Cells Expressing Mutant Huntingtin Research Articles

Disruption of zinc transporter ZnT3 transcriptional activity and synaptic vesicular zinc in the brain of Huntington\u2019s disease transgenic mouse

BackgroundHuntington’s disease (HD) is a neurodegenerative disease that involves a complex combination of psychiatric, cognitive and motor impairments. Synaptic dysfunction has been implicated in HD pathogenesis. However, the mechanisms have not been clearly delineated. Synaptic vesicular zinc is closely linked to modulating synaptic transmission and maintaining cognitive ability. It is significant to assess zinc homeostasis for further revealing the pathogenesis of synaptic dysfunction and cognitive impairment in HD.ResultsHistochemical staining by autometallography indicated that synaptic vesicular zinc was decreased in the hippocampus, cortex and striatum of N171-82Q HD transgenic mice. Analyses by immunohistochemistry, Western blot and RT-PCR found that the expression of zinc transporter 3 (ZnT3) required for transport of zinc into synaptic vesicles was obviously reduced in these three brain regions of the HD mice aged from 14 to 20 weeks and BHK cells expressing mutant huntingtin. Significantly, dual-luciferase reporter gene and chromatin immunoprecipitation assays demonstrated that transcription factor Sp1 could activate ZnT3 transcription via its binding to the GC boxes in ZnT3 promoter. Moreover, mutant huntingtin was found to inhibit the binding of Sp1 to the promoter of ZnT3 and down-regulate ZnT3 expression, and the decline in ZnT3 expression could be ameliorated through overexpression of Sp1.ConclusionsThis is first study to reveal a significant loss of synaptic vesicular zinc and a decline in ZnT3 transcriptional activity in the HD transgenic mice. Our work sheds a novel mechanistic insight into pathogenesis of HD that mutant huntingtin down-regulates expression of ZnT3 through inhibiting binding of Sp1 to the promoter of ZnT3 gene, causing disruption of synaptic vesicular zinc homeostasis. Disrupted vesicular zinc ultimately leads to early synaptic dysfunction and cognitive deficits in HD. It is also suggested that maintaining normal synaptic vesicular zinc concentration is a potential therapeutic strategy for HD.

Energy Metabolism and Mitochondrial Superoxide Anion Production in Pre-symptomatic Striatal Neurons Derived from Human-Induced Pluripotent Stem Cells Expressing Mutant Huntingtin.

In the present study, we investigated whether mutant huntingtin (mHTT) impairs mitochondrial functions in human striatal neurons derived from induced pluripotent stem cells (iPSCs). Striatal neurons and astrocytes derived from iPSCs from unaffected individuals (Ctrl) and Huntington's disease (HD) patients with HTT gene containing increased number of CAG repeats were used to assess the effect of mHTT on bioenergetics and mitochondrial superoxide anion production. The human neurons were thoroughly characterized and shown to express MAP2, DARPP32, GABA, synapsin, and PSD95. In human neurons and astrocytes expressing mHTT, the ratio of mHTT to wild-type huntingtin (HTT) was 1:1. The human neurons were excitable and could generate action potentials, confirming successful conversion of iPSCs into functional neurons. The neurons and astrocytes from Ctrl individuals and HD patients had similar levels of ADP and ATP and comparable respiratory and glycolytic activities. The mitochondrial mass, mitochondrial membrane potential, and superoxide anion production in human neurons appeared to be similar regardless of mHTT presence. The present results are in line with the results obtained in our previous studies with isolated brain mitochondria and cultured striatal neurons from YAC128 and R6/2 mice, in which we demonstrated that mutant huntingtin at early stages of HD pathology does not deteriorate mitochondrial functions. Overall, our results argue against bioenergetic deficits as a factor in HD pathogenesis and suggest that other detrimental processes might be more relevant to the development of HD pathology.

Angiotensins and Huntington's Disease: A Study on Immortalized Progenitor Striatal Cell Lines.

