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Huntington's Disease
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Drosophila genes associated with Huntington's disease
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Position effect variegation
Relevant studies of Huntington's disease

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Babcock, D.T. and Ganetzky, B. (2015). Transcellular spreading of huntingtin aggregates in the Drosophila brain. Proc Natl Acad Sci U S A. 112: E5427-E5433. PubMed ID: 26351672

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 and Ganetzky, 2015).


  • Htt aggregates spread throughout the Drosophila brain.
  • Htt aggregates are taken up by large posterior neurons.
  • Spreading of Htt aggregates causes non–cell-autonomous damage.
  • Spreading of Htt aggregates requires NSF1 and Dynamin.
  • Transmission is not observed in all PolyQ-expanded aggregate-prone proteins.


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 and Ganetzky, 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 and Ganetzky, 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 and Ganetzky, 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 and Ganetzky, 2015).

It was shown that release of Htt aggregates requires 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 and Ganetzky, 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 and Ganetzky, 2015).

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El-Daher, M.T., Hangen, E., Bruyère, J., Poizat, G., Al-Ramahi, I., Pardo, R., Bourg, N., Souquere, S., Mayet, C., Pierron, G., Lévêque-Fort, S., Botas, J, Humbert, S. and Saudou, F. (2015). Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation. EMBO J 34: 2255-2271. PubMed ID: 26165689

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N‐ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full‐length HTT remain elusive. Moreover, the contribution of non‐polyQ C‐terminal fragments is unknown. Using time‐ and site‐specific control of full‐length HTT proteolysis, this study shows that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N‐ter fragments, the C‐ter fragments generated, that do not contain the polyQ stretch, induce toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C‐ter HTT binds to dynamin 1 and subsequently impairs its activity at ER membranes. These findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis‐induced alteration of this function may be relevant to disease (El-Daher, 2015).


  • Sequential proteolysis of huntingtin reveals toxic events.
  • Huntingtin intramolecular interaction is lost upon specific proteolysis.
  • Both N‐ and C‐terminal huntingtin fragments induce toxicity that depends on the size of N‐terminal fragments.
  • C‐terminal huntingtin fragment causes endoplasmic reticulum‐derived vacuolation, stress and toxicity.
  • ER stress and vacuolation are observed in Huntington's disease knock‐in mice.
  • HTT proteolysis and the presence of C‐ter huntingtin lead to neurodegeneration in flies.
  • Dynamin 1 interacts with the C‐terminal part of huntingtin.
  • Dynamin 1 impairment at the ER mediates the effect of C‐ter huntingtin on vacuolation and death.

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Hansen, T., Thant, C., White, J. A., Banerjee, R., Thuamsang, B. and Gunawardena, S. (2019). TExcess active P13K rescues huntingtin-mediated neuronal cell death but has no effect on axonal transport defects. Apoptosis. PubMed ID: 30725352


High levels of oxidative stress is detected in neurons affected by many neurodegenerative diseases, including huntington's disease. Many of these diseases also show neuronal cell death and axonal transport defects. While nuclear inclusions/accumulations likely cause cell death, previous work has shown that cytoplasmic axonal accumulations can also contribute to neuronal death. However, the cellular mechanisms responsible for activating cell death is unclear. One possibility is that perturbations in normal axonal transport alter the function of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-pathway, a signal transduction pathway that promotes survival/growth in response to extracellular signals. To test this proposal in vivo, active PI3K was expressed in the context of pathogenic huntingtin (HTT-138Q) in Drosophila larval nerves, which show axonal transport defects and neuronal cell death. Excess expression of active P13K significantly suppressed HTT-138Q-mediated neuronal cell death, but had no effect on HTT-138Q-mediated axonal transport defects. Expression of active PI3K also rescued Paraquat-mediated cell death. Further, increased levels of pSer9 (inactive) glycogen synthase kinase 3beta was seen in HTT-138Q-mediated larval brains, and in dynein loss of function mutants, indicating the modulation of the pro-survival pathway. Intriguingly, proteins in the PI3K/AKT-pathway showed functional interactions with motor proteins. Taken together these observations suggest that proper axonal transport is likely essential for the normal function of the pro-survival PI3K/AKT-signaling pathway and for neuronal survival in vivo. These results have important implications for targeting therapeutics to early insults during neurodegeneration and death (Hansen, 2019).

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Xu, F., Kula-Eversole, E., Iwanaszko, M., Hutchison, A. L., Dinner, A. and Allada, R (2019). Circadian clocks function in concert with heat shock organizing protein to modulate mutant Huntingtin aggregation and toxicity. Cell Rep 27(1): 59-70. PubMed ID: 30943415


Neurodegenerative diseases commonly involve the disruption of circadian rhythms. Studies indicate that mutant Huntingtin (mHtt), the cause of Huntington's disease (HD), disrupts circadian rhythms often before motor symptoms are evident. Yet little is known about the molecular mechanisms by which mHtt impairs circadian rhythmicity and whether circadian clocks can modulate HD pathogenesis. To address this question, a Drosophila HD model was used. Both environmental and genetic perturbations of the circadian clock were found to alter mHtt-mediated neurodegeneration. To identify potential genetic pathways that mediate these effects, a behavioral platform was applied to screen for clock-regulated HD suppressors, identifying a role for Heat Shock Protein 70/90 Organizing Protein (Hop). Hop knockdown paradoxically reduces mHtt aggregation and toxicity. These studies demonstrate a role for the circadian clock in a neurodegenerative disease model and reveal a clock-regulated molecular and cellular pathway that links clock function to neurodegenerative disease.

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Farago, A., Zsindely, N. and Bodai, L. . (2019) Mutant huntingtin disturbs circadian clock gene expression and sleep patterns in Drosophila. Sci Rep 9(1): 7174. PubMed ID: 31073199


Deficiency of the sleep-wake cycle can accelerate the progression of Huntington's disease (HD) and exacerbate symptoms making it a target of investigation to better understand the molecular pathology of the disorder. This study analyzed sleep defects in a Drosophila model of HD and investigated whether disturbed sleep coincides with alterations in the molecular mechanism controlling circadian rhythm. To analyze sleep defects, the daily activity was recorded of flies in 12:12 hours light:dark entrainment, and in regard to the underlying molecular mechanism circadian "clock" gene expression was measured. In HD flies reduced amount of sleep, sleep fragmentation and prolonged sleep latency were recorded. Changes were found in gene expression patterns of both transcriptional feedback loops of circadian regulation. Prolonged expression of the core feedback loop components period and timeless was detected, whilst the secondary feedback loop member vrille had lower expression rates in general. The results show that the Drosophila HD model recapitulates most of the sleep related symptoms reported in patients therefore it can be a potential tool to study the molecular background of sleep defects in HD. Altered expression of circadian "clock" genes suggests that disturbed sleep pattern in HD might be the consequence of disturbed circadian regulation (Farago, 2019).

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Calpena, E., Lopez Del Amo, V., Chakraborty, M., Llamusi, B., Artero, R., Espinos, C. and Galindo, M. I. (2018). The Drosophila junctophilin gene is functionally equivalent to its four mammalian counterparts and is a modifier of a Huntingtin poly-Q expansion and the Notch pathway. Dis Model Mech 11(1). PubMed ID: 29208631


Members of the Junctophilin (JPH) protein family have emerged as key actors in all excitable cells, with crucial implications for human pathophysiology. In mammals, this family consists of four members (JPH1-JPH4) that are differentially expressed throughout excitable cells. The analysis of knockout mice lacking JPH subtypes has demonstrated their essential contribution to physiological functions in skeletal and cardiac muscles and in neurons. Moreover, mutations in the human JPH2 gene are associated with hypertrophic and dilated cardiomyopathies; mutations in JPH3 are responsible for the neurodegenerative Huntington's disease-like-2 (HDL2), whereas JPH1 acts as a genetic modifier in Charcot-Marie-Tooth 2K peripheral neuropathy. Drosophila melanogaster has a single junctophilin (jp) gene, as is the case in all invertebrates, which might retain equivalent functions of the four homologous JPH genes present in mammalian genomes. Therefore, owing to the lack of putatively redundant genes, a jp Drosophila model could provide an excellent platform to model the Junctophilin-related diseases, to discover the ancestral functions of the JPH proteins and to reveal new pathways. By up- and downregulation of Jp in a tissue-specific manner in Drosophila, this study shows that altering its levels of expression produces a phenotypic spectrum characterized by muscular deficits, dilated cardiomyopathy and neuronal alterations. Importantly, this study has demonstrated that Jp modifies the neuronal degeneration in a Drosophila model of Huntington's disease, and it has allowed uncovery of an unsuspected functional relationship with the Notch pathway. Therefore, this Drosophila model has revealed new aspects of Junctophilin function that can be relevant for the disease mechanisms of their human counterparts (Calpena, 2018).

