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Huntington's disease (HD) is a neurodegenerative polyglutamine (polyQ) disease resulting from the expansion of CAG repeats located in the ORF of the huntingtin gene (HTT). The extent to which mutant mRNA-driven disruptions contribute to HD pathogenesis, particularly in comparison to the dominant mechanisms related to the gain-of-function effects of the mutant polyQ protein, is still debatable. To evaluate this contribution in vivo, we generated two mouse models through a knock-in strategy at the Rosa26 locus. These models expressed distinct variants of human mutant HTT cDNA fragment: a translated variant (HD/100Q model, serving as a reference) and a nontranslated variant (HD/100CAG model). The cohorts of animals were subjected to a broad spectrum of molecular, behavioral, and cognitive analysis for 21 months. Behavioral testing revealed alterations in both models, with the HD/100Q model exhibiting late disease phenotype. The rotarod, static rod, and open-field tests showed some motor deficits in HD/100CAG and HD/100Q model mice during the light phase, while ActiMot indicated hyperkinesis during the dark phase. Both models also exhibited certain gene deregulations in the striatum that are related to disrupted pathways and phenotype alterations observed in HD. In conclusion, we provide in vivo evidence for a minor contributory role of mutant RNA in HD pathogenesis. The separated effects resulting from the presence of mutant RNA in the HD/100CAG model led to less severe but, to some extent, similar types of impairments as in the HD/100Q model. Increased anxiety was one of the most substantial effects caused by mutant HTT RNA., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a single mutation in the huntingtin gene (HTT). Normal HTT has a CAG trinucleotide repeat at its N-terminal within the range of 36. However, once the CAG repeats exceed 37, the mutant gene (mHTT) will encode mutant HTT protein (mHTT), which results in neurodegeneration in the brain, specifically in the striatum and other brain regions. Since the mutation was discovered, there have been many research efforts to understand the mechanism and develop therapeutic strategies to treat HD. HTT is a large protein with many post-translational modification sites (PTMs) and can be modified by phosphorylation, acetylation, methylation, sumoylation, etc. Some modifications reduced mHTT toxicity both in cell and animal models of HD. We aimed to find the known kinase inhibitors that can modulate the toxicity of mHTT. We performed an in vitro kinase assay using HTT peptides, which bear different PTM sites identified by us previously. A total of 368 kinases were screened. Among those kinases, cyclin-dependent kinases (CDKs) affected the serine phosphorylation on the peptides that contain S1181 and S1201 of HTT. We explored the effect of CDK1 and CDK5 on the phosphorylation of these PTMs of HTT and found that CDK5 modified these two serine sites, while CDK5 knockdown reduced the phosphorylation of S1181 and S1201. Modifying these two serine sites altered the neuronal toxicity induced by mHTT. Roscovitine, a CDK inhibitor, reduced the p-S1181 and p-S1201 and had a protective effect against mHTT toxicity. We further investigated the feasibility of the use of roscovitine in HD mice. We confirmed that roscovitine penetrated the mouse brain by IP injection and inhibited CDK5 activity in the brains of HD mice. It is promising to move this study to in vivo for pre-clinical HD treatment.

Short tandem repeat expansions in the human genome are overrepresented in a variety of neurological disorders. It was recently shown that huntingtin (HTT) repeat expansions with full penetrance, i.e. 40 or more CAG repeats, which normally cause Huntington's disease (HD), are overrepresented in patients with amyotrophic lateral sclerosis (ALS). Whether patients carrying HTT repeat expansions with reduced penetrance, (36-39 CAG repeats), or alleles with intermediate penetrance, (27-35 CAG repeats), have an increased risk of ALS has not yet been investigated. Here, we examined the role of HTT repeat expansions in a motor neuron disease (MND) cohort, searched for expanded HTT alleles, and investigated correlations with phenotype and neuropathology. MND patients harboring C9ORF72 hexanucleotide repeat expansions (HREs) were included, to investigate whether HTT repeat expansions were more common in this group. We found a high prevalence of intermediate (range 5.63%-6.61%) and reduced penetrance (range 0.57%-0.66%) HTT gene expansions in this cohort compared to other populations of European ancestry, but no differences between the MND cohort and the control cohort were observed, regardless of C9ORF72HRE status. Upon autopsy of three patients with intermediate or reduced penetrance HTT alleles, huntingtin inclusions were observed in the caudate nucleus and frontal lobe, but no significant somatic mosaicism was detected in different parts of the nervous system. Thus, we demonstrate, for the first time, huntingtin inclusions in individuals with MND and intermediate and reduced penetrance HTT repeat expansions but more clinicopathological investigations are needed to further understand the impact of HTT gene expansion-related pleiotropy., (© The Author(s) 2024. Published by Oxford University Press.)

Fluorescence labeling via fluorescent proteins (FPs) or immunofluorescence has been routinely applied for microscopic imaging of specific proteins. However, due to these over-weight and oversized labels ( e.g. GFP, 238 aa, 27 kDa, ∼4 nm in size), the potential physiological malfunctions of the target proteins are largely underestimated in living cells. Herein, for living cells, we report a small and minimally-invasive Raman reporter (about 2 aa and <1 kDa), which can be site-specifically introduced into proteins by genetic codon expansion. After a single unnatural amino acid (UAA) is precisely incorporated into the target protein, the strained alkyne can rapidly undergo copper-free Diels-Alder cycloaddition reactions with the tetrazine-functionalized Raman reporter, which features a fine vibrational spectrum in contrast to fluorescence. In our experimental results, the UAA-based Raman tag was successfully incorporated into vimentin, histone 3.3 and huntingtin (Htt74Q) proteins in living HeLa cells and further utilized for stimulated Raman imaging. The site-specific bioorthogonal fusion of small Raman tags with intracellular proteins will pave the way for minimally-invasive protein labeling and multi-color imaging in living cells.

