Reducing huntingtin by immunotherapy delays disease progression in a mouse model of Huntington disease.

In Huntington disease (HD), the mutant huntingtin (mtHTT) protein is the principal cause of pathological changes that initiate primarily along the cortico-striatal axis. mtHTT is ubiquitously expressed and there is, accordingly, growing recognition that HD is a systemic disorder with functional interplay between the brain and the periphery. We have developed a monoclonal antibody, C6-17, targeting an exposed region of HTT near the aa586 Caspase 6 cleavage site. As recently published, mAB C6-17 can block cell-to-cell propagation of mtHTT in vitro. In order to reduce the burden of the mutant protein in vivo, we queried whether extracellular mtHTT could be therapeutically targeted in YAC128 HD mice. In a series of proof of concept experiments, we found that systemic mAB C6-17 treatment resulted in the distribution of the mAB C6-17 to peripheral and CNS tissues and led to the reduction of HTT protein levels. Compared to CTRL mAB or vehicle treated mice, the mAB C6-17 treated YAC128 animals showed improved body weight and motor behaviors, a delayed progression in motor deficits and reduced striatal EM48 immunoreactivity. These results provide the first proof of concept for the feasibility and therapeutic efficacy of an antibody-based anti-HTT passive immunization approach and suggest this modality as a potential new HD treatment strategy.

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Cerebrospinal fluid mutant huntingtin is a biomarker for huntingtin lowering in the striatum of Huntington disease mice.

Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. Expanded mutant HTT (mHTT) is toxic and leads to regional atrophy and neuronal cell loss in the brain, which occurs earliest in the striatum. Therapeutic lowering of mHTT in the central nervous system (CNS) has shown promise in preclinical studies, with multiple approaches currently in clinical development for HD. Quantitation of mHTT in the cerebrospinal fluid (CSF) is being used as a clinical pharmacodynamic biomarker of target engagement in the CNS. We have previously shown that the CNS is a major source of mHTT in the CSF. However, little is known about the specific brain regions and cell types that contribute to CSF mHTT. Therefore, a better understanding of the origins of CSF mHTT and whether therapies targeting mHTT in the striatum would be expected to be associated with significant lowering of mHTT in the CSF is needed. Here, we use complementary pharmacological and genetic-based approaches to either restrict expression of mHTT to the striatum or selectively deplete mHTT in the striatum to evaluate the contribution of this brain region to mHTT in the CSF. We show that viral expression of a mHTT fragment restricted to the striatum leads to detectable mHTT in the CSF. We demonstrate that targeted lowering of mHTT selectively in the striatum using an antisense oligonucleotide leads to a significant reduction of mHTT in the CSF of HD mice. Furthermore, using a transgenic mouse model of HD that expresses full length human mHTT and wild type HTT, we show that genetic inactivation of mHTT selectively in the striatum results in a significant reduction of mHTT in the CSF. Taken together, our data supports the conclusion that the striatum contributes sufficiently to the pool of mHTT in the CSF that therapeutic levels of mHTT lowering in the striatum can be detected by this measure in HD mice. This suggests that CSF mHTT may represent a pharmacodynamic biomarker for therapies that lower mHTT in the striatum.

Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.

Super-resolution imaging reveals extrastriatal synaptic dysfunction in presymptomatic Huntington disease mice.

Synaptic structure and function are compromised prior to cell death and symptom onset in a variety of neurodegenerative diseases. In Huntington disease (HD), a CAG repeat expansion in the gene encoding the huntingtin protein results in a presymptomatic stage that typically spans multiple decades and is followed by striking degeneration of striatal tissue and the progression of debilitating motor symptoms. Many lines of evidence demonstrate that the HD presymptomatic window is associated with injurious effects to striatal synapses, many of which appear to be prerequisites to subsequent cell death. While the striatum is the most vulnerable region in the HD brain, it is widely recognized that HD is a brain-wide disease, affecting numerous extrastriatal regions that contribute to debilitating non-motor symptoms including cognitive dysfunction. Currently, we have a poor understanding of the synaptic integrity, or lack thereof, in extrastriatal regions in the presymptomatic HD brain. If early therapeutic intervention seeks to maintain healthy synaptic function, it is important to understand early HD-associated synaptopathy at a brain-wide, rather than striatal-exclusive, level. Here, we focused on the hippocampus as this structure is generally thought to be affected only in manifest HD despite the subtle cognitive deficits known to emerge in prodromal HD. We used super-resolution microscopy and multi-electrode array electrophysiology as sensitive measures of excitatory synapse structure and function, respectively, in the hippocampus of presymptomatic heterozygous HD mice (Q175FDN model). We found clear evidence for enhanced AMPA receptor subunit clustering and hyperexcitability well before the onset of a detectable HD-like behavioral phenotype. In addition, activity-dependent re-organization of synaptic protein nanostructure, and functional measures of synaptic plasticity were impaired in presymptomatic HD mice. These data demonstrate that synaptic abnormalities in the presymptomatic HD brain are not exclusive to the striatum, and highlight the need to better understand the region-dependent complexities of early synaptopathy in the HD brain.

