Knock-in Mouse Model Of Huntington's Disease Research Articles

Altered mitochondrial Ca2+ uptake in presynaptic terminals of cultured striatal and cortical neurons from the zQ175 knock-in mouse model of Huntington's disease

Calcium (Ca2+) in mitochondria plays crucial roles in neurons including modulating metabolic processes. Moreover, excessive Ca2+ in mitochondria can lead to cell death. Thus, altered mitochondrial Ca2+ regulation has been implicated in several neurodegenerative diseases including Huntington's disease (HD). HD is a progressive hereditary neurodegenerative disorder that results from abnormally expanded cytosine-adenine-guanine trinucleotide repeats in the huntingtin gene. One neuropathological hallmark of HD is neuronal loss in the striatum and cortex. However, mechanisms underlying selective loss of striatal and cortical neurons in HD remain elusive. Here, we measured the basal Ca2+ levels and Ca2+ uptake in single presynaptic mitochondria during 100 external electrical stimuli using highly sensitive mitochondria-targeted Ca2+ indicators in cultured cortical and striatal neurons of a knock-in mouse model of HD (zQ175 mice). We observed elevated presynaptic mitochondrial Ca2+ uptake during 100 electrical stimuli in HD cortical neurons compared with wild-type (WT) cortical neurons. We also found the highly elevated presynaptic mitochondrial basal Ca2+ level and Ca2+ uptake during 100 stimuli in HD striatal neurons. The elevated presynaptic mitochondrial basal Ca2+ level in HD striatal neurons and Ca2+ uptake during stimulation in HD striatal and cortical neurons can disrupt neurotransmission and induce mitochondrial Ca2+ overload, eventually leading to neuronal death in the striatum and cortex of HD.

Progressive alterations in polysomal architecture and activation of ribosome stalling relief factors in a mouse model of Huntington's disease

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.

Altered anterograde axonal transport of mitochondria in cultured striatal neurons of a knock-in mouse model of Huntington's disease

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.

Altered exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of cultured striatal neurons in a knock-in mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive dominantly inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine (CAG) trinucleotide repeat in the huntingtin gene, which encodes the mutant huntingtin protein containing an expanded polyglutamine tract. One of neuropathologic hallmarks of HD is selective degeneration in the striatum. Mechanisms underlying selective neurodegeneration in the striatum of HD remain elusive. Neurodegeneration is suggested to be preceded by abnormal synaptic transmission at the early stage of HD. However, how mutant huntingtin protein affects synaptic vesicle exocytosis at single presynaptic terminals of HD striatal neurons is poorly understood. Here, we measured synaptic vesicle exocytosis at single presynaptic terminals of cultured striatal neurons (mainly inhibitory neurons) in a knock-in mouse model of HD (zQ175) during electrical field stimulation using real-time imaging of FM 1-43 (a lipophilic dye). We found a significant decrease in bouton density and exocytosis of synaptic vesicles at single presynaptic terminals in cultured striatal neurons. Real-time imaging of VGAT-CypHer5E (a pH sensitive dye conjugated to an antibody against vesicular GABA transporter (VGAT)) for inhibitory synaptic vesicles revealed a reduction in bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of HD striatal neurons. Thus, our results suggest that the mutant huntingtin protein decreases bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of striatal neurons, causing impaired inhibitory synaptic transmission, eventually leading to the neurodegeneration in the striatum of HD.

