuron-specific GAL4 driver lines, APPL-GAL4 and 179Y-GAL4, and their nerves were stained for synaptic vesicle markers. While httex1-20Q nerves were similar to wild-type, two different httex1-93Q lines had organelle accumulations characteristic of defects in axonal transport (Gunawardena, 2003).
If expression of httex1 with pathogenic polyQ repeats causes axonal transport phenotypes by titrating motor proteins from other cargoes and pathways, then reduction of motor protein gene dosage in larvae overexpressing httex1 is predicted to significantly enhance the axonal transport phenotype by further reducing the available motor protein pool. To test this prediction, larvae were generated that overexpressed httex1-20Q or httex1-93Q and were heterozygous for motor protein gene mutations. A 50% reduction in KHC or DLC with httex1-93Q enhanced the amount of organelle accumulations, while KHC or DLC reductions combined with httex1-20Q were comparable to wild-type (Gunawardena, 2003).
TUNEL analysis was used to test whether perturbations caused by httex1 causes neuronal apoptosis. While the transgenic line expressing httex1-20Q was comparable to wild-type, the transgenic lines expressing httex1-93Q showed some neuronal apoptosis; a strong enhancement of neuronal death was also observed with 50% reduction in KHC and a small increase with 50% reduction in DLC (Gunawardena, 2003).
Huntington's disease (see Drosophila as a Model for Human Diseases: Huntington's disease) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N terminus of the huntingtin (htt) protein. This study describes the generation and characterization of a full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded htt is imported into the nucleus in detectable amounts. Expression of expanded full-length mammalian htt (128QhttFL) in Drosophila leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128QhttFL animals. Partial loss of function in synaptic transmission (syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, the data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length htt during early stages of pathogenesis (Romero, 2008).
Expression of 128QhttFL in the eye using GMR-GAL4 leads to progressive photoreceptor neuron degeneration. Histological examination of the internal eye structure in flies of different ages reveals that the number and arrangement of rhabdomeres in photoreceptor cells is relatively normal in 1-day-old flies, but degeneration is evident at day 20. Expression of 128QhttFL in motor neurons leads to motor impairment phenotypes. The 128QhttFL animals perform as controls do in a climbing assay when they are young, but their motor performance declines prematurely as they age. Moreover, flying ability is impaired in aged 128QhttFL flies, and they also show progressive loss of NMJs at the IFM. In addition, these flies show a reduced survival rate when compared with controls (Romero, 2008).
These neurodegenerative phenotypes are not likely a consequence of transcriptional dysregulation, because they occur in the absence of obvious nuclear htt, even in aged flies. The possibility was investigated that axonal blockages trigger the phenotypes observed in 128QhttFL flies; axonal blockages and impaired fast axonal transport have been reported following expression of polyglutamine tracts alone or in the context of other polypeptides, including expanded htt. However, this study did not detect htt or synaptotagmin accumulation in the axons of 128QhttFL flies, even though the observation of axonal blockages reported with an expanded htt fragment was reproduced. Despite the absence of visible htt or synaptotagmin aggregates, the possibility that intracellular transport is decreased cannot be excluded. However, mislocalization or aberrant distribution of known synaptic markers that rely on vesicular transport for their proper synaptic localization was not observed (Romero, 2008).
All together, these data suggest that the presynaptic accumulation of 128QhttFL impairs the function of factors involved in neurotransmitter release. This hypothesis agrees with abundant data describing protein interactions between htt and components of the synaptic machinery (Smith, 2005) and with findings in R6/1 and R6/2 mouse models that suggested a role for altered neurotransmitter release as a potential mechanism of HD pathogenesis. In R6/2 mice, synapsin phosphorylation is partially defective (Lievens, 2002), and in R6/1 mice glutamate levels are reduced and aspartate and GABA are increased (Nicniocaill, 2001). Moreover, increased NMDA receptor activity has been reported in full-length HD mice (Cepeda, 2001, Zeron, 2002), leading to a postsynaptic increase in Ca2+ influx and abnormal synaptic transmission. In addition, Ca2+ levels were found to be increased by almost 2-fold in CA1 pyramidal neurons in full-length HD mice (Hodgson, 1999). However, no defects were observed in paired-pulse facilitation, which questions the biological relevance of this finding. In addition, mutant htt has been implicated in aberrant mitochondrial Ca2+ buffering (Panov, 2005), and it also increases the sensitivity of the inositol 1,4,5-triphosphate (IP3) receptor to IP3, causing enhanced Ca2+ release following mGluR1/5 activation (Tang, 2003). These data suggest that cytosolic Ca2+ levels play a role in HD pathogenesis (Bezprozvanny, 2004; Romero, 2008 and references therein).
