Drosophila Model Of Huntington's Disease Research Articles

Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease.

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, we have reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, we tested the polyphenolic compound curcumin 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. We found that curcumin supplementation commendably 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. Our results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD.

Copper enhances aggregational toxicity of mutant huntingtin in a Drosophila model of Huntington's Disease

Huntington's disease (HD) is a progressive neurodegenerative disorder with clinical presentations of moderate to severe cognitive, motor, and psychiatric disturbances. HD is caused by the trinucleotide repeat expansion of CAG of the huntingtin (HTT) gene. The mutant HTT protein containing pathological polyglutamine (polyQ) extension is prone to misfolding and aggregation in the brain. It has previously been observed that copper and iron concentrations are increased in the striata of post-mortem human HD brains. Although it has been shown that the accumulation of mutant HTT protein can interact with copper, the underlying HD progressive phenotypes due to copper overload remains elusive. Here, in a Drosophila model of HD, we showed that copper induces dose-dependent aggregational toxicity and enhancement of Htt-induced neurodegeneration. Specifically, we found that copper increases mutant Htt aggregation, enhances the accumulation of Thioflavin S positive β-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases β-amyloid aggregates in the HD pathological model. These findings reveal a direct role of copper in potentiating mutant Htt protein-induced aggregational toxicity, and further indicate the potential impact of environmental copper exposure in the disease onset and progression of HD.

Dysregulated miRNA and mRNA Expression Affect Overlapping Pathways in a Huntington's Disease Model.

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). In this study, we investigate the alterations in miRNA and mRNA expression levels in a Drosophila model of HD by RNA sequencing and assess the functional effects of misregulated miRNAs in vivo. We found that in head samples of HD flies, the level of 32 miRNAs changed significantly; half of these were upregulated, while the other half were downregulated. After comparing miRNA and mRNA expression data, we discovered similarities in the impacted molecular pathways. Additionally, we observed that the putative targets of almost all dysregulated miRNAs were overrepresented among the upregulated mRNAs. We tested the effects of overexpression of five misregulated miRNAs in the HD model and found that while mir-10 and mir-219 enhanced, mir-137, mir-305, and mir-1010 ameliorated mHtt-induced phenotypes. Based on our results, we propose 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.

Acetylation State of Lysine 14 of Histone H3.3 Affects Mutant Huntingtin Induced Pathogenesis.

Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine-coding CAG repeat in the Huntingtin gene. One of the main causes of neurodegeneration in HD is transcriptional dysregulation that, in part, is caused by the inhibition of histone acetyltransferase (HAT) enzymes. HD pathology can be alleviated by increasing the activity of specific HATs or by inhibiting histone deacetylase (HDAC) enzymes. To determine which histone's post-translational modifications (PTMs) might play crucial roles in HD pathology, we investigated the phenotype-modifying effects of PTM mimetic mutations of variant histone H3.3 in a Drosophila model of HD. Specifically, we studied the mutations (K→Q: acetylated; K→R: non-modified; and K→M: methylated) of lysine residues K9, K14, and K27 of transgenic H3.3. In the case of H3.3K14Q modification, we observed the amelioration of all tested phenotypes (viability, longevity, neurodegeneration, motor activity, and circadian rhythm defects), while H3.3K14R had the opposite effect. H3.3K14Q expression prevented the negative effects of reduced Gcn5 (a HAT acting on H3K14) on HD pathology, while it only partially hindered the positive effects of heterozygous Sirt1 (an HDAC acting on H3K14). Thus, we conclude that the Gcn5-dependent acetylation of H3.3K14 might be an important epigenetic contributor to HD pathology.

Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington’s disease

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, we have earlier reported that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. Here, we first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, we checked the transcriptional changes of key metabolic genes in whole fly as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. We found extensive alterations in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, we also assessed lysosome-mediated autophagy in the fatbody of diseased flies in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, we found elevated autophagy in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD.

A phosphoswitch at acinus-serine437 controls autophagic responses to cadmium exposure and neurodegenerative stress.