VGLUT3 Deletion Rescues Motor Deficits and Neuronal Loss in the zQ175 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease characterized by progressive motor and cognitive impairments, with no disease-modifying therapies yet available. HD pathophysiology involves evident impairment in glutamatergic neurotransmission leading to severe striatal neurodegeneration. The vesicular glutamate transporter-3 (VGLUT3) regulates the striatal network that is centrally affected by HD. Nevertheless, current evidence on the role of VGLUT3 in HD pathophysiology is lacking. Here, we crossed mice lacking Slc17a8 gene (VGLUT3 -/-) with heterozygous zQ175 knock-in mouse model of HD (zQ175:VGLUT3 -/-). Longitudinal assessment of motor and cognitive functions from 6 to 15 months of age reveals that VGLUT3 deletion rescues motor coordination and short-term memory deficits in both male and female zQ175 mice. VGLUT3 deletion also rescues neuronal loss likely via the activation of Akt and ERK1/2 in the striatum of zQ175 mice of both sexes. Interestingly, the rescue in neuronal survival in zQ175:VGLUT3 -/- mice is accompanied by a reduction in the number of nuclear mutant huntingtin (mHTT) aggregates with no change in the total aggregate levels or microgliosis. Collectively, these findings provide novel evidence that VGLUT3, despite its limited expression, can be a vital contributor to HD pathophysiology and a viable target for HD therapeutics.SIGNIFICANCE STATEMENT Dysregulation of the striatal network centrally contributes to the pathophysiology of Huntington's disease (HD). The atypical vesicular glutamate transporter-3 (VGLUT3) has been shown to regulate several major striatal pathologies, such as addiction, eating disorders, or L-DOPA-induced dyskinesia. Yet, our understanding of VGLUT3's role in HD remains unclear. We report here that deletion of the Slc17a8 (Vglut3) gene rescues the deficits in both motor and cognitive functions in HD mice of both sexes. We also find that VGLUT3 deletion activates neuronal survival signaling and reduces nuclear aggregation of abnormal huntingtin proteins and striatal neuron loss in HD mice. Our novel findings highlight the vital contribution of VGLUT3 in HD pathophysiology that can be exploited for HD therapeutic management.

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.

Deuterium-reinforced linoleic acid lowers lipid peroxidation and mitigates cognitive impairment in the Q140 knock in mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease which has no effective treatment and is characterized by psychiatric disorders, motor alterations, and dementia, with the cognitive deficits representing a devastating aspect of the disorder. Oxidative stress and elevated levels of lipid peroxidation (LPO) products are found in mouse models and patients with HD, suggesting that strategies to reduce LPO may be beneficial in HD. In contrast with traditional antioxidants, substituting hydrogen with deuterium at bis-allylic sites in polyunsaturated fatty acids (D-PUFA) decreases the rate-limiting initiation step of PUFA autoxidation, a strategy that has shown benefits in other neurodegenerative diseases. Here, we investigated the effect of D-PUFA treatment in a knock-in mouse model of HD (Q140) which presents motor deficits and neuropathology from a few months of age, and progressive cognitive decline. Q140 knock-in mice were fed a diet containing either D- or H-PUFAs for 5 months starting at 1 month of age. D-PUFA treatment significantly decreased F2 -isoprostanes in the striatum by approximately 80% as compared to H-PUFA treatment and improved performance in novel object recognition tests, without significantly changing motor deficits or huntingtin aggregation. Therefore, D-PUFA administration represents a promising new strategy to broadly reduce rates of LPO, and may be useful in improving a subset of the core deficits in HD.

Striatal Mutant Huntingtin Protein Levels Decline with Age in Homozygous Huntington's Disease Knock-In Mouse Models.

Background:Huntington’s disease (HD) is a progressive neurodegenerative disorder associated with aging, caused by an expanded polyglutamine (polyQ) repeat within the Huntingtin (HTT) protein. In HD, degeneration of the striatum and atrophy of the cortex are observed while cerebellum is less affected.Objective:To test the hypothesis that HTT protein levels decline with age, which together with HTT mutation could influence disease progression.Methods:Using whole brain cell lysates, a unique method of SDS-PAGE and western analysis was used to quantitate HTT protein, which resolves as a monomer and as a high molecular weight species that is modulated by the presence of transglutaminase 2. HTT levels were measured in striatum, cortex and cerebellum in congenic homozygous Q140 and HdhQ150 knock-in mice and WT littermate controls.Results:Mutant HTT in both homozygous knock-in HD mouse models and WT HTT in control striatal and cortical tissues significantly declined in a progressive manner over time. Levels of mutant HTT in HD cerebellum remained high during aging.Conclusions:A general decline in mutant HTT levels in striatum and cortex is observed that may contribute to disease progression in homozygous knock-in HD mouse models through reduction of HTT function. In cerebellum, sustained levels of mutant HTT with aging may be protective to this tissue which is less overtly affected in HD.

Early Downregulation of p75NTR by Genetic and Pharmacological Approaches Delays the Onset of Motor Deficits and Striatal Dysfunction in Huntington's Disease Mice.