Huntington's disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington's disease and other polyglutamine diseases., (© 2024 Korsten et al.)

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG tract expansion in the huntingtin gene ( HTT ). HD is characterized by involuntary movements, cognitive decline, and behavioral changes. Pathologically, patients with HD show selective striatal neuronal vulnerability at the early disease stage, although the mutant protein is ubiquitously expressed. Activation of the immune system and glial cell-mediated neuroinflammatory responses are early pathological features and have been found in all neurodegenerative diseases (NDDs), including HD. However, the role of inflammation in HD, as well as its therapeutic significance, has been less extensively studied compared to other NDDs. This review highlights the significantly elevated levels of inflammatory proteins and cellular markers observed in various HD animal models and HD patient tissues, emphasizing the critical roles of microglia, astrocytes, and oligodendrocytes in mediating neuroinflammation in HD. Moreover, it expands on recent discoveries related to the peripheral immune system's involvement in HD. Although current immunomodulatory treatments and inflammatory biomarkers for adjunctive diagnosis in HD are limited, targeting inflammation in combination with other therapies, along with comprehensive personalized treatment approaches, shows promising therapeutic potential.

Huntington's disease (HD) is a fatal, progressive neurodegenerative disorder. Prior studies revealed an increase in extracellular glutamate levels after evoking astrocytic SNARE-dependent exocytosis from cultured primary astrocytes from mutant huntingtin (mHTT)-expressing BACHD mice compared to control astrocytes, suggesting alterations in astrocytic SNARE-dependent exocytosis in HD. We used BACHD and dominant-negative (dn)SNARE mice to decrease SNARE-dependent exocytosis from astrocytes to determine whether reducing SNARE-dependent exocytosis from astrocytes could rescue neuropathological changes in vivo. We observed significant protection against striatal atrophy and no significant rescue of cortical atrophy in BACHD/dnSNARE mice compared to BACHD mice. Amino acid transporters are important for modulating the levels of extracellular neurotransmitters. BACHD mice had no change in GLT1 expression, decreased striatal GAT1 expression and increased levels of GAT3. There was no change in GAT1 after reducing astrocytic SNARE-dependent exocytosis, and increased GAT3 expression in BACHD mice was normalized in BACHD/dnSNARE mice. Thus, modulation of astrocytic SNARE-dependent exocytosis in BACHD mice is protective against striatal atrophy and modulates GABA transporter expression., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Recently, it has been shown that blocking the binding of valosin-containing protein (VCP) to mutant huntingtin (mtHtt) can prevent neuronal mitochondrial autophagy in Huntington's disease (HD) models. Herein, we describe the development and efficacy of a protein-like polymer (PLP) for inhibiting this interaction in cellular and in vivo models of HD. PLPs exhibit bioactivity in HD mouse striatal cells by successfully inhibiting mitochondrial destruction. PLP is notably resilient to in vitro enzyme, serum, and liver microsome stability assays, which render analogous control oligopeptides ineffective. PLP demonstrates a 2000-fold increase in circulation half-life compared to peptides, exhibiting an elimination half-life of 152 hours. In vivo efficacy studies in HD transgenic mice (R6/2) confirm the superior bioactivity of PLP compared to free peptide through behavioral and neuropathological analyses. PLP functions by preventing pathologic VCP/mtHtt binding in HD animal models; exhibits enhanced efficacy over the parent, free peptide; and implicates the PLP as a platform with potential for translational central nervous system therapeutics.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m 6 A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m 6 A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m 6 A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m 6 A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m 6 A in aberrant splicing of Htt mRNA., (© 2024. The Author(s).)

There is now compelling evidence for the presence of pathological forms of Tau in tissues of both patients and animal models of Huntington's disease (HD). While the root cause of this illness is a mutation within the huntingtin gene, a number of studies now suggest that HD could also be considered a secondary tauopathy. However, the contributory role of Tau in the pathogenesis and pathophysiology of this condition, as well as its implications in cellular toxicity and consequent behavioral impairments are largely unknown. We therefore performed intracerebral stereotaxic injections of recombinant human Tau monomers and fibrils into the knock-in zQ175 mouse model of HD. Tau fibrils induced cognitive and anxiety-like phenotypes predominantly in zQ175 mice and increased the number and size of insoluble mutant huntingtin (mHTT) aggregates in the brains of treated animals. To better understand the putative mechanisms through which Tau could initiate and/or contribute to pathology, we incubated StHdh striatal cells, an in vitro model of HD, with the different Tau forms and evaluated the effects on cell functionality and heat shock proteins Hsp70 and Hsp90. Calcium imaging experiments showed functional impairments of HD StHdh cells following treatment with Tau fibrils, as well as significant changes to the levels of both heat shock proteins which were found trapped within mHTT aggregates. The accumulation of Hsp70 and 90 within aggregates was also present in mouse tissue which suggests that alteration of molecular chaperone-dependent protein quality control may influence aggregation, implicating proteostasis in the mHTT-Tau interplay., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microglia, the resident immune cells of the brain, regulate the balance of inflammation in the central nervous system under healthy and pathogenic conditions. Huntington's disease (HD) is a chronic neurodegenerative disease characterized by activated microglia and elevated concentrations of pro-inflammatory cytokines within the brain. Chronic hyperactivation of microglia is associated with brain pathology and eventual neuron death. However, it is unclear which specific cytokines are required for neuron death and whether HD neurons may be hypersensitive to neuroinflammation. We assessed the profile of microglia-secreted proteins in response to LPS and IFNγ, and a conditioned media paradigm was used to examine the effects of these secreted proteins on cultured neuronal cells. STHdh Q7/Q7 and STHdh Q111/Q111 neuronal cells were used to model wild-type and HD neurons, respectively. We determined that STHdh Q111/Q111 cells were hypersensitive to pro-inflammatory factors secreted by microglia, and that TNF was required to induce neuronal death. Microglia-mediated neuronal death could be effectively halted through the use of JAK-STAT or TNF inhibitors which supported the requirement for TNF as well as IFNγ in the process of secondary neurotoxicity. Further data derived from human HD patients as well as HD mice were suggestive of enhanced receptor density for TNF (TNFR1) and IFNγ (IFNGR) which could sensitize the HD brain to these cytokines. This highlights several potential mechanisms by which microglia may induce neuronal death and suggests that these mechanisms may be upregulated in the brain of HD patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eileen M. Denovan-Wright reports financial support was provided by Dalhousie University Faculty of Medicine. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in exon 1 of the HTT gene, leading to the formation of a toxic variant of the huntingtin protein. It is a rare but severe hereditary disease for which no effective treatment method has been found yet. The primary therapeutic targets include the mutant protein and the mutant mRNA of HTT . Current clinical trial approaches in gene therapy involve the application of splice modulation, siRNA, or antisense oligonucleotides for RNA-targeted knockdown of HTT . However, these approaches do not take into account the diversity of HTT transcript isoforms in the normal conditions and in HD. In this review, we discuss the features of transcriptional regulation and processing that lead to the formation of various HTT mRNA variants, each of which may uniquely contribute to the progression of the disease. Furthermore, understanding the role of known transcription factors of HTT in pathology may aid in the development of potentially new therapeutic tools based on endogenous regulators.

