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The
InteractiveFly: Drosophila as a Model for
Human Diseases |
| Drosophila
genes associated with Huntington's disease
cinnabar huntingtin Ku70 mTOR Pcaf nejire Rab5 UCP5 vermillion |
Related
terms Autophagy Glia Position effect variegation |
| Relevant
studies of Huntington's disease 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 Abstract Highlights
Discussion 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). 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 Abstract Highlights
<Deng, N., Wu, Y. Y., Feng, Y., Hsieh, W. C., Song, J. S., Lin, Y. S., Tseng, Y. H., Liao, W. J., Chu, Y. F., Liu, Y. C., Chang, E. C., Liu, C. R., Sheu, S. Y., Su, M. T., Kuo, H. C., Cohen, S. N. and Cheng, T. H. (2022). Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes. Proc Natl Acad Sci U S A 119(32): e2204779119. PubMed ID: 35914128
Expansion of the number of contiguous nucleotide repeats that normally exist within certain human genes is the cause of multiple human diseases. Earlier work has shown that expression of alleles containing nucleotide repeat expansions can be reduced differentially by inhibiting production of SUPT4H or SUPT5H, highly conserved cellular proteins that interact to form the transcription elongation complex, DSIF (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor). DSIF assists in the elongation of mRNA molecules by attaching to RNA polymerase II (RNAPII) via an SUPT5H binding site and forming a structural clamp that maintains RNAPII occupancy of template DNA as the polymerase proceeds along the template . A decrease in production or function of SUPT4H or SUPT5H has been found to decrease synthesis of transcripts encoded by genes containing nucleotide repeat expansions including HTT, the gene that causes Huntington's Disease, the C9orf72 locus associated with amyotrophic lateral sclerosis and frontotemporal dementia, and NOP56, the gene associated with spinocerebellar atrophy type 36 (SCA36), and it has been suggested that SUPT4H or SUPT5H may be a target for treatment of certain diseases caused by nucleotide repeat expansions. As interaction between SUPT4H and SUPT5H to form the DSIF complex is required for these proteins to form the structural clamp that maintains RNAPII on DNA template, this study sought to identify compounds that interfere with the SUPT4H-SUPT5H interaction and to elucidate their effects on mutant HTT gene products. This study describes the results of experiments aimed at: 1) identifying chemicals that can interfere with the SUPT4H/5H interaction, 2) determining whether chemical interference with the interaction recapitulates the effects of decreasing SUPT4H or SUPT5H on expression of genes containing expanded nucleotide repeats, and 3) determining whether chemical interference with the interaction has phenotypic effects (Deng, 2022). Decreasing the expression of the SUPT4H or SUPT5H components of the DSIF complex can lower production of mRNAs encoded by mutant gene alleles containing nucleotide repeat expansions, and also can modify phenotypes associated with repeat expansions. These findings have led to proposals that that chemical or genetic targeting of SUPT4H or SUPT5H may be useful therapeutically. The results reported in this study indicate that chemical interference with the interaction of SUPT4H and SUPT5H is achievable, that such interference -which has been confirmed by two independent reporter assays and a direct biochemical assay-can lower the abundance of mutant HTT gene products in cultured cells and an HD animal model, and that chemical targeting of DSIF complex formation can mitigate phenotypic effects of repeat expansions. However, the broad and essential biochemical functions of DSIF, raise the prospect that therapeutic targeting of DSIF may be challenging. As SUPT4H and SUPT5H can act individually, as well as in complex with each other, the effects of targeting DSIF also may differ from the effects of targeting its individual components (Deng, 2022). Compounds of multiple chemical classes potentially may interfere with the SUPT4H-SUPT5H interaction. Among the compounds identified by the screening assays was 6-azauridine, a previously studied nucleoside inhibitor of de novo uridine-5'-monophosphate productive pathway and consequently of nucleic acid synthesis and cell division. Addition of uridine to cell cultures reversed the effects of approximately equimolar amounts of 6-AZA on global nucleic acid synthesis without affecting mutant HTT expression, demonstrating the distinctness of these two effects of the compound (Deng, 2022). Loss of medium spiny neurons (MSNs) in the striatum is a characteristic feature of HD and other neurodegenerative diseases. This study used CRISPR/Cas9 gene editing methodology to shorten the number of HTT gene CAG repeats in HD patient MSNs to a nonpathological length, and found that shortening of repeats in these congenic cells was associated with diminished sensitivity to H2O2 exposure. Treatment with 6-AZA partially reversed the incremental sensitivity of cells containing expanded repeats, but did not affect H2O2 sensitivity in cells containing shorter repeats (Deng, 2022). Analogous partial reversal of phenotypic effects of mutant HTT expression was observed also in the adult Drosophila compound eye, which has been widely used as a model for Huntington's Disease and other human neurodegenerative disorders. No loss of Drosophila larval viability was detected at a 6-AZA concentration that rescued animals displaying the rough eye phenotype. However, the ability of uridine supplementation to reverse the global effects of 6-AZA on nucleic acid synthesis in cell culture raises the possibility that such supplementation may prove useful also in mammalian models during in vivo studies (Deng, 2022). Whereas the pathogenic effects of repeat expansions in HD and certain other diseases have been observed most clearly in neuronal cells, they are also evident in non-CNS tissues. In the current experiments, they were observed in MSNs, in neuronal cells, in blood cells, and in photoreceptor cells of the eye-and in replicating and nonreplicating cells. Whereas chemical interference with the SUPT4H-SUPT5H interaction has the potential for affecting multiple tissues simultaneously, differences in the length of repeats as well as tissue-specific factors unrelated to DSIF may influence the results of such interference (Deng, 2022). Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620 Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress (Swinter, 2023). Sharma, A., Narasimha, K., Manjithaya, R. and Sheeba, V. (2023) . Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease. J Neurosci 43(26): 4907-4925. PubMed ID: 37268416 Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, this study expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, the contribution was examined of autophagy in mitigating toxicity caused by mutant HTT protein. Targeted overexpression of an autophagy gene, Atg8a inmale flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, it was demonstrated that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. The rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, this study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits (Sharma, 2023). Clabough, E. B. D., Aspili, C., Fussy, W. S., ...., Venton, B. J., Hayes, D., Sipe, C. W.(2023). Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila. Journal of Huntington's disease, 12(3):241-252 PubMed ID: 37661891 Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. To elucidate the role of htt in the context of ethanol exposure, this study investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. Flies lacking htt were tested for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. dhtt-null flies were found to be both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. dhtt was shown to mediate these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. These results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli (Clabough, 2023). Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C.(2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007 Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Drosophila models have been used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction, a negative geotaxis assay was performed to measure locomotor performance and it was found that neuronal mutant huntingtin caused an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain (Roth, 2023). ODhankhar, J., Shrivastava, A., Agrawal, N. (2023). Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease Journal of Huntington's disease, 12(4):335-354 PubMed ID: 37781812 Though primarily classified as a brain disorder, surplus studies direct Huntington's disease (HD) to be a multi-system disorder affecting various tissues and organs, thus affecting overall physiology of host. Recently, it has been reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, the polyphenolic compound curcumin was tested to circumvent the impact of immune dysregulation in Drosophila model of HD. The present study examined the molecular basis underlying immune derangements and immunomodulatory potential of curcumin in HD. UAS-GAL4 system was used to imitate the HD symptoms in Drosophila, and the desired female progenies (elav > Httex1pQ25; control and elav > Httex1pQ93; diseased) were cultured on food mixed without and with 10 μM concentration of curcumin since early development. Effect of curcumin supplementation was investigated by monitoring the hemocytes' count and their functional abilities in diseased condition. Reactive oxygen species (ROS) level in cells was assessed by DHE staining and mitochondrial dysfunction was assessed by CMXros red dye. In addition, transcript levels of pro-inflammatory cytokines and anti-microbial peptides were monitored by qRT-PCR. Curcumin supplementation was found to effectively reduced higher crystal cell count and phenoloxidase activity in diseased flies. Interestingly, curcumin significantly managed altered plasmatocytes count, improved their phagocytic activity by upregulating the expression of key phagocytic receptors in HD condition. Moreover, substantial alleviation of ROS levels and mitochondria dysfunction was observed in plasmatocytes of diseased flies upon curcumin supplementation. Furthermore, curcumin administration effectively attenuated transcriptional expression of pro-inflammatory cytokines and AMPs in diseased flies. These results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD (Dhankhar, 2023). Onkar, A., Sheshadri, D., Rai, A., Gupta, A. K., Gupta, N., Ganesh, S.(2023). Increase in brain glycogen levels ameliorates Huntington's disease phenotype and rescues neurodegeneration in Drosophila Disease models & mechanisms, 16(10) PubMed ID: 37681238 Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, a neuroprotective role for neuronal glycogen and glycogen synthase has been recently demonstrated in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, a Drosophila model of Huntington's disease was used, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. This study, thus, provides in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process (Onkar, 2023). Tandon, S., Sarkar, S. (2023). Glutamine stimulates the S6K/4E-BP branch of insulin signalling pathway to mitigate human poly(Q) disorders in Drosophila disease models Nutritional neuroscience:1-12 PubMed ID: 37658796 Since, the S6K/4E-BP sub-pathway can be stimulated by various amino acids; this study examine if oral feeding of amino acids delivers rescue against human poly(Q) toxicity in Drosophila. Drosophila models of two different poly(Q) disorders were used to test this hypothesis. Glutamine was fed to the test flies orally mixed in the food. Control and treated flies were then tested for different parameters, such as formation of poly(Q) aggregates and neurodegeneration, to evaluate glutamine's proficiency in mitigating poly(Q) neurotoxicity. This study study, for the first time, reports that glutamine feeding stimulates the growth promoting S6K/4E-BP branch of insulin signalling pathway and restricts pathogenesis of poly(Q) disorders in Drosophila disease models. It is noted that glutamine treatment restricts the formation of neurotoxic poly(Q) aggregates and minimises neuronal deaths. Further, glutamine treatment re-establishes the chromatin architecture by improving the histone acetylation which is otherwise compromised in poly(Q) expressing neuronal cells. Since, the insulin signalling pathway as well as mechanism of action of glutamine are fairly conserved between human and Drosophila, this finding strongly suggests that glutamine holds