Deficits in striatal brain-derived neurotrophic factor (BDNF) delivery and/or BDNF/tropomyosin receptor kinase B (TrkB) signaling may contribute to neurotrophic support reduction and selective early degeneration of striatal medium spiny neurons in Huntington's disease (HD). Furthermore, we and others have demonstrated that TrkB/p75NTR imbalance in vitro increases the vulnerability of striatal neurons to excitotoxic insults and induces corticostriatal synaptic alterations. We have now expanded these studies by analyzing the consequences of BDNF/TrkB/p75NTR imbalance in the onset of motor behavior and striatal neuropathology in HD mice. Our findings demonstrate for the first time that the onset of motor coordination abnormalities, in a full-length knock-in HD mouse model (KI), correlates with the reduction of BDNF and TrkB levels, along with an increase in p75NTR expression. Genetic normalization of p75NTR expression in KI mutant mice delayed the onset of motor deficits and striatal neuropathology, as shown by restored levels of striatal-enriched proteins and dendritic spine density and reduced huntingtin aggregation. We found that the BDNF/TrkB/p75NTR imbalance led to abnormal BDNF signaling, manifested as a diminished activation of TrkB-phospholipase C-gamma pathway but upregulation of c-Jun kinase pathway. Moreover, we confirmed the contribution of the proper balance of BDNF/TrkB/p75NTR on HD pathology by a pharmacological approach using fingolimod. We observed that chronic infusion of fingolimod normalizes p75NTR levels, which is likely to improve motor coordination and striatal neuropathology in HD transgenic mice. We conclude that downregulation of p75NTR expression can delay disease progression suggesting that therapeutic approaches aimed to restore the balance between BDNF, TrkB, and p75NTR could be promising to prevent motor deficits in HD.

Cell-Specific Deletion of PGC-1α from Medium Spiny Neurons Causes Transcriptional Alterations and Age-Related Motor Impairment.

Multiple lines of evidence indicate that a reduction in the expression and function of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is associated with neurodegeneration in diseases such as Huntington's disease (HD). Polymorphisms in the PGC-1α gene modify HD progression and PGC-1α expression is reduced in striatal medium spiny neurons (MSNs) of HD patients and mouse models. However, neither the MSN-specific function of PGC-1α nor the contribution of PGC-1α deficiency to motor dysfunction is known. We identified novel, PGC-1α-dependent transcripts involved in RNA processing, signal transduction, and neuronal morphology and confirmed reductions in these transcripts in male and female mice lacking PGC-1α specifically in MSNs, indicating a cell-autonomous effect in this population. MSN-specific PGC-1α deletion caused reductions in previously identified neuronal and metabolic PGC-1α-dependent genes without causing striatal vacuolizations. Interestingly, these mice exhibited a hypoactivity with age, similar to several HD animal models. However, these newly identified PGC-1α-dependent genes were upregulated with disease severity and age in knock-in HD mouse models independent of changes in PGC-1α transcript, contrary to what would be predicted from a loss-of-function etiological mechanism. These data indicate that PGC-1α is necessary for MSN transcriptional homeostasis and function with age and that, whereas PGC-1α loss in MSNs does not replicate an HD-like phenocopy, its downstream genes are altered in a repeat-length and age-dependent fashion. Understanding the additive effects of PGC-1α gene functional variation and mutant huntingtin on transcription in this cell type may provide insight into the selective vulnerability of MSNs in HD.SIGNIFICANCE STATEMENT Reductions in peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)-mediated transcription have been implicated in the pathogenesis of Huntington's disease (HD). We show that, although PGC-1α-dependent transcription is necessary to maintain medium spiny neuron (MSN) function with age, its loss is insufficient to cause striatal atrophy in mice. We also highlight a set of genes that can serve as proxies for PGC-1α functional activity in the striatum for target engagement studies. Furthermore, we demonstrate that PGC-1α-dependent genes are upregulated in a dose- and age-dependent fashion in HD mouse models, contrary to what would be predicted from a loss-of-function etiological mechanism. However, given this role for PGC-1α in MSN transcriptional homeostasis, it is important to consider how genetic variation in PGC-1α could contribute to mutant-huntingtin-induced cell death and disease progression.

