HdhQ150 Mice Research Articles

Reduction in PA28αβ activation in HD mouse brain correlates to increased mHTT aggregation in cell models.

Huntington's disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αβ disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αβ activated proteasomes in various in vitro models showed that PA28αβ improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αβ in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αβ is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

C57BL/6 Background Attenuates mHTT Toxicity in the Striatum of YAC128 Mice.

Mouse models are frequently used to study Huntington’s disease (HD). The onset and severity of neuronal and behavioral pathologies vary greatly between HD mouse models, which results from different huntingtin expression levels and different CAG repeat length. HD pathology appears to depend also on the strain background of mouse models. Thus, behavioral deficits of HD mice are more severe in the FVB than in the C57BL/6 background. Alterations in medium spiny neuron (MSN) morphology and function have been well documented in young YAC128 mice in the FVB background. Here, we tested the relevance of strain background for mutant huntingtin (mHTT) toxicity on the cellular level by investigating HD pathologies in YAC128 mice in the C57BL/6 background (YAC128/BL6). Morphology, spine density, synapse function and membrane properties were not or only subtly altered in MSNs of 12-month-old YAC128/BL6 mice. Despite the mild cellular phenotype, YAC128/BL6 mice showed deficits in motor performance. More pronounced alterations in MSN function were found in the HdhQ150 mouse model in the C57BL/6 background (HdhQ150/BL6). Consistent with the differences in HD pathology, the number of inclusion bodies was considerably lower in YAC128/BL6 mice than HdhQ150/BL6 mice. This study highlights the relevance of strain background for mHTT toxicity in HD mouse models.

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.

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the HdhQ150/Q150 Mouse Model of Huntington's Disease.

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington’s disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the “clinical horizon” of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments.

Longitudinal in vivo MRI in a Huntington's disease mouse model: Global atrophy in the absence of white matter microstructural damage.

Huntington’s disease (HD) is a genetically-determined neurodegenerative disease. Characterising neuropathology in mouse models of HD is commonly restricted to cross-sectional ex vivo analyses, beset by tissue fixation issues. In vivo longitudinal magnetic resonance imaging (MRI) allows for disease progression to be probed non-invasively. In the HdhQ150 mouse model of HD, in vivo MRI was employed at two time points, before and after the onset of motor signs, to assess brain macrostructure and white matter microstructure. Ex vivo MRI, immunohistochemistry, transmission electron microscopy and behavioural testing were also conducted. Global brain atrophy was found in HdhQ150 mice at both time points, with no neuropathological progression across time and a selective sparing of the cerebellum. In contrast, no white matter abnormalities were detected from the MRI images or electron microscopy images alike. The relationship between motor function and MR-based structural measurements was different for the HdhQ150 and wild-type mice, although there was no relationship between motor deficits and histopathology. Widespread neuropathology prior to symptom onset is consistent with patient studies, whereas the absence of white matter abnormalities conflicts with patient data. The myriad reasons for this inconsistency require further attention to improve the translatability from mouse models of disease.

Early white matter abnormalities, progressive brain pathology and motor deficits in a novel knock-in mouse model of Huntington's disease.

White matter abnormalities have been reported in premanifest Huntington's disease (HD) subjects before overt striatal neuronal loss, but whether the white matter changes represent a necessary step towards further pathology and the underlying mechanism of these changes remains unknown. Here, we characterized a novel knock-in mouse model that expresses mouse HD gene homolog (Hdh) with extended CAG repeat- HdhQ250, which was derived from the selective breeding of HdhQ150 mice. HdhQ250 mice manifest an accelerated and robust phenotype compared with its parent line. HdhQ250 mice exhibit progressive motor deficits, reduction in striatal and cortical volume, accumulation of mutant huntingtin aggregation, decreased levels of DARPP32 and BDNF and altered striatal metabolites. The abnormalities detected in this mouse model are reminiscent of several aspects of human HD. In addition, disturbed myelination was evident in postnatal Day 14 HdhQ250 mouse brain, including reduced levels of myelin regulatory factor and myelin basic protein, and decreased numbers of myelinated axons in the corpus callosum. Thinner myelin sheaths, indicated by increased G-ratio of myelin, were also detected in the corpus callosum of adult HdhQ250 mice. Moreover, proliferation of oligodendrocyte precursor cells is altered by mutant huntingtin both in vitro and in vivo. Our data indicate that this model is suitable for understanding comprehensive pathogenesis of HD in white matter and gray matter as well as developing therapeutics for HD.