Neurons from mouse models of Huntington’s disease (HD) exhibit altered electrophysiological properties, potentially contributing to neuronal dysfunction and neurodegeneration. The renin–angiotensin system (RAS) is a potential contributor to the pathophysiology of neurodegenerative diseases. However, the role of angiotensin II (Ang II) and angiotensin (1-7) has not been characterized in HD. We investigated the influence of Ang II and angiotensin (1-7) on total potassium current using immortalized progenitor mutant huntingtin-expressing (Q111) and wild-type (Q7) cell lines. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results showed that (1) the effect of Ang II administered to the bath caused a negligible effect on potassium current in mutant Q111 cells compared with wild-type Q7 cells and that intracellular administration of Ang II reduced the potassium current in wild type but not in mutant cells; (2) the small effect of Ang II was abolished by losartan; (3) intracellular administration of Ang II performed in mutant huntingtin-expressing Q111 cells revealed a negligible effect of the peptide on potassium current; (4) flow cytometer analysis indicated a low expression of Ang II AT1 receptors in mutant Q111 cells; (5) mutant huntingtin-expressing striatal cells are highly sensitive to Ang (1-7) and that the effect of Ang (1-7) is related to the activation of Mas receptors. In conclusion, mutant huntingtin-expressing cells showed a negligible effect of Ang II on potassium current, a result probably due to the reduced expression of AT1 receptors at the surface cell membrane. In contrast, administration of Ang (1-7) to the bath showed a significant decline of the potassium current in mutant cells, an effect dependent on the activation of Mas receptors. Ang II had an intracrine effect in wild-type cells and Ang (1-7) exerted a significant effect in mutant huntingtin-expressing striatal cells.

Resveratrol protects neuronal-like cells expressing mutant Huntingtin from dopamine toxicity by rescuing ATG4-mediated autophagosome formation

Parkinsonian-like motor deficits in Huntington's Disease (HD) patients are associated with abnormal dopamine neurotransmission in the striatum. Dopamine metabolism leads to the formation of oxidized dopamine quinones that exacerbates mitochondrial dysfunction with production of reactive oxygen species (ROS) that eventually lead to neuronal cell death. We have previously shown that dopamine-induced oxidative stress triggers apoptotic cell death in dopaminergic neuroblastoma SH-SY5Y cells hyper-expressing the mutant polyQ Huntingtin (polyQ-Htt) protein. Dopamine toxicity was paralleled by impaired autophagy clearance of the polyQ-Htt aggregates. In this study, we found that Dopamine affects the stability and function of ATG4, a redox-sensitive cysteine-protein involved in the processing of LC3, a key step in the formation of autophagosomes. Resveratrol, a dietary polyphenol with anti-oxidant and pro-autophagic properties, has shown neuroprotective potential in HD. Yet the molecular mechanism through which Resveratrol can protect HD cells against DA is not known. Here, we show that Resveratrol prevents the generation of ROS, restores the level of ATG4, allows the lipidation of LC3, facilitates the degradation of polyQ-Htt aggregates and protects the cells from Dopamine toxicity.The present findings provide a mechanistic explanation of the neuroprotective activity of Resveratrol and support its inclusion in a therapeutic regimen to slow down HD progression.

Polyglutamine tracts regulate beclin 1-dependent autophagy.

Nine neurodegenerative diseases are caused by expanded polyglutamine (polyQ) tracts in different proteins, like huntingtin in Huntington’s disease (HD) and ataxin-3 in spinocerebellar ataxia type 3 (SCA3)1, 2. Age-at-onset decreases with increasing polyglutamine length in these proteins and the normal length is also polymorphic3. PolyQ expansions drive pathogenesis in these diseases, as isolated polyQ tracts are toxic, and an N-terminal huntingtin fragment comprising exon 1, which occurs in vivo due to alternative splicing4, causes toxicity. While such mutant proteins are aggregate-prone5, toxicity is also associated with soluble forms of the proteins6. The function of the polyQ tracts in many normal/wild-type cytoplasmic proteins is unclear. One such protein is the deubiquitinating enzyme ataxin 37, 8, which is widely expressed in the brain9, 10. Here we show that the polyQ domain in wild-type ataxin-3 enables its interaction with beclin 1, a key autophagy initiator11. This interaction allows the deubiquitinase activity of ataxin-3 to protect beclin 1 from proteasome-mediated degradation and thus enables autophagy. Starvation-induced autophagy, which is regulated by beclin 1, was particularly inhibited in ataxin-3-depleted human cell-lines, primary neurons and in-vivo. This activity of ataxin-3 and its interaction with beclin 1 mediated by its polyQ domain was competed by other soluble proteins with polyQ tracts in a length-dependent fashion. This resulted in impaired starvation-induced autophagy in cells expressing mutant huntingtin exon 1, which was also recapitulated in the brain of HD mouse model and in patient cells. A similar phenomenon was also seen with other polyQ disease proteins, including mutant ataxin-3 itself. Our data thus describe a specific function for a wild-type polyQ tract which is abrogated by a competing longer polyQ mutation in a disease protein. This also reveals a deleterious function of such mutations distinct from their aggregation propensity.

