Huntingtin Research Articles

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils implicated in Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report new magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a new understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

Characterization of fission and fusion mitochondrial dynamics in HD fibroblasts according to patient's severity status

Huntington's Disease (HD) is an inheritable neurodegenerative condition caused by an expanded CAG trinucleotide repeat in the HTT gene with a direct correlation between CAG repeats expansion and disease severity with earlier onset-of- disease. Previously we have shown that primary skin fibroblasts from HD patients exhibit unique phenotype disease features, including distinct nuclear morphology and perturbed actin cap linked with cell motility, that are correlated with the HD patient disease severity. Here we provide further evidence that mitochondrial fission-fusion morphology balance dynamics, classified using a custom image-based high-content analysis (HCA) machine learning tool, that improved correlation with HD severity status. This mitochondrial phenotype is supported by appropriate changes in fission-fusion biomarkers (Drp1, MFN1, MFN2, VAT1) levels in the HD patients' fibroblasts. These findings collectively point towards a dysregulation in mitochondrial dynamics, where both fission and fusion processes may be disrupted in HD cells compared to healthy controls. This study shows for the first time a methodology that enables identification of HD phenotype before patient's disease onset (Premanifest). Therefore, we believe that this tool holds a potential for improving precision in HD patient's diagnostics bearing the potential to evaluate alterations in mitochondrial dynamics throughout the progression of HD, offering valuable insights into the molecular mechanisms and drug therapy evaluation underlying biological differences in any disease stage.

Small striatal huntingtin inclusions in patients with motor neuron disease with reduced penetrance and intermediate HTT gene expansions.

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.

Oleuropein enhances proteasomal activity and reduces mutant huntingtin-induced cytotoxicity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder that primarily affects the striatum, a brain region responsible for movement control. The disease is characterized by the mutant huntingtin (mHtt) proteins with an extended polyQ stretch, which are prone to aggregation. These mHtt aggregates accumulate in neurons and are the primary cause of the neuropathology associated with HD. To date, no effective cure for HD has been developed. The immortalized STHdh Q111/Q111 striatal cell line, the mHtt-transfected wild-type STHdh Q7/Q7 striatal cell line, and N2a cells were used as Huntington's disease cell models. Flow cytometry was used to assess cellular reactive oxygen species and transfection efficiency. The CCK-8 assay was used to measure cell viability, while fluorescence microscopy was used to quantify aggregates. Immunoblotting analyses were used to evaluate the effects on protein expression. Polyphenols are natural antioxidants that offer neuroprotection in neurological disorders. In this study, we provide evidence that oleuropein, the primary polyphenol in olive leaves and olive oil, enhances cell viability in HD cell models, including. STHdh Q7/Q7 STHdh Q7/Q7 striatal cells, N2a cells ectopically expressing the truncated mHtt, and STHdh Q111/Q111 striatal cells expressing the full-length mHtt. Oleuropein effectively reduced both soluble and aggregated forms of mHtt protein in these HD model cells. Notably, the reduction of mHtt aggregates associated with oleuropein was linked to increased proteasome activity rather than changes in autophagic flux. Oleuropein seems to modulate proteasome activity through an unidentified pathway, as it did not affect the 20S proteasome catalytic β subunits, the proteasome regulator PA28γ, or multiple MAPK pathways. We demonstrated that oleuropein enhances the degradation of mHtt by increasing proteasomal protease activities and alleviates mHtt-induced cytotoxicity. Hence, we propose that oleuropein and potentially other polyphenols hold promise as a candidate for alleviating Huntington's disease.

A - 122 Neuropsychological Case Study: Late-Onset Huntington’s Disease

Abstract Objective We present the neuropsychological profile of a 65-year-old male diagnosed with late-onset Huntington’s Disease (HD) in April 2024. In the context of very subtle chorea movements over the past several years, he started experiencing mood disturbances (e.g., increased irritability and social withdrawal) starting in June 2023. These changes were followed by cognitive difficulties, which led to a hospitalization for autoimmune encephalitis workup in February 2024. He described difficulties with learning and memory for future events, concentration, and executive functions (e.g., organizing and problem-solving). Method Referral from neurology to conduct a comprehensive neuropsychological evaluation to characterize his cognitive functioning. Exam, chart review, and clinical interviews were conducted. Results The cognitive profile was largely preserved, with mild attentional and executive difficulties, as well as mild challenges retrieving unstructured verbal information. This indicated subtle frontosubcortical network disruption. Self-report questionnaires indicated mild hopelessness but minimal anxiety and mood concerns. During the course of the neuropsychological evaluation, he underwent genetic testing, which was consistent with a diagnosis of HD (41 CAG repeats in the HTT gene). From a neuropsychological perspective, his psychiatric and cognitive disruption were also consistent with a diagnosis of HD. Conclusion The patient’s psychiatric and cognitive decline are now understood in light of his recent HD diagnosis. While cognitive difficulties are prevalent in individuals with HD, the low prevalence of late-onset HD makes the results of this case report particularly informative. This report contributes valuable insights into the neuropsychological presentation of older adults with HD.