To test whether expanded htt impairs the normal function of proteins involved in synaptic transmission, a genetic approach was used, using the 128QhttFL animals. This study found that partial loss of function of Snap, syntaxin, or Rop restores the increased EJP amplitude observed in 128QhttFL larvae to near-normal levels. Moreover, the lack of neurotransmitter release failures is also suppressed by these mutations. These observations suggest that neurodegeneration in 128QhttFL flies is caused by increased synaptic transmission. In agreement with this hypothesis, a progressive neurodegenerative phenotype was found in the NMJ of adult 128QhttFL animals. Most importantly, further support for this hypothesis comes from the observation that the same synaptic transmission mutants that restore the EJP amplitude and release failure abnormalities also suppress motor impairment, photoreceptor degeneration, or both in 128QhttFL adult animals (Romero, 2008).
Ca2+ levels have a bimodal distribution in 128QhttFL flies, with some boutons showing high Ca2+ levels and other boutons within the same neuromuscular junction showing levels in the normal range. This distribution can be correlated with the accumulation pattern of htt, which is present in some boutons and absent in others within a given neuromuscular junction. The hypothesis was tested that Ca2+ levels are relevant for the increased transmission and decreased failures observed in 128QhttFL animals using mutations in voltage-gated Ca2+ channels. It was found that Ca2+ levels are restored within normal range in 128QhttFL flies carrying heterozygous mutations in either Syx or the Dmca1D Ca2+ channel. Furthermore, heterozygous mutants for either the Dmca1A or Dmca1D Ca2+ channels also show suppression of the increased transmission and decreased failure phenotypes. Dmca1D, an L-type voltage-gated Ca2+ channel, was also tested in the context of the eye assay and found that its partial loss of function suppresses photoreceptor degeneration. These data support the hypothesis that increased Ca2+ levels play an important role in the observed increased transmission in neurons of 128QhttFL animals. Interestingly, mutations in K+ channels cause neurodegeneration in flies and in humans, further supporting the idea that the increased release is responsible, at least in part, for neuronal degeneration caused by expanded htt (Romero, 2008).
The findings described in this report unveil a mechanism of pathogenesis for expanded htt that does not require its nuclear accumulation in detectable amounts. The increased synaptic transmission phenotype exerted by full-length htt likely represents a mechanism of pathogenesis taking place at early stages of disease progression. In later stages, cleavage of htt would compound the toxic effects of the full-length protein with fast axonal transport impairments and transcriptional dysregulation caused by N-terminal fragments. These findings point to increased synaptic transmission as a therapeutic target with the potential of delaying HD onset and thus likely impacting disease progression. The genetic data showing suppression of the synaptic transmission and neurodegenerative phenotypes further define specific therapeutic targets and support the idea that Ca2+ channel antagonists, and perhaps other inhibitors of neurotransmission, offer an attractive therapeutic option due to their specificity and wide usage (Romero, 2008).
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. This study examined the role of the Drosophila huntingtin ortholog in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, dhtt was found to act as a suppressor of position effect variegation (PEV), suggesting that it influences chromatin organization. dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, a candidate genetic screen was constructed using RNAi lines targeting known PEV modifier genes. dhtt was found to modify phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the histone demethylase dLsd1, in both the ovary and wing, and dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla (Dietz, 2015).
Previous studies of the inverse relationship between the age at onset of clinical symptoms and the size of the HTT CAG repeat mutation have revealed that the repeat confers on the mutant allele a fully dominant gain-of-function property. However, it is unknown whether this is the acquisition of enhanced normal huntingtin function or the acquisition of a novel opportunistic function, although targeted null and CAG expansion mutations at the mouse homolog provide support for both possibilities. Despite earlier studies, details of the normal function of huntingtin remain relatively elusive, hindering further investigation into the molecular mechanisms underlying the disease (Dietz, 2015).