A hydrocortisone derivative binds to GAPDH and reduces the toxicity of extracellular polyglutamine-containing aggregates

Huntington's disease (HD) has been recently shown to have a horizontally transmitted, prion-like pathology. Thus, the migration of polyglutamine-containing aggregates to acceptor cells is important for the progression of HD. These aggregates contain glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which increases their intracellular transport and their toxicity. Here, we show that RX624, a derivative of hydrocortisone that binds to GAPDH, prevents the formation of aggregates of GAPDH-polyglutamine excreted into the culture medium by PC-12 rat cells expressing mutant huntingtin. RX624 was previously shown to be unable to penetrate cells and, thus, its principal therapeutic action might be the inhibition of polyglutamine-GAPDH complex aggregation in the extracellular matrix. The administration of RX624 to SH-SY5Y acceptor cells that incubated in conditioned medium from PC-12 cells expressing mutant huntingtin caused an approximately 20% increase in survival. This suggests that RX624 might be useful as a drug against polyglutamine pathologies, and that is could be administered exogenously without affecting target cell physiology. This protective effect was validated by the similar effect of an anti-GAPDH specific antibody.

Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease.

Transcriptional deregulation and changes in mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction, have been described in Huntington's disease (HD). We showed previously that the histone deacetylase inhibitors (HDACIs) trichostatin A and sodium butyrate (SB) ameliorate mitochondrial function in cells expressing mutant huntingtin. In this work, we investigated the effect of HDACIs on the regulation of PDH activity in striatal cells derived from HD knock-in mice and YAC128 mice. Mutant cells exhibited decreased PDH activity and increased PDH E1alpha phosphorylation/inactivation, accompanied by enhanced protein levels of PDH kinases 1 and 3 (PDK1 and PDK3). Exposure to dichloroacetate, an inhibitor of PDKs, increased mitochondrial respiration and decreased production of reactive oxygen species in mutant cells, emphasizing PDH as an interesting therapeutic target in HD. Treatment with SB and sodium phenylbutyrate, another HDACI, recovered cell viability and overall mitochondrial metabolism in mutant cells. Exposure to SB also suppressed hypoxia-inducible factor-1 (HIF-1α) stabilization and decreased the transcription of the two most abundant PDK isoforms, PDK2 and PDK3, culminating in increased PDH activation in mutant cells. Concordantly, PDK3 knockdown improved mitochondrial function, emphasizing the role of PDK3 inactivation on the positive effects achieved by SB treatment. YAC128 mouse brain presented higher mRNA levels of PDK1-3 and PDH phosphorylation and decreased energy levels that were significantly ameliorated after SB treatment. Furthermore, enhanced motor learning and coordination were observed in SB-treated YAC128 mice. These results suggest that HDACIs, particularly SB, promote the activity of PDH in the HD brain, helping to counteract HD-related deficits in mitochondrial bioenergetics and motor function.SIGNIFICANCE STATEMENT The present work provides a better understanding of mitochondrial dysfunction in Huntington's disease (HD) by showing that the pyruvate dehydrogenase (PDH) complex is a promising therapeutic target. In particular, the histone deacetylase inhibitor sodium butyrate (SB) may indirectly (through reduced hypoxia-inducible factor 1 alpha stabilization) decrease the expression of the most abundant PDH kinase isoforms (e.g., PDK3), ameliorating PDH activity and mitochondrial metabolism and further affecting motor behavior in HD mice, thus constituting a promising agent for HD neuroprotective treatment.

