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Andreas Weiss - One of the best experts on this subject based on the ideXlab platform.
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mutant Huntingtin impairs immune cell migration in huntington disease
Journal of Clinical Investigation, 2012Co-Authors: Wanda Kwan, Flaviano Giorgini, Andreas Weiss, Ulrike Trager, Dimitrios Davalos, Austin Chou, Jill Bouchard, Ralph Andre, Aaron W Miller, Christine S CheahAbstract:In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant Huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed.
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sustained therapeutic reversal of huntington s disease by transient repression of Huntingtin synthesis
Neuron, 2012Co-Authors: Holly Kordasiewicz, Andreas Weiss, Lisa M Stanek, Edward Wancewicz, Curt Mazur, Melissa Mcalonis, Kimberly A Pytel, Jonathan W Artates, Seng H Cheng, Lamya S ShihabuddinAbstract:Summary The primary cause of Huntington's disease (HD) is expression of Huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant Huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of Huntingtin mRNA, we demonstrate that transient infusion into the cerebrospinal fluid of symptomatic HD mouse models not only delays disease progression but mediates a sustained reversal of disease phenotype that persists longer than the Huntingtin knockdown. Reduction of wild-type Huntingtin, along with mutant Huntingtin, produces the same sustained disease reversal. Similar ASO infusion into nonhuman primates is shown to effectively lower Huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of Huntingtin synthesis.
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proteolysis of mutant Huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in huntington disease
Journal of Biological Chemistry, 2010Co-Authors: Christian Landles, Lisa M Ellerby, Andreas Weiss, Kirupa Sathasivam, Ben Woodman, Hilary Moffitt, Steve Finkbeiner, Juliette Gafni, Yvon Trottier, William G RichardsAbstract:Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-Huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type Huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 Huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.
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single step detection of mutant Huntingtin in animal and human tissues a bioassay for huntington s disease
Analytical Biochemistry, 2009Co-Authors: Andreas Weiss, Dorothée Abramowski, Miriam Bibel, Ruth Bodner, Vanita Chopra, Kimberly Kegel, Stephan Grueninger, Corinna Klein, Marian Difiglia, Steven M HerschAbstract:Abstract The genetic mutation causing Huntington’s disease is a polyglutamine expansion in the Huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant Huntingtin, understanding Huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant Huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Forster resonance energy transfer (FRET) assay for quantification of soluble mutant Huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble Huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant Huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant Huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant Huntingtin. The design of the homogeneous one-step method for Huntingtin detection is such that it can be easily applied to measure other proteins of interest.
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Single-step detection of mutant Huntingtin in animal and human tissues: A bioassay for Huntington's disease
Analytical Biochemistry, 2009Co-Authors: Andreas Weiss, Dorothée Abramowski, Miriam Bibel, Ruth Bodner, Vanita Chopra, Jonathan Fox, Kimberly Kegel, M Difiglia, Corinna Klein, Stephan GrueningerAbstract:The genetic mutation causing Huntington's disease is a polyglutamine expansion in the Huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant Huntingtin, understanding Huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant Huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Förster resonance energy transfer (FRET) assay for quantification of soluble mutant Huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble Huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant Huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant Huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant Huntingtin. The design of the homogeneous one-step method for Huntingtin detection is such that it can be easily applied to measure other proteins of interest. © 2009 Elsevier Inc.
Michael R. Hayden - One of the best experts on this subject based on the ideXlab platform.
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Huntington disease: new insights on the role of Huntingtin cleavage.