Decision cost hypersensitivity underlies Huntington's disease apathy.

The neuropsychiatric syndrome of apathy is now recognized to be a common and disabling condition in Huntington's disease (HD). However, the mechanisms underlying it are poorly understood. One way to investigate apathy is to utilise a theoretical framework of normal motivated behaviour, to determine where breakdown has occurred in people with this behavioural disruption. A fundamental computation underlying motivated, goal-directed behaviour across species is weighing up the costs and rewards associated with actions. Here, we asked whether people with apathy are more sensitive to costs of actions (physical effort and time delay), less sensitive to rewarding outcomes, or both. Based on the unique anatomical substrates associated with HD pathology, we hypothesised that a general hypersensitivity to costs would underpin HD apathy. Genetically confirmed carriers of the expanded Huntingtin gene (premanifest to mild motor manifest disease (n=53) were compared to healthy controls (n = 38). Participants performed a physical effort-based decision-making task (Apple Gathering Task) and a delay discounting task (Money Choice Questionnaire). Choice data was analysed using linear regression and drift diffusion models that also accounted for the time taken to make decisions. Apathetic people with HD accepted fewer offers overall on the Apple Gathering Task, specifically driven by increased sensitivity to physical effort costs, and not explained by motor severity, mood, cognition, or medication. Drift diffusion modelling provided further evidence of effort hypersensitivity, with apathy associated with a faster drift rate towards rejecting offers as a function of varying effort. Increased delay sensitivity was also associated with apathy, both when analysing raw choice and also drift rate, where there was moderate evidence of HD apathy drifting faster towards the immediately available (low cost) option. Furthermore, the effort and delay sensitivity parameters from these tasks were positively correlated. The results demonstrate a clear mechanism for apathy in HD, cost hypersensitivity, which manifests in both the effort and time costs associated with actions towards rewarding goals. This suggests that HD pathology may cause a domain-general disruption of cost processing, which is distinct to apathy occurrence in other brain disorders, and may require different therapeutic approaches.

Protective Proteolysis in Huntington's Disease: Unraveling the Role of Post-Translational Myristoylation of Huntingtin in Autophagy.

Huntington's disease (HD) is a devastating neurodegenerative disorder characterized by impaired motor function and cognitive decline, ultimately leading to death. HD is caused by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein, which is linked to decreased HTT turnover, increased HTT proteolysis, increased HTT aggregation, and subsequent neuronal death. In this review, we explore the mechanism of the protective effect of blocking HTT proteolysis at D586, which has been shown to rescue the HD phenotype in HD mouse models. Until recently, the mechanism remained unclear. Herein, we discuss how blocking HTT proteolysis at D586 promotes HTT turnover by correcting autophagy, and making HTT a better autophagy substrate, through post-translational myristoylation of HTT at G553.

White Matter Microstructure Changes Revealed by Diffusion Kurtosis and Diffusion Tensor Imaging in Mutant Huntingtin Gene Carriers.

Diffusion magnetic resonance imaging (dMRI) has revealed microstructural changes in white matter (WM) in Huntington's disease (HD). To compare the validities of different dMRI, i.e., diffusion kurtosis imaging (DKI) and diffusion tensor imaging (DTI) in HD. 22 mutant huntingtin (mHTT) carriers and 14 controls were enrolled. Clinical assessments and dMRI were conducted. Based on CAG-Age Product (CAP) score, mHTT carriers were categorized into high CAP (hCAP) and medium and low CAP (m& lCAP) groups. Spearman analyses were used to explore correlations between imaging parameters in brain regions and clinical assessments. Receiver operating characteristic (ROC) was used to distinguish mHTT carriers from control, and define the HD patients at advanced stage. Compared to controls, mHTT carriers exhibited WM changes in DKI and DTI. There were 22 more regions showing significant differences in HD detected by MK than FA. Only MK in five brain regions showed significantly difference between any two group, and negatively correlated with the disease burden (r = -0.80 to -0.71). ROC analysis revealed that MK was more sensitive and FA was more specific, while Youden index showed that the integration of FA and MK gave rise to higher authenticities, in distinguishing m& lCAP from controls (Youden Index = 0.786), and discerning different phase of HD (Youden Index = 0.804). Microstructural changes in WM occur at early stage of HD and deteriorate over the disease progression. Integrating DKI and DTI would provide the best accuracies for differentiating early HD from control and identifying advanced HD.