Neuronal health depends on quality control functions of autophagy, but mechanisms regulating neuronal autophagy are poorly understood. Previously, we showed that in Drosophila starvation-independent quality control autophagy is regulated by acinus (acn) and the Cdk5-dependent phosphorylation of its serine437 (Nandi et al., 2017). Here, we identify the phosphatase that counterbalances this activity and provides for the dynamic nature of acinus-serine437 (acn-S437) phosphorylation. A genetic screen identified six phosphatases that genetically interacted with an acn gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-acn levels. Cdk5-dependent phosphorylation of acn-S437 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 Cd2+ inhibits PPM-type phosphatases, Cd2+ exposure elevated acn-S437 phosphorylation which was necessary for increased neuronal autophagy and protection against Cd2+-induced cytotoxicity. Together, our data establish the acn-S437 phosphoswitch as critical integrator of multiple stress signals regulating neuronal autophagy.

Exploding the Repeat Length Paradigm while Exploring Amyloid Toxicity in Huntington's Disease.

Huntington's disease (HD) is a progressive, familial neurodegenerative disease triggered by the expansion of a polyglutamine (polyQ) track in the protein huntingtin (htt). PolyQ sequences up to Q36 in htt are not known to be toxic, while polyQ lengths above Q36 almost invariably lead to increased disease risk and decreased ages of onset. The large number of physical states (monomers, dimers, tetramers, non-β oligomers, nanofibrils, and clustered amyloid fibrils) on the self-association landscape, with their overlapping kinetics of formation, have greatly complicated identification of the molecular species responsible for HD toxicity, drawing attention to the need for innovative approaches.After reports of HD-associated intraneuronal htt inclusions in 1997, we elucidated aggregation mechanisms of both simple polyQ sequences and the more complex polyQ-containing "exon1" fragment of htt (htt-ex1). Grounded in this work, the more recent results described here were made possible by breakthroughs in the molecular design of diagnostic polyQ derivatives and in fluorescence applications for characterizing amyloid assembly intermediates. Thus, insertion of β-turn-promoting mutations into relatively short, disordered polyQ sequences created "pro-β-hairpin" polyQs (βHPs) that exhibit amyloid formation rates comparable to the enhanced rates seen with expanded polyQ peptides. Introduction of "β-breaker" mutations into these βHP polyQ sequences created molecules that are blocked from aggregating into amyloid and also can inhibit amyloid formation by other polyQ proteins. These mutational effects were then successfully transferred into more complex htt-ex1 sequence backgrounds. Insights into the aggregation properties of htt-ex1 derivatives-as well as into the nucleation process itself-were obtained using fluorescence correlation spectroscopy (FCS) and a novel thioflavin-T (ThT) protocol that allows quantitation of htt-ex1 assembly intermediates.Using these tools, we quantified physical states of htt-ex1 at different growth times in mammalian PC12 cells engineered for inducible expression of both normal and expanded polyQ repeat length versions of htt-ex1. For expanded polyQ versions, we found tetramers, oligomers, and fibrils (but no monomers) all populated in these cells at a time when the first indication of toxicity (nuclear DNA damage) was observed. These experiments provided a strong hint that monomeric forms of htt-ex1 are not involved in toxicity, but we were otherwise unable to implicate a specific toxic self-assembled state because of the overlapping kinetics of formation. To gain a more intimate focus and control over the timelines of htt-ex1 self-assembly and the resulting toxic response, we engineered various htt-ex1-βHP molecules-with and without added β-breaker mutations-that could be expressed in rat neuronal and Drosophila models of HD. In both models, novel htt-ex1-βHP analogues exhibiting strong aggregation in spite of their very short polyQ repeat lengths proved to be toxic, dramatically breaking the "repeat length paradigm" and strongly suggesting that the toxic species must be some kind of aggregate. In both models, β-breaker analogues of htt-ex1-βHP that are slow to make amyloid-instead favoring accumulation of non-β oligomers-were nontoxic. In contrast, htt-ex1-βHP analogues that rapidly progress to amyloid states were toxic, suggesting that an aggregate possessing the fundamental amyloid folding motif is very likely the major toxic species in HD.

