Brain-Periphery Interactions in Huntington's Disease: Mediators and Lifestyle Interventions.

Prominent pathological features of Huntington's disease (HD) are aggregations of mutated Huntingtin protein (mHtt) in the brain and neurodegeneration, which causes characteristic motor (such as chorea and dystonia) and non-motor symptoms. However, the numerous systemic and peripheral deficits in HD have gained increasing attention recently, since those factors likely modulate disease progression, including brain pathology. While whole-body metabolic abnormalities and organ-specific pathologies in HD have been relatively well described, the potential mediators of compromised inter-organ communication in HD have been insufficiently characterized. Therefore, we applied an exploratory literature search to identify such mediators. Unsurprisingly, dysregulation of inflammatory factors, circulating mHtt, and many other messenger molecules (hormones, lipids, RNAs) were found that suggest impaired inter-organ communication, including of the gut-brain and muscle-brain axis. Based on these findings, we aimed to assess the risks and potentials of lifestyle interventions that are thought to improve communication across these axes: dietary strategies and exercise. We conclude that appropriate lifestyle interventions have great potential to reduce symptoms and potentially modify disease progression (possibly via improving inter-organ signaling) in HD. However, impaired systemic metabolism and peripheral symptoms warrant particular care in the design of dietary and exercise programs for people with HD.

HAP40 modulates mutant Huntingtin aggregation and toxicity in Huntington's disease mice.

Huntington's disease (HD) is a monogenic neurodegenerative disease, caused by the CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene. The HTT gene encodes a large protein known to interact with many proteins. Huntingtin-associated protein 40 (HAP40) is one that shows high binding affinity with HTT and functions to maintain HTT conformation in vitro. However, the potential role of HAP40 in HD pathogenesis remains unknown. In this study, we found that the expression level of HAP40 is in parallel with HTT but inversely correlates with mutant HTT aggregates in mouse brains. Depletion of endogenous HAP40 in the striatum of HD140Q knock-in (KI) mice leads to enhanced mutant HTT aggregation and neuronal loss. Consistently, overexpression of HAP40 in the striatum of HD140Q KI mice reduced mutant HTT aggregation and ameliorated the behavioral deficits. Mechanistically, HAP40 preferentially binds to mutant HTT and promotes Lysine 48-linked ubiquitination of mutant HTT. Our results revealed that HAP40 is an important regulator of HTT protein homeostasis in vivo and hinted at HAP40 as a therapeutic target in HD treatment.

Progressive alterations in polysomal architecture and activation of ribosome stalling relief factors in a mouse model of Huntington's disease.

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.

BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

Role of autophagy and proteostasis in neurodegenerative diseases: Exploring the therapeutic interventions.

Neurodegenerative disorders are devastating disorders characterized by gradual loss of neurons and cognition or mobility impairment. The common pathological features of these diseases are associated with the accumulation of misfolded or aggregation of proteins. The pivotal roles of autophagy and proteostasis in maintaining cellular health and preventing the accumulation of misfolded proteins, which are associated with neurodegenerative diseases like Huntington's disease (HD), Alzheimer's disease (AD), and Parkinson's disease (PD). This article presents an in-depth examination of the interplay between autophagy and proteostasis, highlighting how these processes cooperatively contribute to cellular homeostasis and prevent pathogenic protein aggregate accumulation. Furthermore, the review emphasises the potential therapeutic implications of targeting autophagy and proteostasis to mitigate neurodegenerative diseases. While advancements in research hold promise for developing novel treatments, the article also addresses the challenges and complexities associated with modulating these intricate cellular pathways. Ultimately, advancing understanding of the underlying mechanism of autophagy and proteostasis in neurodegenerative disorders provides valuable insights into potential therapeutic avenues and future research directions.