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Aron, R., Pellegrini, P., Green, E. W., Maddison, D. C., Opoku-Nsiah, K., Wong, J. S., Daub, A. C., Giorgini, F. and Finkbeiner, S. (2018). Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease. Nat Commun 9(1): 3191. PubMed ID: 30266909


Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). This study shows that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, this study identifies Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration (Aron, 2018).

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Ast, A., et al. (2018). mHTT seeding activity: A marker of disease progression and neurotoxicity in models of Huntington's disease. Mol Cell 71(5): 675-688 PubMed ID: 30193095


Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). This paper reports the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, the neurotoxicity of mHTT seeds was assessed in an inducible Drosophila model transgenic for HTTex1. A strong correlation was found between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo (Ast, 2018).

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Drombosky, K. W., Rode, S., Kodali, R., Jacob, T. C., Palladino, M. J. and Wetzel, R. (2018). Mutational analysis implicates the amyloid fibril as the toxic entity in Huntington's disease. Neurobiol Dis. PubMed ID: 30171891


In Huntington disease (HD), an expanded polyglutamine (polyQ>37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an alpha-helix-rich oligomer, or a beta-sheet-rich amyloid fibril. This study describes an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in these experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents (Drombosky, 2018).

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Donnelly, K. M. and Pearce, M. M. P. (2018). Monitoring cell-to-cell transmission of prion-like protein aggregates in Drosophila melanogaster. J Vis Exp(133). PubMed ID: 29578503


Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. A Drosophila melanogaster model was established that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach described in this study can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention (Donnelly, 2018).

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Al-Ramahi, I., Lu, B., Di Paola, S., Pang, K., de Haro, M., Peluso, I., Gallego-Flores, T., Malik, N. T., Erikson, K., Bleiberg, B. A., Avalos, M., Fan, G., Rivers, L. E., Laitman, A. M., Diaz-Garcia, J. R., Hild, M., Palacino, J., Liu, Z., Medina, D. L. and Botas, J. . (2018). High-Throughput Functional Analysis Distinguishes Pathogenic, Nonpathogenic, and Compensatory Transcriptional Changes in Neurodegeneration. Cell Syst. PubMed ID: 29936182


Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. This study integrates functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. 312 genes were selected whose expression is dysregulated both in HD patients and in HD mice and then replicated and/or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroautophagy, suggesting a mechanism for mitigating pathogenesis. This multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points (Al-Ramahi, 2018).

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Akbergenova, Y. and Littleton, J. T. (2017). Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments. J Neurosci [Epub ahead of print]. PubMed ID: 28235896


Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) stretch within the Huntingtin (Htt) protein. Pathogenic Htt disrupts multiple neuronal processes, including gene expression, axonal trafficking, proteasome and mitochondrial activity, and intracellular vesicle trafficking. However, the primary pathogenic mechanism and subcellular site of action for mutant Htt are still unclear. Using a Drosophila HD model, this study found that pathogenic Htt expression leads to a profound overgrowth of synaptic connections that directly correlates with the levels of Htt at nerve terminals. Branches of the same nerve containing different levels of Htt show distinct phenotypes, indicating Htt acts locally to disrupt synaptic growth. The effects of pathogenic Htt on synaptic growth arise from defective synaptic endosomal trafficking, leading to expansion of a recycling endosomal signaling compartment containing Sorting Nexin 16, and a reduction in late endosomes containing Rab11. The disruption of endosomal compartments leads to elevated BMP signaling within nerve terminals, driving excessive synaptic growth. Blocking aberrant signaling from endosomes or reducing BMP activity (see Wishful thinking) ameliorates the severity of HD pathology and improves viability. Pathogenic Htt is present largely in a non-aggregated form at synapses, indicating cytosolic forms of the protein are likely to be the toxic species that disrupt endosomal signaling. These data indicate that pathogenic Htt acts locally at nerve terminals to alter trafficking between endosomal compartments, leading to defects in synaptic structure that correlate with pathogenesis and lethality in the Drosophila HD model (Akbergenova, 2017).

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Singh, V., Sharma, R. K., Athilingam, T., Sinha, P., Sinha, N. and Thakur, A. K. (2017). NMR spectroscopy-based metabolomics of Drosophila model of Huntington's disease suggests altered cell energetics. J Proteome Res [Epub ahead of print]. PubMed ID: 28871787


Huntington's disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophila model, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, this study has investigated the 1D 1H NMR spectroscopy-based metabolomics profile of Drosophila HD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, this study showed that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics (Singh, 2017).

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Raj, K. and Sarkar, S. (2017). Transactivation domain of human c-Myc Is essential to alleviate poly(Q)-mediated neurotoxicity in Drosophila disease models. J Mol Neurosci 62(1):55-66. PubMed ID: 28316031


Polyglutamine (poly(Q)) disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, represent a group of neurological disorders which arise due to an atypically expanded poly(Q) tract in the coding region of the affected gene. Pathogenesis of these disorders inside the cells begins with the assembly of these mutant proteins in the form of insoluble inclusion bodies (IBs), which progressively sequester several vital cellular transcription factors and other essential proteins, and finally leads to neuronal dysfunction and apoptosis. Earlier studies have shown that targeted upregulation of Drosophila myc (dmyc) dominantly suppresses the poly(Q) toxicity in Drosophila. The present study examines the ability of the human c-myc proto-oncogene and also identifies the specific c-Myc isoform which drives the mitigation of poly(Q)-mediated neurotoxicity, so that it could be further substantiated as a potential drug target. This study reports that similar to dmyc, tissue-specific induced expression of human c-myc also suppresses poly(Q)-mediated neurotoxicity by an analogous mechanism. Among the three isoforms of c-Myc, the rescue potential was maximally manifested by the full-length c-Myc2 protein, followed by c-Myc1, but not by c-MycS which lacks the transactivation domain. This study suggests that strategies focussing on the transactivation domain of c-Myc could be a very useful approach to design novel drug molecules against poly(Q) disorders (Raj, 2017).

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Dietz, K.N., Di Stefano, L., Maher, R.C., Zhu, H., Macdonald, M.E., Gusella, J.F. and Walker, J.A. (2015). The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development. Hum Mol Genet 24: 330-345. PubMed ID: 25168387

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 examines the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, it was found that dhtt acts as a suppressor of position-effect variegation (PEV), suggesting that it influences chromatin organization. It was demonstrated that 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 using RNAi lines targeting known PEV modifier genes was conducted. dhtt was found to modify phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases (HDMs) and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the HDM dLsd1, in both the ovary and wing, and it was demonstrated that 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).


  • Huntingtin is well conserved across different Drosophila species.
  • Generation of a new dhtt allele by homologous recombination.
  • Drosophila huntingtin is a suppressor of PEV.
  • Drosophila huntingtin affects heterochromatin spreading in a PEV model.
  • Genetic interactions between dhtt and genes encoding heterochromatin proteins.
  • Genetic interactions of dhtt with genes encoding chromatin-remodeling complex components.
  • Loss of dhtt enhances dLsd1 mutant phenotypes.
  • Drosophila huntingtin facilitates histone H3K4 demethylation activity of dLsd1.


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).

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Rui, Y.N., Xu, Z., Patel, B., Chen, Z., Chen, D., Tito, A., David, G., Sun, Y., Stimming, E.F., Bellen, H.J., Cuervo, A.M. and Zhang, S. (2015). Huntingtin functions as a scaffold for selective macroautophagy. Nat Cell Biol 17: 262-275. PubMed ID: 25686248

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).


  • Drosophila ​huntingtin genetically interacts with autophagy pathway components.
  • Drosophila ​huntingtin positively regulates autophagy in vivo.
  • Drosophila ​huntingtin is required for intracellular quality control.
  • Huntingtin’s function is conserved from flies to humans.
  • Mammalian ​Huntingtin is required for selective autophagy.
  • Huntingtin physically interacts with ​p62 and ​ULK1 proteins.
  • Huntingtin facilitates ​p62-mediated cargo recognition efficiency.
  • Huntingtin–​ULK1 and ​mTOR–​ULK1 complexes are mutually exclusive.

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 (A​Tau; ​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 atg13, 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).

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Pearce, M.M., Spartz, E.J., Hong, W., Luo, L. and Kopito, R.R. (2015). Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nat Commun 6: 6768. PubMed ID: 25866135

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).


  • Clearance of neuronal Htt aggregates by phagocytic glia.
  • Prion-like transmission of Htt aggregates from ORNs to glia.
  • Antennal injury enhances ORN-to-glial Htt aggregate transfer.
  • Glial Htt aggregates associate with cytoplasmic chaperones.
  • ORN-to-glia Htt aggregate transfer requires Draper signalling.

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).