Journal of Neural Transmission-supplement, 2020Co-Authors: Cheryl L Wellington, Blair R. Leavitt, Michael R. HaydenAbstract:Huntington Disease (HD) results from polyglutamine expansion within the N-terminus of Huntingtin. We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) human Huntingtin in a developmentally appropriate and tissue-specific manner identical to the pattern of expression of endogenous Huntingtin. YAC46 and YAC72 mice show early electrophysiological abnormalities indicating neuronal cytoplasmic dysfunction prior to developing nuclear inclusions or neurodegeneration. YAC72 mice display a hyperkinetic movement disorder by 7 months of age, and have evidence for selective and specific degeneration of medium spiny neurons in the lateral striatum by 12 months of age. A key molecular feature of pathology of these YAC72 mice is cleavage of Huntingtin in the cytoplasm following by translocation of the resulting Huntingtin N-terminal fragments into the nucleus of striatal neurons. Increasing nuclear localization of Huntingtin N-terminal fragments within medium spiny neurons of the striatum occurs concomitantly with the onset of selective neurodegeneration. Because Huntingtin is a caspase substrate and truncated Huntingtin fragments are toxic in vitro, inhibiting caspase cleavage of hunting tin may be of potential therapeutic benefit in HD. We show that caspase inhibitors eliminate Huntingtin cleavage in cells and protects them from an apoptotic stress.
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Altered Regulation of Striatal Neuronal N-Methyl-D-Aspartate Receptor Trafficking by Palmitoylation in Huntington Disease Mouse Model
Frontiers in Synaptic Neuroscience, 2019Co-Authors: Rujun Kang, Shaun S. Sanders, Michael R. Hayden, Liang Wang, Lynn A. RaymondAbstract:N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic signaling, and alterations in the synaptic/extrasynaptic NMDAR balance affect neuronal survival. Studies have shown enhanced extrasynaptic GluN2B-type NMDAR (2B-NMDAR) activity in striatal neurons in the YAC128 mouse model of Huntington disease (HD), resulting in increased cell death pathway activation contributing to striatal vulnerability to degeneration. However, the mechanism(s) of altered GluN2B trafficking remains unclear. Previous work shows that GluN2B palmitoylation on two C-terminal cysteine clusters regulates 2B-NMDAR trafficking to the surface membrane and synapses in cortical neurons. Notably, two palmitoyl acyltransferases (PATs), zDHHC17 and zDHHC13, also called Huntingtin-interacting protein 14 (HIP14) and HIP14-like (HIP14L), directly interact with the Huntingtin protein (Htt), and mutant Htt disrupts this interaction. Here, we investigated whetherGluN2B palmitoylation is involved in enhanced extrasynaptic surface expression of 2B-NMDARs in YAC128 striatal neurons and whether this process is regulated by HIP14 or HIP14L. We found reduced GluN2B palmitoylation in YAC128 striatum, specifically on cysteine cluster II. Consistent with that finding, the palmitoylation-deficient GluN2B Cysteine cluster II mutant exhibited enhanced, extrasynaptic surface expression in striatal neurons from wild-type mice, mimicking increased extrasynaptic 2B-NMDAR observed in YAC128 cultures. We also found that HIP14L palmitoylated GluN2B cysteine cluster II. Moreover, GluN2B palmitoylation levels were reduced in striatal tissue from HIP14L-deficient mice, and siRNA-mediated HIP14L knockdown in cultured neurons enhanced striatal neuronal GluN2B surface expression and susceptibility to NMDA toxicity. Thus, altered regulation of GluN2B palmitoylation levels by the Huntingtin-associated PAT HIP14L may contribute to the cell death-signaling pathways underlying HD.
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Therapeutic approaches to Huntington disease: from the bench to the clinic
Nature Reviews Drug Discovery, 2018Co-Authors: Nicholas S. Caron, E. Ray Dorsey, Michael R. HaydenAbstract:The 25 years since the identification of the gene responsible for Huntington disease (HD) have stood witness to profound discoveries about the nature of the disease and its pathogenesis. Despite this progress, however, the development of disease-modifying therapies has thus far been slow. Preclinical validation of the therapeutic potential of disrupted pathways in HD has led to the advancement of pharmacological agents, both novel and repurposed, for clinical evaluation. The most promising therapeutic approaches include Huntingtin (HTT) lowering and modification as well as modulation of neuroinflammation and synaptic transmission. With clinical trials for many of these approaches imminent or currently ongoing, the coming years are promising not only for HD but also for more prevalent neurodegenerative disorders, such as Alzheimer and Parkinson disease, in which many of these pathways have been similarly implicated. New therapies, including RNA-based and gene therapies, are poised to change the therapeutic landscape for Huntington disease. In this article, Hayden and colleagues review the progress that has been made in the past 25 years in developing therapies for this disease and highlight the pitfalls and potential of future treatments.