Dietary fasting and time-restricted eating in Huntington's disease: therapeutic potential and underlying mechanisms.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by aggregation of the mutant huntingtin (mHTT) protein, resulting from a CAG repeat expansion in the huntingtin gene HTT. HD is characterized by a variety of debilitating symptoms including involuntary movements, cognitive impairment, and psychiatric disturbances. Despite considerable efforts, effective disease-modifying treatments for HD remain elusive, necessitating exploration of novel therapeutic approaches, including lifestyle modifications that could delay symptom onset and disease progression. Recent studies suggest that time-restricted eating (TRE), a form of intermittent fasting involving daily caloric intake within a limited time window, may hold promise in the treatment of neurodegenerative diseases, including HD. TRE has been shown to improve mitochondrial function, upregulate autophagy, reduce oxidative stress, regulate the sleep-wake cycle, and enhance cognitive function. In this review, we explore the potential therapeutic role of TRE in HD, focusing on its underlying physiological mechanisms. We discuss how TRE might enhance the clearance of mHTT, recover striatal brain-derived neurotrophic factor levels, improve mitochondrial function and stress-response pathways, and synchronize circadian rhythm activity. Understanding these mechanisms is critical for the development of targeted lifestyle interventions to mitigate HD pathology and improve patient outcomes. While the potential benefits of TRE in HD animal models are encouraging, future comprehensive clinical trials will be necessary to evaluate its safety, feasibility, and efficacy in persons 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 ß-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases ß-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.

Solid-state nuclear magnetic resonance in the structural study of polyglutamine aggregation.

The aggregation of proteins into amyloid-like fibrils is seen in many neurodegenerative diseases. Recent years have seen much progress in our understanding of these misfolded protein inclusions, thanks to advances in techniques such as solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, multiple repeat-expansion-related disorders have presented special challenges to structural elucidation. This review discusses the special role of ssNMR analysis in the study of protein aggregates associated with CAG repeat expansion disorders. In these diseases, the misfolding and aggregation affect mutant proteins with expanded polyglutamine segments. The most common disorder, Huntington's disease (HD), is connected to the mutation of the huntingtin protein. Since the discovery of the genetic causes for HD in the 1990s, steady progress in our understanding of the role of protein aggregation has depended on the integrative and interdisciplinary use of multiple types of structural techniques. The heterogeneous and dynamic features of polyQ protein fibrils, and in particular those formed by huntingtin N-terminal fragments, have made these aggregates into challenging targets for structural analysis. ssNMR has offered unique insights into many aspects of these amyloid-like aggregates. These include the atomic-level structure of the polyglutamine core, but also measurements of dynamics and solvent accessibility of the non-core flanking domains of these fibrils' fuzzy coats. The obtained structural insights shed new light on pathogenic mechanisms behind this and other protein misfolding diseases.

Dysregulation of extracellular potassium distinguishes healthy ageing from neurodegeneration.

Progressive neuronal loss is a hallmark feature distinguishing neurodegenerative diseases from normal ageing. However, the underlying mechanisms remain unknown. Extracellular K+ homeostasis is a potential mediator of neuronal injury as K+ elevations increase excitatory activity. The dysregulation of extracellular K+ and potassium channel expressions during neurodegeneration could contribute to this distinction. Here we measured the cortical extracellular K+ concentration ([K+]e) in awake wild-type mice as well as murine models of neurodegeneration using K+-sensitive microelectrodes. Unexpectedly, aged wild-type mice exhibited significantly lower cortical [K+]e than young mice. In contrast, cortical [K+]e was consistently elevated in Alzheimer's disease (APP/PS1), amyotrophic lateral sclerosis (ALS) (SOD1G93A) and Huntington's disease (R6/2) models. Cortical resting [K+]e correlated inversely with neuronal density and the [K+]e buffering rate but correlated positively with the predicted neuronal firing rate. Screening of astrocyte-selective genomic datasets revealed a number of potassium channel genes that were downregulated in these disease models but not in normal ageing. In particular, the inwardly rectifying potassium channel Kcnj10 was downregulated in ALS and Huntington's disease models but not in normal ageing, while Fxyd1 and Slc1a3, each of which acts as a negative regulator of potassium uptake, were each upregulated by astrocytes in both Alzheimer's disease and ALS models. Chronic elevation of [K+]e in response to changes in gene expression and the attendant neuronal hyperexcitability may drive the neuronal loss characteristic of these neurodegenerative diseases. These observations suggest that the dysregulation of extracellular K+ homeostasis in a number of neurodegenerative diseases could be due to aberrant astrocytic K+ buffering and as such, highlight a fundamental role for glial dysfunction in neurodegeneration.

Ribosome Profiling and Mass Spectrometry Reveal Widespread Mitochondrial Translation Defects in a Striatal Cell Model of Huntington Disease.