Inhibiting cellular uptake of mutant huntingtin using a monoclonal antibody: Implications for the treatment of Huntington's disease.

Huntington's disease (HD) is caused by a highly polymorphic CAG trinucleotide expansion in the gene encoding for the huntingtin protein (HTT). The resulting mutant huntingtin protein (mutHTT) is ubiquitously expressed but also exhibits the ability to propagate from cell-to-cell to disseminate pathology; a property which may serve as a new therapeutic focus. Accordingly, we set out to develop a monoclonal antibody (mAB) targeting a particularly exposed region close to the aa586 caspase-6 cleavage site of the HTT protein. This monoclonal antibody, designated C6-17, effectively binds mutHTT and is able to deplete the protein from cell culture supernatants. Using cell-based assays, we demonstrate that extracellular secretion of mutHTT into cell culture media and its subsequent uptake in recipient HeLa cells can be almost entirely blocked by mAB C6-17. Immunohistochemical stainings of post-mortem HD brain tissue confirmed the specificity of mAB C6-17 to human mutHTT aggregates. These findings demonstrate that mAB C6-17 not only successfully engages with its target, mutHTT, but also inhibits cell uptake suggesting that this antibody could interfere with the pathological processes of mutHTT spreading in vivo.

HACE1 is essential for astrocyte mitochondrial function and influences Huntington disease phenotypes in vivo.

Oxidative stress is a prominent feature of Huntington disease (HD), and we have shown previously that reduced levels of hace1 (HECT domain and Ankyrin repeat containing E3 ubiquitin protein ligase 1) in patient striatum may contribute to the pathogenesis of HD. Hace1 promotes the stability of Nrf2 and thus plays an important role in antioxidant response mechanisms, which are dysfunctional in HD. Moreover, hace1 overexpression mitigates mutant huntingtin (mHTT)-induced oxidative stress in vitro through promotion of the Nrf2 antioxidant response. Here, we show that the genetic ablation of hace1 in the YAC128 mouse model of HD accelerates motor deficits and exacerbates cognitive and psychiatric phenotypes in vivo. We find that both the expression of mHTT and the ablation of hace1 alone are sufficient to cause deficits in astrocytic mitochondrial respiration. We confirm the crucial role of hace1 in astrocytes in vivo, since its ablation is sufficient to cause dramatic astrogliosis in wild-type FVB/N mice. Astrogliosis is not observed in the presence of mHTT but a strong dysregulation in the expression of astrocytic markers in HACE1-/- x YAC128 striatum suggests an additive effect of mHTT expression and hace1 loss on this cell type. HACE1-/- x YAC128 mice and primary cells derived from these animals therefore provide model systems that will allow for the further dissection of Nrf2 pathways and astrocyte dysfunction in the context of HD.

Childhood Cat Bites Relate to Increased Adulthood Severity of Schizotypy, Psychotic-Like Experiences, and Social Anhedonia in a Transdiagnostic Psychiatric Sample.