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models

Most neurodegenerative disorders are associated with accumulation of disease-relevant proteins. Among them, Huntington disease (HD) is of particular interest because of its monogenetic nature. HD is mainly caused by cytotoxicity of the defective protein encoded by the mutant Huntingtin gene (HTT). Thus, lowering mutant HTT protein (mHTT) levels would be a promising treatment strategy for HD. Here we report two kinases HIPK3 and MAPK11 as positive modulators of mHTT levels both in cells and in vivo. Both kinases regulate mHTT via their kinase activities, suggesting that inhibiting these kinases may have therapeutic values. Interestingly, their effects on HTT levels are mHTT-dependent, providing a feedback mechanism in which mHTT enhances its own level thus contributing to mHTT accumulation and disease progression. Importantly, knockout of MAPK11 significantly rescues disease-relevant behavioral phenotypes in a knockin HD mouse model. Collectively, our data reveal new therapeutic entry points for HD and target-discovery approaches for similar diseases.

Differential Alteration in Expression of Striatal GABAAR Subunits in Mouse Models of Huntington's Disease.

Huntington’s disease (HD) is a neurodegenerative disorder characterized by progressive motor symptoms that are preceded by cognitive deficits and is considered as a disorder that primarily affects forebrain striatal neurons. To gain a better understanding of the molecular and cellular mechanisms associated with disease progression, we analyzed the expression of proteins involved in GABAergic neurotransmission in the striatum of the R6/1 transgenic mouse model. Western blot, quantitative PCR and immunohistochemical analyses were conducted on male R6/1 mice and age-matched wild type littermates. Analyses were performed on 2 and 6 month-old animals, respectively, before and after the onset of motor symptoms. Expression of GAD 67, GAD 65, NL2, or gephyrin proteins, involved in GABA synthesis or synapse formation did not display major changes. In contrast, expression of α1, α3 and α5 GABAAR subunits was increased while the expression of δ was decreased, suggesting a change in tonic- and phasic inhibitory transmission. Western blot analysis of the striatum from 8 month-old Hdh Q111, a knock-in mouse model of HD with mild deficits, confirmed the α1 subunit increased expression. From immunohistochemical analyses, we also found that α1 subunit expression is increased in medium-sized spiny projection neurons (MSN) and decreased in parvalbumin (PV)-expressing interneurons at 2 and 6 months in R6/1 mice. Moreover, α2 subunit labeling on the PV and MSN cell membranes was increased at 2 months and decreased at 6 months. Alteration of gene expression in the striatum and modification of GABAA receptor subtypes in both interneurons and projection neurons suggested that HD mutation has a profound effect on synaptic plasticity at an early stage, before the onset of motor symptoms. These results also indicate that cognitive and other behavioral deficits may be associated with changes in GABAergic neurotransmission that consequently could be a relevant target for early therapeutic treatment.

Compromised Dopaminergic Encoding of Reward Accompanying Suppressed Willingness to Overcome High Effort Costs Is a Prominent Prodromal Characteristic of the Q175 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a heritable neurodegenerative disorder caused by expansion of CAG (glutamine) repeats in the HTT gene. A prodromal stage characterized by psychiatric disturbances normally precedes primary motor symptoms and suppressed motivation represents one of the earliest and most common psychiatric symptoms. Although dopamine in the nucleus accumbens (NAc) critically regulates motivation and altered dopamine signaling is implicated in HD, the nature of dopaminergic deficits and contribution to symptoms in HD is poorly understood. We therefore tested whether altered NAc dopamine release accompanies motivational deficits in the Q175 knock-in HD mouse model. Q175 mice express a CAG expansion of the human mutant huntingtin allele in the native mouse genome and gradually manifest symptoms late in life, closely mimicking the genotypic context and disease progression in human HD. Sub-second extracellular dopamine release dynamics were monitored using fast-scan cyclic voltammetry, whereas motivation was assessed using a progressive ratio reinforcement schedule. As the response ratio (lever presses per reward) escalated, Q175 mice exerted less effort to earn fewer rewards versus wild-type (WT). Moreover, dopamine released at reward delivery dynamically encoded increasing reward cost in WT but not Q175 mice. Deficits were specific to situations of high effortful demand as no difference was observed in locomotion, free feeding, hedonic processing, or reward seeking when the response requirement was low. This compromised dopaminergic encoding of reward delivery coincident with suppressed motivation to work for reward in Q175 mice provides novel, neurobiological insight into an established and clinically relevant endophenotype of prodromal HD. Psychiatric impairments in Huntington's disease (HD) typically manifest early in disease progression, before motor deficits. However, the neurobiological factors contributing to psychiatric symptoms are poorly understood. We used a mouse HD model and assessed whether impaired dopamine release in the nucleus accumbens (NAc), a brain region critical to goal-directed behaviors, accompanies motivational deficits, one of the most common early HD symptoms. HD mice exhibited blunted motivation to work for food reward coincident with diminished dopamine release to reward receipt. Motivational and NAc dopaminergic deficits were not associated with gross motor deficits or impaired food seeking when effortful demands were low. This work identifies a specific prodromal HD phenotype associated with a prominent and previously unidentified neurobiological impairment.