Nmnat restores neuronal integrity by neutralizing mutant Huntingtin aggregate-induced progressive toxicity

Accumulative aggregation of mutant Huntingtin (Htt) is a primary neuropathological hallmark of Huntington's disease (HD). Currently, mechanistic understanding of the cytotoxicity of mutant Htt aggregates remains limited, and neuroprotective strategies combating mutant Htt-induced neurodegeneration are lacking. Here, we show that in Drosophila models of HD, neuronal compartment-specific accumulation of mutant Htt aggregates causes neurodegenerative phenotypes. In addition to the increase in the number and size, we discovered an age-dependent acquisition of thioflavin S+, amyloid-like adhesive properties of mutant Htt aggregates and a concomitant progressive clustering of aggregates with mitochondria and synaptic proteins, indicating that the amyloid-like adhesive property underlies the neurotoxicity of mutant Htt aggregation. Importantly, nicotinamide mononucleotide adenylyltransferase (NMNAT), an evolutionarily conserved nicotinamide adenine dinucleotide (NAD+) synthase and neuroprotective factor, significantly mitigates mutant Htt-induced neurodegeneration by reducing mutant Htt aggregation through promoting autophagic clearance. Additionally, Nmnat overexpression reduces progressive accumulation of amyloid-like Htt aggregates, neutralizes adhesiveness, and inhibits the clustering of mutant Htt with mitochondria and synaptic proteins, thereby restoring neuronal function. Conversely, partial loss of endogenous Nmnat exacerbates mutant Htt-induced neurodegeneration through enhancing mutant Htt aggregation and adhesive property. Finally, conditional expression of Nmnat after the onset of degenerative phenotypes significantly delays the progression of neurodegeneration, revealing the therapeutic potential of Nmnat-mediated neuroprotection at advanced stages of HD. Our study uncovers essential mechanistic insights to the neurotoxicity of mutant Htt aggregation and describes the molecular basis of Nmnat-mediated neuroprotection in HD.

Biodegradable Nanoparticles Containing Mechanism Based Peptide Inhibitors Reduce Polyglutamine Aggregation in Cell Models and Alleviate Motor Symptoms in a Drosophila Model of Huntington's Disease.

Detailed study of the molecular mechanism behind the pathogenesis of Huntington's disease (HD) suggests that polyglutamine aggregation is one of the fundamental reasons for HD. Despite the discovery of many potential molecules, HD therapy is still limited to symptomatic relief. Among these molecules, few mechanism based peptide inhibitors of polyglutamine aggregation (QBP1, NT17 and PGQ9P2) have shown promising activity; however, poor blood-brain barrier (BBB) penetration, low bioavailability, and low half-life may hinder their therapeutic potential. Hence, to deliver them to the brain for assessing their efficacy, we have designed and synthesized peptide loaded poly-d,l-lactide- co-glycolide (PLGA) nanoparticles of less than 200 nm in size by carbodiimide chemistry and nanoprecipitation protocols. For brain delivery, PLGA nanoparticles were coated with polysorbate 80 which aids receptor mediated internalization. Using the in vitro BBB model of Madin-Darby canine kidney cells and healthy mice, the translocation of polysorbate 80 coated fluorescent nanoparticles was confirmed. Moreover, QBP1, NT17, and PGQ9P2 loaded PLGA nanoparticles showed dose dependent inhibition of polyglutamine aggregation in cell models of HD (Neuro 2A and PC12 cells) and improved motor performance in Drosophila model of HD. Additionally, no toxicity in cells and animals confirmed biocompatibility of the nanoparticulate formulations. Based on this work, future studies can be designed in higher animal models to test peptide loaded nanoparticles in HD and other polyglutamine expansion related diseases.

High-Throughput Functional Analysis Distinguishes Pathogenic, Nonpathogenic, and Compensatory Transcriptional Changes in Neurodegeneration.

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. Here we integrate functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. We selected 312 genes 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. Our multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points.

NMR Spectroscopy-based Metabolomics of Drosophila Model of Huntington's Disease Suggests Altered Cell Energetics.

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, we have 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, we show 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.

Pathogenic Huntington Alters BMP Signaling and Synaptic Growth through Local Disruptions of Endosomal Compartments.