The accumulation of mutant huntingtin protein aggregates in neurons is a pathological hallmark of Huntington's disease (HD). The glymphatic system, a brain-wide perivascular network, facilitates the exchange of interstitial fluid and cerebrospinal fluid (CSF), supporting interstitial solute clearance of brain wastes. In this study, we employed dynamic glucose-enhanced (DGE) MRI to measure d-glucose clearance from CSF as a tool to predict glymphatic function in a mouse model of HD. We found significantly diminished CSF clearance efficiency in HD mice before phenotypic onset. The impairment of CSF clearance efficiency worsened with disease progression. These DGE MRI findings in compromised glymphatic function were further verified with fluorescence-based imaging of CSF tracer influx, suggesting an impaired glymphatic function in premanifest HD. Moreover, expression of the astroglial water channel aquaporin-4 in the perivascular compartment, a key mediator of glymphatic function, was significantly diminished in both HD mouse brain and human HD brain. Our data, acquired using a clinically translatable MRI, indicate a perturbed glymphatic network in the HD brain. Further validation of these findings in clinical studies will provide insights into the potential of glymphatic clearance as a therapeutic target as well as an early biomarker in HD.

Cockayne syndrome (CS) is an autosomal recessive disorder of developmental delay, multiple organ system degeneration and signs of premature ageing. We show here, using the RNA-seq data from two CS mutant cell lines, that the CS key transcriptional signature displays significant enrichment of neurodegeneration terms, including genes relevant in Huntington disease (HD). By using deep learning approaches and two published RNA-Seq datasets, the CS transcriptional signature highly significantly classified and predicted HD and control samples. Neurodegeneration is one hallmark of CS disease, and fibroblasts from CS patients with different causative mutations display disturbed ribosomal biogenesis and a consecutive loss of protein homeostasis - proteostasis. Encouraged by the transcriptomic data, we asked whether this pathomechanism is also active in HD. In different HD cell-culture models, we showed that mutant Huntingtin impacts ribosomal biogenesis and function. This led to an error-prone protein synthesis and, as shown in different mouse models and human tissue, whole proteome instability, and a general loss of proteostasis., Competing Interests: Declaration of competing interest G.B.L. has provided consulting services, advisory board functions, clinical trial services and/or lectures for Acadia Pharmaceuticals, Affiris, Allergan, Alnylam, Amarin, AOP Orphan Pharmaceuticals AG, Bayer Pharma AG, Boehringer-Ingelheim, CHDI-Foundation, Deutsche Huntington-Hilfe, Desitin, Genentech, Genzyme, GlaxoSmithKline, F. Hoffmann-LaRoche, Ipsen, ISIS Pharma (IONIS), Lilly, Lundbeck, Medesis, Medivation, Medtronic, NeuraMetrix, Neurosearch Inc., Novartis, Pfizer, Prana Biotechnology, Prilenia, PTC Therapeutics, Raptor, Remix Therapeutics, Rhône-Poulenc Rorer, Roche Pharma AG Deutschland, Sage Therapeutics, Sanofi-Aventis, Sangamo/Shire, Siena Biotech, Takeda, Temmler Pharma GmbH, Teva, Triplet Therapeutics, Trophos, UniQure and Wave Life Sciences. The other authors declare no conflict of interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA., (© 2024. The Author(s).)