immense potential to be developed as an intervention therapy against the incurable human poly(Q) disorders (Tandon, 2023). Zsindely, N., Nagy, G., Siagi, F., Farkas, A. and Bodai, L. (2023). Dysregulated miRNA and mRNA Expression Affect Overlapping Pathways in a Huntington's Disease Model. Int J Mol Sci 24(15). PubMed ID: 37569316 Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the Huntingtin gene. Transcriptional dysregulation is one of the main cellular processes affected by mutant Huntingtin (mHtt). This study investigated the alterations in miRNA and mRNA expression levels in a Drosophila model of HD by RNA sequencing and assessed the functional effects of misregulated miRNAs in vivo. in head samples of HD flies, the level of 32 miRNAs were found to change significantly; half of these were upregulated, while the other half were downregulated. After comparing miRNA and mRNA expression data, similarities were discovered in the impacted molecular pathways. Additionally, it was observed that the putative targets of almost all dysregulated miRNAs were overrepresented were tested among the upregulated mRNAs. The effects were tested of overexpression of five misregulated miRNAs in the HD model, and it was found that while mir-10 and mir-219 enhanced, mir-137, mir-305, and mir-1010 ameliorated mHtt-induced phenotypes. Based on these results, it is proposed that while altered expression of mir-10, mir-137, and mir-1010 might be part of HD pathology, the upregulation of mir-305 might serve as a compensatory mechanism as a response to mHtt-induced transcriptional dysregulation (Zsindely, 2023) Haga-Yamanaka, S., Nunez-Flores, R., Scott, C. A., Perry, S., Chen, S. T., Pontrello, C., Nair, M. G. and Ray, A.(2023). Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs. bioRxiv. PubMed ID: 36865229 Eukaryotes are often exposed to microbes and respond to their secreted metabolites, such as the microbiome in animals or commensal bacteria in roots. Little is known about the effects of long-term exposure to volatile chemicals emitted by microbes, or other volatiles that humans are exposed to over a long duration. Using the model system Drosophila melanogaster this study evaluated a yeast emitted volatile, diacetyl, found in high levels around fermenting fruits where they spend long periods of time. Exposure to just the headspace containing the volatile molecules was shown to alter gene expression in the antenna. Experiments showed that diacetyl and structurally related volatile compounds inhibited human histone-deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused wide changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure causes modulation of gene expression in the brain, therefore has potential as a therapeutic. Using two separate disease models known to be responsive to HDAC-inhibitors, this study evaluated physiological effects of volatile exposure. First, it was found that the HDAC inhibitor also halts proliferation of a neuroblastoma cell line in culture as predicted. Next, exposure to vapors slows progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression and physiology in animals (Haga-Yamanaka, 2023). Bhatnagar, A., Parmar, V., Barbieri, N., Bearoff, F., Elefant, F. and Kortagere, S. (2023). Novel EAAT2 activators improve motor and cognitive impairment in a transgenic model of Huntington's disease. Front Behav Neurosci 17: 1176777. PubMed ID: 37351153
Glutamate excitotoxicity is causal in striatal neurodegeneration underlying motor dysfunction and cognitive deficits in Huntington's disease (HD). Excitatory amino acid transporter 2 (EAAT2), the predominant glutamate transporter accounting for >90% of glutamate transport, plays a key role in preventing excitotoxicity by clearing excess glutamate from the intrasynaptic cleft. Accordingly, EAAT2 has emerged as a promising therapeutic target for prevention of neuronal excitotoxicity underlying HD and other neurodegenerative diseases. Previously novel EAAT2 positive allosteric modulators were designed, GT951, GTS467, and GTS551, with low nanomolar efficacy in glutamate uptake and favorable pharmacokinetic properties. In this study, the neuroprotective abilities of these novel EAAT2 activators was tested in vivo using the robust Drosophila HD transgenic model expressing human huntingtin gene with expanded repeats (Htt128Q). All three compounds significantly restored motor function impaired under HD pathology over a wide dose range. Additionally, treatment with all three compounds significantly improved HD-associated olfactory associative learning and short-term memory defects, while GT951 and GTS551 also improved middle-term memory in low-performing group. Similarly, treatment with GT951 and GTS551 partially protected against early mortality observed in the HD model. Further, treatment with all three EAAT2 activators induced epigenetic expression of EAAT2 Drosophila homolog and several cognition-associated genes. Together, these results highlight the efficacy of GT951, GTS467 and GTS551 in treating motor and cognitive impairments under HD pathology and support their development for treatment of HD (Bhatnagar, 2023). Barwell, T., Raina, S., Page, A., MacCharles, H. and Seroude, L. (2023). Juvenile and adult expression of polyglutamine expanded huntingtin produce distinct aggregate distributions in Drosophila muscle. Hum Mol Genet 32(16): 2656-2668. PubMed ID: 37369041
Haga-Yamanaka, S., Nunez-Flores, R., Scott, C. A., Perry, S., Chen, S. T., Pontrello, C., Nair, M. G. and Ray, A. (2023). Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs. bioRxiv. PubMed ID: 36865229
Nandi, N., Zaidi, Z., Tracy, C. and Kramer, H. (2022) A phospho-switch at Acinus-Serine(437) controls autophagic responses to Cadmium exposure and neurodegenerative stress. Elife 11. PubMed ID: 35037620