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, we found that pathogenic Htt expression leads to a profound overgrowth of synaptic connections that correlates directly with the levels of Htt at nerve terminals. Branches of the same nerve containing different levels of Htt show distinct phenotypes, indicating that 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 ameliorates the severity of HD pathology and improves viability. Pathogenic Htt is present largely in a nonaggregated form at synapses, indicating that cytosolic forms of the protein are likely to be the toxic species that disrupt endosomal signaling. Our 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.SIGNIFICANCE STATEMENT Huntington's disease (HD) is the most commonly inherited polyglutamine expansion disorder, but how mutant Huntingtin (Htt) disrupts neuronal function is unclear. In particular, it is unknown within what subcellular compartment pathogenic Htt acts and whether the pathogenesis is associated with aggregated or more soluble forms of the protein. Using a Drosophila HD model, we find that nonaggregated pathogenic Htt acts locally at synaptic terminals to disrupt endosomal compartments, leading to aberrant wiring defects. Genetic manipulations to increase endosomal trafficking of synaptic growth receptors from signaling endosomes or to reduce BMP signaling reduce pathology in this HD model. These data indicate that pathogenic Htt can act locally within nerve terminals to disrupt synaptic endosomal signaling and induce neuropathology.

SIRT2- and NRF2-Targeting Thiazole-Containing Compound with Therapeutic Activity in Huntington's Disease Models

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in exvivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactiveoxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.

AUTEN-67 (Autophagy Enhancer-67) Hampers the Progression of Neurodegenerative Symptoms in a Drosophila model of Huntington's Disease.

Autophagy, a lysosome-mediated self-degradation process of eukaryotic cells, serves as a main route for the elimination of cellular damage [1-3]. Such damages include aggregated, oxidized or misfolded proteins whose accumulation can cause various neurodegenerative pathologies, including Huntington's disease (HD). Here we examined whether enhanced autophagic activity can alleviate neurophatological features in a Drosophila model of HD (the transgenic animals express a human mutant Huntingtin protein with a long polyglutamine repeat, 128Q). We have recently identified an autophagy-enhancing small molecule, AUTEN-67 (autophagy enhancer 67), with potent neuroprotective effects [4]. AUTEN-67 was applied to induce autophagic activity in the HD model used in this study. We showed that AUTEN-67 treatment interferes with the progressive accumulation of ubiquitinated proteins in the brain of Drosophila transgenic for the pathological 128Q form of human Huntingtin protein. The compound significantly improved the climbing ability and moderately extended the mean life span of these flies. Furthermore, brain tissue samples from human patients diagnosed for HD displayed increased levels of the autophagy substrate SQSTM1/p62 protein, as compared with controls. These results imply that AUTEN-67 impedes the progression of neurodegenerative symptoms characterizing HD, and that autophagy is a promising therapeutic target for treating this pathology. In humans, AUTEN-67 may have the potential to delay the onset and decrease the severity of HD.

A Drosophila model of Huntington disease-like 2 exhibits nuclear toxicity and distinct pathogenic mechanisms from Huntington disease.

Huntington disease-like 2 (HDL2) and Huntington disease (HD) are adult-onset neurodegenerative diseases characterized by movement disorders, psychiatric disturbances and cognitive decline. Brain tissue from HD and HDL2 patients shows degeneration of the striatum and ubiquitinated inclusions immunoreactive for polyglutamine (polyQ) antibodies. Despite these similarities, the diseases result from different genetic mutations. HD is caused by a CAG repeat expansion in the huntingtin (HTT) gene, while HDL2 results from an expansion at the junctophilin 3 (JPH3) locus. Recent evidence indicates that the HDL2 expansion may give rise to a toxic polyQ protein translated from an antisense mRNA derived from the JPH3 locus. To investigate this hypothesis, we generated and characterized a Drosophila HDL2 model and compared it with a previously established HD model. We find that neuronal expression of HDL2-Q15 is not toxic, while the expression of an expanded HDL2-Q138 protein is lethal. HDL2-Q138 forms large nuclear aggregates, with only smaller puncta observed in the cytoplasm. This is in contrast to what is observed in a Drosophila model of HD, where polyQ aggregates localize exclusively to the cytoplasm. Altering localization of HLD2 with the addition of a nuclear localization or nuclear export sequence demonstrates that nuclear accumulation is required for toxicity in the Drosophila HDL2 model. Directing HDL2-Q138 to the nucleus exacerbates toxicity in multiple tissue types, while confining HDL2-Q138 to the cytoplasm restores viability to control levels. We conclude that while HD and HDL2 have similar clinical profiles, distinct pathogenic mechanisms are likely to drive toxicity in Drosophila models of these disorders.