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.

Fragments of HdhQ150 Mutant Huntingtin Form a Soluble Oligomer Pool That Declines with Aggregate Deposition upon Aging

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington’s Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.

B08 Differential sensitivity of aggregate markers in HdhQ150 and YAC128 HD mouse models

Huntington's disease (HD), an inherited neurodegenerative disorder that is characterised histologically by the presence of intranuclear inclusions (NIIs) and the loss of neurons in the striatum and cortex. Protein aggregates are a hallmark of several neurodegenerative disorders including HD. These aggregates contain a variety of other proteins including ubiquitin. The present study was designed to determine the sensitivity of the different antibody in inclusion formations in knock-in HdhQ150 and transgenic YAC128 mouse lines. In order to fully assess the different protein deposition patterns in these mouse models, brains were assessed using S830 or ubiquitin immunohistochemistry at 8 and 18 months of age. In 8 months old HdhQ150 and YAC128 carrier mice, ubiquitin antibody showed more diffuse staining with minimum NIIs in the olfactory tubercle, striatum, piriform cortex, cortex and hippocampus. However, at the same age, the S830 antibody showed more specific binding to NIIs in the HdhQ150 but not in YAC128 mice. As would be predicted the deposition of aggregated protein becomes more widespread at later ages. At 18 months of age the HdhQ150 mice showed increased ubiquitin immunoactivity, however there was little difference found in the YAC128 mice. NIIs were visible with the S830 antibody in the HdhQ150 and YAC128 mice at this stage. The present study demonstrates that the differential sensitivity of different antibodies in identifying NIIs between mouse lines, which may reflect confirmatinal changes in protein aggregates.

B09 Caspase-6 does not contribute to the proteolysis of mutant huntingtin in the HDHQ150 knock-in mouse model of Huntington's disease

Huntington9s 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 further explore the role that CASP6 plays in HTT proteolysis, we have 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 has no effect on the proteolysis of HTT, and does 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 wild type or mutant HTT in the brains of presymptomatic mice. Therefore, we conclude that CASP6 inhibition does not modify 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.

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.

Comparative analysis of pathology and behavioural phenotypes in mouse models of Huntington's disease

The longitudinal characterisation of Huntington's disease (HD) mouse lines is essential for the understanding of the differential developmental time course, nature and severity of phenotype progression over time. This overview outlines detailed behavioural, neuropathological and gene expression studies in four HD mouse lines: R6/1, YAC128, HdhQ92 and HdhQ150 and outlines their relevance to human HD. The review describes the similarities and differences between the models at the behavioural, anatomical and genetic levels of pathology and how these phenotypes interact in the development of disease in the lines. The HdhQ150 mouse demonstrates the most similarities to the functional deficits observed in human HD. The neuropathological profile with early cortical development of intense aggregate/inclusion pathology in the YAC128 mouse suggests that this line most resembles the development of inclusion pathology in the human disease. The gene expression analyses of the mouse lines find significant similarities between each of the lines and human HD, which converge as the mice age. In the YAC128 and HdhQ92 mouse lines some severe functional deficits are progressive whilst others are not, despite the concomitant ongoing development of neuropathological and gene expression changes. We suggest that the YAC128 and R6/1 lines may be more representative of the juvenile form of HD. The suitability of the different mouse models studied here for different types of pre-clinical therapeutic trials is discussed.