SMAD transcription factors are altered in cell models of HD and regulate HTT expression

Transcriptional dysregulation is observable in multiple animal and cell models of Huntington's disease, as well as in human blood and post-mortem caudate. This contributes to HD pathogenesis, although the exact mechanism by which this occurs is unknown. We therefore utilised a dynamic model in order to determine the differential effect of growth factor stimulation on gene expression, to highlight potential alterations in kinase signalling pathways that may be in part responsible for the transcriptional dysregulation observed in HD, and which may reveal new therapeutic targets. We demonstrate that cells expressing mutant huntingtin have a dysregulated transcriptional response to epidermal growth factor stimulation, and identify the transforming growth factor-beta pathway as a novel signalling pathway of interest that may regulate the expression of the Huntingtin (HTT) gene itself. The dysregulation of HTT expression may contribute to the altered transcriptional phenotype observed in HD.

Metformin Protects Cells from Mutant Huntingtin Toxicity Through Activation of AMPK and Modulation of Mitochondrial Dynamics.

Huntington's disease (HD) is a devastating neurodegenerative disease caused by the pathological elongation of the CAG repeats in the huntingtin gene. Caloric restriction (CR) has been the most reproducible environmental intervention to improve health and prolong life span. We have demonstrated that CR delayed onset and slowed disease progression in a mouse model of HD. Metformin, an antidiabetic drug, mimics CR by acting on cell metabolism at multiple levels. Long-term administration of metformin improved health and life span in mice. In this study, we showed that metformin rescued cells from mutant huntingtin (HTT)-induced toxicity, as indicated by reduced lactate dehydrogenase (LDH) release from cells and preserved ATP levels in cells expressing mutant HTT. Further mechanistic study indicated that metformin activated AMP-activated protein kinase (AMPK) and that inhibition of AMPK activation reduced its protective effects on mutant HTT toxicity, suggesting that AMPK mediates the protection of metformin in HD cells. Furthermore, metformin treatment prevented mitochondrial membrane depolarization and excess fission and modulated the disturbed mitochondrial dynamics in HD cells. We confirmed that metformin crossed the blood-brain barrier after oral administration and activated AMPK in the mouse brain. Our results urge further evaluation of the clinical potential for use of metformin in HD treatment.

Interactome network analysis identifies multiple caspase-6 interactors involved in the pathogenesis of HD.

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.

Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-014-8974-4) contains supplementary material, which is available to authorized users.

D10 Pharmacodynamic Biomarkers For Selisistat: The Paddington Project

Background Siena Biotech is developing a highly selective SIRT1 inhibitor, selisistat, as a potentially disease-modifying therapy for Huntington’s disease. Selisistat has shown to ameliorate HD phenotypes in HD preclinical models, from cells expressing mutant huntingtin to transgenic Drosophila and R6/2 mice before entering the clinical trials. Selisistat recently completed the Phase II Study, assessing safety and tolerability in HD patients. Aim Evaluate possible selisistat pharmacodynamic biomarkers to support further clinical progression. Within the EC funded project PADDINGTON, a transcriptional profile and huntingtin protein levels were evaluated in biological samples from HD patients treated with placebo or selisistat at different doses. Methods HD patients were randomised to receive different doses of selisistat or placebo. Serial blood sampling was collected at different time points after selisistat administration. Peripheral blood mononuclear cells (PBMCs) were employed to detect HTT protein levels by a validated ELISA bioassay. Whole blood samples were analysed through RT-qPCR to validate a previous identified transcriptional signature. Results Four transcripts were found specifically modulated by selisistat in whole blood samples, confirming the transcriptional signature. Moreover, the ELISA in PBMCs has proven to reliably measure of circulating HTT despite the low protein levels and high individual variability. Conclusions The quantisation of circulating HTT levels as well as gene expression evaluation represents a promising avenue towards monitoring disease progression and therapeutic interventions. Additionally, transcriptional signatures could be useful to draw hypotheses for target engagement and mechanism of action through the pathway analysis of the modulated genes. Final results for both investigations will be presented.