αB-Crystallin overexpression in astrocytes modulates the phenotype of the BACHD mouse model of Huntington's disease.

Huntington's disease (HD) is caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. The polyQ expansion increases the propensity of htt to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins are predicted to slow disease progression in HD models. αB-crystallin (αBc) or HspB5 is a well-characterized member of the small heat shock protein (sHsp) family that reduces mutant htt (mhtt) aggregation and toxicity in vitro and in Drosophila models of HD. Here, we determined if overexpressing αBc in vivo modulates aggregation and delays the onset and progression of disease in a full-length model of HD, BACHD mice. Expression of sHsps in neurodegenerative disease predominantly occurs in non-neuronal cells, and in the brain, αBc is mainly found in astrocytes and oligodendrocytes. Here, we show that directed αBc overexpression in astrocytes improves motor performance in rotarod and balance beam tests and improves cognitive function in the BACHD mice. Improvement in behavioral deficits correlated with mitigation of neuropathological features commonly observed in HD. Interestingly, astrocytic αBc overexpression was neuroprotective against neuronal cell loss in BACHD brains, suggesting αBc might be acting in a non-cell-autonomous manner. At the protein level, αBc decreased the level of soluble mhtt and decreased the size of mhtt inclusions in BACHD brain. Our results support a model in which elevating astrocytic αBc confers neuroprotection through a potential non-cell-autonomous pathway that modulates mhtt aggregation and protein levels.

Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease

We have previously demonstrated amelioration of Huntington's disease (HD)-related phenotypes in R6/2 transgenic mice in response to treatment with the novel histone deacetylase (HDAC) inhibitor 4b. Here we have measured the selectivity profiles of 4b and related compounds against class I and class II HDACs and have tested their ability to restore altered expression of genes related to HD pathology in mice and to rescue disease effects in cell culture and Drosophila models of HD. R6/2 transgenic and wild-type (wt) mice received daily injections of HDAC inhibitors for 3days followed by real-time PCR analysis to detect expression differences for 13 HD-related genes. We find that HDACi 4b and 136, two compounds showing high potency for inhibiting HDAC3 were most effective in reversing the expression of genes relevant to HD, including Ppp1r1b, which encodes DARPP-32, a marker for medium spiny striatal neurons. In contrast, compounds targeting HDAC1 were less effective at correcting gene expression abnormalities in R6/2 transgenic mice, but did cause significant increases in the expression of selected genes. An additional panel of 4b-related compounds was tested in a Drosophila model of HD and in STHdhQ111 striatal cells to further distinguish HDAC selectivity. Significant improvement in huntingtin-elicited Drosophila eye neurodegeneration in the fly was observed in response to treatment with compounds targeting human HDAC1 and/or HDAC3. In STHdhQ111 striatal cells, the ability of HDAC inhibitors to improve huntingtin-elicited metabolic deficits correlated with the potency at inhibiting HDAC1 and HDAC3, although the IC50 values for HDAC1 inhibition were typically 10-fold higher than for inhibition of HDAC3. Assessment of HDAC protein localization in brain tissue by Western blot analysis revealed accumulation of HDAC1 and HDAC3 in the nucleus of HD transgenic mice compared to wt mice, with a concurrent decrease in cytoplasmic localization, suggesting that these HDACs contribute to a repressive chromatin environment in HD. No differences were detected in the localization of HDAC2, HDAC4 or HDAC7. These results suggest that inhibition of HDACs 1 and 3 can relieve HD-like phenotypes in model systems and that HDAC inhibitors targeting these isotypes might show therapeutic benefit in human HD.

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines - sites of synaptogenesis - are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.

Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1α and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

B04 A genetic screen targeting suppressors of aggregation and neurodegeneration in a Drosophila Huntington's disease model

BackgroundHuntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the huntingtin protein (Htt). Polyglutamine expansion in Htt leads to intracellular aggregates within neurons although it...