Two novel DnaJ chaperone proteins CG5001 and P58IPK regulate the pathogenicity of Huntington’s disease related aggregates

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.

Glymphatic influx and clearance are perturbed in Huntington's disease.

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.

Global huntingtin knockout in adult mice leads to fatal neurodegeneration that spares the pancreas.

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.

In-vitro Neuro-2a cytotoxicity analysis and molecular docking investigation on potential anti-amyloid agents from Adiantumlunulatum

In neurodegenerative diseases, amyloid formation by some proteins cause neuronal damage and loss. To prevent this neuronal damage and loss certain pharmaceuticals are available. Many of these pharmaceuticals act on the neurodegenerative disease symptoms but not on the root cause. This study helps to detect more effective agents which directly act on the root cause and reduce the risk of neurodegenerative diseases. To identify new anti-amyloid agents, the folk medicinally important plant Adiantum lunulatum was collected, authenticated, dried, extracted with ethanol and analyzed by GC-MS method. The screening of the identified phytochemicals was done using the webservers swissADME and ProTox-II. In-vitro MTT assay using Neuro-2a cell lines was carried out to determine the cytotoxicity of the extract. The interactions of these phytochemicals with the amyloid forming peptides and proteins were predicted using the molecular docking tools such as AutoDock Vina and BIOVIA discovery studio visualizer 2020. Through GC-MS analysis, 18 different volatile phytochemicals were identified from the ethanol extract. From this, 7 phytochemicals were selected based on the computational non-toxicity prediction. In-vitro cytotoxicity analysis of the ethanol extract using Neuro-2a cell lines detected the IC50 value of 0.09 mg/ml. Of these, the phytochemical P1 (trans, trans-9, 12-Octadecadienoic acid, propyl ester) interacts with tau, and huntingtin proteins, P2 (2-Pentadecanone, 6, 10, 14-trimethyl-) interacts with prion protein. The phytochemicals P1, P3 (Ethyl oleate), P4 (Octadecanoic acid, ethyl ester), and P5 (Phytol) interact with acetylcholinesterase. P2, P4, P5 and P6 (Henicosanal), interact with BACE-1. The phytochemical P3 interacts with γ- Secretase. The interaction of P2 and P5 with BACE-1 and P3 with γ- Secretase show better inhibition in inhibitory constant (Ki) analysis. These phytochemicals have been predicted to show significant potential against the formation or breakdown of peptide/protein amyloids, and further in-vitro studies are necessary to develop them into anti-amyloid agents.

Psychiatric symptoms of Huntington's disease

Huntington's disease (HD) is an autosomal dominant inherited disease, which leads to motor, cognitive and psychiatric symptoms. The diagnosis can be confirmed by genetic testing for extended CAG repeats in the Huntingtin gene. Mental and behavioral symptoms are common in HD and can appear several years before the onset of motor symptoms. The psychiatric symptoms include apathy, depression, anxiety, obsessive-compulsive symptoms and, in some cases, psychoses and aggression. These are currently restricted to symptomatic treatment as disease-modifying treatment approaches are still under investigation. The current clinical practice is based on expert opinions as well as experience with the treatment of similar symptoms in other neurological and mental health diseases. This article provides an overview of the complex psychiatric manifestations of HD, the diagnostic options and the established pharmacological and nonpharmacological treatment approaches.

Developmental and physiological impacts of pathogenic human huntingtin protein in the nervous system.