B31 Striatal Deregulation Of Cdk5 Alters Mitochondrial Dynamics In Huntington's Disease

Huntington’s disease (HD) is characterised by motor disturbances associated to dysfunction and degeneration of the medium spiny neurons within the striatum. 1,2 The molecular mechanisms underlying striatal vulnerability are still unknown, but growing evidences suggest that mitochondrial dysfunction occurs during the pathogenesis of the disease. 3 We previously described that deregulation of cyclin-dependent kinase 5 (Cdk5) activity induced by mutant huntingtin increases the susceptibility of striatal neurons to dopamine via D1 receptor activation. 4 Interestingly, Cdk5 has been shown to act as a mitochondrial regulator during neuronal apoptosis. 5 Here, we investigated whether this aberrant Cdk5 signalling contributes to the striatal neurodegeneration by altering mitochondrial dynamics processes. We first observed that striatal cells expressing mutant huntingtin (mHtt) present increased mitochondrial fragmentation that worsens after dopaminergic stimuli. These mitochondrial defects can be widely rescued by Cdk5 inhibition with roscovitine or Cdk5 knockdown with siRNA transfection. Furthermore we found that mHtt deregulates the levels and the subcellular distribution of fission/fusion proteins while activation of D1 receptors promotes an increase in the levels of the fission protein Drp1 and its translocation to the mitochondria. We demonstrated that mHtt-induced Cdk5 activation is involved in the deregulation of the Drp1 GTPase activity since its inhibition prevents the aberrant activation of this fission protein. Altogether, our findings support the hypothesis that Cdk5 plays a crucial role in mitochondrial defects involved in the striatal neurodegeneration in HD. This study was supported by Ministerio de Innovacion y Ciencia SAF2012–39142, CHDI Ministerio deSanidad y Consumo (CIBERNED CB06/05/0054), Fundacion Ramon Areces. References 1Ferrante RJ, Kowall NW, Beal MF, Martin JB, Bird ED, Richardson EPJr. Morphologic and histochemical characteristics of a spared subset of striatal neurons in Huntington’s disease. J Neuropathol Exp Neurol. 1987; 46 :12–27 2Myers RH, Vonsattle JP, Stevens TJ, Cupples LA, Richardson EP, Martin JB, Bird ED. Clinical and neuropathologic assesment of severity in Huntington’s disease. Neurology 1988; 38 :341–347 3Damiano M, Galvan L, Deglon N, Brouillet E. Mitochondria in Huntington’s disease. Biochim Biophys Acta 2010;1802:52–61 4Paoletti P, Vila I, Rife M, Lizcano JM, Alberch J, Gines S. Dopaminergic and glutamatergic signaling crosstalk in Huntington’s disease neurodegeneration: the role of p25/cyclin dependent kinase 5. J Neurosci 2008; 28 (40):10090–10101 5Meuer K, Suppanz IE, Lingor P, Planchamp V, Goricke B, Fichtner L, Braus GH, Dietz GP, Jakobs S, Bahr M, Weishaupt JH. Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis. Cell Death Differ 2007; 14 (4):651–61

Maintenance of chaperone-mediated autophagy activity in cultured cells expressing mutant huntingtin

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutant huntingtin (Htt) with an expanded polyglutamine tract. It has been reported that Htt regulates autophagy. However, it remains unclear whether mutant Htt affects chaperone-mediated autophagy (CMA). Our study aimed to investigate the effect of mutant Htt on CMA activity in cultured HEK293T cells. A HEK293T cell model of HD was produced by transient transfection of wild-type (20Q) or mutant (120Q) Htt plasmids. The effect of mutant Htt on two CMA components, lysosomal-associated membrane protein 2a (Lamp2a) and heat-shock cognate protein 70 (Hsc70), was determined by western blotting and immunofluorescent staining. We observed that mutant Htt did not significantly alter the expression of Lamp2a and Hsc70 when compared to normal Htt. These findings suggest that mutant Htt does not reduce CMA activity and that enhancing CMA activity to clear mutant Htt may be a novel strategy for the management of HD.