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by cognitive, motor, and psychiatric symptoms. Despite significant advances in understanding the underlying molecular mechanisms of HD, there is currently no cure or disease-modifying treatment available. Emerging pharmacological approaches offer promising strategies to alleviate symptoms and slow down disease progression. This comprehensive review aims to provide a critical appraisal of the latest developments in pharmacological interventions for HD. The review begins by discussing the pathogenesis of HD, focusing on the role of mutant huntingtin protein, mitochondrial dysfunction, excitotoxicity, and neuro-inflammation. It then explores emerging therapeutic targets, including the modulation of protein homeostasis, mitochondrial function, neuro-inflammation, and neurotransmitter systems. Pharmacological agents targeting these pathways are discussed, including small molecules, gene-based therapies, and neuroprotective agents. In recent years, several clinical trials have been conducted to evaluate the safety and efficiency of novel compounds for HD. This review presents an update on the outcomes of these trials, highlighting promising results and challenges encountered. Additionally, it discusses the potential of repurposing existing drugs approved for other indications as a cost-effective approach for HD treatment. The review concludes by summarizing the current state of pharmacological approaches for HD and outlining future directions in drug development. The integration of multiple therapeutic strategies, personalized medicine approaches, and combination therapies are highlighted as potential avenues to maximize treatment effectiveness., Competing Interests: Declaration of competing interest No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my-coauthors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Huntington's disease (HD) is a devastating neurodegenerative disease that is manifested by a gradual loss of physical, cognitive, and mental abilities. As the disease advances, age has a major impact on the pathogenic signature of mutant huntingtin (mHTT) protein aggregation. This review aims to explore the intricate relationship between aging, mHTT toxicity, and cellular senescence in HD. Scientific data on the interplay between aging, mHTT, and cellular senescence in HD were collected from several academic databases, including PubMed, Google Scholar, Google, and ScienceDirect. The search terms employed were "AGING," "HUNTINGTON'S DISEASE," "MUTANT HUNTINGTIN," and "CELLULAR SENESCENCE." Additionally, to gather information on the molecular mechanisms and potential therapeutic targets, the search was extended to include relevant terms such as "DNA DAMAGE," "OXIDATIVE STRESS," and "AUTOPHAGY." According to research, aging leads to worsening HD pathophysiology through some processes. As a result of the mHTT accumulation, cellular senescence is promoted, which causes DNA damage, oxidative stress, decreased autophagy, and increased inflammatory responses. Pro-inflammatory cytokines and other substances are released by senescent cells, which may worsen the neuronal damage and the course of the disease. It has been shown that treatments directed at these pathways reduce some of the HD symptoms and enhance longevity in experimental animals, pointing to a new possibility of treating the condition. Through their amplification of the harmful effects of mHTT, aging and cellular senescence play crucial roles in the development of HD. Comprehending these interplays creates novel opportunities for therapeutic measures targeted at alleviating cellular aging and enhancing HD patients' quality of life., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Huntington disease (HD) is a genetic neurodegenerative disease caused by cytosine, adenine, guanine (CAG) expansion in the Huntingtin ( HTT ) gene, translating to an expanded polyglutamine tract in the HTT protein. Age at disease onset correlates to CAG repeat length but varies by decades between individuals with identical repeat lengths. Genome-wide association studies link HD modification to DNA repair and mitochondrial health pathways. Clinical studies show elevated DNA damage in HD, even at the premanifest stage. A major DNA repair node influencing neurodegenerative disease is the PARP pathway. Accumulation of poly adenosine diphosphate (ADP)-ribose (PAR) has been implicated in Alzheimer and Parkinson diseases, as well as cerebellar ataxia. We report that HD mutation carriers have lower cerebrospinal fluid PAR levels than healthy controls, starting at the premanifest stage. Human HD induced pluripotent stem cell-derived neurons and patient-derived fibroblasts have diminished PAR response in the context of elevated DNA damage. We have defined a PAR-binding motif in HTT, detected HTT complexed with PARylated proteins in human cells during stress, and localized HTT to mitotic chromosomes upon inhibition of PAR degradation. Direct HTT PAR binding was measured by fluorescence polarization and visualized by atomic force microscopy at the single molecule level. While wild-type and mutant HTT did not differ in their PAR binding ability, purified wild-type HTT protein increased in vitro PARP1 activity while mutant HTT did not. These results provide insight into an early molecular mechanism of HD, suggesting possible targets for the design of early preventive therapies., Competing Interests: Competing interests statement:E.J.W. reports consultancy/ advisory board memberships with Annexon, Remix Therapeutics, Hoffman La Roche Ltd., Ionis Pharmaceuticals, PTC Therapeutics, Takeda, Teitur Trophics, Triplet Therapeutics and Vico Therapeutics. All honoraria for these consultancies were paid through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London. F.B.R. is a Medpace UK Ltd. employee and was a University College London employee during the conduct of this study. F.B.R. has provided consultancy services to GLG and F. Hoffmann-La Roche Ltd. L.M.B. currently holds consultancy contracts with Annexon Biosciences, Remix Therapeutics, and LoQus23 Therapeutics Ltd. via UCL Consultants Ltd. R.T. has past consultancy with Novartis AG, PTC Therapeutics and Mitokinin LLC.

Despite growing descriptions of wild-type Huntingtin (wt-HTT) roles in both adult brain function and, more recently, development, several clinical trials are exploring HTT-lowering approaches that target both wt-HTT and the mutant isoform (mut-HTT) responsible for Huntington's disease (HD). This non-selective targeting is based on the autosomal dominant inheritance of HD, supporting the idea that mut-HTT exerts its harmful effects through a toxic gain-of-function or a dominant-negative mechanism. However, the precise amount of wt-HTT needed for healthy neurons in adults and during development remains unclear. In this study, we address this question by examining how wt-HTT loss affects human neuronal network formation, synaptic maturation, and homeostasis in vitro. Our findings establish a role of wt-HTT in the maturation of dendritic arborization and the acquisition of network-wide synchronized activity by human cortical neuronal networks modeled in vitro. Interestingly, the network synchronization defects only became apparent when more than two-thirds of the wt-HTT protein was depleted. Our study underscores the critical need to precisely understand wt-HTT role in neuronal health. It also emphasizes the potential risks of excessive wt-HTT loss associated with non-selective therapeutic approaches targeting both wt- and mut-HTT isoforms in HD patients., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2024. Published by Elsevier Inc.)