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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 24(25): 7182-7195. 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 (White, 2015).

This study has identified a novel role for HTT in the regulation of the axonal transport of a particular sub-set of Rab-containing vesicles under physiological conditions. In vivo observations have led to two major findings; (1) HTT differentially regulates the movement of a specific sub-set of Rab-containing vesicles within axons, and (2) HTT is present on these Rab-containing vesicles during axonal transport. At least two possible mechanisms could exist by which HTT exerts a differential control on Rab motility, (1) by associations with specific Rab-containing vesicles, and/or (2) by regulating the motors on moving Rab-containing vesicles, although these two pathways may not be mutually exclusive. Collectively, these findings provide new insight into the normal physiological role of HTT which, when disrupted, may contribute to disease pathology observed in HD (White, 2015).

Different Rab-containing vesicles, even those within the same sub-cellular compartment, move at varying velocities, suggesting that Rab proteins found in the same compartment are differentially regulated. Indeed, different regulatory mechanisms could exist as Rab proteins control trafficking in both the secretory and endocytic pathways. Some Rab proteins are in distinct sub-sets of neurons suggesting that Rabs have roles in diversely regulated mechanisms, and HTT may function to differentially regulate the motility of these neuronal Rab proteins via Rab protein specific associations. Previously, work found that reduction of HTT perturbed the motility of Rab11-containing vesicles but not Rab5-containing vesicles. Since Rab11 is a marker for recycling endosomes and Rab5 is a marker for early endosomes, it is hypothesized that HTT influences the axonal motility of all recycling endosomes. Contrary to this, however, the current systematic in vivo analysis found that HTT does not influence the motility of all Rab proteins found in recycling endosomes, but rather, HTT only affects the movement of particular Rab proteins located in many different compartments. Reduction of HTT function perturbed the bi-directional motility of Rab19, a recycling endosomal Rab, while no effect was seen in the motility of other recycling endosomal Rab proteins. Additionally, reduction of HTT perturbed the bi-directional motility of Rab3, a Rab protein known to be present in synaptic vesicles. Intriguingly, reduction of HTT perturbed the retrograde movement of Rab7 (present on late endosomes), while the anterograde movement of Rab2 (present in ER-Golgi associated compartments) was stimulated by reduction of HTT. Perhaps this differential regulation that is observed with reduction of HTT may be caused by the existence of different HTT-Rab-containing motor complexes. Either several HTT-Rab-containing vesicle complexes may exist or alternatively more than one Rab could be present with a single HTT-Rab-containing vesicle. Perhaps, during long distance transport within axons, HTT-mediated regulation of specific Rab-containing vesicles is required for particular functions at the synapse. Indeed, similar to many Rab proteins, roles for HTT in endocytosis, intracellular trafficking and membrane recycling have also been proposed (White, 2015).

Specific associations between HTT, Rab proteins and linker proteins could perhaps dictate one potential mechanism by which HTT-mediated differential regulation of Rab-containing vesicle motility occurs. It is thought that associations between HTT and motors are facilitated by HTT associated proteins (HAPs). Pal (2006). showed that HTT can mediate the transport of a Rab5-HAP40-HTT-containing early endosome on actin filaments via associations with myosin, the actin motor. HTT can also interact with myosin via optineurin. Optineurin is a binding partner that links both myosin and HTT to the Golgi network via Rab8 for ER-Golgi trafficking in the secretory pathway. HTT can also associate with Rab8 through FIP-2 for regulated cell polarization and morphogenesis. Since reduction of HTT altered the sub-cellular localization of Rab8 the current observations suggest that HTT can play a role in linking Rab8 to vesicles; enabling associations with MT motors during axonal transport. While the involvement of optineurin or FIP-2 in the association of HTT and Rab8 in the context of axonal movement is still unclear, what is clear is that HTT is likely required for the membrane-bound state of Rab8 during axonal transport under physiological conditions (White, 2015).

It has been proposed that a HTT-Rab11-motor complex likely exists during axonal transport. The motility of Rab11-containing vesicles was perturbed with reduction of HTT. Both kinesin-1 and dynein motors were required for MT motility of Rab11. Additionally, membrane binding of Rab11 was decreased in HD knock-in mice, suggesting that similar to Rab8, HTT is also likely required for the membrane-bound state of Rab 11. The Rab11 effector Rip11 regulates the endocytic recycling pathway by forming a complex with Rab11 and kinesin II. Rip11 is also important for the trafficking of Rab11 from apical recycling endosomes to the apical membrane. Perhaps Rip11 may act as a linker that connects Rab11 and HTT similar to optineurin linking Rab8 and HTT. Rabphilin-3A, a Rab3 effector molecule may link Rab3 vesicles to HTT during axonal transport. Studies have shown that Rab3 and Rabphilin-3A are both transported by fast anterograde transport and associate with synaptic vesicles. Thus, although further study is needed, Rab-associated proteins could aid in linking specific Rab-containing vesicles with HTT during axonal transport (White, 2015).

Alternatively, HTT-mediated differential regulation of Rab protein motility could result due to changes in motor protein regulation. Indeed, previous work postulated HTT as a molecular switch that determines the direction of movement during axonal transport. HTT is phosphorylated by Akt (protein kinase B) (a serine-threonine kinase) at serine 421. Constitutively phosphorylated (S421D) HTT can recruit kinesin-1 to the dynactin complex to facilitate anterograde transport while disruption of phosphorylation at S421 (S421A) prevents kinesin association with HTT and the motor complex, enabling retrograde transport. Perhaps HTT's role as a molecular regulator during axonal transport could result in the HTT-mediated motility changes were observe in this study, since reduction of HTT not only perturbed the bi-directional motility of Rab3 and 19, and the retrograde motility of Rab7, but also stimulated the anterograde motility of Rab2, via specific changes to motility parameters; vesicle velocities, pause duration/frequencies and run lengths. While the functional significance of the differential regulation of Rab motility and the mechanistic steps of how HTT controls motors in the context of the different Rab-containing complexes are still unclear, perhaps particular Rab proteins could also exert a regulatory function during vesicle motility by affecting the phosphorylation state of HTT and changing the direction of vesicle movement. Interestingly, several Rabs have been shown to be effectors of kinases. Rab5 and Rab7 are thought to be effectors of PI3K, which is an upstream activator of Akt. Additionally, it has been shown that HTT can act as a scaffold to transport glycolytic machinery down the axon that is required for vesicular motility. Reduction of HTT could decrease glycolysis disrupting the motility of Rab-containing vesicles. Further experiments will be needed to dissect the mechanistic steps involved in the differential regulation of these different HTT-Rab-containing complexes during axonal transport under physiological conditions (White, 2015).

An intriguing result from this analysis was that reduction of HTT influenced the retrograde transport of Rab7, although Rab7-containing vesicles moved bi-directionally. Previous work has implicated Rab7 in neurotrophin receptor trafficking, particularly in the retrograde transport of TrkB/p75NTR-positive signaling endosomes in motor neurons. Consistent with this, CMT2B Rab7 mutants altered trafficking and signaling of retrograde NGF/TrkA trophic signal. Thus, since HTT and Rab7 co-localize on moving vesicles during axonal transport a HTT-Rab7-containing signaling endosome could exist during axonal transport. Alternatively, since Rab7 is a marker for late endosomal and lysosomal compartments, and HTT and dynein were found to be required for the perinuclear positioning of lysosomes, perhaps a HTT-Rab7-containing lysosome could exist during axonal transport. Work has also shown that Rab7 and LC3 (a marker for autophagosomes) are together during the transport of autophagosomes at growth cones and that the retrograde movement of autophagosomes is required for their maturation. Interestingly Rab7 interacting lysosomal protein (RILP) was shown to control lysosomal transport by recruiting dynein-dynactin to Rab7-containing late endosomes/lysosomes. The FYVE (Fab1-YotB-Vac1p-EEA1) and coiled-coil domain-containing 1 protein (FYCO1) was found to function as an adaptor to link autophagosomes to kinesin via Rab7. Additionally, both HTT and HAP1 were identified as regulators of autophagosome transport in neurons. Thus, the current results are consistent with these observations and suggest that perhaps a HTT-Rab7-authophagosome complex and/or a HTT-Rab7-signaling endosomal complex could exist under physiological conditions(White, 2015).

Surprisingly this analysis also revealed that reduction of HTT stimulated the anterograde velocity of Rab2, although Rab2-containing vesicles moved bi-directionally. Rab2 is known to regulate the anterograde and retrograde trafficking of vesicles between the Golgi, the ER-Golgi intermediate compartment and the ER. Rab2 was also one of the Rab proteins that showed the most neuronal sub-cellular localization behaviors: synaptic enrichment with expression of a CA form and loss of synaptic localization with the dominant negative form , suggesting a role for Rab2 at the synapse. While roles for HTT at synapses have been documented, perhaps HTT may function to regulate the anterograde motility of a Rab2-containing complex, although the functional significance for this complex at the synapse is still unknown (White, 2015).