HACE1 reduces oxidative stress and mutant Huntingtin toxicity by promoting the NRF2 response

Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.

Deciphering the Roles of Trehalose and Hsp104 in the Inhibition of Aggregation of Mutant Huntingtin in a Yeast Model of Huntington’s Disease

Despite the significant amount of experimental data available on trehalose, the molecular mechanism responsible for its intracellular stabilising properties has not emerged yet. The repair of cellular homeostasis in many protein-misfolding diseases by trehalose is credited to the disaccharide being an inducer of autophagy, a mechanism by which aggregates of misfolded proteins are cleared by the cell. In this work, we expressed the pathogenic N-terminal fragment of huntingtin in Δnth1 mutant (unable to degrade trehalose) of Saccharomyces cerevisiae BY4742 strain. We show that the presence of trehalose resulted in the partitioning of the mutant huntingtin in the soluble fraction of the cell. This led to reduced oxidative stress and improved cell survival. The beneficial effect was independent of the expression of the major cellular antioxidant enzyme, superoxide dismutase. Additionally, trehalose led to the overexpression of the heat shock protein, Hsp104p, in mutant huntingtin-expressing cells, and resulted in rescue of the endocytotic defect in the yeast cell. We propose that at least in the initial stages of aggregation, trehalose functions as a stabiliser, increasing the level of monomeric mutant huntingtin protein, with its concomitant beneficial effects, in addition to its role as an inducer of autophagy.

Association of age at onset in Huntington disease with functional promoter variations in NPY and NPY2R

Huntington disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the HTT gene. Although the variation in age at onset (AO) is partly explained by the lengths of the expanded repeats, the unexplained variation is highly heritable, emphasizing the role of the so-called genetic background on disease expression. Neuropeptide Y (NPY) has been implicated in the modulation of neuroprotection, neurogenesis, and neuroinflammation. Therefore, the aim of the present study was to analyze different single nucleotide polymorphisms (SNPs) in order to test the possibility that genetic variation in NPY or three of its receptor genes (NPY1R, NPY2R, and NPY5R) may explain some of the variation in AO of HD motor manifestations, in a comprehensive cohort of 487 German HD patients. We found modest association of the AO with two NPY promoter variations and a highly significant association with a NPY2R promoter SNP (rs2234759; p = 0.0004). Investigating the functional impact of rs2234759 by luciferase assays revealed that the high-expression NPY2R genotypes were associated with later AO in HD. Additionally, treatment of PC12 cells expressing mutant huntingtin (htt) exon 1 with NPY and the NPY2R agonist NPY(3-36) has a protective effect against mutant htt-induced cell death. Thus, NPY might act through Y2 receptors to slow down the course of HD, and hence, this peptide could be of interest as a possible therapeutic agent.

Oxidizing Effects of Exogenous Stressors in Huntington’s Disease Knock-in Striatal Cells—Protective Effect of Cystamine and Creatine

Huntington's disease (HD) is a polyglutamine-expansion disease associated to degeneration of striatal and cortical neurons. Previously, we showed that oxidative stress occurs in HD knock-in striatal cells, but little is known regarding cell antioxidant response against exogenous stimuli. Therefore, in the present study we analyzed cellular antioxidant profile following hydrogen peroxide (H2O2) and staurosporine (STS) exposure and tested the protective effect of cystamine and creatine in striatal cells expressing mutant huntingtin with 111 glutamines (STHdh (Q111/Q111); mutant cells) versus wild-type cells (STHdh (Q7/Q7)). Mutant cells displayed increased mitochondrial reactive oxygen species (ROS) and decreased NADPH oxidase and xanthine oxidase (XO) activities, reflecting lower superoxide cytosolic generation, along with increased superoxide dismutases (SODs) and components of glutathione redox cycle. Exposure to H2O2 and STS enhanced ROS in mutant cells and largely increased XO activity; STS further boosted the generation of mitochondrial ROS and caspase-3 activity. Both stimuli slightly increased SOD1 activity, without affecting SOD2 activity, and decreased glutathione reductase with a consequent rise in oxidized glutathione or glutathione disulfide in mutant cells, whereas H2O2 only increased glutathione peroxidase activity. Additionally, creatine and cystamine increased mutant cells viability and prevented ROS formation in HD cells subjected to H2O2 and STS. These results indicate that elevation of the antioxidant systems accompanies mitochondrial-driven ROS generation in mutant striatal cells and that exposure to noxious stimuli induces a higher susceptibility to oxidative stress by increasing XO activity and lowering the antioxidant response. Furthermore, creatine and cystamine are efficient in preventing H2O2- and STS-evoked ROS formation in HD striatal cells.

