Huntington's Disease Knock-in Mice Research Articles

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and guide RNAs (gRNAs) efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased in the liver by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion strategies in HD and potentially other repeat expansion disorders.

Identification of genetic modifiers of Huntington's disease somatic CAG repeat instability by in vivo CRISPR-Cas9 genome editing.

Huntington's disease (HD), one of >50 inherited repeat expansion disorders (Depienne and Mandel, 2021), is a dominantly-inherited neurodegenerative disease caused by a CAG expansion in HTT (The Huntington's Disease Collaborative Research Group, 1993). Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the property driving disease is the CAG length-dependent propensity of the repeat to further expand in brain (Swami et al ., 2009; GeM-HD, 2015; Hensman Moss et al ., 2017; Ciosi et al ., 2019; GeM-HD, 2019; Hong et al ., 2021). Routes to slowing somatic CAG expansion therefore hold great promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair (MMR) pathway, modify somatic expansion in HD mouse models (Wheeler and Dion, 2021). To identify novel modifiers of somatic expansion, we have used CRISPR-Cas9 editing in HD knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This has included testing of HD onset modifier genes emerging from human genome-wide association studies (GWAS), as well as interactions between modifier genes, thereby providing new insight into pathways underlying CAG expansion and potential therapeutic targets.

Comparing HD knockin pigs and mice reveals the pathological role of IL-17

Our previous work has established a knockin (KI) pig model of Huntington's disease (HD) that can replicate the typical pathological features of HD, including selective striatal neuronal loss, reactive gliosis, and axonal degeneration. However, HD KI mice exhibit milder neuropathological phenotypes and lack overt neurodegeneration. By performing RNA sequencing to compare the gene expression profiles between HD KI pigs and mice, we find that genes related to interleukin-17 (IL-17) signaling are upregulated in the HD pig brains compared to the mouse brains. Delivery of IL-17 into the brain striatum of HD KI mice causes greater reactive gliosis and synaptic deficiency compared to HD KI mice that received PBS. These findings suggest that the upregulation of genes related to IL-17 signaling in HD pig brains contributes to severe glial pathology in HD and identify this as a potential therapeutic target for treating HD.

A Specific Mini-Intrabody Mediates Lysosome Degradation of Mutant Huntingtin.

Accumulation of misfolded proteins leads to many neurodegenerative diseases that can be treated by lowering or removing mutant proteins. Huntington's disease (HD) is characterized by the intracellular accumulation of mutant huntingtin (mHTT) that can be soluble and aggregated in the central nervous system and causes neuronal damage and death. Here, an intracellular antibody (intrabody) fragment is generated that can specifically bind mHTT and link to the lysosome for degradation. It is found that delivery of this peptide by either brain injection or intravenous administration can efficiently clear the soluble and aggregated mHTT by activating the lysosomal degradation pathway, resulting in amelioration of gliosis and dyskinesia in HD knock-in (KI-140Q) mice. These findings suggest that the small intrabody peptide linked to lysosomes can effectively lower mutant proteins and provide a new approach for treating neurodegenerative diseases that are caused by the accumulation of mutant proteins.

Rhes depletion promotes striatal accumulation and aggregation of mutant huntingtin in a presymptomatic HD mouse model.