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Huntington disease
Nature Reviews Disease Primers, 2015Co-Authors: Gillian P. Bates, Blair R. Leavitt, Ray Dorsey, James F. Gusella, Michael R. Hayden, Martha Nance, Christopher A. Ross, Rachael I. Scahill, Ronald Wetzel, Edward J. WildAbstract:Huntington disease is devastating to patients and their families — with autosomal dominant inheritance, onset typically in the prime of adult life, progressive course, and a combination of motor, cognitive and behavioural features. The disease is caused by an expanded CAG trinucleotide repeat (of variable length) in HTT , the gene that encodes the protein Huntingtin. In mutation carriers, Huntingtin is produced with abnormally long polyglutamine sequences that confer toxic gains of function and predispose the protein to fragmentation, resulting in neuronal dysfunction and death. In this Primer, we review the epidemiology of Huntington disease, noting that prevalence is higher than previously thought, geographically variable and increasing. We describe the relationship between CAG repeat length and clinical phenotype, as well as the concept of genetic modifiers of the disease. We discuss normal Huntingtin protein function, evidence for differential toxicity of mutant Huntingtin variants, theories of Huntingtin aggregation and the many different mechanisms of Huntington disease pathogenesis. We describe the genetic and clinical diagnosis of the condition, its clinical assessment and the multidisciplinary management of symptoms, given the absence of effective disease-modifying therapies. We review past and present clinical trials and therapeutic strategies under investigation, including impending trials of targeted Huntingtin-lowering drugs and the progress in development of biomarkers that will support the next generation of trials. For an illustrated summary of this Primer, visit: http://go.nature.com/hPMENh Huntington disease is an autosomal dominant neurological disorder caused by mutation in HTT . The disease typically manifests in adulthood and is characterized by progressive motor, cognitive and behavioural impairment. This Primer discusses the current knowledge of this disease.
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Aberrant palmitoylation in Huntington disease
Biochemical Society Transactions, 2015Co-Authors: Shaun S. Sanders, Michael R. HaydenAbstract:Huntington disease (HD) is an adult-onset neurodegenerative disease caused by a CAG expansion in the HTT gene. HD is characterized by striatal atrophy and is associated with motor, cognitive and psychiatric deficits. In the presence of the HD mutation, the interactions between Huntingtin (HTT) and Huntingtin interacting protein 14 (HIP14 or DHHC17) and HIP14-like (DHHC13, a HIP14 orthologue), palmitoyl acyltransferases for HTT, are disturbed, resulting in reduced palmitoylation of HTT. Genetic ablation of either Hip14 or Hip14l recapitulates many features of HD, including striatal atrophy and motor deficits. However, there are no changes in palmitoylation of HTT in either mouse model and, subsequently, the similarities between the phenotypes of these two mouse models and the HD mouse model are believed to result from underpalmitoylation of other HIP14 and HIP14L substrates. HTT acts as a modulator of HIP14 activity such that in the presence of the HD mutation, HIP14 is less active. Consequently, HIP14 substrates are less palmitoylated, leading to neuronal toxicity. This suggests that altered HIP14–HTT and HIP14L–HTT interactions in the presence of the HD mutation reduces palmitoylation and promotes mislocalization of HTT and other HIP14/HIP14L substrates. Ultimately, HD may be, in part, a disease of altered palmitoylation. Abbreviations: HD, Huntington disease; HTT, Huntingtin; wtHTT, wild type HTT; mHTT, mutant HTT; YAC128, full-length human HTT transgenic mouse model of HD; HIP14, Huntingtin interacting protein 14; HIP14L, Huntingtin interacting protein 14-like; SNAP25, synaptosomal-associated protein 25; PSD-95, post-synaptic density protein 95; APT, acyl-protein thioesterase; MSN, medium spiny neuron
David Craufurd - One of the best experts on this subject based on the ideXlab platform.