Pathogenic mutant huntingtin (mHTT) infiltrates the adult Huntington's disease (HD) brain and impairs fetal corticogenesis. However, most HD animal models rarely recapitulate neuroanatomical alterations in adult HD and developing brains. Thus, the human cortical organoid (hCO) is an alternative approach to decode mHTT pathogenesis precisely during human corticogenesis. Here, we replicated the altered corticogenesis in the HD fetal brain using HD patient-derived hCOs. Our HD-hCOs had pathological phenotypes, including deficient junctional complexes in the neural tubes, delayed postmitotic neuronal maturation, dysregulated fate specification of cortical neuron subtypes, and abnormalities in early HD subcortical projections during corticogenesis, revealing a causal link between impaired progenitor cells and chaotic cortical neuronal layering in the HD brain. We identified novel long, oriented, and enriched polyQ assemblies of HTTs that hold large flat Golgi stacks and scaffold clathrin+ vesicles in the neural tubes of hCOs. Flat Golgi stacks conjugated polyQ assemblies by ADP-ribosylation factor 1 (ARF1). Inhibiting ARF1 activation with Brefeldin A (BFA) disassociated polyQ assemblies from Golgi. PolyQ assembles with mHTT scaffolded fewer ARF1 and formed shorter polyQ assembles with fewer and shorter Golgi and clathrin vesicles in neural tubes of HD-hCOs compared with those in hCOs. Inhibiting the activation of ARF1 by BFA in healthy hCOs replicated impaired junctional complexes in the neural tubes. Together, endogenous polyQ assemblies with mHTT reduced the Golgi recruiting ARF1 in the neuroepithelium, impaired the Golgi structure and activities, and altered the corticogenesis in HD-hCO., (© 2024. The Author(s).)

To evaluate the artificial intelligence (AI)-guided AlphaFold algorithm for studying the binding interactions of human huntingtin and the aggregation of huntingtin peptides. Variants of huntingtin protein implicated in Huntington's disease were used as a model system to evaluate AlphaFold. Variants of huntingtin and huntingtin peptides with polyglutamine tracts (PQT) containing 21, 31, 51, or 78 glutamines were studied. The 3-dimensional structures of huntingtin variants and their interactions with huntingtin-associated protein-40 (HAP40) were obtained. Aggregation experiments were conducted with peptide sequences corresponding to variants of PQT, amino terminal sequence (NTS) plus PQT, NTS plus PQT plus proline rich region (PRR), and the 300 amino acid sequence from the NTS through HEAT3 of huntingtin. Oligomerization experiments with 1, 3, 6, or 12 peptide sequences were used to assess the quaternary structures of aggregates. The PQT and PQT plus NTS peptides formed a helical secondary structure that formed a central core in the quaternary structure of the aggregates The PRR formed an extended type II polyproline helix that did not participate in central core the aggregates. The distance between the amino and carboxyl termini of disease-linked 31Q, 51Q, and 78Q variants of full-length huntingtin was prominently decreased compared to the 21Q huntingtin. The interaction of HAP40 with the 78Q variant increased the distance between the amino and carboxyl termini. AlphaFold identified key tertiary structure changes in human huntingtin that have been independently corroborated in experimental models. The results highlight the utility of AlphaFold for hypothesis generation in pharmaceutical research., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30 % flexible, 30-40 % aliphatic and 20-30 % aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Huntington's disease (HD) is a rare neurodegenerative disease caused due to aggregation of Huntingtin (HTT) protein. This study involves the cloning of 40 DnaJ chaperones from Drosophila, and overexpressing them in yeasts and fly models of HD. Accordingly, DnaJ chaperones were catalogued as enhancers or suppressors based on their growth phenotypes and aggregation properties. 2 of the chaperones that came up as targets were CG5001 and P58IPK. Protein aggregation and slow growth phenotype was rescued in yeasts, S2 cells, and Drosophila transgenic lines of HTT103Q with these overexpressed chaperones. Since DnaJ chaperones have protein sequence similarity across species, they can be used as possible tools to combat the effects of neurodegenerative diseases., (© 2024. The Author(s).)

The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization., (open access.)

Protein aggregation is a common feature of many neurodegenerative diseases. In Huntington's disease, mutant huntingtin is the primary aggregating protein, but the aggregation of other proteins, such as TDP43, is likely to further contribute to toxicity. Moreover, mutant huntingtin is also a risk factor for TDP pathology in ALS. Despite this co-pathology of huntingtin and TDP43, it remains unknown whether these amyloidogenic proteins directly interact with each other. Using a combination of biophysical methods, we show that the aggregation-prone regions of both proteins, huntingtin exon-1 (Httex1) and the TDP43 low complexity domain (TDP43-LCD), interact in a conformationally specific manner. This interaction significantly slows Httex1 aggregation, while it accelerates TDP43-LCD aggregation. A key intermediate responsible for both effects is a complex formed by liquid TDP43-LCD condensates and Httex1 fibrils. This complex shields seeding competent surfaces of Httex1 fibrils from Httex1 monomers, which are excluded from the condensates. In contrast, TDP43-LCD condensates undergo an accelerated liquid-to-solid transition upon exposure to Httex1 fibrils. Cellular studies show co-aggregation of untagged Httex1 with TDP43. This interaction causes mislocalization of TDP43, which has been linked to TDP43 toxicity. The protection from Httex1 aggregation in lieu of TDP43-LCD aggregation is interesting, as it mirrors what has been found in disease models, namely that TDP43 can protect from huntingtin toxicity, while mutant huntingtin can promote TDP43 pathology. These results suggest that direct protein interaction could, at least in part, be responsible for the linked pathologies of both proteins., Competing Interests: Conflict of interests The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While protein aggregation is a hallmark of many neurodegenerative diseases, acquiring structural information on protein aggregates inside live cells remains challenging. Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter-propagating mid-infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid-infrared photothermal spectra of label-free and GFP-tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub-micrometer distances, we reveal that huntingtin inclusions partition into a β-sheet-rich core and a ɑ-helix-rich shell. We further demonstrate that this structural partition exists only in cells with the [RNQ + ] prion state, while [rnq - ] cells only carry smaller β-rich non-toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high-throughput structural screenings of macromolecular assemblies., (© 2024 Wiley-VCH GmbH.)