Rab dysfunction has been implicated in many neuronal diseases. For example, a missense mutation in Rab7 was demonstrated in the myelin and axonal disorder Charcot-Marie Tooth disease Type 2B. Altered expression of Rab1, Rab8, and Rab2 was shown to cause Golgi fragmentation in Parkinson's disease. Expansion of a hexanucleotide repeat in C9ORF72, a Rab-associated GEF, was seen in both Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD), suggesting that this mutant form of the GEF may contribute to the physiology of the disease through Rab dysfunction. Defects in the recycling of Rab7 from lysosomes to early endosomes impaired the transport and degradation of amyloid beta (Aβ) in Alzheimer's disease (AD). Rab6 was shown to modulate the unfolded protein response due to ER stress in AD. Interestingly, defects in Rab11 function were recently observed in HD. Expression of Rab11 was decreased in HD mouse models, and Rab11 activation was impaired by mutant HTTA. Over expression of Rab11 rescued neurodegeneration, dendritic spine loss, synaptic defects and behavioral defects in HD models in both mice and Drosophila. Perhaps defects to HTT-mediated axonal transport of a specific sub-set of Rab-containing vesicles could contribute to neurodegeneration and synaptic defects observed in HD. Thus this work could highlight a potential novel therapeutic pathway for early treatment of HD pathology (White, 2015).

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Maheshwari, M., Bhutani, S., Das, A., Mukherjee, R., Sharma, A., Kino, Y., Nukina, N. and Jana, N.R. (2014). Dexamethasone induces heat shock response and slows down disease progression in mouse and fly models of Huntington's disease. Hum Mol Genet 23: 2737-2751. PubMed ID: PubMed

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by abnormal expansion of glutamine repeats in the protein huntingtin. In HD brain, mutant huntingtin undergoes proteolytic processing, and its N-terminal fragment containing poly-glutamine repeats accumulate as insoluble aggregates leading to the defect in cellular protein quality control system and heat shock response (HSR). This study demonstrates that the defective HSR in the brain is due to the down-regulation of heat shock factor 1 (HSF1) in both mice and fly models of HD. Interestingly, treatment of dexamethasone (a synthetic glucocorticoid) to HD mice or flies significantly increases the expression and transactivation of HSF1 and induction of HSR and these effects are mediated through the down-regulation of HSP90. Dexamethasone treatment also significantly decreases the aggregate load and transient recovery of HD-related behavioural phenotypes in both disease models. These results suggest that dexamethasone could be a potential therapeutic molecule for the treatment of HD and related poly-glutamine disorders (Maheshwari, 2014).


  • HSF1 is reduced in HD mice brain.
  • HSF1 is induced upon dexamethasone treatment.
  • Dexamethasone treatment induces transactivation of HSF1 and up-regulation of HSP70.
  • Dexamethasone treatment reduces aggregate load and improves motor performance of HD mice.
  • Effects of dexamethasone in HD are recapitulated in a fly model.
  • Dexamethasone treatment down-regulates HSP90.

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Melkani, G.C., Trujillo, A.S., Ramos, R., Bodmer, R., Bernstein, S.I. and Ocorr, K. (2013). Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart. PLoS Genet 9: e1004024. PubMed ID: 24367279 

Amyloid-like inclusions have been associated with Huntington's disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. This study generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs results in PolyQ length-dependent functional defects that include increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells reveals reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, expanded PolyQ-72 was co-expressed with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppresses PolyQ-72 induced mitochondrial defects and partially suppresses aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppresses PolyQ-72 induced aggregation and partially suppresses myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppresses GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. The study demonstrates that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increased oxidative stress in cardiomyocytes leading to abnormal cardiac function. The study concludes that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients (Melkani, 2013).


  • Expression of disease causing PolyQ in the heart causes cardiac dilation and reduced contractility.
  • Cardiac expression of disease-causing PolyQ causes accumulation of aggregates and myofibrillar defects.
  • Ultrastructural analysis revels mitochondrial and myofibrillar defects upon expression of mutant PolyQ.
  • Oxidative stress plays a role in PolyQ-induced cardiac defects.
  • Transgenic over-expression of SODs rescues the Poly-Q induced cardiac mitochondrial and functional defects.
  • Over-expression of the chaperone UNC-45 suppresses Poly-Q induced cardiomyopathy.
  • Combined UNC-45 chaperone and antioxidant treatment is required for efficient suppression of PolyQ-induced aggregation and cardiac defects.

Huntingtin protein is expressed in many tissues including the heart and epidemiological studies suggest that HD patients have a higher susceptibility to cardiac failure compared to age-matched controls without HD. However, the cellular mechanisms underlying the cardiac dysfunction in HD have yet to be studied in the heart. Using the genetically tractable model system Drosophila, this study shows a direct correlation between the levels of amyloid accumulation, overall ROS production and the severity of cardiac dysfunction. Cardiac-specific expression of disease-causing Htt-PolyQ (PolyQ-46, PolyQ-72 and PolyQ-102) all elicit cardiac dysfunction compared to hearts expressing the non-disease-causing PolyQ-25. In addition the qualitative as well as quantitative defects that were observed in response to PolyQ expression were dose-dependent. Since mutant Htt-PolyQ protein was expressed specifically in the heart, it is unlikely that there is a neuronal contribution to these cardiac defects. It was suggested that the increased risk of cardiac disease in HD patients is possibly due to cardiac amyloid accumulation, mitochondrial defects as well as oxidative stress and that the severity of disease depended upon the length of the PolyQ repeat (Melkani, 2013).

The likely cause of the observed functional defects was demonstrated to be severe myofibrillar disorganization and reduced myosin and actin content in myocardial cells resulting from cardiac-specific expression of disease causing PolyQ. It has earlier been shown that the chaperone UNC-45 is required for preserving myosin accumulation/folding in Drosophila cardiomyocytes, as its reduction leads to severe disorganization of myosin-actin containing myofibrils and thus sarcomeres. This study extended this observation and demonstrated a role for UNC-45 in amyloidosis-induced cardiac defects for the first time. In support of this hypothesis, it was previously shown that nuclear or cytoplasmic aggregates (inclusion bodies) of polyglutamine proteins contained chaperones involved in protein folding. Furthermore, and consistent with this study's results, over-expression of the chaperone αB-crystallin reduced PolyQ-induced aggregation in rat neonatal cardiomyocytes; however, over-expression of αB-crystallin enhanced amyloid oligomer formation and toxicity (Melkani, 2013).

Co-over-expression of UNC-45 with disease-causing PolyQ-72 dramatically reduces amyloid aggregate density and ameliorates cardiac dysfunction by decreasing the incidence of cardiac arrhythmia, suppressing the mutant-Htt-induced cardiac dilation and improving cardiac contractility to a dramatic extent. Importantly, over-expression of UNC-45 in the presence of PolyQ-72 restores myosin-containing myofibrils, suggesting that one effect of amyloid aggregation is to interfere with proper folding of muscle myosin in cardiomyocytes (Melkani, 2013).

The fact that UNC-45 over-expression does not completely suppress the mutant Htt-PolyQ-induced cardiac physiological defects and lifespan reduction is consistent with the idea that amyloid accumulation affects additional cellular pathways that result in cardiac abnormalities. As reported for αB-crystallin, suppression of aggregates is not sufficient to reduce toxicity and this possibility may also exist in the case of UNC-45. It is also possible that the overall high level of oxidative stress produced by mutant PolyQ is the main determinant for lethality. Expression of mutant PolyQ leads to mitochondrial defects due to increased oxidative stress. Several neuronal studies have shown that expression of mutant polyQ affects SOD expression. Manipulation of SOD seems to be directly correlated with levels of oxidative stress in several neurodegenerative diseases. Additionally, SOD over-expression reduces diabetic cardiomyopathy and some forms of neurodegeneration by reducing oxidative stress. However, neither UNC-45 nor SOD has been shown previously to suppress the PolyQ-induced phenotypes in either neuronal or cardiac animal disease models (Melkani, 2013).

Data from this study also support a role for oxidative stress pathways in amyloid-induced cardiac dysfunction and lethality. Treatment with oxidants aggravates the moderate effects of PolyQ-46 on heart function, causing an increase in amyloid aggregate density and more severe cardiac defects. This suggests a possibly causal relationship between oxidative stress, the formation of aggregates and cardiac dysfunction. Furthermore, ultrastructural analysis clearly shows mutant PolyQ-induced mitochondrial defects, while DHE staining indicates that excess ROS production occurrs upon expression of mutant PolyQ. Interestingly, some of the PolyQ aggregates co-localize with concentrated DHE staining. Significantly, the size and density of mutant PolyQ-aggregates as well as the severity of the PolyQ-72-induced cardiac defects are reduced by over-expression of SOD or by feeding the anti-oxidant resveratrol. This is consistent with findings that resveratrol provides protection in neuronal models of Huntington's disease. Interestingly, the anti-oxidant resveratrol has been shown to affect expression of anti-oxidative enzymes, including enhanced expression of SOD-1 (Melkani, 2013).