This study uses a novel dhtt allele to examine the normal function of Drosophila huntingtin, focusing on its potential role in chromatin function during development. Although dhtt flies are viable and appear grossly normal, genetic findings indicate that dhtt influences chromatin regulation: (i) dhtt acts as a suppressor of PEV, suggesting that it is involved in heterochromatin formation; (ii) dhtt affects heterochromatin spreading in a PEV model; (iii) dhtt genetically interacts with a number of genes encoding proteins known to affect chromatin organization and function and (iv) dhtt genetically interacts with the HDM dLsd1 and facilitates its ability in demethylating histone H3K4 (Dietz, 2015).
PEV is a powerful genetic assay that has been used previously to identify genes that can regulate chromatin structure. In PEV models, a chromosomal rearrangement or transposition abnormally juxtaposes a reporter gene with heterochromatin. A variegated phenotype is produced since the gene is stochastically silenced in some of the cells in which it is normally active. The silencing that occurs in PEV is attributed to the ‘spreading’ of heterochromatin along the chromosome into a region that would normally be in a euchromatic form. Thus, since the reporter gene is on the boundary between these two states, PEV provides a sensitive system in which to test genetic modifiers of heterochromatin formation. This study uses two independent PEV assays [T(2;3)Sbv and In(1)y3P] and demonstrates that dhtt facilitates heterochromatin formation, thereby suppressing variegated phenotypes. To date, approximately 500 dominant Su(var) and E(var) mutations have been isolated from PEV screens and it is estimated that these affect about 150 unique genes. Those that have been molecularly characterized so far have been revealed to generally encode chromosomal proteins or modifiers of chromosomal proteins (Dietz, 2015).
Histone post-translational modifications (PTMs) play essential roles in the transition between active (euchromatin) and inactive (heterochromatin) chromatin states. In particular, histone methylation has been widely studied in nearly all model systems and is generally recognized as an epigenetic marker for transcriptionally silent heterochromatin. High levels of methylated histone H3K9me2 are associated with heterochromatin loci. Using the established PEV model, wm4, this study demonstrates that the dhtt allele dominantly reduces the level of histone H3K9me2 at the white locus and the adjacent CG12498 gene at the heterochromatin–euchromatin boundary. This level of histone H3K9me2 reduction is comparable to that caused by a dLsd1/Su(var)3-3 null allele, an established suppressor of variegation. The loss of dhtt therefore significantly influences chromatin structure, thereby shifting the euchromatin–heterochromatin boundary (Dietz, 2015).
Where in the cell might huntingtin function to affect chromatin structure and act as a suppressor of variegation? Although the majority of huntingtin in human and mouse cells has been shown to reside within the cytoplasm, about 5% is estimated to be nuclear. A previous report suggests that Drosophila huntingtin is solely cytoplasmic, but this was based solely on ectopic dhtt overexpression. Since both fly and mouse loss-of-function huntingtin models show defects in mitotic spindle orientation in neuroblast precursors, it is clear that huntingtin does have a nuclear function. However, it is possible that dhtt could also influence chromatin structure by acting in the cytoplasm (Dietz, 2015).
Based on the findings that dhtt dominantly suppresses PEV and affects chromatin function, it was hypothesized that it may genetically interact with previously identified PEV modifiers. The approach to screening for possible dhtt interactors utilized a collection of RNAi lines targeting known suppressors and enhancers of PEV. Such a screen has a number of caveats: first, it relies on RNAi producing a modifiable phenotype in a relevant tissue. Secondly, due to the nature of the screen, it is largely limited to looking for interactions in the adult eye and wing. Interestingly, in some cases, interactions between dhtt and genes in the wing were found, but not in eye and vice versa. This may reflect tissue-specific requirements for different genes, or that dhtt functions only within certain complexes in certain tissues. Nevertheless, a number of strong genetic interactors of dhtt were identified, which included central regulators of chromatin architecture and function, such as the heterochromatin proteins, HP1a and HP1b, brm—the ATPase subunit of the SWI/SNF (Brm) complex, the transcription factor dE2F1 and various HDMs and HMTs. Next, the interaction between dhtt and dLsd1 was evaluated for the following reasons: dLsd1 interacts with both the dhtt allele and dhtt RNAi and loss of dhtt causes enhancement of dLsd1 phenotypes in both wing and ovary. Additionally, it was found that dhtt and dLsd1 both affect PEV and heterochromatin formation to comparable extents (Dietz, 2015).