Huntington's disease (HD) is caused by CAG trinucleotide repeats in the HTT gene. Selective neurodegeneration in the striatum is prominent in HD, despite widespread expression of mutant HTT (mHTT). Ras homolog enriched in the striatum (Rhes) is a GTP-binding protein enriched in the striatum, involved in dopamine-related behaviors and autophagy regulation. Growing evidence suggests Rhes plays a critical role in the selective striatal degeneration in HD, but its specific function in this context remains complex and controversial. In this study, we utilized CRISPR/Cas9 to knockdown Rhes at different disease stages through adeno-associated virus (AAV) transduction in HD knock-in (KI) mice. Immunoblotting and immunofluorescence were employed to assess the impact of Rhes depletion on mHTT levels, neuronal loss, astrogliosis and autophagy activity. Rhes depletion in 22-week-old HD KI mice (representing the presymptomatic stage) led to mHTT accumulation, reduced neuronal cell staining, and increased astrogliosis. However, no such effects were observed in 36-week-old HD KI mice (representing the symptomatic stage). Additionally, Rhes deletion in 22-week-old HD KI mice resulted in increased P62 levels, reduced LC3-II levels, and unchanged phosphorylation of mTOR and beclin-1, unchanged mTOR protein level, except for a decrease in beclin-1. Our findings suggest that knockdown Rhes promotes striatal aggregation of mutant huntingtin by reducing autophagy activity in a mTOR-independent manner. Rhes plays a protective role during the presymptomatic stage of HD KI mice.

Mutant HTT does not affect glial development but impairs myelination in the early disease stage.

Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) and is characterized by late-onset neurodegeneration that primarily affects the striatum. Several studies have shown that mutant HTT can also affect neuronal development, contributing to the late-onset neurodegeneration. However, it is currently unclear whether mutant HTT impairs the development of glial cells, which is important for understanding whether mutant HTT affects glial cells during early brain development. Using HD knock-in mice that express full-length mutant HTT with a 140 glutamine repeat at the endogenous level, we analyzed the numbers of astrocytes and oligodendrocytes from postnatal day 1 to 3 months of age via Western blotting and immunocytochemistry. We also performed electron microscopy, RNAseq analysis, and quantitative RT-PCR. The numbers of astrocytes and oligodendrocytes were not significantly altered in postnatal HD KI mice compared to wild type (WT) mice. Consistently, glial protein expression levels were not significantly different between HD KI and WT mice. However, at 3 months of age, myelin protein expression was reduced in HD KI mice, as evidenced by Western blotting and immunocytochemical results. Electron microscopy revealed a slight but significant reduction in myelin thickness of axons in the HD KI mouse brain at 3 months of age. RNAseq analysis did not show significant reductions in myelin-related genes in postnatal HD KI mice. These data suggest that cytoplasmic mutant HTT, rather than nuclear mutant HTT, mediates myelination defects in the early stages of the disease without impacting the differentiation and maturation of glial cells.

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington’s disease

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing (“early treatment”) or reversing (“late treatment”) HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic (“early treatment”) or symptomatic (“late treatment”) stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments (“2 cycle treatment”) lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. Data availabilityThis study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Lack of association of somatic CAG repeat expansion with striatal neurodegeneration in HD knock-in animal models.

Our previous work has established a huntingtin knock-in (KI) pig model that displays striatal neuronal loss, allowing us to examine if somatic CAG expansion in striatum accounts for the preferential neurodegeneration in Huntington disease (HD). We found that HD KI pigs do not display somatic CAG expansion in striatum as HD KI mice and that the majority of polyQ repeats in exon 1 HTT in the striatum of HD KI mice are fairly stable. We also found that striatal MSH2 and MLH3, which are involved in DNA repair, are more abundant in mouse brains than pig brains. Consistently inhibiting MSH2 and MLH3 reduced the somatic CAG expansion in HD KI mouse striatum with no influence on neuropathology. Our findings suggest that somatic CAG expansion is species-dependent, occurs in a small fraction of the HD gene in mice, and does not critically contribute to HD neuropathology.

Increasing brain palmitoylation rescues behavior and neuropathology in Huntington disease mice.