Expression of mutant PolyQ may both induce oxidative stress and interfere with protein folding pathways. A study using cultured mouse neurons showed that oxidative stress increases PolyQ aggregation and that over-expression of SOD1 in conjunction with the chaperone HSP-70/HSP-40 could suppress Htt-polyQ-induced aggregation and toxicity. However, simultaneous manipulation of both of these genetic pathways has not previously been attempted in vivo. In addition to neurons, expression of the mutated Htt protein or expression of pre-amyloid oligomers cause cardiac defects by affecting several pathways including oxidative stress, mitochondrial abnormalities, presence of protein aggregates and increased autophagosomal content. However, no attempt has thus far been made to suppress PolyQ-induced cardiac defects, a crucial step for understanding the mechanistic basis of disease progression and amelioration. Indeed, co-expression of UNC-45 and SOD-1 or expression of UNC-45 in the presence of resveratrol had a tendency to suppress the PolyQ-72-induced amyloid aggregation and concomitant cardiac dilation more efficiently than either treatment alone. Thus, suppression of both protein aggregates and ROS may be required for the amelioration of PolyQ-induced cardiomyopathy. As HD is primarily a neurological disease, the effect of such suppression is worth exploring in neural tissues. (Melkani, 2013).

In addition to interfering with protein folding pathways, expression of mutant PolyQ may lead to myofibril loss by directly interacting with muscle proteins. Previous studies have suggested that mutant PolyQ may bind directly to contractile proteins and disturb their function. Integrity of contractile proteins is also required for maintaining mitochondrial organization and cardiomyocyte function. Additionally, expression of aggregation-prone mutant PolyQ may induce oxidative stress due to mitochondrial damage in the cardiomyocytes, which are heavily dependent on mitochondrial function and are vulnerable to oxidative stress. For example, knockdown of SOD results in mitochondrial defects and severe dilated cardiomyopathy phenotype in a mouse model. In this study, a dramatic increase in overall ROS levels in mutant PolyQ expressing hearts was observed. Moreover, the GFP-positive PolyQ aggregates co-localized with areas of strong DHE staining and the observation that antioxidant treatments partially rescued the cardiac defects further support this hypothesis (Melkani, 2013).

Overall, accumulation of amyloid in the cardiomyocytes could induce mechanical deficits by affecting the integrity of contractile proteins as well as mitochondria and lead to cardiomyocyte death, possibly through activation of autophagy. Consistent with data from this study, a similar mechanism has been proposed for cardiomyopathy associated with amyloid producing mutant αB-crystallin. Both mutant αB-crystallin and mutant PolyQ caused aggregate formation in cardiomyocytes suggesting a common mechanism for underlying cardiomyocyte degeneration. It is unclear at this point whether the presence of toxic aggregates in cardiomyocytes is directly interfering with mitochondrial organization leading to cardiac defects or whether oxidative stress produced by mutant PolyQ leads to mitochondrial dysfunction that triggers cardiomyocyte dysfunction (Melkani, 2013).

A full understanding of all the molecular details involved in mutant PolyQ induced cardiomyopathy will require additional study but some of the key players and interactions in vivo have been identified. Although dense granular deposits, immunoreactive to an anti-Huntingtin antibody, have been found in muscle tissue from an HD patient, no such study has been performed on HD heart biopsy samples. Thus, this study suggests that it would be useful to look for accumulation of amyloid protein in the hearts of HD patients, especially those with heart disease. Delineating how these aggregates might be toxic to cells will be critical not only for an understanding of PolyQ-induced cardiomyopathy but also for gaining insights into aggregation-based neural degeneration. The Drosophila heart model provides a genetically tractable system whereby these interactions can be examined in the context of a functioning organ. Indeed, elucidating the genetics underlying PolyQ-induced cardiomyopathy should also have an impact on our understanding of other cardiac diseases associated with oxidative stress, mitochondrial dysfunction, the unfolded protein response and proteostasis in general (Melkani, 2013).

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Mason, R.P., Casu, M., Butler, N., Breda, C., Campesan, S., Clapp, J., Green, E.W., Dhulkhed, D., Kyriacou, C.P. and Giorgini, F. (2013). Glutathione peroxidase activity is neuroprotective in models of Huntington's disease. Nat Genet 45: 1249-1254. PubMed ID: 23974869

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (htt) protein. This study reports a genome-wide overexpression suppressor screen which identified 317 open reading frames that ameliorate toxicity of a mutant htt fragment in yeast, and play roles in diverse cellular processes, including mitochondrial import and copper metabolism. Two suppressors encode glutathione peroxidases (GPxs), conserved antioxidant enzymes that reduce hydrogen peroxide and lipid hydroperoxides. Using genetic and pharmacological approaches in yeast, mammalian cells and Drosophila, it was found that GPx activity robustly ameliorates HD-relevant metrics and is more protective than other antioxidant approaches tested. Importantly, it was observed that GPx activity – unlike many antioxidant treatments – does not inhibit autophagy, an important mechanism for clearing mutant htt. As previous clinical trials indicate GPx mimetics are well-tolerated in humans, this study may have important implications for the treatment of HD (Mason, 2013).


  • Suppression of Htt103Q toxicity in yeast by ORF overexpression.
  • mGpx1 and ebselen improve HD-relevant phenotypes in PC12 cells.
  • mGpx1 and ebselen ameliorate phenotypes in HD flies.
  • Ebselen does not inhibit basal or induced autophagy in PC12 cells.

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Bodai, L., Pallos, J., Thompson, L.M. and Marsh, J.L. (2012). Pcaf modulates polyglutamine pathology in a Drosophila model of Huntington's disease. Neurodegener Dis 9: 104-106. PubMed ID: 21912091

Huntingtin peptides with elongated polyglutamine domains, the root causes of Huntington's disease, hinder histone acetylation, which leads to transcriptional dysregulation. However, the range of acetyltransferases interacting with mutant Huntingtin has not been systematically evaluated. This study used genetic interaction tests in Drosophila to determine whether specific acetyltransferases belonging to distinct protein families influence polyglutamine pathology. It was found that flies expressing a mutant form of the Huntingtin protein (Httex1pQ93) exhibit reduced viability, which was further decreased by partial loss of Pcaf or nejire, while the tested MYST family acetyltransferases do not affect pathology. Reduced levels of Pcaf also lead to the increased degeneration of photoreceptor neurons in the retina. Overexpression of Pcaf, however, was not sufficient to ameliorate these phenotypes, and the level of soluble Pcaf was unchanged in Httex1pQ93-expressing flies. These results indicate that while Pcaf has a significant impact on Huntington's disease pathology, therapeutic strategies aimed at elevating its levels are likely to be ineffective in ameliorating Huntington's disease pathology; however, strategies that aim to increase the specific activity of Pcaf remain to be tested (Bodai, 2012).


  • Mutant Huntingtin does not reduce the level of soluble Pcaf.

To investigate the consequences of reduced HAT activity on polyQ pathology, this study used a Drosophila model of HD, which was previously shown to be sensitive to acetylation levels. The phenotypes of flies that expressed Httex1pQ93 in the nervous system were compared with their siblings that, in addition, carried a HAT mutation as well. It was found that partial loss of Pcaf (the single fly homolog of human Pcaf and Gcn5) or nejire/dCBP (nej, homolog of human CBP) by the PcafE333St or nej3 mutations, respectively, significantly reduces the viability of Htt-expressing flies, while reducing the MYST family acetyltransferases enok, mof or CG1894 did not have a significant effect on pathology (Bodai, 2012).

Since the role of CBP is well established in HD pathogenesis, the effect of Pcaf was investigated. First, it was tested whether Df(3L)iro-2, an independent deletion that removed the Pcaf gene but did not share the same genetic background as the PcafE333St null allele, has the same effect. It was found that Htt-expressing flies heterozygous for Df(3L)iro-2 also exhibit significantly reduced viability. By comparing Htt-expressing control flies with siblings expressing Htt and also carrying the PcafE333St allele or the Df(3L)iro-2 deletion, it was found that the average number of rhabdomeres (light gathering structures of photoreceptor neurons) per ommatidium are decreased indicating that reduced Pcaf levels enhance neurodegeneration (Bodai, 2012).