Early Down-Regulation of PKCδ as a Pro-Survival Mechanism in Huntington’s Disease

A balance between cell survival and apoptosis is crucial to avoid neurodegeneration. Here, we analyzed whether the pro-apoptotic protein PKCδ, and the pro-survival PKCα and βII, were dysregulated in the brain of R6/1 mouse model of Huntington's disease (HD). Protein levels of the three PKCs examined were reduced in all the brain regions analyzed being PKCδ the most affected isoform. Interestingly, PKCδ protein levels were also decreased in the striatum and cortex of R6/2 and Hdh(Q111/Q111) mice, and in the putamen of HD patients. Nuclear PKCδ induces apoptosis, but we detected reduced PKCδ in both cytoplasmic and nuclear enriched fractions from R6/1 mouse striatum, cortex and hippocampus. In addition, we show that phosphorylation and ubiquitination of PKCδ are increased in 30-week-old R6/1 mouse brain. All together these results suggest a pro-survival role of reduced PKCδ levels in response to mutant huntingtin-induced toxicity. In fact, we show that over-expression of PKCδ increases mutant huntingtin-induced cell death in vitro, whereas over-expression of a PKCδ dominant negative form or silencing of endogenous PKCδ partially blocks mutant huntingtin-induced cell death. Finally, we show that the analysis of lamin B protein levels could be a good marker of PKCδ activity, but it is not involved in PKCδ-mediated cell death in mutant huntingtin-expressing cells. In conclusion, our results suggest that neurons increase the degradation of PKCδ as a compensatory pro-survival mechanism in response to mutant huntingtin-induced toxicity that can help to understand why cell death appears late in the disease.

Neuroprotective effects of PPAR‐γ agonist rosiglitazone in N171‐82Q mouse model of Huntington's disease

Huntington's disease (HD) is a devastating genetic neurodegenerative disease caused by CAG trinucleotide expansion in the exon-1 region of the huntingtin gene. Currently, no cure is available. It is becoming increasingly apparent that mutant Huntingtin (HTT) impairs metabolic homeostasis and causes transcriptional dysregulation. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a transcriptional factor that plays a key role in regulating genes involved in energy metabolism; recent studies demonstrated that PPAR-γ activation prevented mitochondrial depolarization in cells expressing mutant HTT and attenuated neurodegeneration in various models of neurodegenerative diseases. PPAR-γ-coactivator 1α (PGC-1 α) transcription activity is also impaired by mutant HTT. We now report that the PPAR-γ agonist, rosiglitazone (RSG), significantly attenuated mutant HTT-induced toxicity in striatal cells and that the protective effect of RSG is mediated by activation of PPAR-γ. Moreover, chronic administration of RSG (10 mg/kg/day, i.p) significantly improved motor function and attenuated hyperglycemia in N171-82Q HD mice. RSG administration rescued brain derived neurotrophic factor(BDNF) deficiency in the cerebral cortex, and prevented loss of orexin-A-immunopositive neurons in the hypothalamus of N171-82Q HD mice. RSG also prevented PGC-1α reduction and increased Sirt6 protein levels in HD mouse brain. Our results suggest that modifying the PPAR-γ pathway plays a beneficial role in rescuing motor function as well as glucose metabolic abnormalities in HD.