INTRODUCTION: Previous research has linked childhood cat scratches and bites to an increased risk for depression, and childhood cat ownership to increased risk of schizophrenia, bipolar disorder, and social anhedonia. Our group previously reported that childhood cat bites, but not ownership, related to increased schizotypy severity in an undergraduate sample. METHODS: The current study expands this research by inquiring about cat bites and ownership in a transdiagnostic adult sample (N = 162; 51% female; mean age = 38.15, SD = 10.65), composed of schizophrenia spectrum disorders (n = 30), bipolar disorders (n = 30), unipolar depression (n = 23), and nonpsychiatric participants (n = 79). Participants completed a diagnostic interview, scales of symptom severity, and a cat interaction history interview. RESULTS: Across the entire sample, self-report of cat bites prior to age 13, but not after, related to greater current severity of overall schizotypy, self-reported and clinician-rated psychotic-like symptoms, and social anhedonia, when compared to individuals who reported no lifetime cat bites. Cat bites prior to age 13 did not relate to severity of depression, non-social anhedonia, or clinician-rated negative symptoms. Self-report of residing with a cat prior to age 13, or a first cat bite after age 12, did not relate to any symptom severity measure examined. CONCLUSIONS: One theory for these findings is that an unknown infectious agent common in cat saliva interacted with brain development in childhood to increase the likelihood of these symptoms. A novel theory for the infectious agent Pasteurella multocidais discussed. Future research can examine candidate infectious agents to identify potential causal mechanisms for these relationships.

A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent.

An enhanced Q175 knock-in mouse model of Huntington disease with higher mutant huntingtin levels and accelerated disease phenotypes.

Huntington disease (HD) model mice with heterozygous knock-in (KI) of an expanded CAG tract in exon 1 of the mouse huntingtin (Htt) gene homolog genetically recapitulate the mutation that causes HD, and might be favoured for preclinical studies. However, historically these mice have failed to phenotypically recapitulate the human disease. Thus, homozygous KI mice, which lack wildtype Htt, and are much less relevant to human HD, have been used. The zQ175 model was the first KI mouse to exhibit significant HD-like phenotypes when heterozygous. In an effort to exacerbate HD-like phenotypes and enhance preclinical utility, we have backcrossed zQ175 mice to FVB/N, a strain highly susceptible to neurodegeneration. These Q175F mice display significant HD-like phenotypes along with sudden early death from fatal seizures. The zQ175 KI allele retains a floxed neomycin resistance cassette upstream of the Htt gene locus and produces dramatically reduced mutant Htt as compared to the endogenous wildtype Htt allele. By intercrossing with mice expressing cre in germ line cells, we have excised the neo cassette from Q175F mice generating a new line, Q175FΔneo (Q175FDN). Removal of the neo cassette resulted in a ∼2 fold increase in mutant Htt and rescue of fatal seizures, indicating that the early death phenotype of Q175F mice is caused by Htt deficiency rather than by mutant Htt. Additionally, Q175FDN mice exhibit earlier onset and a greater variety and severity of HD-like phenotypes than Q175F mice or any previously reported KI HD mouse model, making them valuable for preclinical studies.

HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity.

Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics.

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

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

Sudden death due to paralysis and synaptic and behavioral deficits when Hip14/Zdhhc17 is deleted in adult mice.

BACKGROUND: Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic mice have behavioral and synaptic deficits. However, the phenotype is developmental; thus, a model of post-developmental loss of Hip14 was generated to examine the role of HIP14 in synaptic function in the adult. RESULTS: Ten weeks after Hip14 deletion (iHip14 Δ/Δ ), mice die suddenly from rapidly progressive paralysis. Prior to death the mice exhibit motor deficits, increased escape response during tests of anxiety, anhedonia, a symptom indicative of depressive-like behavior, and striatal synaptic deficits, including reduced probability of transmitter release and increased amplitude but decreased frequency of spontaneous post-synaptic currents. The mice also have increased brain weight due to microgliosis and astrogliosis in the cortex. CONCLUSIONS: Behavioral changes and electrophysiological measures suggest striatal dysfunction in iHip14 Δ/Δ mice, and increased cortical volume due to astrogliosis and microgliosis suggests a novel role for HIP14 in glia. These data suggest that HIP14 is essential for maintenance of life and neuronal integrity in the adult mouse.

Huntingtin Haplotypes Provide Prioritized Target Panels for Allele-specific Silencing in Huntington Disease Patients of European Ancestry.