It was also found that partial loss of Pcaf significantly enhances polyglutamine pathology, but the level of Pcaf was not reduced by mutant Htt, and HD phenotypes could not be rescued by the overexpression of Pcaf, indicating that the interaction of mutant Htt and Pcaf does not involve the depletion of soluble Pcaf by either degradation or sequestration to insoluble aggregates. This result, however, does not exclude the possibility that a soluble toxic form of Htt may inhibit the function of either Pcaf itself or of Pcaf-containing complexes. Since Pcaf acts as a catalytic subunit in large multiprotein complexes in metazoans, it is speculated that its interaction with a polyQ peptide may cripple entire complexes, which can not be rescued by overexpression of Pcaf alone (Bodai, 2012).

The study concludes that although Pcaf has a significant impact on HD pathology, therapeutic strategies aimed at elevating the levels of Pcaf protein are unlikely to be effective in ameliorating HD pathology. The question, however, remains open whether strategies that aim to increase the specific activity of Pcaf might be useful. Interestingly, of the three HAT families of proteins tested, only the GNAT and the CBP/p300 families exhibit a strong influence over HD pathology, while the MYST family members have decidedly less impact (Bodai, 2012).

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Campesan, S., Green, E.W., Breda, C., Sathyasaikumar, K.V., Muchowski, P.J., Schwarcz, R., Kyriacou, C.P. and Giorgini, F. (2011). The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease. Curr Biol 21: 961-966.  PubMed ID: 21636279

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD). A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein. This study exploits a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. It was observed that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. It was also found that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, it was demonstrated that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted at two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders (Campesan, 2011).


  • HD model flies exhibit perturbed flux through the KP.
  • cinnabar and vermillion mutations are neuroprotective in HD model flies.
  • KMO inhibitors ameliorate neurodegeneration in HD model flies.
  • 3-HK and KYNA modulate neurodegeneration.
  • Genetic manipulation of the KP modulates neurodegeneration.

This study observed that both genetic and chemical inhibition of KMO ameliorates neurodegeneration in HD model flies, supporting previous observations that inhibition of KMO is protective in a yeast model of mutant htt toxicity. It was also found that this neuroprotection correlates with decreases in 3-HK relative to KYNA. Importantly, this protection could be abrogated by 3-HK feeding, showing the causative nature of this metabolite. Furthermore, a KYNA feeding regime that forced a neuroprotective ratio of these metabolites also reduced neurodegeneration in HD flies. Therefore, it was shown for the first time by this study that KP metabolites directly modulate neurodegeneration in an animal model of neurodegenerative disease. Finally, genetic dissection of the KP with vermillion and cardinal mutants further underscores the critical relationship between imbalances in the KP and neurodegeneration (Campesan, 2011).

3-HK generates free radicals by auto-oxidation, and this mechanism is believed to underlie 3-HK-dependent toxicity observed in neuronal cell lines and in primary neurons. It is therefore likely that 3-HK was contributing to neurodegeneration by generation of free radicals in experminents in this study. At endogenous concentrations in the mammalian brain, KYNA functions as a competitive inhibitor of the glycine coagonist site of the NMDA receptor and a potent, noncompetitive inhibitor of the α7 nicotinic acetylcholine receptor. KYNA treatment results in dose-dependent behavioral changes in rodents, mimicking properties of NMDA receptor antagonists. Moreover, KYNA is neuroprotective against excitotoxic lesions and inhibits the release of glutamate at concentrations in the physiological range. In another study that used a novel KMO inhibitor, it was found that glutamate reduction by KYNA is the likely mechanism for neuroprotection in the central nervous system (CNS) of HD mice. Because flies express orthologs of both NMDA and α7 nicotinic acetylcholine receptors, KYNA could be conferring neuroprotection by antagonizing these receptors and decreasing glutamate-dependent excitotoxicity, as well as by scavenging free radicals (Campesan, 2011).

Interestingly, it was found that increased 3-HK relative to KYNA was only neurotoxic in the presence of Htt93Q, suggesting that general cellular dysfunction due to mutant htt expression confers a sensitized background for KP modulation. In a related fashion, it was found that expression of Htt20Q was sufficient to induce increased 3-HK/KYNA ratios but did not confer neurodegeneration—providing further support that additional Htt93Q-dependent cellular defects are required to uncover KP-dependent toxicity. A similar observation was made in another study with flies expressing full-length htt constructs where it was found that whereas expression of either an expanded construct (128QhttFL) or a nonexpanded construct (16QhttFL) leads to elevated resting synaptic Ca2+ levels, this perturbation is associated with pathogenesis only in the case of 128QhttFL flies. (Campesan, 2011).

The finding in this study that inhibition of TDO is protective in the fly validates this protein (TDO) as a novel therapeutic target for HD. TDO and IDO, which are both expressed in the brain, have distinct structural and biochemical characteristics, and selective inhibitors are being actively explored as potential therapeutic compounds. It is now imperative that such compounds be tested in animal models of HD, and perhaps other neurodegenerative disorders, to characterize their efficacy and therapeutic potential (Campesan, 2011).

In summary, results of this study are consistent with the view that modulation of the KP is central to mutant htt toxicity. The observations provide genetic and pharmacological support for the “kynurenine hypothesis,” underscoring the important role that Drosophila plays in the understanding and possible development of therapy for human neurodegenerative disorders. The study favors a model in which increased flux toward KYNA synthesis is neuroprotective in HD, provides unequivocal evidence that 3-HK, independent of QUIN, is pathogenic, and shows that reduction of 3-HK relative to KYNA is therapeutic (Campesan, 2011).

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Tamura, T., Sone, M., Iwatsubo, T., Tagawa, K., Wanker, E.E. and Okazawa, H. (2011). Ku70 alleviates neurodegeneration in Drosophila models of Huntington's disease. PLoS One 6: e27408.  PubMed ID: 22096569

DNA damage accumulates in genome DNA during the long life of neurons, thus DNA damage repair is indispensable to keep normal functions of neurons. It was previously reported that Ku70, a critical molecule for DNA double strand break (DSB) repair, is involved in the pathology of Huntington's disease (HD). Mutant huntingtin (Htt) impairs Ku70 function via direct interaction, and Ku70 supplementation recovers phenotypes of a mouse HD model. In this study, multiple Drosophila HD models were generated that expressed mutant huntingtin (Htt) in eye or motor neuron by different drivers and showed various phenotypes. In such fly models, Ku70 co-expression recovers lifespan, locomotive activity and eye degeneration. In contrast, Ku70 reduction by heterozygous null mutation or siRNA-mediated knock down accelerates lifespan shortening and locomotion disability. These results collectively support that Ku70 is a critical mediator of the HD pathology and a candidate therapeutic target in HD (Tamura, 2011).


  • Ku70 rescues mutant Htt-induced eye degeneration in Drosophila.
  • Ku70 elongates lifespan of Drosophila HD model.
  • Ku70 reduction affects the lifespan of HD model fly.
  • Ku70 reduction accelerates locomotion disability of HD model fly.

This study shows that Ku70 alleviates the HD phenotypes in Drosophila models and that the functional deficiency of Ku70 accelerates the lifespan shortening and locomotion disability of HD model flies. These results are consistent with earlier observation that Ku70 remarkably elongates the lifespan of R6/2 mice, the severest mouse model of HD, and strongly support the significance of Ku70 as a disease mediator/modifier gene and a therapeutic target (Tamura, 2011).

Mutant Htt reduces DNA-PK activity and impairs the DNA repair function of Ku70 in vitro. In the double transgenic mice generated from R6/2 and Ku70 transgenic mice, Ku70 reduces DNA damage of striatal neurons. These earlier findings provide the idea that the functional defect of Ku70 underlies the HD pathology. However, it is also possible to speculate that Ku70 blocks mutant Htt to prevent interaction between mutant Htt and other target molecules and/or to inhibit aggregation of mutant Htt. The two hypotheses are inseparable and just like two sides of a coin. The two stories are identical if one focuses on DNA repair. However, if there is certain pathology other than DNA repair, the blocking effect might be more important (Tamura, 2011).

With Drosophila, DSBs can not be directly evaluated in a few motor neurons because there is no fly orthologue of mammalian H2AX, a direct indicator of DSB, and there is no alternative method to detect DSB in neurons of Drosophila. However, results in this study are basically consistent with earlier observation that impairment of DSB repair is a critical component in the HD pathology and with previous reports showing DNA damage signal activation in HD cell models (Tamura, 2011).

The concept of the linkage between DNA damage repair and neurodegeneration is further supported by multiple autosomal recessive cerebellar atrophies, in which mutations of DNA repair genes such as AOA1/EAOH, AOA2, and SCAN1 cause neuronal dysfunction and cell death. Therefore, impairment of DNA damage repair can be considered as a common pathological component across categories of neurodegenerative disorder (Tamura, 2011).