In Drosophila starvation-independent quality control autophagy is regulated by Acinus and the Cdk5-dependent phosphorylation of its serine(437). This study identified the phosphatase that counterbalances this activity. A genetic screen identified six phosphatases that genetically interacted with an Acinus gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-Acinus levels. Cdk5-dependent phosphorylation of Acinus serine(437) in nil1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington's disease model. Consistent with previous findings that Cd(2+) inhibits PPM-type phosphatases, Cd(2+)-exposure elevated Acinus-serine(437) phosphorylation which was necessary for increased neuronal autophagy and protection against Cd(2+)-induced cytotoxicity. Together, these data establish the Acinus-S437 phospho-switch as critical integrator of multiple stress signals regulating neuronal autophagy. <Hernandez, S. J., Lim, R. G., Onur, T., Dane, M. A., Smith, R., Wang, K., Jean, G. E., Devlin, K., Miramontes, R., Wu, J., Casale, M., Kilburn, D., Heiser, L. M., Korkola, J. E., Van Vactor, D., Botas, J., Thompson-Peer, K. L. and Thompson, L. M. (2022). An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells. Hum Mol Genet. PubMed ID: 36547263 Abstract <Tandon, S. and Sarkar, S. (2023). Glipizide ameliorates human poly(Q) mediated neurotoxicity by upregulating insulin signalling in Drosophila disease models. Biochem Biophys Res Commun 645: 88-96. PubMed ID: 36680941 Abstract <Hernandez, S. J., Lim, R. G., Onur, T., Dane, M. A., Smith, R., Wang, K., Jean, G. E., Devlin, K., Miramontes, R., Wu, J., Casale, M., Kilburn, D., Heiser, L. M., Korkola, J. E., Van Vactor, D., Botas, J., Thompson-Peer, K. L. and Thompson, L. M. (2022). An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells. Hum Mol Genet. PubMed ID: 36547263 Abstract <Farago, A., Zsindely, N., Farkas, A., Neller, A., Siagi, F., Szabo, M. R., Csont, T. and Bodai, L. (2022). Acetylation State of Lysine 14 of Histone H3.3 Affects Mutant Huntingtin Induced Pathogenesis. Int J Mol Sci 23(23). PubMed ID: 36499499 Abstract <Prakash, P., Pradhan, A. K. and Sheeba, V. (2022) (2022) Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease. Dis Model Mech 15(6). PubMed ID: 35645202 Abstract Circadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, it was asked whether known neurotoxic modifiers can suppress circadian dysfunction. A screen was performed of neurotoxicity-modifier genes to suppress circadian behavioural arrhythmicity in a Drosophila circadian HD model. The molecular chaperones Hsp40 and HSP70 emerged as significant suppressors in the circadian context, with Hsp40 being the more potent mitigator. Upon Hsp40 overexpression in the Drosophila circadian ventrolateral neurons (LNv), the behavioural rescue was associated with neuronal rescue of loss of circadian proteins from small LNv soma. Specifically, there was a restoration of the molecular clock protein Period and its oscillations in young flies and a long-lasting rescue of the output neuropeptide Pigment dispersing factor. Significantly, there was a reduction in the expanded Huntingtin inclusion load, concomitant with the appearance of a spot-like Huntingtin form. Thus, this study provided evidence implicating the neuroprotective chaperone Hsp40 in circadian rehabilitation. The involvement of molecular chaperones in circadian maintenance has broader therapeutic implications for neurodegenerative diseases. <Chongtham, A., Yoo, J. H., Chin, T. M., Akingbesote, N. D., Huda, A., Marsh, J. L. and Khoshnan, A. (2022) Gut Bacteria Regulate the Pathogenesis of Huntington's Disease in Drosophila Model. Front Neurosci 16: 902205. PubMed ID: 35757549 Abstract Changes in the composition of gut microbiota are implicated in the pathogenesis of several neurodegenerative disorders. This study investigated whether gut bacteria affect the progression of Huntington's disease (HD) in transgenic Drosophila melanogaster models expressing full-length or N-terminal fragments of human mutant huntingtin (HTT) protein. Elimination of commensal gut bacteria by antibiotics was found to reduce the aggregation of amyloidogenic N-terminal fragments of HTT and delays the development of motor defects. Conversely, colonization of HD flies with Escherichia coli (E. coli), a known pathobiont of human gut with links to neurodegeneration and other morbidities, accelerates HTT aggregation, aggravates immobility, and shortens lifespan. Similar to antibiotics, treatment of HD flies with small compounds such as luteolin, a flavone, or crocin a beta-carotenoid, ameliorates disease phenotypes, and promotes survival. Crocin prevents colonization of E. coli in the gut and alters the levels of commensal bacteria, which may be linked to its protective effects. The opposing effects of E. coli and crocin on HTT aggregation, motor defects, and survival in transgenic Drosophila models support the involvement of gut-brain networks in the pathogenesis of HD. <Singh, A. and Agrawal, N (2022) Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease. Metab Brain Dis 37(8): 2783-2792. PubMed ID: 36121619 Abstract Dhankhar, J., Agrawal, N. and Shrivastava, A. (2022). Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes. J Neuroimmunol 363: 577801. PubMed ID: 34973473
Deng, X., Sun, X., Yue, W., Duan, Y., Hu, R., Zhang, K., Ni, J., Cui, J., Wang, Q., Chen, Y., Li, A. and Fang, Y. (2021). CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1. J Cell Biol 221(1). PubMed ID: 34726688. Abstract The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. This study identified CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, this study found that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, this study uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, these findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation (Deng, 2021). Aditi, K., Singh, A., Shakarad, M. N. and Agrawal, N. (2021). Management of altered metabolic activity in Drosophila model of Huntington's disease by curcumin. Exp Biol Med (Maywood): 15353702211046927. PubMed ID: 34743577 Abstract Martin, E., Heidari, R., Monnier, V. and Tricoire, H. (2021). Genetic Screen in Adult Drosophila Reveals That dCBP Depletion in Glial Cells Mitigates Huntington Disease Pathology through a Foxo-Dependent Pathway. Int J Mol Sci 22(8). PubMed ID: 33918672 Abstract Huntington's disease (HD) is a progressive and fatal autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the first exon of the huntingtin gene (HTT). In spite of considerable efforts, there is currently no treatment to stop or delay the disease. Although HTT is expressed ubiquitously, most of the current knowledge has been obtained on neurons. More recently, the impact of mutant huntingtin (mHTT) on other cell types, including glial cells, has received growing interest. It is currently unclear whether new pathological pathways could be identified in these cells compared to neurons. To address this question, an in vivo screen was performed for modifiers of mutant huntingtin (HTT-548-128Q) induced pathology in Drosophila adult glial cells and several putative therapeutic targets were identified. Among them, it was discovered that partial nej/dCBP depletion in these cells was protective, as revealed by strongly increased lifespan and restored locomotor activity. Thus, dCBP promotes the HD pathology in glial cells, in contrast to previous opposite findings in neurons. Further investigations implicated the transcriptional activator Foxo as a critical downstream player in this glial protective pathway. These data suggest that combinatorial approaches combined to specific tissue targeting may be required to uncover efficient therapies in HD. Casale, A. M., Liguori, F., Ansaloni, F., Cappucci, U., Finaurini, S., Spirito, G., Persichetti, F., Sanges, R., Gustincich, S. and Piacentini, L. (2022). Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease. iScience 25(1): 103702. PubMed ID: 35036881
Ghosh, B., Karmakar, S., Prasad, M. and Mandal, A. K. (2021). Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity. Mol Biol Cell: mbcE20110747. PubMed ID: 34161122 Abstract A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), this study aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, these findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates (Ghosh, 2021). Singh, A. and Agrawal, N. (2021). Deciphering the key mechanisms leading to alteration of lipid metabolism in Drosophila model of Huntington's disease. Biochim Biophys Acta Mol Basis Dis 1867(7): 166127. PubMed ID: 33722743 Abstract Huntington's disease (HD) is an inherited, progressively debilitating disorder marked by prominent degeneration in striatal and cortical brain regions. HD is caused by (CAG)(n) repeat expansion in huntingtin (HTT) gene that translates into a mutant form of the ubiquitously present Huntingtin (HTT) protein. Extensive metabolic dysfunction coexisting with overt neuropathies has been evidenced in clinical and experimental settings of HD. Body weight loss despite normal to high caloric intake remains a critical determinant of the disease progression and a challenge for therapeutic interventions. This study, monitored the cellular and molecular perturbations in Drosophila, caused by pan-neuronal expression of mHTT (mutant Huntingtin) protein. Aberrant transcription profile of key lipolytic and lipogenic genes was found in whole-body of the fly with disease progression. Interestingly, fat body undergoes extensive alteration of vital cellular processes and eventually surrenders to increased apoptotic cell death in terminal stage of the disease. Extensive mitochondrial dysfunction from early disease stage along with calcium derangement at terminal stage were observed in fat body, which contribute to its deteriorating integrity. All the mechanisms were monitored progressively, at different disease stages, and many alterations were documented in the early stage itself. This study hence provides insight into the mechanisms through which neuronal expression of mHTT might be inflicting the profound systemic effects, specifically on lipid metabolism, and may open new therapeutic avenues for alleviation of the multidimensional disease (Singh, 2021). Onur, T. S., Laitman, A., Zhao, H., Keyho, R., Kim, H., Wang, J., Mair, M., Wang, H., Li, L., Perez, A., de Haro, M., Wan, Y. W., Allen, G., Lu, B., Al-Ramahi, I., Liu, Z. and Botas, J. (2021). Downregulation of glial genes involved in synaptic function mitigates Huntington's Disease pathogenesis. Elife 10. PubMed ID: 33871358 Abstract Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntington's disease (HD), this study profiled the gene expression alterations of Drosophila expressing human mutant Huntingtin (mHTT) in either glia or neurons and compared these changes to what is observed in HD human and HD mice striata. A large portion of conserved genes are concordantly dysregulated across the three species; these genes were tested in a high-throughput behavioral assay, and it was found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. Surprisingly, reducing dNRXN3 (also known as nrx-1) function in glia was sufficient to improve the phenotype of flies expressing mHTT in neurons, suggesting that mHTT's toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD (Onur, 2021). Suart, C. E., Perez, A. M., Al-Ramahi, I., Maiuri, T., Botas, J. and Truant, R. (2021). Spinocerebellar Ataxia Type 1 protein Ataxin-1 is signalled to DNA damage by Ataxia Telangiectasia Mutated kinase. Hum Mol Genet. PubMed ID: 33772540 Abstract Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the ataxin-1 protein. Recent genetic correlational studies have implicated DNA damage repair pathways in modifying the age at onset of disease symptoms in SCA1 and Huntington's Disease, another polyglutamine expansion disease. This study demonstrates that both endogenous and transfected ataxin-1 localizes to sites of DNA damage, which is impaired by polyglutamine expansion. This response is dependent on ataxia telangiectasia mutated (ATM) kinase activity. Further, an ATM phosphorylation motif within ataxin-1 at serine 188 was characterized. Reduction of the Drosophila ATM homolog levels in a ATXN1[82Q] Drosophila model through shRNA or genetic cross ameliorates motor symptoms. These findings offer a possible explanation as to why DNA repair was implicated in SCA1 pathogenesis by past studies. The similarities between the ataxin-1 and the huntingtin responses to DNA damage provide further support for a shared pathogenic mechanism for polyglutamine expansion diseases (Suart, 2021). Tandon, S. and Sarkar, S. (2021). The S6k/4E-BP mediated growth promoting sub-pathway of insulin signalling cascade is essential to restrict pathogenesis of poly(Q) disorders in Drosophila. Life Sci 275: 119358. PubMed ID: 33744321 Abstract Human neurodegenerative polyglutamine [poly(Q)] disorders, such as Huntington's disease (HD) and spinocerebellar ataxias (SCA), are characterised by an abnormal expansion of CAG repeats in the affected gene. The mutated proteins misfold and aggregate to form inclusion bodies that sequester important factors involved in cellular transcription, growth, stress and autophagic response and other essential functions. The insulin signalling