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targeting Huntingtin expression in patients with huntington s disease
The New England Journal of Medicine, 2019Co-Authors: Sarah J Tabrizi, Carsten Saft, David Craufurd, Blair R. Leavitt, Josef Priller, Edward J. Wild, Bernhard G Landwehrmeyer, Roger A Barker, Nick F Blair, Hugh RickardsAbstract:BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant Huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant Huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of Huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant Huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant Huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant Huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).
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n02 safety and tolerability of selisistat for the treatment of huntington s disease results from a randomised double blind placebo controlled phase ii trial
Journal of Neurology Neurosurgery and Psychiatry, 2014Co-Authors: Ralf Reilmann, Sigurd D. Süssmuth, Carsten Saft, Chiara Mariotti, Oliver Quarrell, Andrea H Nemeth, Josef Priller, Ferdinando Squitieri, Sarah J Tabrizi, David CraufurdAbstract:Background Selisistat is a first-in-class SirT1 inhibitor shown to be safe and well tolerated in healthy volunteers and HD patients in short-term studies. Objective To evaluate safety and tolerability of selisistat over 12 weeks in patients with Huntington’s disease (HD). Design/methods This was a double-blind, placebo-controlled, international multi-centre study of selisistat in individuals with Stage I-III HD. Participants (30–70 yrs) with genetically confirmed HD, a Unified Huntington Disease Rating Scale (UHDRS) Total Motor Score of ≥ 5 and a Total Functional Capacity ≥ 5 were randomised (1:1:1) to selisistat 50 or 200 mg or placebo once daily for 12 weeks. Safety and tolerability were evaluated by monitoring adverse events, vital signs, ECG and laboratory safety data throughout the study. Blood sampling for pharmacokinetics and soluble mutant Huntingtin levels were collected throughout. Results/outcome A total of 144 patients were randomised and 125 patients (87%) completed the study. There were 9 serious adverse events, three in each treatment group, including one death in the placebo group. The most common adverse events were reversible increases in liver function tests without accompanying increases in bilirubin. All of these occurred in the selisistat groups; while most of these increases were Conclusions Apart from increases in liver function tests in a subset of patients, selisistat was safe and well tolerated, and a trend for modulation of the levels of soluble mutant Huntingtin was observed. Acknowledgement Supported by Siena Biotech SpA.
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N02 Safety And Tolerability Of Selisistat For The Treatment Of Huntington’s Disease: Results From A Randomised, Double-blind, Placebo-controlled Phase Ii Trial
Journal of Neurology Neurosurgery and Psychiatry, 2014Co-Authors: Ralf Reilmann, Sigurd D. Süssmuth, Carsten Saft, Chiara Mariotti, Oliver Quarrell, Andrea H Nemeth, Josef Priller, Ferdinando Squitieri, Sarah J Tabrizi, David CraufurdAbstract:Background Selisistat is a first-in-class SirT1 inhibitor shown to be safe and well tolerated in healthy volunteers and HD patients in short-term studies. Objective To evaluate safety and tolerability of selisistat over 12 weeks in patients with Huntington’s disease (HD). Design/methods This was a double-blind, placebo-controlled, international multi-centre study of selisistat in individuals with Stage I-III HD. Participants (30–70 yrs) with genetically confirmed HD, a Unified Huntington Disease Rating Scale (UHDRS) Total Motor Score of ≥ 5 and a Total Functional Capacity ≥ 5 were randomised (1:1:1) to selisistat 50 or 200 mg or placebo once daily for 12 weeks. Safety and tolerability were evaluated by monitoring adverse events, vital signs, ECG and laboratory safety data throughout the study. Blood sampling for pharmacokinetics and soluble mutant Huntingtin levels were collected throughout. Results/outcome A total of 144 patients were randomised and 125 patients (87%) completed the study. There were 9 serious adverse events, three in each treatment group, including one death in the placebo group. The most common adverse events were reversible increases in liver function tests without accompanying increases in bilirubin. All of these occurred in the selisistat groups; while most of these increases were Conclusions Apart from increases in liver function tests in a subset of patients, selisistat was safe and well tolerated, and a trend for modulation of the levels of soluble mutant Huntingtin was observed. Acknowledgement Supported by Siena Biotech SpA.