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD., (© 2024. The Author(s).)

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death., (© The Author(s) 2024. Published by Oxford University Press.)

Huntington's disease (HD) is caused by a glutamine repeat expansion in the protein huntingtin. Mutated huntingtin (mHtt) forms aggregates whose impacts on neuronal survival are still debated. Using weeks-long, continual imaging of cortical neurons, we find that mHtt is gradually sequestrated into peripheral, mainly axonal aggregates, concomitant with dramatic reductions in cytosolic mHtt levels and enhanced neuronal survival. in-situ pulse-chase imaging reveals that aggregates continually gain and lose mHtt, in line with these acting as mHtt sinks at equilibrium with cytosolic pools. Mutating two N-terminal lysines found to be ubiquitinated in HD animal models suppresses peripheral aggregate formation and reductions in cytosolic mHtt, promotes nuclear aggregate formation, stabilizes aggregates and leads to pervasive neuronal death. These findings demonstrate the capacity of aggregates formed at peripheral locations to sequester away cytosolic, presumably toxic mHtt forms and support a crucial role for N-terminal ubiquitination in promoting these processes and delaying neuronal death., (© 2024. The Author(s).)

Huntington's disease (HD) is a fatal neurodegenerative disorder resulting from an abnormal expansion of polyglutamine (polyQ) repeats in the N-terminus of the huntingtin protein. When the polyQ tract surpasses 35 repeats, the mutated protein undergoes misfolding, culminating in the formation of intracellular aggregates. Research in mouse models suggests that HD pathogenesis involves the aggregation of N-terminal fragments of the huntingtin protein (htt). These early oligomeric assemblies of htt, exhibiting diverse characteristics during aggregation, are implicated as potential toxic entities in HD. However, a consensus on their specific structures remains elusive. Understanding the heterogeneous nature of htt oligomers provides crucial insights into disease mechanisms, emphasizing the need to identify various oligomeric conformations as potential therapeutic targets. Employing coarse-grained molecular dynamics, our study aims to elucidate the mechanisms governing the aggregation process and resultant aggregate architectures of htt. The polyQ tract within htt is flanked by two regions: an N-terminal domain (N17) and a short C-terminal proline-rich segment. We conducted self-assembly simulations involving five distinct N17 + polyQ systems with polyQ lengths ranging from 7 to 45, utilizing the ProMPT force field. Prolongation of the polyQ domain correlates with an increase in β-sheet-rich structures. Longer polyQ lengths favor intramolecular β-sheets over intermolecular interactions due to the folding of the elongated polyQ domain into hairpin-rich conformations. Importantly, variations in polyQ length significantly influence resulting oligomeric structures. Shorter polyQ domains lead to N17 domain aggregation, forming a hydrophobic core, while longer polyQ lengths introduce a competition between N17 hydrophobic interactions and polyQ polar interactions, resulting in densely packed polyQ cores with outwardly distributed N17 domains. Additionally, at extended polyQ lengths, we observe distinct oligomeric conformations with varying degrees of N17 bundling. These findings can help explain the toxic gain-of-function that htt with expanded polyQ acquires.

The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD., (© 2024. The Author(s).)