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive deficits, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo-Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded oxidative phosphorylation mRNAs (mt-Nd1, mt-Nd2, mt-Nd4, mt-Nd4l, mt-Nd5, mt-Nd6, mt-Co1, mt-Cytb, and mt-ATP8). We also applied tandem mass tag-based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein abundance of mitochondria-related genes in HD.

Patient-derived neuron model: Capturing age-dependent adult-onset degenerative pathology in Huntington's disease.

MicroRNAs play a crucial role in directly reprogramming (converting) human fibroblasts into neurons. Specifically, miR-9/9* and miR-124 (miR-9/9*-124) display neurogenic and cell fate-switching activities when ectopically expressed in human fibroblasts by erasing fibroblast identity and inducing a pan-neuronal state. These converted neurons maintain the biological age of the starting fibroblasts and thus provide a human neuron-based platform to study cellular properties in aged neurons and model adult-onset neurodegenerative disorders using patient-derived cells. Furthermore, the expression of striatal-enriched transcription factors in conjunction with miR-9/9*-124 guides the identity of medium spiny neurons (MSNs), the primary targets in Huntington's disease (HD). Converted MSNs from HD patient-derived fibroblasts (HD-MSNs) can replicate HD-related phenotypes including neurodegeneration associated with age-related declines in critical cellular functions such as autophagy. Here, we review the role of microRNAs in the direct conversion of patient-derived fibroblasts into MSNs and the practical application of converted HD-MSNs as a model for studying adult-onset neuropathology in HD. We provide valuable insights into age-related, cell-intrinsic changes contributing to neurodegeneration in HD-MSNs. Ultimately, we address a comprehensive understanding of the complex molecular landscape underlying HD pathology, offering potential avenues for therapeutic application.

Intravenous MSC-Treatment Improves Impaired Brain Functions in the R6/2 Mouse Model of Huntington's Disease via Recovered Hepatic Pathological Changes.

Huntington's disease (HD), a congenital neurodegenerative disorder, extends its pathological damages beyond the nervous system. The systematic manifestation of HD has been extensively described in numerous studies, including dysfunction in peripheral organs and peripheral inflammation. Gut dysbiosis and the gut-liver-brain axis have garnered greater emphasis in neurodegenerative research, and increased plasma levels of pro-inflammatory cytokines have been identified in HD patients and various in vivo models, correlating with disease progression. In the present study, we investigated hepatic pathological markers in the liver of R6/2 mice which convey exon 1 of the human mutant huntingtin gene. Furthermore, we evaluated the impact of intravenously administered Mesenchymal Stromal Cells (MSCs) on the liver enzymes, changes in hepatic inflammatory markers, as well as brain pathology and behavioral deficits in R6/2 mice. Our results revealed altered enzyme expression and increased levels of inflammatory mediators in the liver of R6/2 mice, which were significantly attenuated in the MSC-treated R6/2 mice. Remarkably, neuronal pathology and altered motor activities in the MSC-treated R6/2 mice were significantly ameliorated, despite the absence of MSCs in the postmortem brain. Our data highlight the importance of hepatic pathological changes in HD, providing a potential therapeutic approach. Moreover, the data open new perspectives for the search in blood biomarkers correlating with liver pathology in HD.

Effects of an Angiotensin IV Analog on 3-Nitropropionic Acid-Induced Huntington's Disease-Like Symptoms in Rats.

Background: Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and psychiatric dysfunction caused by a mutant huntingtin protein. Compromised metabolic activity resulting from systemic administration of the mitochondrial toxin, 3-nitropropionic acid (3-NP), is known to mimic the pathology of HD and induce HD-like symptoms in rats. N-hexanoic-Tyr-Ile-(6)-amino hexanoic amide (PNB-0408), also known as Dihexa, has been shown to have neuroprotective and procognitive properties in animal models of Alzheimer's and Parkinson's diseases. Given the mechanism of action and success in other neurodegenerative diseases, we felt it an appropriate compound to investigate further for HD. Objective: The present study was designed to test if PNB-0408, an angiotensin IV analog, could attenuate 3-NP-induced HD-like symptoms in rats and serve as a potential therapeutic agent. Methods: Forty male Wistar rats were randomized into three groups consisting of a "vehicle" group, a "3-NP" group, and a "3-NP + PNB-0408" group. PNB-0408 was administered along with chronic exposure to 3-NP. Animal body weight, motor function, and cognitive abilities were measured for five weeks, before euthanasia and histopathological analysis. Results: Exposure to 3-NP decreased the amount of weight rats gained, impaired spatial learning and memory consolidation, and led to marked motor dysfunction. From our observations and analysis, PNB-0408 did not protect rats from the deficits induced by 3-NP neurotoxicity. Conclusions: Our findings suggest that PNB-0408 may not be an efficacious treatment strategy for preventing 3-NP-induced HD-like symptoms in a preclinical model. These data highlight the need for further research of this compound in alternate models and/or alternative approaches to managing this disorder.

