Impact of caspase-6 modulation on Huntington disease phenotypes in the YAC128 mouse model

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. HD is caused by a CAG repeat expansion in the huntingtin (HTT) gene leading to the production of the mutant huntingtin protein (mHTT). Caspase-6 (C6) is a cysteine aspartyl protease that plays a central role in apoptosis and has been postulated to play a role in inflammation. Increased C6 activation is observed in human HD brains and mouse models and the inhibition of C6-mediated cleavage of mHTT protects against neuropathology and behavioural deficits in the YAC128 mouse model of HD. Additionally, alterations in inflammation are a feature of many neurodegenerative diseases, including HD. Hyperactive inflammatory responses are observed in both HD patients and mouse models and C6 has been postulated to play a role in mediating inflammation. Constitutive deletion of the Casp6 gene (denoted as C6) in YAC128 mice results in a partial rescue of some features of HD; however, the continued presence of the 586 cleavage fragment in the absence of C6 suggests possible compensation by other proteases. The goal of this thesis was to investigate the impact of modulating C6 in the adult YAC128 mouse and to further characterize the role of C6 in inflammation. To that end, the C6 gene was partially deleted in the adult YAC128 mouse and characterization of these mice reveals no amelioration in motor or cognitive phenotypes but a modest improvement in certain psychiatric behaviours. Neuropathological assessment shows no attenuation in canonical brain pathology but peripherally, the loss of C6 attenuates the overactive inflammatory response observed in YAC128 mice. These data suggest that partial loss of C6 in the brain is not sufficient to improve most behavioural and neuropathological phenotypes but implicate C6 in the regulation of inflammation. Furthermore, loss of C6 results in a blunted inflammatory response characterized by reduced cytokine release. As the presence of elevated cytokine levels have been suspected to cause psychiatric behaviours such as depression, this finding provides a possible mechanistic link between C6 activity and the onset of affective behaviours.Medicine, Faculty ofMedical Genetics, Department ofGraduat

A quantitative method for the specific assessment of caspase-6 activity in cell culture.

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders

Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease

Huntington disease (HD) is caused by the expression of mutant huntingtin (mHTT) bearing a polyglutamine expansion. In HD, mHTT accumulation is accompanied by a dysfunction in basal autophagy, which manifests as specific defects in cargo loading during selective autophagy. Here we show that the expression of mHTT resistant to proteolysis at the caspase cleavage site D586 (C6R mHTT) increases autophagy, which may be due to its increased binding to the autophagy adapter p62. This is accompanied by faster degradation of C6R mHTT in vitro and a lack of mHTT accumulation the C6R mouse model with age. These findings may explain the previously observed neuroprotective properties of C6R mHTT. As the C6R mutation cannot be easily translated into a therapeutic approach, we show that a scheduled feeding paradigm is sufficient to lower mHTT levels in YAC128 mice expressing cleavable mHTT. This is consistent with a previous model, where the presence of cleavable mHTT impairs basal autophagy, while fasting-induced autophagy remains functional. In HD, mHTT clearance and autophagy may become increasingly impaired as a function of age and disease stage, because of gradually increased activity of mHTT-processing enzymes. Our findings imply that mHTT clearance could be enhanced by a regulated dietary schedule that promotes autophagy.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofReviewedFacult

Additional file 2: of Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease

Figure S2. Autophagy pathways are not altered in MEFs derived from YAC18 mice. A Primary MEF cultures from YAC18 or wt littermate embryos were seeded onto coverslips and treated with bafilomycin. Cells were fixed and stained for p62 and LC3, Hoechst dye was used for nuclear counterstaining. Samples were imaged on a confocal microscope and the density of punctae as well as the co-localization of LC3 and p62 staining were analyzed. B Primary MEF cultures from YAC18 or wt littermate embryos were seeded onto coverslips and treated with MG132 or DMSO as a control. Cells were fixed and stained for p62, Hoechst dye was used for nuclear counterstaining. Samples were imaged on a confocal microscope and the density of punctae were analyzed. Representative images and pooled quantification data with S.E.M. are shown, 3 independent cultures were analyzed. Number of replicates is shown as insets for Western blot experiments, for imaging experiments 24-30 cells per condition were analyzed. Statistical significance was determined by Student’s t-test. No statistically significant differences were found. (TIFF 5239 kb

Additional file 4: of Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease

Figure S4. Full blots corresponding to Fig. 3b. (TIFF 4590 kb

Additional file 7: of Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease

Figure S7. Cortical p62, LC3-I and wt HTT protein levels as well as mHTT mRNA are not altered by scheduled feeding. A YAC128 and C6R mice as well as their wt littermates were subjected to one week of scheduled feeding and compared to littermates with ad libitum access to food. Protein levels of p62 were analyzed by Western blotting in cortical tissues. 2way-ANOVA, genotype p=0.2138, feeding p=0.5807. B YAC128 and C6R mice as well as their wt littermates were subjected to one week of scheduled feeding and compared to littermates with ad libitum access to food. Protein levels of LC3-I were analyzed by Western blotting in cortical tissues. 2way-ANOVA, genotype p=0.5798, feeding p=0.2548. C + D YAC128 mice and their wt littermates were subjected to one week of scheduled feeding and compared to littermates with ad libitum access to food. C Protein levels of wt HTT were analyzed by Western blotting with antibody MAB2166 in cortical tissues. 2way-ANOVA genotype p=0.6115, feeding p=0.1818. D mRNA levels for transgenic human mHTT were analyzed by qRT-PCR in cortical tissues derived from YAC128 mice. Representative blots and pooled quantification data with S.E.M. are shown, number of replicates is shown as insets. The blot corresponding to panel B is shown in Fig. 6a, the blot corresponding to panel C is shown in Fig. 6d. Statistical significance was determined by 2way ANOVA (A-C) or Student’s t-test (D). (TIFF 1304 kb