Sophie A. Franklin

SAHA Decreases HDAC 2 and 4 Levels In Vivo and Improves Molecular Phenotypes in the R6/2 Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a progressive neurological disorder for which there are no disease-modifying treatments. Transcriptional dysregulation is a major molecular feature of HD, which significantly contributes to disease progression. Therefore, the development of histone deacetylase (HDAC) inhibitors as therapeutics for HD has been energetically pursued. Suberoylanilide hydroxamic acid (SAHA) – a class I HDAC as well an HDAC6 inhibitor, improved motor impairment in the R6/2 mouse model of HD. Recently it has been found that SAHA can also promote the degradation of HDAC4 and possibly other class IIa HDACs at the protein level in various cancer cell lines. To elucidate whether SAHA is a potent modifier of HDAC protein levels in vivo, we performed two independent mouse trials. Both WT and R6/2 mice were chronically treated with SAHA and vehicle. We found that prolonged SAHA treatment causes the degradation of HDAC4 in cortex and brain stem, but not hippocampus, without affecting its transcript levels in vivo. Similarly, SAHA also decreased HDAC2 levels without modifying the expression of its mRNA. Consistent with our previous data, SAHA treatment diminishes Hdac7 transcript levels in both wild type and R6/2 brains and unexpectedly was found to decrease Hdac11 in R6/2 but not wild type. We investigated the effects of SAHA administration on well-characterised molecular readouts of disease progression. We found that SAHA reduces SDS-insoluble aggregate load in the cortex and brain stem but not in the hippocampus of the R6/2 brains, and that this was accompanied by restoration of Bdnf cortical transcript levels.

HDAC4 Reduction: A Novel Therapeutic Strategy to Target Cytoplasmic Huntingtin and Ameliorate Neurodegeneration

HDAC4 histone deacetylase is found to associate with huntingtin in a polyQ-length dependent manner. Reduction of HDAC4 levels in mouse models of Huntingtons disease (HD) delays cytoplasmic aggregation in the brain and improves the molecular pathology of HD, providing a potential new therapeutic target.

Dysfunction of the CNS-heart axis in mouse models of Huntington's disease.

Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimers, Parkinsons and Huntingtons disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.

HDAC4-Myogenin Axis As an Important Marker of HD-Related Skeletal Muscle Atrophy

Skeletal muscle remodelling and contractile dysfunction occur through both acute and chronic disease processes. These include the accumulation of insoluble aggregates of misfolded amyloid proteins that is a pathological feature of Huntington’s disease (HD). While HD has been described primarily as a neurological disease, HD patients’ exhibit pronounced skeletal muscle atrophy. Given that huntingtin is a ubiquitously expressed protein, skeletal muscle fibres may be at risk of a cell autonomous HD-related dysfunction. However the mechanism leading to skeletal muscle abnormalities in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that symptomatic animals developed a progressive impairment of the contractile characteristics of the hind limb muscles tibialis anterior (TA) and extensor digitorum longus (EDL), accompanied by a significant loss of motor units in the EDL. In symptomatic animals, these pronounced functional changes were accompanied by an aberrant deregulation of contractile protein transcripts and their up-stream transcriptional regulators. In addition, HD mouse models develop a significant reduction in muscle force, possibly as a result of a deterioration in energy metabolism and decreased oxidation that is accompanied by the re-expression of the HDAC4-DACH2-myogenin axis. These results show that muscle dysfunction is a key pathological feature of HD.

HDAC4 Does Not Act as a Protein Deacetylase in the Postnatal Murine Brain In Vivo