In conclusion, results from this study support that Ku70 is a critical regulatory factor of Htt toxicity and a candidate for therapeutic target of HD. This study provides a rationale to proceed to the next step for translational approaches with Ku70 such as using viral vectors expressing Ku70 or low molecular weight chemicals against DSB in HD (Tamura, 2011).

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Besson, M.T., Dupont, P., Fridell, Y.W. and Liévens, J.C. (2010). Increased energy metabolism rescues glia-induced pathology in a Drosophila model of Huntington's disease. Hum Mol Genet 19: 3372-3382. PubMed ID: 20566711

Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by an expanded CAG tract within the coding region of Huntingtin protein. Mutant Huntingtin (mHtt) is ubiquitously expressed, abundantly in neurons but also significantly in glial cells. Neuron-intrinsic mechanism and alterations in glia-to-neuron communication both contribute to the neuronal dysfunction and death in HD pathology. However, the role of glial cells in HD pathogenesis remains to be determined. In recent years, development of Drosophila models have facilitated the dissection of the cellular and molecular events in polyQ-related diseases. By using genetic approaches in Drosophila, this study manipulated the expression levels of mitochondrial uncoupling proteins (UCPs) that regulate production of both ATP and reactive oxygen species in mitochondria. It was discovered that enhanced levels of UCPs alleviate the HD phenotype when mHtt was selectively expressed in glia, including defects in locomotor behavior and early death of Drosophila. In contrast, UCPs fail to prevent the HD toxicity in neurons. Increased oxidative stress defense was found to rescue neuron but not glia-induced pathology. Evidence is now emerging that UCPs are fundamental to adapt the energy metabolism in order to meet the metabolic demand. Thus, the study proposes that UCPs are glioprotective by rescuing energy-dependent functions in glia that are challenged by mHtt. In support of this, increasing glucose entry in glia was found to alleviate glia-induced pathology. Altogether, data from this study emphasizes the importance of energy metabolism in the glial alterations in HD and may lead to a new therapeutic avenue (Besson, 2010).


  • Drosophila UCP5 rescues glial pathology in HD flies.
  • Drosophila UCP5 fails to rescue neuronal pathology in HD flies.
  • Human UCP2 alleviates glial pathology in HD flies.
  • Superoxide dismutase and catalase do not rescue glia-induced pathology in HD flies.
  • Drosophila glucose transporter rescues glial pathology in HD flies.

The main outcome of this study is that manipulating energy metabolism alleviates the glial-induced pathology in a Drosophila model of HD. Neuron–glia interactions are crucial to ensure optimal brain function and accumulation of mHtt compromises crosstalk between the two cell types. Interestingly, whereas both neuronal and glial expression of mHtt trigger decreased climbing performance, only flies expressing mHtt in glia suffer from bang-sensitivity following a mechanical stress. Thus, the bang-sensitive phenotype likely reflects the disruption of glial-specific functions by expanded polyQ proteins. The exact mechanism that triggers the bang-sensitive behavior in Drosophila is not well understood. So far, bang-sensitivity has been related to depletion in ATP levels and impairment of the Na+/K+ APTase pump. Interestingly, two crucial functions of glia are to supply neurons with energy metabolites and to maintain ionic composition of the extracellular environment (Besson, 2010).

It was reported that DmUCP5 rescues the bang-sensitive phenotype and the climbing performance when co-expressed with mHtt in glia. Moreover, DmUCP5 significantly prolongs the survival of flies expressing mHtt in glia. Similarly, the exogenous expression of hUCP2 conferrs protection against mHtt toxicity in glia. In contrast, DmUCP5 fails to alleviate the deleterious phenotype when co-expressed with mHtt in neurons. Thus, this study provides the first evidence that UCPs mediate differential effects on the glial- and neuronal-specific physiology (Besson, 2010).

Numerous studies have raised the possibility that UCPs can confer neuroprotection in models of brain injury or neurodegenerative diseases. Evidence comes mainly from transgenic mice containing a copy of human UCP2 and UCP3 genes cloned under the regulation of their endogenous promoters, respectively. Accordingly to the regional distribution of UCPs, those mice show a moderate overexpression of UCP2 into the brain. UCP2/UCP3 transgenic mice exhibit a reduction in cell death occurring after experimental induction of epileptic seizures or following ischemia and traumatic brain injury. Acute treatment with 1-methyl-4-phenyl-1,2,5,6-methyl-phenyl-tetrahydropyridine (MPTP) is currently used to induce dopaminergic neuron degeneration and reproduce a syndrome clinically similar to Parkinson's disease in animal models. Whereas UCP2 knock-out mice display an enhanced sensitivity to MPTP exposure, UCP2/UCP3 transgenic mice show a lower MPTP-induced loss of dopaminergic neurons in the substantia nigra (Besson, 2010).

Similarly, selective overexpression of UCP2 in catecholaminergic neurons by using the tyrosine hydroxylase promoter protects nigral neurons from acute MPTP toxicity. Stroke, ischemia and acute MPTP treatment appear to lead to neuronal death by a common pathway: they all alter mitochondrial metabolism and increase ROS release. Accordingly, in the aforementioned studies, the neuroprotective effect of UCP2 is correlated with the reduction of oxidative stress in these models. Since mHtt is known to interact with mitochondria and leads to increased ROS production, it is tempting to propose that UCPs are glioprotective by reducing oxidative stress in glia. In this study, the potential benefit of increasing ROS defense was evaluated when mHtt was selectively expressed in Drosophila glia or neurons. Whereas increasing MnSOD and catalase levels rescued the early death of flies expressing mHtt in neurons, no improvement was found when mHtt was present selectively in glia. Thus, this study further confirms that ROS overproduction is a crucial pathway by which mHtt leads to neuronal dysfunction and/or death in HD. However, despite the impact of UCPs on ROS production, this may not be sufficient to counteract the mHtt-induced alterations in neurons. The data also reveals that oxidative stress is likely not the primary cause of HD glial pathogenesis and the beneficial effects of UCPs on glial cells are likely not due to the UCP-mediated proton leak and the subsequent reduction of ROS (Besson, 2010).

In recent years, an increasing amount of data indicate that UCPs may not only act as uncoupling agents. They may be fundamental for metabolic sensing and adaptive energetic metabolism in order to meet the energy demand. For instance, it was reported that UCP2 knock-out fibroblasts display enhanced proliferation associated with a higher pyruvate oxidation rate and a reduced fatty acid oxidation in mitochondria. On the contrary, UCP2 overexpression decreases the glucose-dependent proliferation of CHO cells. This is consistent with a ‘metabolic hypothesis’ whereby the role of UCP2 would be to promote oxidation of fatty acids rather than that of glucose-derived pyruvate. Earlier data also suggests that UCPs may physically conduct free fatty acid anions and thereby, actively participate to the fatty acid cycling. Finally, since UCPs shunt the oxidation of pyruvate in mitochondria, this may give rise to increased glycolysis followed by lactate production: a process known as the Warburg effect. In respect to this, overexpression of UCP3 and UCP4 in muscle and neuronal cultured cells, respectively, was found to stimulate glucose transport and shifting the way of ATP production from mitochondrial production to glycolysis. In the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidation is primarily neuronal. Therefore, this study proposes that UCP overexpression in the presence of mHtt would ameliorate the glial-induced alterations by enhancing glycolysis. In support of this hypothesis, overexpression of the Drosophila glucose transporter DmGluT1 ameliorated the locomotor deficiency and the lifespan of flies expressing mHtt in glia. A role for glucose metabolism as a modulator for mHtt toxicity was previously suggested on HD cell models as cell death was significantly reduced by glucose transporter overexpression. This study provides the first in vivo evidence that increased entry of glucose is beneficial against mHtt-induced glial dysfunction. The exact steps whereby enhanced glucose metabolism rescues glia-induced alterations in HD flies remain to be clarified. As a possible mechanism, raised intracellular glucose levels may induce autophagy via mTOR signaling (Besson, 2010).

In conclusion, this study proposes that defects in energetic metabolism are involved in mHtt-induced glial alterations and that increasing glucose metabolism may be beneficial to rescue abnormal glia-to-neuron communication in HD. Further work is required to fully understand the importance of energy metabolism in the abnormal neuron–glia crosstalk in HD pathogenesis (Besson, 2010).