Although dhtt-deficient flies are fertile and display no obvious ovarian phenotype, loss of dhtt strongly enhances the dLsd1 ovary defects. It was hypothesized that dLsd1 and dhtt collaborat in the regulation of histone H3K4 methylation at specific loci to control gene expression critical for oogenesis. Similarly, in contrast to dLsd1 mutant flies which show elevated levels of histone H3K4me1 and H3K4me2, any changes in the global levels of these modifications in dhtt-deficient flies could not be detected. However, simultaneous knockdown of dLsd1 and dhtt results in a significant increase in histone H3K4me1 and H3K4me2 levels over that of the dLsd1 knockdown alone. Human LSD1 is a component of the CoREST/REST (repressor element silencing transcription factor) complex, which represses the transcription of neuronal genes in non-neuronal cell lineages. Within this complex, LSD1 acts to demethylate histone H3K4 residues in nucleosomes at REST target genes, thereby contributing to their transcriptional repression. Mammalian full-length huntingtin has been shown to physically interact with this complex and contribute to its regulation, and it will therefore be interesting to determine whether dLsd1 and dhtt similarly associate with each other. Unfortunately due to the lack of phenotype upon knocking down the Drosophila ortholog of CoREST, dCoREST, with the available RNAi lines, testing for a potential interaction with dhtt could not be performed by this study. The genetic interaction between dhtt and dLsd1 could potentially account for the strong effect of dhtt seen on H3K9 methylation at the heterochromatin/euchromatin boundary in the wm4 PEV model. dLsd1 has been shown to physically associate with Su(var)3-9 and to control Su(var)3-9-dependent spreading of histone H3K9 methylation along euchromatin (Dietz, 2015).
There is considerable evidence suggesting a link between aberrant acetylation and methylation marks and HD. Mouse Htt has been implicated in facilitating the trimethylation of histone H3K27 in developing murine embryoid bodies. The levels of histone H3K4me3 have been shown to change at dysregulated promoters in a mouse HD model (R6/2) and human HD postmortem brain tissue. The screen in this study uncovered interactions between dhtt and Drosophila HDM and HMTs with a variety of different histone H3 specificities (H3K4, H3K27, H3K9 and H3K79). It is therefore possible that dhtt has a general role, possibly as a scaffold protein, in facilitating a number of complexes containing histone-modifying enzymes with different specificities. Since mammalian full-length huntingtin has been implicated in the trimethylation of histone H3K27 by facilitating PRC2 function, it is surprising that the histone H3K27 methyltransferase, esc, is the only component of PRC2 found to interact with dhtt. Although there was no effect of the dhtt null mutation on the global levels of histone H3K27me levels, it is possible that dhtt may play a similar role to mouse Htt in modulating histone H3K27me during development, with histone H3K27me differences only observed at specific loci (Dietz, 2015).
A number of the dhtt interacting genes found in the screen encode for important chromatin regulating proteins that have previously been found to genetically interact with each other. For example, a strong interaction between dhtt and brm, the central subunit of the Brm SWI/SNF complex, was detected. brm is known to interact with E(Pc), dE2F1, Asx and Rpd3, which were also found in the screen. The SIN3 corepressor complex is a class I HDAC complex conserved from Drosophila to humans and regulates gene transcription through deacetylation of nucleosomes. Loss of dhtt suppresses both eye and wing phenotypes caused by Sin3A RNAi. Drosophila Sin3A has been shown to interact with the HDAC Rpd3 and the HDM, lid—both of which were also scored as hits in the screen. Furthermore, mammalian Sin3A was previously reported as a huntingtin N-terminal yeast two-hybrid interactor (Dietz, 2015).
Drosophila has proved to be a useful model to investigate polyglutamine-fragment toxicity. Expression of an N-terminal fragment with an expanded polyglutamine tract in the fly has been shown to accumulate in the nucleus. It would therefore be interesting to evaluate whether the normal chromatin regulatory functions of dhtt are perturbed in the fly polyglutamine-fragment models. Although the Drosophila screen in this study was designed to look initially for phenotypes in visible external structures of the adult fly (wing and eye), many of the genes that were found to interact with dhtt are known to also be expressed and function in the developing nervous system. It is therefore possible that dhtt may also exert a role in regulating chromatin function during neurogenesis and neural function in the fly, leading to subtle behavioral mutant phenotypes that have been described previously. This study establishes Drosophila as a system in which to investigate the normal role of dhtt in chromatin regulation. It will be particularly useful in examining dhtt functions that are evolutionarily conserved as these provide assays with which to determine the impact of the expanded polyglutamine region on full-length huntingtin function, thereby deepening our understanding of the mechanism that initiates the HD disease process (Dietz, 2015).