pathway has been demonstrated as a major modifier and a potential drug target to ameliorate the poly(Q) mediated neurotoxicity in various model systems. Insulin signalling cascade harbours several downstream sub-pathways, which are synergistically involved in discharging indispensable biological functions such as growth and proliferation, metabolism, autophagy, regulation of cell death pathways etc. Hence, it is difficult to conclude whether the mitigation of poly(Q) neurotoxicity is an accumulative outcome of the insulin cascade, or the result of a specific sub-pathway. This study reports that the ligand binding domain of insulin receptor mediated downstream growth promoting sub-pathway plays the pivotal role in operating the rescue event. The growth promoting activity of insulin cascade is essential to minimize the abundance of inclusion bodies, to restrict neurodegeneration, and to restore the cellular transcriptional balance. Subsequently, the involvement of the mTOR/S6k/4E-BP candidates in mitigating poly(Q) mediated neurotoxicity was noted. Due to the conserved cellular functioning of the insulin cascade across species, and availability of several growth promoting molecules, these results in Drosophila poly(Q) models indicate towards a possibility of designing novel therapeutic strategies to restrict the pathogenesis of devastating human poly(Q) disorders (Tandon, 2021). Delfino, L., Mason, R. P., Kyriacou, C. P., Giorgini, F. and Rosato, E. (2020). Rab8 Promotes Mutant HTT Aggregation, Reduces Neurodegeneration, and Ameliorates Behavioural Alterations in a Drosophila Model of Huntington's Disease. J Huntingtons Dis 9(3): 253-263. PubMed ID: 33044189 Abstract Altered cellular vesicle trafficking has been linked to the pathogenesis of Huntington's disease (HD), a fatal, inherited neurodegenerative disorder caused by mutation of the huntingtin (HTT) protein. The Rab GTPase family of proteins plays a key role in regulation of vesicle trafficking. This study investigated whether Rab8 (see Drosophila Rab8), which regulates post-Golgi vesicle trafficking, is able to improve HD-relevant phenotypes in a well-characterised model. Rab8 was overexpressed in a Drosophila model of HD, testing cellular, behavioural, and molecular phenotypes. Rab8 overexpression ameliorated several disease-related phenotypes in fruit flies expressing a mutant HTT (see Drosophila Huntingtin) fragment throughout the nervous system, including neurodegeneration of photoreceptor neurons, reduced eclosion of the adult fly from the pupal case and shortened lifespan. Rab8 overexpression also normalised aberrant circadian locomotor behaviour in flies expressing mutant HTT in a specific population of neurons that regulate the circadian clock. Intriguingly, expression of Rab8 increased the accumulation of SDS-insoluble aggregated species of mutant HTT. Collectively, these findings demonstrate that increased Rab8 levels protect against mutant HTT toxicity and potentiate its aggregation, likely reducing the accumulation of downstream toxic soluble species (Delfino, 2020). Khyati, Malik, I., Agrawal, N. and Kumar, V. (2020). Melatonin and curcumin reestablish disturbed circadian gene expressions and restore locomotion ability and eclosion behavior in Drosophila model of Huntington's disease. Chronobiol Int: 1-18. PubMed ID: 33334207 Abstract Deficit in locomotion (motor) ability and disturbance of the circadian behavior and sleep-wake pattern characterize Huntington's disease (HD). This study examined the disturbance of circadian timing with the progression of HD pathogenesis and tested the efficacy of melatonin and curcumin in preventing the motor deficit and disturbed eclosion behavior in the Drosophila model of HD. To examine circadian timing, mRNA expression was examined of genes of the transcriptional feedback (TF) loop that generates the near 24-h rhythmicity. qPCR was performed of the Period, Timeless, Clock, Cycle, Clockwork, and Cryptochrome genes in transgenic fly heads from elav-Gal4 (pan neuronal) and PDF-Gal4 (PDF-specific neurons) driver lines through the progression of HD disease post-eclosion, from day 1 to its terminal stage on day 13. Cycle was arrhythmic from day 1, but Period and Timeless became arrhythmic on day 13 of the HD pathogenesis in elav, but not PDF, neurons. Twenty-four-hour mRNA rhythms showed alteration in the waveform properties (mesor and amplitude, not acrophase), but not in the persistence, in both elav-Gal4 and PDF-Gal4 HD flies; however, disturbance of the clock gene rhythm was delayed in PDF-Gal4 flies. To assess the preventive effects on HD pathogenesis, flies of both driver lines were provided with melatonin (50, 100, or 150 μg) or curcumin (10 μM) in the diet commencing from the larval stage. Both melatonin (100 μg) and curcumin reestablished the 24-h pattern in mRNA expression of Period and Timeless to normal (control) levels, and significantly improved both locomotion ability and eclosion behavior of HD flies. It is suggested that the disturbance of circadian timekeeping progressively accelerated HD pathogenesis, possibly via modulation of the transcriptional state that resulted in the modification of the Huntington gene. These findings suggest melatonin and curcumin might be potential therapeutic agents for the treatment of HD in humans, although this needs specific investigation (Khyati, 2020). Chatterjee, M., Steffan, J. S., Lukacsovich, T., Marsh, J. L. and Agrawal, N. (2020). Serine residues 13 and 16 are key modulators of mutant huntingtin induced toxicity in Drosophila. Exp Neurol: 113463. PubMed ID: 32941796 Abstract Donnelly, K. M., DeLorenzo, O. R., Zaya, A. D., Pisano, G. E., Thu, W. M., Luo, L., Kopito, R. R. and Panning Pearce, M. M. (2020). Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses. Elife 9. PubMed ID: 32463364 Abstract Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. This study shows that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain (Donnelly, 2020). Beaver, M., Bhatnagar, A., Panikker, P., Zhang, H., Snook, R., Parmar, V., Vijayakumar, G., Betini, N., Akhter, S. and Elefant, F. (2020). Disruption of Tip60 HAT mediated neural histone acetylation homeostasis is an early common event in neurodegenerative diseases. Sci Rep 10(1): 18265. PubMed ID: 33106538 Abstract Epigenetic dysregulation is a common mechanism shared by molecularly and clinically heterogenous neurodegenerative diseases (NDs). Histone acetylation homeostasis, maintained by the antagonistic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs), is necessary for appropriate gene expression and neuronal function. Disruption of neural acetylation homeostasis has been implicated in multiple types of NDs including Alzheimer's disease (AD), yet mechanisms underlying alterations remain unclear. This study shows that like AD, disruption of Tip60 HAT/HDAC2 balance with concomitantm epigenetic repression of common Tip60 target neuroplasticity genes occurs early in multiple types of Drosophila ND models such as Parkinson's Disease (PD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Repressed neuroplasticity genes show reduced enrichment of Tip60 and epigentic acetylation signatures at all gene loci examined with certain genes showing inappropriate HDAC2 repressor enrichment. Functional neuronal consequences for these disease conditions are reminiscent of human pathology and include locomotion, synapse morphology, and short-term memory deficits. Increasing Tip60 HAT levels specifically in the mushroom body learning and meory center in the Drosophila brain protects against locomotion and short-term memory function deficits in multiple NDs. Together, these results support a model by which Tip60 protects against neurological impairments in different NDs via similar modes of action (Beaver, 2020). Xue, J., Wang, H. L. and Xiao, G (2020). Transferrin1 modulates rotenone-induced Parkinson's disease through affecting iron homeostasis in Drosophila melanogaster. Biochem Biophys Res Commun 531(3): 305-311. PubMed ID: 32800558 Abstract Mitochondrial dysfunction and oxidative stress are pathophysiologic mechanisms implicated in Parkinson's disease (PD). In recent years, environmental toxins are employed to increase oxidative stress mediated neuropathology and sporadic PD. Disruption of iron homeostasis has been implicated in PD patients for many years, but the functional role of iron in sporadic PD pathogenesis is still not well clarified in vivo. To address this question, the effect of iron on a Drosophila rotenone model of sporadic PD was investigated. Iron homeostasis is maintained by many transporters. Inhibition of transferrin1 (Tsf1) expression in the central nervous system (CNS) results in reduced iron levels in brains and significantly ameliorates the neurodegenerative phenotypes of rotenone exposure Drosophila; moreover, the rotenone induced reactive oxygen species (ROS) levels in the brain, the damaged complex I activity and the decreased ATP generation were dramatically rescued by Tsf1 knockdown. Further study indicated that all the rescue effects of Tsf1 knockdown on sporadic PD could be inhibited by malvolio (Mvl) overexpression, an iron transporter responsible for iron uptake. These results imply that Tsf1 knockdown in the CNS could attenuate rotenone toxicity by decreasing the ROS levels in brains through reducing iron levels, and manipulation of iron transporters in brains may provide a novel therapeutic strategy for sporadic PD (Xue, 2020). White, J. A., 2nd, Krzystek, T. J., Hoffmar-Glennon, H., Thant, C., Zimmerman, K., Iacobucci, G., Vail, J., Thurston, L., Rahman, S. and Gunawardena, S. (2020). Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease. Acta Neuropathol Commun 8(1): 97. PubMed ID: 32611447 Abstract Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. This study shows that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, these observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD (White, 2020). Lin, Y. H., Maaroufi, H. O., Ibrahim, E., Kucerova, L. and Zurovec, M. (2019). Expression of human mutant Huntingtin protein in Drosophila hemocytes impairs immune responses. Front Immunol 10: 2405. PubMed ID: 31681295 Abstract The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. This study used transgenic Drosophila melanogaster expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, it was observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in Drosophila macrophage-like S2 cells in vitro reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in Drosophila hemocytes causes deficient cellular and humoral immune responses against invading pathogens. These findings provide the insight into the pathogenic effects of mHTT in the immune cells (Lin, 2019). Vernizzi, L., Paiardi, C., Licata, G., Vitali, T., Santarelli, S., Raneli, M., Manelli, V., Rizzetto, M., Gioria, M., Pasini, M. E., Grifoni, D., Vanoni, M. A., Gellera, C., Taroni, F. and Bellosta, P. (2020). Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease. Cells 9(1). PubMed ID: 31941072 Abstract Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). This study reports that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that was associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that it associated with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies (Vernizzi, 2020). Chongtham, A. et al. (2020). Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT. Hum Mol Genet. PubMed ID: 31943010 Abstract Huntington's Disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the Huntingtin protein (HTT) and is necessary to cause pathology and formation of HTT aggregates. This study asked whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, the effects were compared of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ (Chongtham, 2020) Lin, Y. H., Maaroufi, H. O., Ibrahim, E., Kucerova, L. and Zurovec, M. (2019). Expression of human mutant Huntingtin protein in Drosophila hemocytes impairs immune responses. Front Immunol 10: 2405. PubMed ID: 31681295 Abstract The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. This study used transgenic Drosophila melanogaster expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, it was observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in Drosophila macrophage-like S2 cells in vitro reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in Drosophila hemocytes causes deficient cellular and humoral immune responses against invading pathogens. These findings provide the insight into the pathogenic effects of mHTT in the immune cells (Lin, 2019). Lin, Y. H., Maaroufi, H. O., Ibrahim, E., Kucerova, L. and Zurovec, M. (2019). Expression of human mutant Huntingtin protein in Drosophila