Large animal models for Huntington's disease research.

Huntington's disease (HD) is a hereditary neurodegenerative disorder for which there is currently no effective treatment available. Consequently, the development of appropriate disease models is critical to thoroughly investigate disease progression. The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin ( HTT) gene, leading to the expansion of a polyglutamine repeat in the HTT protein. Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain, which precipitate selective neuronal loss in specific brain regions. Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets. Due to the marked species differences between rodents and larger animals, substantial efforts have been directed toward establishing large animal models for HD research. These models are pivotal for advancing the discovery of novel therapeutic targets, enhancing effective drug delivery methods, and improving treatment outcomes. We have explored the advantages of utilizing large animal models, particularly pigs, in previous reviews. Since then, however, significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD. In the current review, we provide a comprehensive overview of large animal models of HD, incorporating recent findings regarding the establishment of HD knock-in (KI) pigs and their genetic therapy. We also explore the utilization of large animal models in HD research, with a focus on sheep, non-human primates (NHPs), and pigs. Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS-STING-dependent apoptosis.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues.

BACKGROUND: The disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues. METHODS: To answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes. RESULTS: In summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range. CONCLUSIONS: Our results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

The polyglutamine domain is the primary driver of seeding in huntingtin aggregation.

Huntington's Disease (HD) is a fatal, neurodegenerative disease caused by aggregation of the huntingtin protein (htt) with an expanded polyglutamine (polyQ) domain into amyloid fibrils. Htt aggregation is modified by flanking sequences surrounding the polyQ domain as well as the binding of htt to lipid membranes. Upon fibrillization, htt fibrils are able to template the aggregation of monomers into fibrils in a phenomenon known as seeding, and this process appears to play a critical role in cell-to-cell spread of HD. Here, exposure of C. elegans expressing a nonpathogenic N-terminal htt fragment (15-repeat glutamine residues) to preformed htt-exon1 fibrils induced inclusion formation and resulted in decreased viability in a dose dependent manner, demonstrating that seeding can induce toxic aggregation of nonpathogenic forms of htt. To better understand this seeding process, the impact of flanking sequences adjacent to the polyQ stretch, polyQ length, and the presence of model lipid membranes on htt seeding was investigated. Htt seeding readily occurred across polyQ lengths and was independent of flanking sequence, suggesting that the structured polyQ domain within fibrils is the key contributor to the seeding phenomenon. However, the addition of lipid vesicles modified seeding efficiency in a manner suggesting that seeding primarily occurs in bulk solution and not at the membrane interface. In addition, fibrils formed in the presence of lipid membranes displayed similar seeding efficiencies. Collectively, this suggests that the polyQ domain that forms the amyloid fibril core is the main driver of seeding in htt aggregation.

TYROBP/DAP12 knockout in Huntington's disease Q175 mice cell-autonomously decreases microglial expression of disease-associated genes and non-cell-autonomously mitigates astrogliosis and motor deterioration.