The gene defective in Huntington's disease encodes a protein of unknown function: huntingtin. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking (DiFiglia, 1995).
The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD (DiFiglia, 1997).
Aggregation of N-terminal mutant huntingtin within nuclear inclusions and dystrophic neurites occurs in the cortex and striatum of Huntington disease (HD) patients and may be involved in neurodegeneration. The prevalence of inclusions and dystrophic neurites has been examined in the cortex and striatum of 15 adult onset HD patients who had mild to severe striatal cell loss (grades 1, 2 or 3) using an antibody that detects the N-terminal region of huntingtin. Nuclear inclusions were more frequent in the cortex than the striatum and were sparse or absent in the striatum of patients with low-grade striatal pathology. Dystrophic neurites occurred in both regions. Patients with low-grade striatal pathology had numerous fibers with immunoreactive puncta and large swellings within the striatal neuropil, the subcortical white matter, and the internal and external capsules. In the globus pallidus of 3 grade 1 cases, N-terminal huntingtin markedly accumulated in the perinuclear cytoplasm and in some axons but not in the nucleus. Findings suggest that in the earlier stages of HD, accumulation of N-terminal mutant huntingtin occurs in the cytoplasm and is associated with degeneration of the corticostriatal pathway (Sapp, 1999).
Huntington's disease is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. Mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients (Davies, 1997).
Yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) have been produced in a developmental and tissue-specific manner identical to that observed in Huntington's disease. YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or micro-aggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration (Hodgson, 1999).
Several lines of mice have been developed that are transgenic for exon 1 of the HD gene containing an expanded CAG sequence. These mice exhibit a defined neurological phenotype along with neuronal changes that are pathognomonic for the disease. Neuronal intranuclear inclusions have been demonstrated in these lines, but no evidence has been found for neurodegeneration. All lines of these mice develop a late onset neurodegeneration within the anterior cingulate cortex, dorsal striatum, and of the Purkinje neurons of the cerebellum. Dying neurons characteristically exhibit neuronal intranuclear inclusions, condensation of both the cytoplasm and nucleus, and ruffling of the plasma membrane while maintaining ultrastructural preservation of cellular organelles. These cells do not develop blebbing of the nucleus or cytoplasm, apoptotic bodies, or fragmentation of DNA. Neuronal death occurs over a period of weeks, not hours. Degenerating cells of similar appearance are found within these same regions in brains of patients who had died with HD. It is therefore suggested that the mechanism of neuronal cell death in both HD and a transgenic mouse model of HD is neither by apoptosis nor by necrosis (Turmaine, 2000).
Despite its widespread expression, mutant huntingtin induces selective neuronal loss in striatal neurons. In mutant mice expressing HD repeats, the production and aggregation of N-terminal huntingtin fragments preferentially occur in HD-affected neurons and their processes and axonal terminals. N-terminal fragments of mutant huntingtin form aggregates and induce neuritic degeneration in cultured striatal neurons. N-terminal mutant huntingtin also binds to synaptic vesicles and inhibits their glutamate uptake in vitro. The specific processing and accumulation of toxic fragments of N-terminal huntingtin in HD-affected striatal neurons, especially in their neuronal processes and axonal terminals, may contribute to the selective neuropathology of HD (Li, 2000).
Huntington's disease (HD) is characterized by the selective loss of striatal projection neurons. In early stages of HD, neurodegeneration preferentially occurs in the lateral globus pallidus (LGP) and substantia nigra (SN), two regions in which the axons of striatal neurons terminate. In mice expressing full-length mutant huntingtin and modeling early stages of HD, neuropil aggregates form preferentially in the LGP and SN. The progressive formation of these neuropil aggregates follows intranuclear accumulation of mutant huntingtin and becomes prominent from 11 to 27 months after birth. Neuropil aggregates, but no intranuclear inclusions, were observed in the LGP and SN, suggesting that huntingtin aggregates are formed in the axons of striatal projection neurons. In the LGP and SN, degenerated axons were observed in which huntingtin aggregates were associated with dark, swollen organelles that resemble degenerated mitochondria. Neuritic aggregates also form in cultured striatal neurons expressing mutant huntingtin, block protein transport in neurites, and cause neuritic degeneration before nuclear DNA fragmentation occurs. These findings suggest that the early neuropathology of HD originates from axonal dysfunction and degeneration associated with huntingtin aggregates (Li, 2001).
Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease. Mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. The co-aggregation of mutant and wild-type htt fragments. Mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. These data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis (Busch, 2003).
Cytoplasmic huntingtin aggregates found in axonal terminals and electrophysiological studies show that mutant huntingtin affects synaptic neurotransmission. However, the biochemical basis for huntingtin-mediated synaptic dysfunction is unclear. Using electron microscopy on sections of HD mouse brains, it was found that axonal terminals containing huntingtin aggregates often had fewer synaptic vesicles than did normal axonal terminals. Subcellular fractionation and electron microscopy revealed that mutant huntingtin is co-localized with huntingtin-associated protein-1 (HAP1) in axonal terminals in the brains of HD transgenic mice. Mutant huntingtin binds more tightly to synaptic vesicles than does normal huntingtin, and it decreases the association of HAP1 with synaptic vesicles in HD mouse brains. Brain slices from HD transgenic mice that had axonal aggregates showed a significant decrease in (3H)glutamate release, suggesting that neurotransmitter release from synaptic vesicles was impaired. Taken together, these findings suggest that mutant huntingtin has an abnormal association with synaptic vesicles and this association impairs synaptic function (Li, 2004).
Huntingtin, the protein product of the Huntington's disease gene, associates with vesicle membranes and microtubules in neurons. Analysis of axonal transport with a stop-flow, double crush ligation approach in rat sciatic nerve showed that full length huntingtin (350 kDa) and an N-terminal cleavage product (50 kD) were increased within 6-12 h on both the proximal and distal sides of the crush site when compared with normal unligated nerve. The huntingtin associated protein HAP 1 and the retrograde motor protein dynein also accumulated on both sides of the crush, whereas the vesicle docking protein SNAP-25 was elevated only proximally. The cytoskeletal protein alpha-tubulin was unaffected. The rapid anterograde accumulation of huntingtin and HAP 1 is compatible with their axonal transport on vesicular membranes. Retrograde movement of both proteins, as seen by accumulation distal to the nerve crush, may be necessary for their degradation at the soma or for a function in retrograde membrane trafficking (Block-Galarza, 1997).
Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates (Szebenyi, 2003).
The pathological hallmark of HD is the degeneration of subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of affected cells have not been identified in patients with HD, although a unique intranuclear inclusion was recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part (including the glutamine repeat) of huntingtin. In order to understand the importance of this finding, comparable nuclear abnormalities were sought in autopsy material from patients with HD. In all 20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified itranuclear inclusions in neurons and the frequency of these lesions correlated with the length of the CAG repeat in IT15. In addition, examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions containing protein aggregates may be common feature of the pathogenesis of glutamine repeat neurodegenerative disorders (Becher, 1999).
To distinguish between 'loss of function' and 'gain of function' models of HD, the murine HD homolog Hdh was inactivated by gene targeting. Mice heterozygous for Hdh inactivation were phenotypically normal, whereas homozygosity resulted in embryonic death. Homozygotes displayed abnormal gastrulation at embryonic day 7.5 and were resorbing by day 8.5. Thus, huntingtin is critical early in embryonic development, before the emergence of the nervous system. That Hdh inactivation does not mimic adult HD neuropathology suggests that the human disease involves a gain of function (Duyao, 1995).
Huntington's disease is an incurable neuropsychiatric disease associated with CAG repeat expansion within a widely expressed gene that causes selective neuronal death. To understand its normal function, a targeted disruption in exon 5 of Hdh (Hdhex5), the murine homolog of the HD gene, has been created. Homozygotes die before embryonic day 8.5, initiate gastrulation, but do not proceed to the formation of somites or to organogenesis. Mice heterozygous for the Hdhex5 mutation display increased motor activity and cognitive deficits. Neuropathological assessment of two heterozygous mice shows significant neuronal loss in the subthalamic nucleus. These studies show that the HD gene is essential for postimplantation development and that it may play an important role in normal functioning of the basal ganglia (Nasir, 1995).
Targeted disruption of the mouse huntingtin gene (Hdh), carried out to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, it is proposed that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway (Zeitlin, 1995).
Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), the Cre/loxP site-specific recombination strategy was used to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, it is proposed that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis (Dragatsis, 2000).