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Ravikumar, B., Imarisio, S., Sarkar, S., O'Kane, C.J. and Rubinsztein, D.C. (2008). Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease. J Cell Sci 121: 1649-1660. PubMed ID: 18430781

Huntington disease (HD) is caused by a polyglutamine-expansion mutation in huntingtin (HTT) that makes the protein toxic and aggregate-prone. The subcellular localisation of huntingtin and many of its interactors suggest a role in endocytosis, and it has been shown that huntingtin interacts indirectly with the early endosomal protein Rab5 through HAP40. This study shows that Rab5 inhibition enhances polyglutamine toxicity, whereas Rab5 overexpression attenuates toxicity in cell and fly models of HD. The study tries to identify a mechanism for the Rab5 effects in HD model systems, and it was found that Rab5 acts at an early stage of autophagosome formation in a macromolecular complex that contains Beclin-1 (BECN1) and Vps34. Interestingly chemical or genetic inhibition of endocytosis also impedes macroautophagy, and enhances aggregation and toxicity of mutant huntingtin. However, in contrast to Rab5, inhibition of endocytosis by various means suppresses autophagosome-lysosome fusion (the final step in the macroautophagy pathway) similar to Bafilomycin A1. Thus, Rab5, which was previously thought to be exclusively involved in endocytosis, has a new role in macroautophagy. It was previously shown that macroautophagy is an important clearance route for several aggregate-prone proteins including mutant huntingtin. Thus, better understanding of Rab5-regulated autophagy might lead to rational therapeutic targets for HD and other protein-conformation diseases. (Ravikumar, 2008).


  • Rab5 modifies polyglutamine toxicity and aggregation.
  • Rab5 regulates autophagosome formation in mammalian cells.
  • Rab5 and its effector Vps34 regulate early steps of autophagosome formation.
  • Both Rab5 and Vps34 regulate Atg5-Atg12 conjugation.
  • Rab5 is found in a macromolecular complex containing Vps34 and Beclin-1.
  • Rab5 effect on autophagy is not due to a general inhibition of the endocytic pathway.
  • Rab5 effect on autophagy is not due to mTOR signalling.

This study shows that Rab5 is a modifier of polyglutamine toxicity in cell and fly models. These modifying effects are due to Rab5 regulating autophagy-dependent clearance of the mutant toxic protein. It is suggested that Rab5, previously considered as a “specific” endosome marker, also influences mammalian autophagy. Inhibition of Rab5 results in a decrease in LC3-positive vesicles suggesting a defect in the formation of autophagic vacuoles. Rab5 is an activator of Vps34, a PI-3-kinase essential for autophagy initiation, and it was shown that Rab5 is a new member of a complex containing Vps34 and Beclin-1 that is associated with autophagosome precursors. Thus, the most parsimonious explanation for these findings is that Rab5 acts as an activator for Vps34 in autophagy as it is known to do in endocytosis. This hypothesis is consistent with the various similarities observed in cells with loss-of-function of Rab5 or Vps34. Loss of either of these activities decreased Atg5-Atg12 conjugation, a critical step in early phagophore elongation. It is speculated that the accumulation of Atg5 structures with Rab5 or Vps34 inhibition may be due to a block in the progression from early Atg5-positive autophagosomal structures to the formation of autophagosomes. This block may be due to a defect in Atg12 recruitment. Indeed, decreasing Atg5 conjugation (which is known to block autophagosome formation) also results in increased numbers of Atg5-positive autophagosome precursors (Ravikumar, 2008).

The study does not exclude the possibility that membranes for autophagosome biogenesis may be derived from endosomes. However, the effect of Rab5 inactivation on Atg5-Atg12 conjugation and autophagosome synthesis are not seen with a wide range of other endocytosis inhibitors (β–CD, DN-Dyn, DN-Vps4 and siRNA for clathrin), which instead impede autophagic flux by inhibiting autophagosome-lysosome fusion directly or by inhibiting the autophagosome-amphisome fusion step. This does suggests that endocytosis inhibition through different mechanisms would also enhance polyglutamine aggregation/toxicity by blocking autophagy. It is possible that loss of Rab5 activity has effects on autophagy by perturbing other unrelated/unknown membrane trafficking pathways (distinct from endocytosis). However, it is important to point out that overexpression of CA-Rab5 or WT-Rab5 enhances autophagosome synthesis and suppresses huntingtin aggregation/toxicity in cells and in vivo (Ravikumar, 2008).

The study suggests a hypothetical sequential model in mammalian cells where PI-3-P generated by Vps34 in a complex comprising at least Beclin-1 and active Rab5, is a key regulator of Atg12 conjugation to Atg5, a rate-limiting step in the conversion of Atg5-positive autophagosome precursors to Atg5-negative autophagosomes. On the one hand, inhibition of this putative cascade at a number of points would lead to impaired autophagy and enhance polyglutamine toxicity. On the other hand, better understanding of the initial rate-limiting steps of autophagy may provide opportunities for rational therapeutic design of more specific and safer autophagy-inducing drugs than rapamycin (which affects many pathways). This may have relevance to HD and also to a range of related neurodegenerative diseases caused by intracytosolic aggregate-prone proteins (Ravikumar, 2008).

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Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O'Kane, C.J. and Rubinsztein, D.C. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36: 585-595. PubMed ID: PubMed

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. This study shows that 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, as 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 improves performance on four different behavioral tasks and decreases aggregate formation in a mouse model of Huntington disease. The data in this study provides proof-of-principle for the potential of inducing autophagy to treat Huntington disease (Ravikumar, 2004).


  • mTOR is sequestered into huntingtin aggregates.
  • Polyglutamine expansion impairs mTOR kinase activity.
  • Polyglutamine expansion impairs mTOR-dependent translation.
  • Decreased mTOR activity in other polyglutamine diseases.
  • Polyglutamine expansion induces autophagy.
  • Increasing mTOR activity enhances polyglutamine toxicity.
  • Rapamycin reduces neurodegeneration in a fly model.

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Kazemi-Esfarjani, P. and Benzer, S. (2000). Genetic suppression of polyglutamine toxicity in Drosophila. Science. 287: 1837-1840. PubMed ID: 10710314 

A Drosophila model for Huntington's and other polyglutamine diseases was used to screen for genetic factors modifying the degeneration caused by expression of polyglutamine in the eye. Among 7000 P-element insertions, several suppressor strains were isolated, two of which led to the discovery of the suppressor genes described in this study. The predicted product of one, dHDJ1, is homologous to human heat shock protein 40/HDJ1. That of the second, dTPR2, is homologous to the human tetratricopeptide repeat protein 2. Each of these molecules contains a chaperone-related J domain. Their suppression of polyglutamine toxicity was verified in transgenic flies (Kazemi-Esfarjani, 2000).

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Chongtham, A. and Agrawal, N. (2016). Sci Rep 6: 18736. PubMed ID: 26728250

Huntington's disease (HD) is a progressive, dominantly inherited neurological disorder caused by an abnormal expansion of polyglutamine (polyQ) repeat within the Huntingtin (Htt) protein with no disease modifying treatments. In a Drosophila model of HD, expression of mutant Huntingtin (Htt) protein with expanded polyQ leads to formation of inclusion bodies (IBs), increase in cellular toxicity, progression of motor disabilities and reduced viability. Multiple cellular events such as oxidative stress, mitochondrial dysfunction, inflammation and transcriptional dysregulation are reported to contribute to pathology, however, till date there are no disease-modifying treatments with least side effects. Therefore, this study investigated effect of the phytochemical curcumin on HD pathogenesis. Curcumin, a phytochemical and commonly used ingredient in Asian food has a wide spectrum of anti-oxidant, anti-inflammatory and anti-fibrilogenic properties. This study provides evidence that curcumin significantly ameliorates disease symptoms in a Drosophila model of HD by suppressing cell death and can be a key to halting the progression of Huntington's disease with least side effects (Chongtham, 2016). .

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Rincon-Limas, D.E., Jensen, K. and Fernandez-Funez, P. (2012). Drosophila models of proteinopathies: the little fly that could. Curr Pharm Des 18: 1108-1122. PubMed ID: 22288402 

Lewis, E. A. and Smith, G. A. (2015). Using Drosophila models of Huntington's disease as a translatable tool. J Neurosci Methods. PubMed ID: 26241927

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More in IF

Interaction of Huntington disease protein with transcriptional activator Sp1

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Recent Updates

Lewis, E.A. and Smith, G.A. (2015). Using Drosophila models of Huntington's disease as a translatable tool. J Neurosci Methods [Epub ahead of print]. PubMed ID: 26241927

Gonzales, E.D., Tanenhaus, A.K., Zhang, J., Chaffee, R.P. and Yin, J.C. (2015). Early onset sleep defects in Drosophila models of Huntington's Disease reflect alterations of PKA/CREB Signaling. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26604145

Heidari, R., Monnier, V., Martin, E. and Tricoire, H. (2015). Methylene blue partially rescues heart defects in a Drosophila model of Huntington's disease. J Huntingtons Dis 4: 173-186. PubMed ID: 26397898

White, J.A. 2nd, 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 24: 7182-7195. PubMed ID: 26450517

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Date revised: 15 Dec 2015

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