INTRODUCTION: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the Huntingtin gene (HTT). Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of Tyrobp, a microglial protein, ameliorates neuronal dysfunction in Alzheimer's disease amyloidopathy and tauopathy mouse models while decreasing components of the complement subnetwork. OBJECTIVE: While TYROBP/DAP12-mediated microglial activation is detrimental for some diseases such as peripheral nerve injury, it is beneficial for other diseases. We sought to determine whether the TYROBP network is implicated in HD and whether Tyrobp deletion impacts HD striatal function and transcriptomics. METHODS: To test the hypothesis that Tyrobp deficiency would be beneficial in an HD model, we placed the Q175 HD mouse model on a Tyrobp-null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the gene signature in isolated Q175 striatal microglia, with and without Tyrobp. RESULTS: Comprehensive analysis of publicly available human HD transcriptomic data revealed that the TYROBP network is overactivated in the HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the Tyrobp-null background. Gene expression analysis revealed that lack of Tyrobp in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain, including genes identified as detrimental in neurodegenerative diseases, e.g. C1q and members of the Ccr5 signaling pathway. Integration of transcriptomic and proteomic data revealed that astrogliosis and complement system pathway were reduced after Tyrobp deletion, which was further validated by immunofluorescence analysis. CONCLUSIONS: Our data provide molecular and functional support demonstrating that Tyrobp deletion prevents many of the abnormalities in the HD Q175 mouse model, suggesting that the Tyrobp pathway is a potential therapeutic candidate for Huntington's disease.

Mono- and Biallelic Inactivation of Huntingtin Gene in Patient-Specific Induced Pluripotent Stem Cells Reveal HTT Roles in Striatal Development and Neuronal Functions.

Background: Mutations in the Huntingtin (HTT) gene cause Huntington's disease (HD), a neurodegenerative disorder. As a scaffold protein, HTT is involved in numerous cellular functions, but its normal and pathogenic functions during human forebrain development are poorly understood. Objective: To investigate the developmental component of HD, with a specific emphasis on understanding the functions of wild-type and mutant HTT alleles during forebrain neuron development in individuals carrying HD mutations. Methods: We used CRISPR/Cas9 gene-editing technology to disrupt the ATG region of the HTT gene via non-homologous end joining to produce mono- or biallelic HTT knock-out human induced pluripotent stem cell (iPSC) clones. Results: We showed that the loss of wild-type, mutant, or both HTT isoforms does not affect the pluripotency of iPSCs or their transition into neural cells. However, we observed that HTT loss causes division impairments in forebrain neuro-epithelial cells and alters maturation of striatal projection neurons (SPNs) particularly in the acquisition of DARPP32 expression, a key functional marker of SPNs. Finally, young post-mitotic neurons derived from HTT-/- human iPSCs display cellular dysfunctions observed in adult HD neurons. Conclusions: We described a novel collection of isogenic clones with mono- and biallelic HTT inactivation that complement existing HD-hiPSC isogenic series to explore HTT functions and test therapeutic strategies in particular HTT-lowering drugs. Characterizing neural and neuronal derivatives from human iPSCs of this collection, we show evidence that HTT loss or mutation has impacts on neuro-epithelial and striatal neurons maturation, and on basal DNA damage and BDNF axonal transport in post-mitotic neurons.

Efficacy of therapy by MK-28 PERK activation in the Huntington's disease R6/2 mouse model.

There is currently no disease-modifying therapy for Huntington's disease (HD). We recently described a small molecule, MK-28, which restored homeostasis in HD models by specifically activating PKR-like ER kinase (PERK). This activation boosts the unfolded protein response (UPR), thereby reducing endoplasmic reticulum (ER) stress, a central cytotoxic mechanism in HD and other neurodegenerative diseases. Here, we have tested the long-term effects of MK-28 in HD model mice. R6/2 CAG (160) mice were treated by lifetime intraperitoneal injections 3 times a week. CatWalk measurements of motor function showed strong improvement compared to untreated mice after only two weeks of MK-28 treatment and continued with time, most significantly at 1 â€‹mg/kg MK-28, approaching WT values. Seven weeks treatment significantly improved paw grip strength. Body weight recovered and glucose levels, which are elevated in HD mice, were significantly reduced. Treatment with another PERK activator, CCT020312 at 1 â€‹mg/kg, also caused amelioration, consistent with PERK activation. Lifespan, measured in more resilient R6/2 CAG (120) mice with daily IP injection, was much extended by 16 days (20%) with 0.3 â€‹mg/kg MK-28, and by 38 days (46%) with 1 â€‹mg/kg MK-28. No toxicity, measured by weight, blood glucose levels and blood liver function markers, was detectable in WT mice treated for 6 weeks with 6 â€‹mg/kg MK-28. Boosting of PERK activity by long-term treatment with MK-28 could be a safe and promising therapeutic approach for HD.