Huntington disease (HD) damages the corticostriatal circuitry in large part by impairing transport of brain-derived neurotrophic factor (BDNF). We hypothesized that improving vesicular transport of BDNF could slow or prevent disease progression. We therefore performed selective proteomic analysis of vesicles transported within corticostriatal projecting neurons followed by in silico screening and identified palmitoylation as a pathway that could restore defective huntingtin-dependent trafficking. Using a synchronized trafficking assay and an HD network-on-a-chip, we found that increasing brain palmitoylation via ML348, which inhibits the palmitate-removing enzyme acyl-protein thioesterase 1 (APT1), restores axonal transport, synapse homeostasis, and survival signaling to wild-type levels without toxicity. In human HD induced pluripotent stem cell-derived cortical neurons, ML348 increased BDNF trafficking. In HD knock-in mice, it efficiently crossed the blood-brain barrier to restore palmitoylation levels and reverse neuropathology, locomotor deficits, and anxio-depressive behaviors. APT1 and its inhibitor ML348 thus hold therapeutic interest for HD.

Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via splice redirection.

Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.

Gossypol, a novel modulator of VCP, induces autophagic degradation of mutant huntingtin by promoting the formation of VCP/p97-LC3-mHTT complex.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by toxic aggregates of mutant huntingtin protein (mHTT) in the brain. Decreasing mHTT is a potential strategy for therapeutic purpose of HD. Valosin-containing protein (VCP/p97) is a crucial regulator of proteostasis, which regulates the degradation of damaged protein through proteasome and autophagy pathway. Since VCP has been implicated in pathogenesis of HD as well as other neurodegenerative diseases, small molecules that specifically regulate the activity of VCP may be of therapeutic benefits for HD patients. In this study we established a high-throughput screening biochemical assay for VCP ATPase activity measurement and identified gossypol, a clinical approved drug in China, as a novel modulator of VCP. Gossypol acetate dose-dependently inhibited the enzymatic activity of VCP in vitro with IC50 of 6.53±0.6 μM. We further demonstrated that gossypol directly bound to the interface between the N and D1 domains of VCP. Gossypol acetate treatment not only lowered mHTT levels and rescued HD-relevant phenotypes in HD patient iPS-derived Q47 striatal neurons and HD knock-in mouse striatal cells, but also improved motor function deficits in both Drosophila and mouse HD models. Taken together, gossypol acetate acted through a gain-of-function way to induce the formation of VCP-LC3-mHTT ternary complex, triggering autophagic degradation of mHTT. This study reveals a new strategy for treatment of HD and raises the possibility that an existing drug can be repurposed as a new treatment of neurodegenerative diseases.

Progression of basal ganglia pathology in heterozygous Q175 knock-in Huntington's disease mice.

We used behavioral testing and morphological methods to detail the progression of basal ganglia neuron type‐specific pathology and the deficits stemming from them in male heterozygous Q175 mice, compared to age‐matched WT males. A rotarod deficit was not present in Q175 mice until 18 months, but increased open field turn rate (reflecting hyperkinesia) and open field anxiety were evident at 6 months. No loss of striatal neurons was seen out to 18 months, but ENK+ and DARPP32+ striatal perikarya were fewer by 6 months, due to diminished expression, with further decline by 18 months. No reduction in SP+ striatal perikarya or striatal interneurons was seen in Q175 mice at 18 months, but cholinergic interneurons showed dendrite attenuation by 6 months. Despite reduced ENK expression in indirect pathway striatal perikarya, ENK‐immunostained terminals in globus pallidus externus (GPe) were more abundant at 6 months and remained so out to 18 months. Similarly, SP‐immunostained terminals from striatal direct pathway neurons were more abundant in globus pallidus internus and substantia nigra at 6 months and remained so at 18 months. FoxP2+ arkypallidal GPe neurons and subthalamic nucleus neurons were lost by 18 months but not prototypical PARV+ GPe neurons or dopaminergic nigral neurons. Our results show that striatal projection neuron abnormalities and behavioral abnormalities reflecting them develop between 2 and 6 months of age in Q175 male heterozygotes, indicating early effects of the HD mutation. The striatal pathologies resemble those in human HD, but are less severe at 18 months than even in premanifest HD.

