Georgina F. Osborne

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.

UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome

Summary Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.

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 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.

Genetic deletion of transglutaminase 2 does not rescue the phenotypic deficits observed in R6/2 and zQ175 mouse models of Huntington's disease.

Huntingtons disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene. Tissue transglutaminase 2 (TG2), a multi-functional enzyme, was found to be increased both in HD patients and in mouse models of the disease. Furthermore, beneficial effects have been reported from the genetic ablation of TG2 in R6/2 and R6/1 mouse lines. To further evaluate the validity of this target for the treatment of HD, we examined the effects of TG2 deletion in two genetic mouse models of HD: R6/2 CAG 240 and zQ175 knock in (KI). Contrary to previous reports, under rigorous experimental conditions we found that TG2 ablation had no effect on either motor or cognitive deficits, or on the weight loss. In addition, under optimal husbandry conditions, TG2 ablation did not extend R6/2 lifespan. Moreover, TG2 deletion did not change the huntingtin aggregate load in cortex or striatum and did not decrease the brain atrophy observed in either mouse line. Finally, no amelioration of the dysregulation of striatal and cortical gene markers was detected. We conclude that TG2 is not a valid therapeutic target for the treatment of HD.

Reducing Igf-1r Levels Leads To Paradoxical and Sexually Dimorphic Effects in HD Mice

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called “proteinopathies” include Alzheimer’s disease (AD) and Huntington’s disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.

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.

B3 Comparison of the effect of a pure CAG repeat and mixed cagcaa repeat on the extent to which the htt gene is aberrantly spliced in knock-in mice

Background We have previously shown that the HTT gene is incompletely spliced to generated a small exon 1 – intron 1 polyadenylated mRNA that is translated to produce an exon 1 HTT protein. This occurs in all knock-in mouse models of HD, YAC128 mice, BACHD mice and in patient tissues. Through bioinformatics, we predicted that the splicing factor SRSF6 binds to a degenerative motif that includes both the (CAG)n and (CAGCAA)n sequences and have proposed that the ectopic recruitment of SRSF6 is related to this aberrant splicing event. Aims To compare the effect of (CAG)n and (CAGCAACAGCAACAA)n repeats on the level of expression of the mutant Htt exon1 - intron1 splice product (Htt exon 1 mRNA). Methods Knock-in mice were generated that carried either a (CAG)n repeat or a (CAGCAACAGCAACAA)n repeat that encoded matched polyQ tracts of 45, 80 and 105 glutamines. In both cases the genetic manipulation of the Htt locus left a loxP site located approximately 200 bp into intron 1. Results Colonies of the (CAG)n and (CAGCAACAGCAACAA)n knock-in mice were established on a C57BL/6 background. The levels of the exon 1 HttmRNA and the full length Htt transcript were measured by quantitative qPCR in the cortex and striatum of heterozygous mice at 2 and 10 months of age. At 2 months of age levels of the exon 1 HttmRNA were higher in the (CAGCAACAGCAACAA)n mice than in their matched (CAG)n counterparts, and in both cases, the level of this small transcript increased with the length of the repeat. Conclusions A mixed (CAGCAACAGCAACAA)n repeat promotes the aberrant splicing of mutant Htt to a greater extent than a pure (CAG)n repeat. At 10 months, somatic instability of the (CAG)n but not the (CAGCAACAGCAACAA)n repeat contributes to the comparative levels of this small transcript. Funding CHDI Foundation