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD., Competing Interests: Competing interests statement:The authors declare no competing interest.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene ( HTT ). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence huntingtin mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a divalent scaffold and delivered to two mouse models of HD. In both models, divalent siRNA sustained lowering of wild-type Htt , but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type HTT protein was observed in both models. Subsequent fluorescent in situ hybridization analysis shows that divalent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric symptoms. Currently, there is no cure, and only limited treatments are available to manage the symptoms and to slow down the disease's progression. The molecular and cellular mechanisms of HD's pathogenesis are complex, involving immune cell activation, altered protein turnover, and disturbance in brain energy homeostasis. Microglia have been known to play a dual role in HD, contributing to neurodegeneration through inflammation but also enacting neuroprotective effects by clearing mHTT aggregates. However, little is known about the contribution of microglial metabolism to HD progression. This study explores the impact of a microglial metabolite transporter, equilibrative nucleoside transporter 3 (ENT3), in HD. Known as a lysosomal membrane transporter protein, ENT3 is highly enriched in microglia, with its expression correlated with HD severity. Using the R6/2 ENT3 -/- mouse model, we found that the deletion of ENT3 increases microglia numbers yet worsens HD progression, leading to mHTT accumulation, cell death, and disturbed energy metabolism. These results suggest that the delicate balance between microglial metabolism and function is crucial for maintaining brain homeostasis and that ENT3 has a protective role in ameliorating neurodegenerative processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A number of post-mortem studies conducted in transplanted Huntington's disease (HD) patients from various trials have reported the presence of pathological and misfolded proteins, in particular mutant huntingtin (mHtt) and phosphorylated tau neuropil threads, in the healthy grafted tissue. Here, we extended these observations with histological analysis of post-mortem tissue from three additional HD patients who had received similar striatal allografts from the fetal tissue transplantation trial conducted in Los Angeles in 1998. Immunohistochemical staining was performed using anti-mHtt antibodies, EM48 and MW7, as well as anti-hyperphosphorylated tau antibodies, AT8 and CP13. Immunofluorescence was used to assess the colocalization of EM48 + mHtt aggregates with the neuronal marker MAP2 and/or the extracellular matrix protein phosphacan in both the host and grafts. We confirmed the presence of mHtt aggregates within grafts of all three cases as well as tau neuropil threads in the grafts of two of the three transplanted HD patients. Phosphorylated tau was also variably expressed in the host cerebral cortex of all three subjects. While mHtt inclusions were present within neurons (immunofluorescence co-localization of MAP2 and EM48) as well as within the extracellular matrix of the host (immunofluorescence co-localization of phosphacan and EM48), their localization was limited to the extracellular matrix in the grafted tissue. This study corroborates previous findings that both mHtt and tau pathology can be found in the host and grafts of HD patients years post-grafting., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Huntington's Disease (HD) is caused by an abnormal expansion of the polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). This expansion promotes disease-related htt aggregation into amyloid fibrils and the formation of proteinaceous inclusion bodies within neurons. Fibril formation is a complex heterogenous process involving an array of aggregate species such as oligomers, protofibrils, and fibrils. In HD, structural abnormalities of membranes of several organelles develop. In particular, the accumulation of htt fibrils near the endoplasmic reticulum (ER) impinges upon the membrane, resulting in ER damage, altered dynamics, and leakage of Ca 2+ . Here, the aggregation of htt at a bilayer interface assembled from ER-derived liposomes was investigated, and fibril formation directly on these membranes was enhanced. Based on these observations, simplified model systems were used to investigate mechanisms associated with htt aggregation on ER membranes. As the ER-derived liposome fractions contained residual Ca 2+ , the role of divalent cations was also investigated. In the absence of lipids, divalent cations had minimal impact on htt structure and aggregation. However, the presence of Ca 2+ or Mg 2+ played a key role in promoting fibril formation on lipid membranes despite reduced htt insertion into and association with lipid interfaces, suggesting that the ability of divalent cations to promote fibril formation on membranes is mediated by induced changes to the lipid membrane physicochemical properties. With enhanced concentrations of intracellular calcium being a hallmark of HD, the ability of divalent cations to influence htt aggregation at lipid membranes may play a role in aggregation events that lead to organelle abnormalities associated with disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG tract in the huntingtin (HTT) gene, leading to toxic gains of function. HTT-lowering treatments are in clinical trials, but the risks imposed are unclear. Recent studies have reported on the consequences of widespread HTT loss in mice, where one group described early HTT loss leading to fatal pancreatitis, but later loss as benign. Another group reported no pancreatitis but found widespread neurological phenotypes including subcortical calcification. To better understand the liabilities of widespread HTT loss, we knocked out Htt with two separate tamoxifen-inducible Cre lines. We find that loss of HTT at 2 mo of age leads to progressive tremors and severe subcortical calcification at examination at 14 mo of age but does not result in acute pancreatitis or histological changes in the pancreas. We, in addition, report that HTT loss is followed by sustained induction of circulating neurofilament light chain. These results confirm that global loss of HTT in mice is associated with pronounced risks, including progressive subcortical calcification and neurodegeneration., (© 2024 Bragg et al.)

Huntingtin protein, mutated in Huntington's disease, is implicated in nucleic acid-mediated processes, yet the evidence for direct huntingtin-nucleic acid interaction is limited. Here, we show wild-type and mutant huntingtin copurify with nucleic acids, primarily RNA, and interact directly with G-rich RNAs in in vitro assays. Huntingtin RNA-immunoprecipitation sequencing from patient-derived fibroblasts and neuronal progenitor cells expressing wild-type and mutant huntingtin revealed long noncoding RNA NEAT1 as a significantly enriched transcript. Altered NEAT1 levels were evident in Huntington's disease cells and postmortem brain tissues, and huntingtin knockdown decreased NEAT1 levels. Huntingtin colocalized with NEAT1 in paraspeckles, and we identified a high-affinity RNA motif preferred by huntingtin. This study highlights NEAT1 as a huntingtin interactor, demonstrating huntingtin's involvement in RNA-mediated functions and paraspeckle regulation.

Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called "Tianma" in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington's disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1-100 μM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24-48 h of incubation. The ubiquitin-proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 μM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy-lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose-response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.

Huntington's disease (HD) is an incurable hereditary disease caused by expansion of the CAG repeats in the HTT gene encoding the mutant huntingtin protein (mHTT). Despite numerous studies in cellular and animal models, the mechanisms underlying the biological role of mHTT and its toxicity to striatal neurons have not yet been established and no effective therapy for HD patients has been developed so far. We produced and characterized a new line of dermal fibroblasts (HDDF, Huntington's disease dermal fibroblasts) from a patient with a confirmed HD diagnosis. We also studied the growth characteristics of HDDF cells, stained them for canonical markers, karyotyped these cells, and investigated their phenotype. HDDF cells was successfully reprogrammed into induced striatal neurons via transdifferentiation. The new fibroblast line can be used as a cell model to study the biological role of mHTT and manifestations of HD pathogenesis in both fibroblasts and induced neuronal cells obtained from them by reprogramming techniques.