Loss of Hap1 selectively promotes striatal degeneration in Huntington disease mice

Huntington disease (HD) is an ideal model for investigating selective neurodegeneration, as expanded polyQ repeats in the ubiquitously expressed huntingtin (HTT) cause the preferential neurodegeneration in the striatum of the HD patient brains. Here we report that adeno-associated virus (AAV) transduction-mediated depletion of Hap1, the first identified huntingtin-associated protein, in adult HD knock-in (KI) mouse brains leads to selective neuronal loss in the striatum. Further, Hap1 depletion-mediated neuronal loss via AAV transduction requires the presence of mutant HTT. Rhes, a GTPase that is enriched in the striatum and sumoylates mutant HTT to mediate neurotoxicity, binds more N-terminal HTT when Hap1 is deficient. Consistently, more soluble and sumoylated N-terminal HTT is presented in HD KI mouse striatum when HAP1 is absent. Our findings suggest that both Rhes and Hap1 as well as cellular stress contribute to the preferential neurodegeneration in HD, highlighting the involvement of multiple factors in selective neurodegeneration.

Protein changes in synaptosomes of Huntington's disease knock-in mice are dependent on age and brain region

Molecular changes at synapses are thought to underly the deficits in motor and cognitive dysfunction seen in Huntington's disease (HD). Previously we showed in synaptosome preparations age dependent changes in levels of selected proteins examined by western blot assay in the striatum of Q140/Q140 HD mice. To assess if CAG repeat length influenced protein changes at the synapse, we examined synaptosomes from 6-month old heterozygote HD mice with CAG repeat lengths ranging from 50 to 175. Analysis of 19 selected proteins showed that increasing CAG repeat length in huntingtin (HTT) increased the number of affected proteins in HD striatal synaptosomes. Moreover, SDS-soluble total HTT (WT plus mutant HTT) and pThr3 HTT were reduced with increasing CAG repeat length, and there was no pSer421 mutant HTT detected in any HD mice. A LC-MS/MS and bioinfomatics study of synaptosomes from 2 and 6-month old striatum and cortex of Q140/Q7 HD mice showed enrichment of synaptic proteins and an influence of age, gender and brain region on the number of protein changes. HD striatum at 6 months had the most protein changes that included many HTT protein interactors, followed by 2-month old HD striatum, 2-month old HD cortex and 6-month HD cortex. SDS-insoluble mutant HTT was detected in HD striatal synaptosomes consistent with the presence of aggregates. Proteins changed in cortex differed from those in striatum. Pathways affected in HD striatal synaptosomes that were not identified in whole striatal lysates of the same HD mouse model included axon guidance, focal adhesion, neurotrophin signaling, regulation of actin cytoskeleton, endocytosis, and synaptic vesicle cycle. Results suggest that synaptosomes prepared from HD mice are highly informative for monitoring protein changes at the synapse and may be preferred for assessing the effects of experimental therapies on synaptic function in HD.

Loss of huntingtin function slows synaptic vesicle endocytosis in striatal neurons from the httQ140/Q140 mouse model of Huntington's disease

Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (httQ140/Q140). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in httQ140/Q140 neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in httQ140/Q140 neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous httQ140/Q140 neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.

Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains

Huntington's disease is caused by a polyglutamine repeat expansion in the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington's disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804, and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804, and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction whereas K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared with wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold changes in the soluble fraction revealed that diGly site abundances of most of the proteins were not related to protein fold changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington's disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin and ubiquitin-regulated processes in Huntington's disease.

The Onset and Progression of Hippocampal Synaptic Plasticity Deficits in the Q175FDN Mouse Model of Huntington Disease.