Huntington's Disease (HD) is a neurodegenerative disorder, part of the nine identified inherited polyglutamine (polyQ) diseases. Most commonly, HD pathophysiology manifests in middle-aged adults with symptoms including progressive loss of motor control, cognitive decline, and psychiatric disturbances. Associated with the pathophysiology of HD is the formation of insoluble fragments of the huntingtin protein (htt) that tend to aggregate in the nucleus and cytoplasm of neurons. To track both the intracellular progression of the aggregation phenotype as well as the physiological deficits associated with mutant htt, two constructs of human HTT were expressed with varying polyQ lengths, non-pathogenic-htt (Q15, NP-htt) and pathogenic-htt (Q138, P-htt), with an N-terminal RFP tag for in vivo visualization. P-htt aggregates accumulate in the ventral nerve cord cell bodies as early as 24 hours post hatching and significant aggregates form in the segmental nerve branches at 48 hours post hatching. Organelle trafficking up-and downstream of aggregates formed in motor neurons showed severe deficits in trafficking dynamics. To explore putative downstream deficits of htt aggregation, ultrastructural changes of presynaptic motor neurons and muscles were assessed, but no significant effects were observed. However, the force and kinetics of muscle contractions were severely affected in P-htt animals, reminiscent of human chorea. Reduced muscle force production translated to altered locomotory behavior. A novel HD aggregation model was established to track htt aggregation throughout adulthood in the wing, showing similar aggregation patterns with larvae. Expressing P-htt in the adult nervous system resulted in significantly reduced lifespan, which could be partially rescued by feeding flies the mTOR inhibitor rapamycin. These findings advance our understanding of htt aggregate progression as well the downstream physiological impacts on the nervous system and peripheral tissues.

Pathogenic TDP-43 accelerates the generation of toxic exon1 HTT in Huntington's disease knock-in mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in exon1 of the HTT gene that encodes a polyglutamine tract in huntingtin protein. The formation of HTT exon1 fragments with an expanded polyglutamine repeat has been implicated as a key step in the pathogenesis of HD. It was reported that the CAG repeat length-dependent aberrant splicing of exon1 HTT results in a short polyadenylated mRNA that is translated into an exon1 HTT protein. Under normal conditions, TDP-43 is predominantly found in the nucleus, where it regulates gene expression. However, in various pathological conditions, TDP-43 is mislocalized in the cytoplasm. By investigating HD knock-in mice, we explore whether the pathogenic TDP-43 in the cytoplasm contributes to HD pathogenesis, through expressing the cytoplasmic TDP-43 without nuclear localization signal. We found that the cytoplasmic TDP-43 is increased in the HD mouse brain and that its mislocalization could deteriorate the motor and gait behavior. Importantly, the cytoplasmic TDP-43, via its binding to the intron1 sequence (GU/UG)n of the mouse Htt pre-mRNA, promotes the transport of exon1-intron1 Htt onto ribosome, resulting in the aberrant generation of exon1 Htt. Our findings suggest that cytoplasmic TDP-43 contributes to HD pathogenesis via its binding to and transport of nuclear un-spliced mRNA to the ribosome for the generation of a toxic protein product.

Mutant huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

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.

Trial to assess the tolerability of using felodipine to upregulate autophagy as a treatment of Huntington’s disease (FELL-HD): a phase II, single-centre, open-label, dose-finding trial protocol

IntroductionHuntington’s disease (HD) is an autosomal dominant neurodegenerative disorder that presents with a progressive movement disorder along with cognitive and psychiatric problems. It is caused by a Cytosine-adenin-guanine (CAG) expansion...

NAC guides a ribosomal multienzyme complex for nascent protein processing.

Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.

Pharmacokinetics and pharmacodynamics of PTC518, an oral huntingtin lowering splicing modifier: A first-in-human study.

PTC518 is an orally administered, centrally and peripherally distributed huntingtin (HTT) pre-mRNA splicing modifier being developed for the treatment of Huntington's disease (HD) for which there is a high unmet medical need as there are currently no approved disease-modifying treatments. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PTC518 in healthy volunteers. This phase 1, single-centre, randomized study in 77 healthy male and female volunteers evaluated the safety and tolerability and PK of PTC518 following single ascending doses and multiple ascending doses, PD as assessed by HTT mRNA and HTT protein levels after single and multiple doses, and food effects. PTC518 demonstrated a favourable safety profile. The majority of treatment-emergent adverse events were mild and transient. PTC518 Tmax was reached at 6-7h and the terminal T1/2 was 54.0-75.3h following a single oral dose. Exposure increased with dose though less than dose proportionally. The PTC518 concentrations in cerebrospinal fluid were approximately 2.6-fold higher than the unbound free-drug concentrations in plasma. A significant dose-dependent reduction of up to approximately 60% in HTT mRNA and a significant dose-dependent, time-dependent and sustained reduction in HTT protein levels of up to 35% were observed after PTC518 treatment. PTC518 was well tolerated, and proof of mechanism of this novel splicing modifier was demonstrated by the dose-dependent decrease in systemic HTT mRNA and HTT protein levels. Results from this first-in-human study support further studies in patients with HD and demonstrate the potential for PTC518 as a breakthrough treatment for HD.

Peripheral sequestration of huntingtin delays neuronal death and depends on N-terminal ubiquitination

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.