Sarah J Tabrizi - One of the best experts on this subject based on the ideXlab platform.
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targeting Huntingtin expression in patients with huntington s disease
The New England Journal of Medicine, 2019Co-Authors: Sarah J Tabrizi, Carsten Saft, David Craufurd, Blair R. Leavitt, Josef Priller, Edward J. Wild, Bernhard G Landwehrmeyer, Roger A Barker, Nick F Blair, Hugh RickardsAbstract:BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant Huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant Huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of Huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant Huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant Huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant Huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).
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quantification of mutant Huntingtin protein in cerebrospinal fluid from huntington s disease patients
Journal of Clinical Investigation, 2015Co-Authors: Edward J. Wild, Blair R. Leavitt, Douglas R Langbehn, Roberto Boggio, Nicola Robertson, Salman Haider, James R Miller, Henrik Zetterberg, Rainer Kuhn, Sarah J TabriziAbstract:BACKGROUND: Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant Huntingtin protein (mHTT), the cause of Huntington’s disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. METHODS: We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. RESULTS: This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. CONCLUSIONS: We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD. TRIAL REGISTRATION: Not applicable. FUNDING: CHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.
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n02 safety and tolerability of selisistat for the treatment of huntington s disease results from a randomised double blind placebo controlled phase ii trial
Journal of Neurology Neurosurgery and Psychiatry, 2014Co-Authors: Ralf Reilmann, Sigurd D. Süssmuth, Carsten Saft, Chiara Mariotti, Oliver Quarrell, Andrea H Nemeth, Josef Priller, Ferdinando Squitieri, Sarah J Tabrizi, David CraufurdAbstract:Background Selisistat is a first-in-class SirT1 inhibitor shown to be safe and well tolerated in healthy volunteers and HD patients in short-term studies. Objective To evaluate safety and tolerability of selisistat over 12 weeks in patients with Huntington’s disease (HD). Design/methods This was a double-blind, placebo-controlled, international multi-centre study of selisistat in individuals with Stage I-III HD. Participants (30–70 yrs) with genetically confirmed HD, a Unified Huntington Disease Rating Scale (UHDRS) Total Motor Score of ≥ 5 and a Total Functional Capacity ≥ 5 were randomised (1:1:1) to selisistat 50 or 200 mg or placebo once daily for 12 weeks. Safety and tolerability were evaluated by monitoring adverse events, vital signs, ECG and laboratory safety data throughout the study. Blood sampling for pharmacokinetics and soluble mutant Huntingtin levels were collected throughout. Results/outcome A total of 144 patients were randomised and 125 patients (87%) completed the study. There were 9 serious adverse events, three in each treatment group, including one death in the placebo group. The most common adverse events were reversible increases in liver function tests without accompanying increases in bilirubin. All of these occurred in the selisistat groups; while most of these increases were Conclusions Apart from increases in liver function tests in a subset of patients, selisistat was safe and well tolerated, and a trend for modulation of the levels of soluble mutant Huntingtin was observed. Acknowledgement Supported by Siena Biotech SpA.