Longitudinal analysis of gene expression and behaviour in the HdhQ150 mouse model of Huntington's disease

Substantial transcriptional changes are seen in Huntington's disease (HD) brain and parallel early changes in gene expression are observed in mouse models of HD. Analysis of behaviour in such models also shows substantial deficits in motor, learning and memory tasks. We examined the changes in the transcriptional profile in the HdhQ150 mouse model of HD at 6, 12 and 18 months and correlated these changes with the behavioural tasks the animals had undertaken. Changes in gene expression over time showed a significant enrichment of RNAs altered in abundance that related to cognition in both HdhQ150 and wild-type animals. The most significantly down-regulated mRNA between genotypes over the whole time-course was Htt itself. Other changes between genotypes identified at 6 months related to chromatin organization and structure, whilst at 18 months changes related mainly to intracellular signalling. Correlation of the changes in gene product abundance with phenotypic changes revealed that weight and detection of the opposite position of the platform in the water maze seemed to correlate with the chromatin alterations whereas changes in the rotarod performance related mainly to intracellular signalling and homeostasis. These results implicate alterations in specific molecular pathways that may underpin changes in different behavioural tasks.

Early autophagic response in a novel knock-in model of Huntington disease

The aggregation of mutant polyglutamine (polyQ) proteins has sparked interest in the role of protein quality-control pathways in Huntington's disease (HD) and related polyQ disorders. Employing a novel knock-in HD mouse model, we provide in vivo evidence of early, sustained alterations of autophagy in response to mutant huntingtin (mhtt). The HdhQ200 knock-in model, derived from the selective breeding of HdhQ150 knock-in mice, manifests an accelerated and more robust phenotype than the parent line. Heterozygous HdhQ200 mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age and striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Striatal NIIs accumulate earlier in HdhQ200 mice than in HdhQ150 mice. The earlier appearance of aggregate pathology in HdhQ200 mice is paralleled by earlier and more rapidly progressive motor deficits: progressive imbalance and decreased motor coordination by 50 weeks, gait deficits by 60 weeks and gross motor impairment by 80 weeks of age. At 80 weeks, heterozygous HdhQ200 mice exhibit striatal and cortical astrogliosis and a approximately 50% reduction in striatal dopamine receptor binding. Increased LC3-II protein expression, which is noted early and sustained throughout the disease course, is paralleled by increased expression of the autophagy-related protein, p62. Early and sustained expression of autophagy-related proteins in this genetically precise mouse model of HD suggests that the alteration of autophagic flux is an important and early component of the neuronal response to mhtt.

Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington’s disease

A CAG-repeat gene expansion translated into a pathogenic polyglutamine stretch at the N-terminus of huntingtin triggers Huntington's Disease. Mutated huntingtin is predicted to adopt toxic properties mainly if aggregation-prone N-terminal fragments are released by proteolysis. Huntingtin-aggregates are indeed a major hallmark of this disorder and could represent useful markers of disease-onset or progression. We designed a simple method for qualitative and quantitative characterization of aggregates. For this, we analyzed samples from in vitro and in vivo Huntington's Disease models by agarose gel electrophoresis and showed that in the brain of transgenic mice huntingtin-aggregates became larger as a function of disease progression. This appears to be a property of cytoplasmic but not nuclear aggregates. In cell cultures, treatment with Congo Red inhibited aggregate growth but not total load. Finally, we showed that in primary striatal neurons and in brains of R6/2 and HdhQ150 mice, the presence of aggregates preceded initiation of any other functional deficits. This observation argues for a pathogenic role of huntingtin-aggregation in Huntington's Disease. Our results emphasize that thorough analysis of huntingtin metabolism and aggregation is now feasible, thus significantly improving the power of studies assessing therapies designed to lower huntingtin levels or to interfere with its aggregation.