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene (HTT). Heterozygous polymorphisms in cis with the mutation allow for allele-specific suppression of the pathogenic HTT transcript as a therapeutic strategy. To prioritize target selection, precise heterozygosity estimates are needed across diverse HD patient populations. Here we present the first comprehensive investigation of all common target alleles across the HTT gene, using 738 reference haplotypes from the 1000 Genomes Project and 2364 haplotypes from HD patients and relatives in Canada, Sweden, France, and Italy. The most common HD haplotypes (A1, A2, and A3a) define mutually exclusive sets of polymorphisms for allele-specific therapy in the greatest number of patients. Across all four populations, a maximum of 80% are treatable using these three target haplotypes. We identify a novel deletion found exclusively on the A1 haplotype, enabling potent and selective silencing of mutant HTT in approximately 40% of the patients. Antisense oligonucleotides complementary to the deletion reduce mutant A1 HTT mRNA by 78% in patient cells while sparing wild-type HTT expression. By suppressing specific haplotypes on which expanded CAG occurs, we demonstrate a rational approach to the development of allele-specific therapy for a monogenic disorder.

Bidirectional control of postsynaptic density-95 (PSD-95) clustering by Huntingtin.

Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution.

Anti-semaphorin 4D immunotherapy ameliorates neuropathology and some cognitive impairment in the YAC128 mouse model of Huntington disease.

Huntington disease (HD) is an inherited, fatal neurodegenerative disease with no disease-modifying therapy currently available. In addition to characteristic motor deficits and atrophy of the caudate nucleus, signature hallmarks of HD include behavioral abnormalities, immune activation, and cortical and white matter loss. The identification and validation of novel therapeutic targets that contribute to these degenerative cellular processes may lead to new interventions that slow or even halt the course of this insidious disease. Semaphorin 4D (SEMA4D) is a transmembrane signaling molecule that modulates a variety of processes central to neuroinflammation and neurodegeneration including glial cell activation, neuronal growth cone collapse and apoptosis of neural precursors, as well as inhibition of oligodendrocyte migration, differentiation and process formation. Therefore, inhibition of SEMA4D signaling could reduce CNS inflammation, increase neuronal outgrowth and enhance oligodendrocyte maturation, which may be of therapeutic benefit in the treatment of several neurodegenerative diseases, including HD. To that end, we evaluated the preclinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody, which prevents the interaction between SEMA4D and its receptors, in the YAC128 transgenic HD mouse model. Anti-SEMA4D treatment ameliorated neuropathological signatures, including striatal atrophy, cortical atrophy, and corpus callosum atrophy and prevented testicular degeneration in YAC128 mice. In parallel, a subset of behavioral symptoms was improved in anti-SEMA4D treated YAC128 mice, including reduced anxiety-like behavior and rescue of cognitive deficits. There was, however, no discernible effect on motor deficits. The preservation of brain gray and white matter and improvement in behavioral measures in YAC128 mice treated with anti-SEMA4D suggest that this approach could represent a viable therapeutic strategy for the treatment of HD. Importantly, this work provides in vivo demonstration that inhibition of pathways initiated by SEMA4D constitutes a novel approach to moderation of neurodegeneration.

Hip14l-deficient mice develop neuropathological and behavioural features of Huntington disease.

Palmitoylation, the dynamic post-translational addition of the lipid, palmitate, to proteins by Asp-His-His-Cys-containing palmitoyl acyltransferase (PAT) enzymes, modulates protein function and localization and plays a key role in the nervous system. Huntingtin-interacting protein 14 (HIP14), a well-characterized neuronal PAT, has been implicated in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disease associated with motor, psychiatric and cognitive symptoms, caused by a CAG expansion in the huntingtin gene (HTT). Mice deficient for Hip14 expression develop neuropathological and behavioural features similar to HD, and the catalytic activity of HIP14 is impaired in HD mice, most likely due to the reduced interaction of HIP14 with HTT. Huntingtin-interacting protein 14-like (HIP14L) is a paralog of HIP14, with identical domain structure. Together, HIP14 and HIP14L are the major PATs for HTT. Here, we report the characterization of a Hip14l-deficient mouse model, which develops adult-onset, widespread and progressive neuropathology accompanied by early motor deficits in climbing, impaired motor learning and reduced palmitoylation of a novel HIP14L substrate: SNAP25. Although the phenotype resembles that of the Hip14(-/-) mice, a more progressive phenotype, similar to that of the YAC128 transgenic mouse model of HD, is observed. In addition, HIP14L interacts less with mutant HTT than the wild-type protein, suggesting that reduced HIP14L-dependent palmitoylation of neuronal substrates may contribute to the pathogenesis of HD. Thus, both HIP14 and HIP14L may be dysfunctional in the disease.

A fully humanized transgenic mouse model of Huntington disease.

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh(-/-) background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT.

In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.