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N02 Safety And Tolerability Of Selisistat For The Treatment Of Huntington’s Disease: Results From A Randomised, Double-blind, Placebo-controlled Phase Ii Trial
Journal of Neurology Neurosurgery and Psychiatry, 2014Co-Authors: Ralf Reilmann, Sigurd D. Süssmuth, Carsten Saft, Chiara Mariotti, Oliver Quarrell, Andrea H Nemeth, Josef Priller, Ferdinando Squitieri, Sarah J Tabrizi, David CraufurdAbstract:Background Selisistat is a first-in-class SirT1 inhibitor shown to be safe and well tolerated in healthy volunteers and HD patients in short-term studies. Objective To evaluate safety and tolerability of selisistat over 12 weeks in patients with Huntington’s disease (HD). Design/methods This was a double-blind, placebo-controlled, international multi-centre study of selisistat in individuals with Stage I-III HD. Participants (30–70 yrs) with genetically confirmed HD, a Unified Huntington Disease Rating Scale (UHDRS) Total Motor Score of ≥ 5 and a Total Functional Capacity ≥ 5 were randomised (1:1:1) to selisistat 50 or 200 mg or placebo once daily for 12 weeks. Safety and tolerability were evaluated by monitoring adverse events, vital signs, ECG and laboratory safety data throughout the study. Blood sampling for pharmacokinetics and soluble mutant Huntingtin levels were collected throughout. Results/outcome A total of 144 patients were randomised and 125 patients (87%) completed the study. There were 9 serious adverse events, three in each treatment group, including one death in the placebo group. The most common adverse events were reversible increases in liver function tests without accompanying increases in bilirubin. All of these occurred in the selisistat groups; while most of these increases were Conclusions Apart from increases in liver function tests in a subset of patients, selisistat was safe and well tolerated, and a trend for modulation of the levels of soluble mutant Huntingtin was observed. Acknowledgement Supported by Siena Biotech SpA.
Steven M Hersch - One of the best experts on this subject based on the ideXlab platform.
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single step detection of mutant Huntingtin in animal and human tissues a bioassay for huntington s disease
Analytical Biochemistry, 2009Co-Authors: Andreas Weiss, Dorothée Abramowski, Miriam Bibel, Ruth Bodner, Vanita Chopra, Kimberly Kegel, Stephan Grueninger, Corinna Klein, Marian Difiglia, Steven M HerschAbstract:Abstract The genetic mutation causing Huntington’s disease is a polyglutamine expansion in the Huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant Huntingtin, understanding Huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant Huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Forster resonance energy transfer (FRET) assay for quantification of soluble mutant Huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble Huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant Huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant Huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant Huntingtin. The design of the homogeneous one-step method for Huntingtin detection is such that it can be easily applied to measure other proteins of interest.
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Huntingtin aggregates may not predict neuronal death in huntington s disease
Annals of Neurology, 1999Co-Authors: Claire-anne Gutekunst, Shihua Li, Xiaojiang Li, Steven M Hersch, Stefan Kuemmerle, Autumn M Klein, Flint M Beal, Robert J FerranteAbstract:The mechanism by which polyglutamine expansion in Huntington's disease (HD) results in selective neuronal degeneration remains unclear. We previously reported that the immunohistochemical distribution of N-terminal Huntingtin in HD does not correspond to the severity of neuropathology, such that significantly greater numbers of Huntingtin aggregates are present within the cortex than in the striatum. We now show a dissociation between Huntingtin aggregation and the selective pattern of striatal neuron loss observed in HD. Aggregate formation was predominantly observed in spared interneurons, with few or no aggregates found within vulnerable spiny striatal neurons. Multiple perikaryal aggregates were present in almost all cortical NADPH-diaphorase neurons and in approximately 50% of the spared NADPH-diaphorase striatal neurons from early grade HD cases. In severe grade HD patients, aggregates were more prominent as nuclear inclusions in NADPH-diaphorase neurons, with less perikaryal and neuropil aggregation. In contrast, nuclear or perikaryal Huntingtin aggregates were present in less than 4% of the vulnerable calbindin striatal neurons in all HD cases. These findings support the hypothesis that polyglutamine aggregation may not be a predictor of cell loss. Rather than a harbinger of neuronal death, mutant Huntingtin aggregation may be a cytoprotective mechanism against polyglutamine-induced neurotoxicity.