Huntington disease (HD) is an inherited neurodegenerative disease characterized by a clinical triad of motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Homozygosity for the HD-causing mutation is extremely rare; thus, the majority of HD patients express the mutant huntingtin protein in addition to reduced levels of the non-pathogenic huntingtin protein. Deficits in synaptic plasticity, including hippocampal long-term potentiation (LTP), have been identified in various mouse models of HD and are thought to contribute to the debilitating cognitive symptoms associated with the disease. However, the bulk of these studies used N-terminal fragment or homozygous knock-in mouse models of HD at symptomatic ages, and our understanding of the onset and progression of synaptic plasticity deficits in the HD brain is lacking. To better understand the time-course of synaptic plasticity deficits in HD, as well as the impact of heterozygous and homozygous huntingtin mutations, we quantified basal synaptic connectivity, presynaptic release probability, presynaptically mediated post-tetanic potentiation (PTP) and postsynaptically mediated LTP at presymptomatic, early symptomatic and late symptomatic ages in heterozygous and homozygous Q175FDN knock-in HD mice. Our results demonstrate clear age-dependent effects of the HD-causing mutation on both short and long-term plasticity that generally emerge earlier in homozygous mice. Interestingly, deficits in presynaptic short-term plasticity were more closely linked to disease progression than deficits in postsynaptic LTP, and heterozygous mice were more susceptible to an LTP deficit when induced by high frequency stimulation compared to theta burst stimulation. To the best of our knowledge, the present study represents the most thorough characterization to date of the onset and progression of hippocampal synaptic plasticity deficits in a mouse model of HD, and should prove valuable to future studies exploring cellular mechanisms underlying the debilitating cognitive decline in HD.

Phenotype onset in Huntington's disease knock-in mice is correlated with the incomplete splicing of the mutant huntingtin gene.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat within the huntingtin (HTT) gene. The Q140 and HdhQ150 knock‐in HD mouse models were generated such that HdhQ150 mice have an expanded CAG repeat inserted into the mouse Htt gene, whereas in the Q140s, mouse exon 1 Htt was replaced with a mutated version of human exon 1. By standardizing mouse strain background, breeding to homozygosity and employing sensitive behavioral tests, we demonstrate that the onset of behavioral phenotypes occurs earlier in the Q140 than the HdhQ150 knock‐in mouse models and that huntingtin (HTT) aggregation appears earlier in the striata of Q140 mice. We have previously found that the incomplete splicing of mutant HTT from exon 1 to exon 2 results in the production of a small polyadenylated transcript that encodes the highly pathogenic mutant HTT exon 1 protein. In this report, we have identified a functional consequence of the sequence differences between these two models at the RNA level, in that the level of incomplete splicing, and of the mutant exon 1 HTT protein, are greater in the brains of Q140 mice. While differences in the human and mouse exon 1 HTT proteins (e.g., proline rich sequences) could also contribute to the phenotypic differences, our data indicate that the incomplete splicing of HTT and approaches to lower the levels of the exon 1 HTT transcript should be pursued as therapeutic targets.

Inhibition of Drp1 hyperactivation reduces neuropathology and behavioral deficits in zQ175 knock-in mouse model of Huntington's disease.

Mitochondrial dysfunction manifests in the pathogenesis of Huntington's disease (HD), a fatal and inherited neurodegenerative disease. Dynamin-related protein 1 (Drp1) is the primary component of mitochondrial fission and becomes hyperactivated in various models of HD. We previously reported that inhibition of Drp1 hyperactivation by P110, a rationally designed peptide inhibitor of Drp1-Fis1 interaction, is protective in the HD R6/2 mouse model, which expresses a fragment of mutant Huntingtin (mHtt). In this study, we expand our work to test the effect of P110 treatment in HD knock-in (zQ175 KI) mice that express full-length mtHtt and exhibit progressive disease symptoms, reminiscent of human HD. We find that subcutaneously sustained treatment with P110 reduces movement deficits of mice. Moreover, the treatment attenuates striatal neuronal loss, microglial hyperactivity and white matter disorganization in zQ175 KI mice. These findings provide an additional line of evidence that inhibition of Drp1 hyperactivation is sufficient to reduce HD-associated neuropathology and behavioral deficits. We propose that manipulation of Drp1 hyperactivation might be a useful strategy to develop therapeutics for treating HD.

The long non-coding RNA NEAT1 is elevated in polyglutamine repeat expansion diseases and protects from disease gene-dependent toxicities.

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.