hemocytes impairs immune responses. Front Immunol 10: 2405. PubMed ID: 31681295 Abstract The pathogenic effect of mutant HTT (mHTT) which causes Huntington disease (HD) are not restricted to nervous system. Such phenotypes include aberrant immune responses observed in the HD models. However, it is still unclear how this immune dysregulation influences the innate immune response against pathogenic infection. This study used transgenic Drosophila melanogaster expressing mutant HTT protein (mHTT) with hemocyte-specific drivers and examined the immune responses and hemocyte function. mHTT expression in the hemocytes did not affect fly viability, but the numbers of circulating hemocytes were significantly decreased. Consequently, it was observed that the expression of mHTT in the hemocytes compromised the immune responses including clot formation and encapsulation which lead to the increased susceptibility to entomopathogenic nematode and parasitoid wasp infections. In addition, mHTT expression in Drosophila macrophage-like S2 cells in vitro reduced ATP levels, phagocytic activity and the induction of antimicrobial peptides. Further effects observed in mHTT-expressing cells included the altered production of cytokines and activation of JAK/STAT signaling. The present study shows that the expression of mHTT in Drosophila hemocytes causes deficient cellular and humoral immune responses against invading pathogens. These findings provide the insight into the pathogenic effects of mHTT in the immune cells (Lin, 2019). Hansen, T., Thant, C., White, J. A., Banerjee, R., Thuamsang, B. and Gunawardena, S. (2019). Excess active P13K rescues huntingtin-mediated neuronal cell death but has no effect on axonal transport defects. Apoptosis. PubMed ID: 30725352 Abstract 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). 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 Abstract 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. Xu, F., Kula-Eversole, E., Iwanaszko, M., Lim, C. and Allada, R. (2019). Ataxin2 functions via CrebA to mediate Huntingtin toxicity in circadian clock neurons. PLoS Genet 15(10): e1008356. PubMed ID: 31593562 Abstract Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington's disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, this study discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. These results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis (Xu, 2019). 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 Abstract 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). 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 Abstract 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). 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 Abstract 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). 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 Abstract 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). 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 Abstract 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).
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
Abstract 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).
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
Abstract 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).
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 Abstract 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).
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
Abstract 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).
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 Abstract 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).
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 Abstract Highlights Discussion 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). Abstract Highlights Discussion Although heterozygous dhttko/+ flies expressing Tau
(ATau; dhttko/+) seem normal, removing a single copy
of the fly LC3 gene, atg8a
(atg8ad4 mutant), in these flies also induces a collapsed
thorax and muscle loss, which can be phenocopied by expressing Tau
in homozygous atg8ad4−/− flies alone. Four
additional components of the early steps of the autophagy pathway,
atg1
(ULK1), atg7
and 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). 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). 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 Abstract Highlights Discussion 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). 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). 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
Abstract 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).
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 Abstract Highlights 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
Abstract Highlights Discussion 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). 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). 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
Abstract Highlights 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
Abstract Highlights Discussion 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). 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
Abstract Highlights Discussion 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). 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
Abstract Highlights Discussion 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). 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
Abstract Highlights Discussion 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). 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 Highlights
Discussion 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). 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 Abstract Highlights
Kazemi-Esfarjani, P. and Benzer, S. (2000). Genetic suppression of polyglutamine toxicity in Drosophila. Science. 287: 1837-1840. PubMed ID: 10710314 Abstract Chongtham, A. and Agrawal, N. (2016). Sci Rep 6: 18736. PubMed ID: 26728250 Abstract 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 Interaction of Huntington disease protein with transcriptional activator Sp1 Go to topLewis, 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 Back to Drosophila as a Model for Human Diseases Date revised: 15 Dec 2015 Home page: The Interactive Fly © 2015 Thomas Brody, Ph.D. The Interactive Fly resides on the web server of the Society for Developmental Biology |