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A Human HAP1 Homologue CLONING, EXPRESSION, AND INTERACTION WITH Huntingtin
Journal of Biological Chemistry, 1998Co-Authors: Shihua Li, Claire-anne Gutekunst, Robert J Ferrante, Steven M Hersch, Seyed H. Hosseini, Xiaojiang LiAbstract:Abstract Huntington’s disease (HD) is caused by the expansion of a glutamine repeat in the protein Huntingtin. The expanded glutamine repeat is thought to mediate a gain of function by causing Huntingtin to abnormally interact with other proteins. We previously identified a rat Huntingtin-associated protein (HAP1) that binds to Huntingtin; HAP1 binds more tightly to Huntingtin with an expanded glutamine repeat than to wild type Huntingtin. Identification of the human homologue of HAP1 is necessary for investigation of the potential role of HAP1 in HD pathology. Here, we report the cloning of a human HAP1 homologue (hHAP) that shares 62% identity with rat HAP1 over its entire sequence and 82% amino acid identity in the putative Huntingtin-binding region. The hHAP gene encodes a 4.1-kilobase transcript and a 75-kDa protein which are specifically expressed in human brain tissues. Its expression in Huntington’s disease brains is reduced in parallel with a decreased expression of Huntingtin. While two isoforms of rat HAP1 are expressed at similar levels in rat brain, only a single major form of hHAP is found in primate brains. In vitro binding, immunoprecipitation, and coexpression studies confirm the interaction of hHAP with Huntingtin. The in vitro binding of hHAP to Huntingtin is enhanced by lengthening the glutamine repeat. Despite similar binding properties of rat HAP1 and hHAP, differences in the sequences and expression of hHAP may contribute to a specific role for its interaction with Huntingtin in humans.
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Interaction of Huntingtin-Associated Protein with Dynactin P150Glued
The Journal of Neuroscience, 1998Co-Authors: Shihua Li, Claire-anne Gutekunst, Steven M Hersch, Xiaojiang LiAbstract:Huntingtin is the protein product of the gene for Huntington’s disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to Huntingtin in association with the polyglutamine repeat. Like Huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, Huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150Glued (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both Huntingtin and P150 selectively bound to a glutathione transferase (GST)–HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and Huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and Huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and Huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and Huntingtin are present in a protein complex that may participate in dynein–dynactin-associated intracellular transport.
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heterogeneous topographic and cellular distribution of Huntingtin expression in the normal human neostriatum
The Journal of Neuroscience, 1997Co-Authors: Robert J Ferrante, Claire-anne Gutekunst, James F. Gusella, Marcy E Macdonald, Francesca Persichetti, Sandra M Mcneil, Neil W Kowall, M F Beal, Steven M HerschAbstract:A striking heterogeneous distribution of topographic and cellular Huntingtin immunoreactivity was observed within the human neostriatum using three distinct Huntingtin antibodies. Patchy areas of low Huntingtin immunoreactivity were present in both the caudate nucleus and putamen, surrounded by an intervening area of greater immunoreactivity. Comparison of Huntingtin immunoreactivity with contiguous serial sections stained for enkephalin and calbindin D28k immunoreactivities showed that the topographic heterogeneity of Huntingtin immunostaining corresponded to the patch (striosome) and matrix compartments within the striatum. Huntingtin immunoreactivity was confined primarily to neurons and neuropil within the matrix compartment, whereas little or no neuronal or neuropil Huntingtin immunostaining was observed within the patch compartment. There was marked variability in the intensity of Huntingtin immunolabel among medium-sized striatal neurons, whereas a majority of large striatal neurons were only faintly positive or without any immunoreactivity. Combined techniques for NADPH-diaphorase enzyme histochemistry and Huntingtin immunocytochemistry, as well as double immunofluorescence for either nitric oxide synthase or calbindin D28k in comparison with Huntingtin expression, revealed a striking correspondence between calbindin D28k and Huntingtin immunoreactivities, with little or no colocalization between NADPH-diaphorase or nitric oxide synthase neurons and Huntingtin expression. These observations suggest that the selective vulnerability of spiny striatal neurons and the matrix compartment observed in Huntington’s disease is associated with higher levels of Huntingtin expression, whereas the relative resistance of large and medium-sized aspiny neurons and the patch compartments to degeneration is associated with low levels of Huntingtin expression.