Differential loss of thalamostriatal and corticostriatal input to striatal projection neuron types prior to overt motor symptoms in the Q140 knock-in mouse model of Huntington's disease.

Motor slowing and forebrain white matter loss have been reported in premanifest Huntington's disease (HD) prior to substantial striatal neuron loss. These findings raise the possibility that early motor defects in HD may be related to loss of excitatory input to striatum. In a prior study, we showed that in the heterozygous Q140 knock-in mouse model of HD that loss of thalamostriatal axospinous terminals is evident by 4 months, and loss of corticostriatal axospinous terminals is evident at 12 months, before striatal projection neuron pathology. In the present study, we specifically characterized the loss of thalamostriatal and corticostriatal terminals on direct (dSPN) and indirect (iSPN) pathway striatal projection neurons, using immunolabeling to identify thalamostriatal (VGLUT2+) and corticostriatal (VGLUT1+) axospinous terminals, and D1 receptor immunolabeling to distinguish dSPN (D1+) and iSPN (D1−) synaptic targets. We found that the loss of corticostriatal terminals at 12 months of age was preferential for D1+ spines, and especially involved smaller terminals, presumptively of the intratelencephalically projecting (IT) type. By contrast, indirect pathway D1− spines showed little loss of axospinous terminals at the same age. Thalamostriatal terminal loss was comparable for D1+ and D1− spines at both 4 and 12 months. Regression analysis showed that the loss of VGLUT1+ terminals on D1+ spines was correlated with a slight decline in open field motor parameters at 12 months. Our overall results raise the possibility that differential thalamic and cortical input loss to SPNs is an early event in human HD, with cortical loss to dSPNs in particular contributing to premanifest motor slowing.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Loss of caveolin-1 expression in knock-in mouse model of Huntington's disease suppresses pathophysiology in vivo

Loss of cholesterol homeostasis and altered vesicle trafficking have been detected in Huntington's disease (HD) cellular and animal models, yet the role of these dysfunctions in pathophysiology of HD is unknown. We demonstrate here that defects in caveolar-related cholesterol trafficking directly contribute to the mechanism of HD in vivo. We generated new mouse models that express mutant Huntington's protein (mhtt), but have partial or total loss of caveolin-1 (Cav1) expression. Fluorescence resonance energy transfer dequenching confirms a direct interaction between mhtt and Cav1. Mhtt-expressing neurons exhibited cholesterol accumulation and suppressed caveolar-related post-Golgi trafficking from endoplasmic reticulum/Golgi to plasma membrane. Loss or reduction of Cav1 expression in a knock-in HD mouse model rescues the cholesterol phenotype in neurons and significantly delays the onset of motor decline and development of neuronal inclusions. We propose that aberrant interaction between Cav1 and mhtt leads to altered cholesterol homeostasis and plays a direct causative role in the onset of HD pathophysiology in vivo.

Purkinje cell dysfunction and loss in a knock-in mouse model of Huntington Disease

Huntington Disease (HD) is an autosomal dominant neurological disorder characterized by motor, psychiatric and cognitive disturbances. Recent evidence indicates that the viability and function of cerebellar Purkinje cells (PCs) are compromised in an aggressive mouse model of HD. Here we investigate whether this is also the case in the HdhQ200 knock-in mouse model of HD. Using quantitative-real time-PCR and immunofluorescence, we observed a loss of the PC marker and calcium buffer calbindin in 50week-old symptomatic mice. Reductions were also observed in parvalbumin and glutamic acid decarboxylase protein expression, most markedly in the molecular cell layer. Stereological analysis revealed an overall reduction in the PC population in HdhQ200/Q200 mice by nearly 40%, and loose patch electrophysiology of remaining PCs indicated a reduction in firing rate in HD mice compared to control littermates. Taken together, these data demonstrate that PC survival and function are compromised in a mouse model of adult-onset HD and suggest that further experiments should investigate the contribution of PC death and dysfunction to HD-associated motor impairment.