Protein mislocalization is a key initial step in neurodegeneration, regardless of etiology, and has been linked to changes in the dynamic addition of saturated fatty acids to proteins, a process known as S-acylation. With the advent of new techniques to study S-acylation and the recent discovery of new enzymes that facilitate protein deacylation, novel small molecules are emerging as potential new therapeutic treatments. Huntington disease (HD) is a devastating, fatal neurodegenerative disease characterized by motor, cognitive, and psychiatric deficits caused by a CAG repeat expansion in the HTT gene. The protein that is mutated in HD, huntingtin, is less S-acylated which is associated with mutant HTT aggregation and cytotoxicity. Recent exciting findings indicate that restoring S-acylation in HD models using small molecule inhibitors of the deacylation enzymes is protective. Herein, we set out to describe the known roles of S-acylation in HD and how it can be targeted for therapeutic design., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hht ex1 ) with a 35 polyglutamine repeat, htt ex1 Q 35 , are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of htt ex1 Q 35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1 H- 15 N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" htt ex1 Q 35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, htt ex1 Q 7 , lend confidence to the conclusions drawn from the fits to the htt ex1 Q 35 aggregation kinetics.

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-β core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington's disease (HD), the htt NT segment on the polyQ's N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich htt NT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the htt NT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple htt NT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the htt NT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer - either the tetramer or a larger multimer - plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein-protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

An expanded CAG repeat in the huntingtin gene ( HTT ) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and guide RNAs (gRNAs) efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased in the liver by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion strategies in HD and potentially other repeat expansion disorders., Competing Interests: DC, JS, SZ, EH, JJ, JL, HS No competing interests declared, VW V.C.W. was a founding scientific advisory board member with financial interest in Triplet Therapeutics Inc, Her financial interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. V.C.W. is a scientific advisory board member of LoQus23 Therapeutics Ltd. and has provided paid consulting services to Acadia Pharmaceuticals Inc, Alnylam Inc, Biogen Inc and Passage Bio. V.C.W. has received research support from Pfizer Inc, BK B.P.K is an inventor on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital and is a scientific advisory board member of Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine, JL J-ML consults for GenKOre and serves in the advisory board of GenEdit Inc, (© 2023, Choi et al.)

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability., Competing Interests: Declaration of interests In the past year, through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London, S.J.T. has undertaken consultancy services for Alnylam Pharmaceuticals, Annexon, Ascidian Therapeutics, Arrowhead Pharmaceuticals, Atalanta Therapeutics, Design Therapeutics, F. Hoffman-La Roche, HCD Economics, IQVIA, Iris Medicine, Latus Bio, LifeEdit, Novartis Pharma, Pfizer, Prilenia Neurotherapeutics, PTC Therapeutics, Rgenta Therapeutics, Takeda Pharmaceuticals, UniQure Biopharma, and Vertex Pharmaceuticals. In the past 12 months, University College London Hospitals NHS Foundation Trust, S.J.T.’s host clinical institution, received funding to run clinical trials for F. Hoffman-La Roche, Novartis Pharma, PTC Therapeutics, and UniQure Biopharma., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) predominantly affects the brain, causing a mixed movement disorder, cognitive decline and behavioural abnormalities. It also causes a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been reported in tissues of HD models, including skeletal muscle, and lymphoblast and fibroblast cultures from patients with HD. Mutant huntingtin protein (mutHTT) expression can impair mitochondrial quality control and accelerate mitochondrial ageing. Here, we obtained fresh human skeletal muscle, a post-mitotic tissue expressing the mutated HTT allele at physiological levels since birth, and primary cell lines from HTT CAG repeat expansion mutation carriers and matched healthy volunteers to examine whether such a mitochondrial phenotype exists in human HD. Using ultra-deep mitochondrial DNA (mtDNA) sequencing, we showed an accumulation of mtDNA mutations affecting oxidative phosphorylation. Tissue proteomics indicated impairments in mtDNA maintenance with increased mitochondrial biogenesis of less efficient oxidative phosphorylation (lower complex I and IV activity). In full-length mutHTT expressing primary human cell lines, fission-inducing mitochondrial stress resulted in normal mitophagy. In contrast, expression of high levels of N-terminal mutHTT fragments promoted mitochondrial fission and resulted in slower, less dynamic mitophagy. Expression of high levels of mutHTT fragments due to somatic nuclear HTT CAG instability can thus affect mitochondrial network dynamics and mitophagy, leading to pathogenic mtDNA mutations. We show that life-long expression of mutant HTT causes a mitochondrial phenotype indicative of mtDNA instability in fresh post-mitotic human skeletal muscle. Thus, genomic instability may not be limited to nuclear DNA, where it results in somatic expansion of the HTT CAG repeat length in particularly vulnerable cells such as striatal neurons. In addition to efforts targeting the causative mutation, promoting mitochondrial health may be a complementary strategy in treating diseases with DNA instability such as HD., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Huntington's disease results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mHTT to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by c-Jun N-terminal kinases (JNKs) and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNKs. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem Huntington's disease patients. Together, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in Huntington's disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Positron emission tomography imaging of misfolded proteins with high-affinity and selective radioligands has played a vital role in expanding our knowledge of neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The pathogenesis of Huntington's disease, a CAG trinucleotide repeat disorder, is similarly linked to the presence of protein fibrils formed from mutant huntingtin (mHTT) protein. Development of mHTT fibril-specific radioligands has been limited by the lack of structural knowledge around mHTT and a dearth of available hit compounds for medicinal chemistry refinement. Over the past decade, the CHDI Foundation, a non-for-profit scientific management organisation has orchestrated a large-scale screen of small molecules to identify high affinity ligands of mHTT, with lead compounds now reaching clinical maturity. Here we describe the mHTT radioligands developed to date and opportunities for further improvement of this radiotracer class., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)