Reversible protein acetylation provides a central mechanism for controlling gene expression and cellular signaling events. It is governed by the antagonistic commitment of two enzymes families: the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). HDAC4, like its class IIa counterparts, is a potent transcriptional repressor through interactions with tissue specific transcription factors via its N-terminal domain. Whilst the lysine deacetylase activity of the class IIa HDACs is much less potent than that of the class I enzymes, HDAC4 has been reported to influence protein deacetylation through its interaction with HDAC3. To investigate the influence of HDAC4 on protein acetylation we employed the immunoaffinity-based AcetylScan proteomic method. We identified many proteins known to be modified by acetylation, but found that the absence of HDAC4 had no effect on the acetylation profile of the murine neonate brain. This is consistent with the biochemical data suggesting that HDAC4 may not function as a lysine deacetylase, but these in vivo data do not support the previous report showing that the enzymatic activity of HDAC3 might be modified by its interaction with HDAC4. To complement this work, we used Affymetrix arrays to investigate the effect of HDAC4 knock-out on the transcriptional profile of the postnatal murine brain. There was no effect on global transcription, consistent with the absence of a differential histone acetylation profile. Validation of the array data by Taq-man qPCR indicated that only protamine 1 and Igfbp6 mRNA levels were increased by more than one-fold and only Calml4 was decreased. The lack of a major effect on the transcriptional profile is consistent with the cytoplasmic location of HDAC4 in the P3 murine brain.

The Huntington's disease-related cardiomyopathy prevents a hypertrophic response in the R6/2 mouse model.

Huntingtons disease (HD) is neurodegenerative disorder for which the mutation results in an extra-long tract of glutamines that causes the huntingtin protein to aggregate. It is characterized by neurological symptoms and brain pathology that is associated with nuclear and cytoplasmic aggregates and with transcriptional deregulation. Despite the fact that HD has been recognized principally as a neurological disease, there are multiple epidemiological studies showing that HD patients exhibit a high rate of cardiovascular events leading to heart failure. To unravel the mechanistic basis of cardiac dysfunction in HD, we employed a wide range of molecular techniques using the well-established genetic R6/2 mouse model that develop a considerable degree of the cardiac atrophy at end stage disease. We found that chronic treatment with isoproterenol, a potent beta-adrenoreceptor agonist, did not change the overall gross morphology of the HD murine hearts. However, there was a partial response to the beta-adrenergenic stimulation by the further re-expression of foetal genes. In addition we have profiled the expression level of Hdacs in the R6/2 murine hearts and found that the isoproterenol stimulation of Hdac expression was partially blocked. For the first time we established the Hdac transcriptional profile under hypertrophic conditions and found 10 out of 18 Hdacs to be markedly deregulated. Therefore, we conclude that R6/2 murine hearts are not able to respond to the chronic isoproterenol treatment to the same degree as wild type hearts and some of the hypertrophic signals are likely attenuated in the symptomatic HD animals.

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

Huntingtons 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 Huntingtons 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 Huntingtons 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.

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice

Huntington’s disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington’s disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.

B24 Assessment of immune system activation status during the course of disease in huntington’s disease mouse model

Background A number of neurodegenerative diseases, characterised by progressive and selective death of neurons in the CNS, are accompanied by activation of the peripheral immune system. In Huntington’s disease (HD), clinical and animal studies show elevated immune factors that are hallmarks of immune activity and the use of immunosuppressive regimens have shown beneficial effects in HD mice. These results suggest a contributory role of the immune system in HD pathology, with immune based interventions offering potential therapeutic strategy to disease. Aim To assess peripheral and central nervous system (CNS) immune system activity in HD mouse model during disease course to determine if/when peripheral immunomodulation will be relevant for HD treatment. Method R6/2 mice were investigated pre and post-symptomatic stages for immune activity in the brain and periphery through the assessment of gene expression and protein levels of interleukin (IL)−1β, IL-6, IL-10, IL-17 and tumour necrosis factor (TNF)α cytokines. Gene and cell surface (flow cytometry) expression of monocyte and macrophage activation (CD40 and OX40l) and T cell activation (OX40 and CD25) markers were also measured. Results At 14 weeks old (late-stage disease) cytokines and cell surface markers are elevated in several peripheral compartments as well as the brain. At 8 weeks (pre-symptomatic stage) however, immune activity is detectable in the periphery but not in the brain. Conclusion Immune activity in the periphery precedes immune activation in the CNS suggesting the peripheral immune system may promote activation of the CNS immune system during HD, possibly through the secretion of pro-inflammatory factors that cross the blood brain barrier. Additionally, myeloid immune cells in the CNS and periphery display differing activated phenotypes during late stage HD with microglial cells presenting a predominantly M1 phenotype while monocyte/macrophages show a M2 predominating phenotype. Funding Medical Research Council and the CHDI Foundation

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

Huntingtons 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.