A molecular base of the HD gene transcription has not been elucidated as yet. Two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene have been identified using a yeast one-hybrid system. Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/proline-rich region, and highly conserved C-terminal region. In vitro DNA binding assay indicates that the C-terminal conserved regions of the proteins are responsible for binding to the unique promoter DNA sequences of the HD gene. The DNA sequence protected from DNase I digestion is a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region. The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32). In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals. Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells (Tanaka, 2004).
To explore polyQ-mediated neuronal toxicity, the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins have been expressed in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produces touch insensitivity in young adults. Noticeably, only 28 +/- 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction (Parker, 2001).
Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. A brain-specific protein that associates with huntingtin has been designated huntingtin-associated protein (HAP1). Selective neuronal localizations of HAP1 is described. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy (Li, 1996).
A protein HAP1 has been identified that binds to huntingtin in a glutamine repeat length-dependent manner. HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. The interaction of HAP1 with p150 Glued was examined. Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo. Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD (Engelender, 1997).
Huntingtin-associated protein is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, it was found that HAP1 also binds to dynactin P150Glued (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bind to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitate with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 is enriched in rat brain microtubules and comigrates with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalizes with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. It is proposed that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport (Li, 1998).
The Huntington's disease mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are found to be resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD (Kim, 2001).
Huntingtin has several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, it has been demonstrated that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. Caspase-cleaved htt is detected in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments are detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD (Wellington, 2002).
Huntington's disease (HD) mouse models that express N-terminal huntingtin fragments show rapid disease progression and have been used for developing therapeutics. However, light microscopy reveals no significant neurodegeneration in these mice. It remains unclear how mutant huntingtin induces neurodegeneration. Using caspase staining, TUNEL labelling, and electron microscopy, it has been observed that N171-82Q mice, which express the first 171 aa of mutant huntingtin, display more degenerated neurons than do other HD mouse models. The neurodegeneration was evidenced by increased immunostaining for glial fibrillary acidic protein and ultrastructural features of apoptosis. R6/2 mice, which express exon 1 of mutant huntingtin, showed dark, nonapoptotic neurons and degenerated mitochondria associated with mutant huntingtin. In HD repeat knock-in mice (HdhCAG150), which express full-length mutant huntingtin, degenerated cytoplasmic organelles were found in both axons and neuronal cell bodies in association with mutant huntingtin that was not labeled by an antibody to huntingtin amino acids 342-456. Transfection of cultured cells with mutant huntingtin revealed that an N-terminal huntingtin fragment (amino acids 1-208 plus a 120 glutamine repeat) causes a greater increase in caspase activity than does exon 1 huntingtin and longer huntingtin fragments. These results suggest that context-dependent neurodegeneration in HD may be mediated by different N-terminal huntingtin fragments. In addition, this study has identified neurodegenerative markers for the evaluation of therapeutic treatments in HD mouse models (Yu, 2003).
Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. Interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels have been reported. These associations give rise to a tonic G protein inhibition of the channels. The effects on the CSP chaperone system are reported of huntingtin fragments (exon 1) with [huntingtin(exon1/exp)] and without [huntingtin(exon1/nonexp)] the occurance of expanded polyglutamine (polyQ) tracts. In vitro huntingtin(exon1/exp) sequesters CSP and blocks the association of CSP with G proteins. In contrast, huntingtin(exon1/nonexp) does not interact with CSP and does not alter the CSP/G protein association. Similarly, co-expression of huntingtin(exon1/exp) with CSP and N-type calcium channels eliminates CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin(exon1/nonexp) does not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts (Miller, 2003).
Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, since the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. These data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease (Ravikumar, 2004).
A pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 (see Drosophila SUMO) or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation (Steffan, 2004).
Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of HD. Supporting this view, administration of histone deacetylase (HDAC) inhibitors has been shown to rescue lethality and photoreceptor neurodegeneration in a Drosophila model of polyglutamine disease. To further explore the therapeutic potential of HDAC inhibitors, preclinical trials were conducted with suberoylanilide hydroxamic acid (SAHA), a potent HDAC inhibitor, in the R6/2 HD mouse model. SAHA crosses the blood-brain barrier and increases histone acetylation in the brain. SAHA can be administered orally in drinking water when complexed with cyclodextrins. SAHA dramatically improves the motor impairment in R6/2 mice, clearly validating the pursuit of this class of compounds as HD therapeutics (Hockly, 2003).
Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. Potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) have been investigated including aggregation, protein-protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p was also found to interact in vitro with CREB-binding protein (CBP) and mSin3a (see Drosophila Sin3A), and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors which may result in neuronal dysfunction and cell death in HD (Steffan, 2000).
The polyglutamine-containing domain of Htt, Htt exon 1 protein (Httex1p), directly binds the acetyltransferase domains of two distinct proteins: CREB-binding protein (CBP) and p300/CBP-associated factor (P/CAF). In cell-free assays, Httex1p also inhibits the acetyltransferase activity of at least three enzymes: p300, P/CAF and CBP. Expression of Httex1p in cultured cells reduces the level of the acetylated histones H3 and H4, and this reduction can be reversed by administering inhibitors of histone deacetylase (HDAC). In vivo, HDAC inhibitors arrest ongoing progressive neuronal degeneration induced by polyglutamine repeat expansion, and they reduce lethality in two Drosophila models of polyglutamine disease. These findings raise the possibility that therapy with HDAC inhibitors may slow or prevent the progressive neurodegeneration seen in Huntington's disease and other polyglutamine-repeat diseases, even after the onset of symptoms (Steffan, 2001).
The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. In vitro and in cells it has been shown that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner. FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin. Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates. Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP. These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones (Schaffar, 2004).
Cystamine, a small disulfide-containing chemical, is neuroprotective in a transgenic mouse and a Drosophila model of Huntington's disease (HD) and decreases huntingtin aggregates in an in vitro model of HD. The mechanism of action of cystamine in these models is widely thought to involve inhibition of transglutaminase mediated cross-linking of mutant huntingtin in the process of aggregate formation/stabilization. Cystamine, both in vitro and in a transgenic mouse model of HD (R6/2), increases levels of the cellular antioxidant L-cysteine. Several oxidative stress markers increase in HD brain. Evidence is provided of oxidative stress in mouse HD by demonstrating compensatory responses in R6/2 HD brains. Age-dependent increases in forebrain glutathione (GSH) were found, and increased levels of transcripts coding for proteins involved in GSH synthesis and detoxification pathways, as revealed by quantitative PCR analysis. Given the general importance of oxidative stress as a mediator of neurodegeneration, it is proposed that an increase in brain L-cysteine levels could be protective in HD. Furthermore, cystamine was dramatically protective against 3-nitropropionic acid-induced striatal injury in mice. It is suggested that cystamine's neuroprotective effect in HD transgenic mice results from pleiotropic effects that include transglutaminase inhibition and antioxidant activity (Fox, 2004).
The mutant huntingtin protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. This beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD (Zuccato, 2001).
Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. This study reports that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes (Li, 2002).
Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. This study reports that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms (Dunah, 2002).
Huntington's disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine tract within the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis; recent evidence suggests a defect in Sp1-mediated transcription. Chromatin immunoprecipitation (ChIP) assays followed by real-time PCR were used to quantify the association of Sp1 with individual genes. Despite normal protein levels and normal to increased overall nuclear binding activity, Sp1 has decreased binding to specific promoters of susceptible genes in transgenic HD mouse brain, in striatal HD cells, and in human HD brain. Genes whose mRNA levels are decreased in HD have abnormal Sp1-DNA binding, whereas genes with unchanged mRNA levels have normal levels of Sp1 association. Moreover, the altered binding seen with Sp1 is not found with another transcription factor, NF-Y. These findings suggest that mutant huntingtin dissociates Sp1 from target promoters, inhibiting transcription of specific genes (Chen-Plotkin, 2006).
Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntingtons disease (HD), while htt-/- mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, more than 3700 compounds were compared in three syngeneic mouse embryonic stem cell (mESC) lines [htt-/-, extended poly-Q (Htt-Q140/7), and wild-type mESCs (Htt-Q7/7)] using untargeted metabolite profiling. While Htt-Q140/7 cells did not show major differences in cellular bioenergetics, extensive metabolic aberrations were found in in htt-/- mESCs, including (1) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (2) near-maximal glycolysis, with little or no glycolytic reserve; (3) marked ketogenesis; (4) depletion of intracellular NTPs; (5) accelerated purine biosynthesis and salvage; and (6) loss of mitochondrial structural integrity. Together, these findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt-/- early embryonic lethality (Ismailoglu, 2014).
Search PubMed for articles about Drosophila huntingtin
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date revised: 15 December 2015
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