Caspase-6 does not contribute to the proteolysis of mutant huntingtin in the HdhQ150 knock-in mouse model of Huntington’s disease

Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease. Abstract Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease. Abstract Huntington's disease (HD) is a late-onset progressive neurodegenerative disorder characterised by irrepressible motor dysfunction, cognitive decline and psychiatric disturbances for which there is no effective disease-modifying treatment. The proteolytic cleavage of huntingtin (HTT) to generate N-terminal fragments has been proposed to be a key aspect of HD pathogenesis. In particular, it has been shown that HTT can be cleaved at amino acid 586 by caspase-6 (CASP6) and that prevention of cleavage at this site is neuroprotective and can rescue HD-related phenotypes in YAC transgenic HD mouse models. To determine the role that CASP6 plays in HTT proteolysis, we evaluated the effects of the genetic ablation of Casp6 in the HdhQ150 knock-in mouse model of HD. Here we show that the loss of CASP6 had no effect on the proteolysis of HTT, and did not modify the pattern of N-terminal HTT fragments that are present in the brains of these animals. Furthermore, we show that CASP6 ablation does not influence the steady-state levels of soluble HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 is not necessary for HTT proteolysis in the HdhQ150 mouse model of HD, and that targeting CASP6 as a therapeutic strategy should be approached with caution in the context of this complex disease.

Age-Dependent Resistance to Excitotoxicity in Htt CAG140 Mice and the Effect of Strain Background

Mouse strain background can influence vulnerability to excitotoxic neuronal cell death and potentially modulate phenotypes in transgenic mouse models of human disease. Evidence supports a contribution of excitotoxicity to the selective death of medium spiny neurons in Huntington's disease (HD). Here, we assess whether strain differences in excitotoxic vulnerability influence striatal cell death in a knock-in mouse model of HD. Previous studies that evaluated resistance to excitotoxic lesions in several mouse models of HD had variable outcomes. In the present study, we directly compare one model on two different background strains to test the contribution of strain to excitotoxicity-mediated neurodegeneration. Mice of the FVB/N strain, which are highly vulnerable to excitotoxicity, become extremely resistant to quinolinic acid-induced striatal neurodegeneration with age, when carrying a huntingtin (Htt) allele expressing a HD transgene (CAG140). The resistance is much greater than the age-dependent resistance that has been previously reported in YAC128 mice. By 12 months of age, both heterozygous and homozygous FVB.CAG140 mice displayed virtually complete resistance to quinolinic acid-induced striatal neurodegeneration. A similar resistance develops in CAG140 mice on a C57BL/6N background although the effect size is smaller because C57BL/6N mice are already resistant due to genetic background. In a direct comparison with the YAC128 mice, FVB.CAG140 mice have greater resistance. FVB.CAG140 mice are also resistant to neurodegeneration following kainic acid-induced status epilepticus suggesting the existence of a common cellular mechanism that provides protection against multiple types of excitotoxic insult. These findings establish FVB.CAG140 mice as a useful model to investigate the cellular and molecular mechanisms that confer neuroprotection against excitotoxicity.

Longitudinal behavioral, cross-sectional transcriptional and histopathological characterization of a knock-in mouse model of Huntington's disease with 140 CAG repeats

The discovery of the gene mutation responsible for Huntington's disease (HD), huntingtin, in 1993 allowed for a better understanding of the pathology of and enabled the development of animal models. HD is caused by the expansion of a polyglutamine repeat region in the N-terminal of the huntingtin protein. Here we examine the behavioral, transcriptional, histopathological and anatomical characteristics of a knock-in HD mouse model with a 140 polyglutamine expansion in the huntingtin protein. This CAG 140 model contains a portion of the human exon 1 with 140 CAG repeats knocked into the mouse huntingtin gene. We have longitudinally examined the rearing behavior, accelerating rotarod, constant speed rotarod and gait for age-matched heterozygote, homozygote and non-transgenic mice and have found a significant difference in the afflicted mice. However, while there were significant differences between the non-transgenic and the knock-in mice, these behaviors were not progressive. As in HD, we show that the CAG 140 mice also have a significant decrease in striatally enriched mRNA transcripts. In addition, striatal neuronal intranuclear inclusion density increases with age. Lastly these CAG 140 mice show slight cortical thinning compared to non-transgenic mice, similarly to the cortical thinning recently reported in HD.