Agnesska C. Benjamin

Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease

Huntington disease (HD) is a devastating, late-onset, inherited neurodegenerative disorder that manifests with personality changes, movement disorders, and cognitive decline. It is caused by a CAG repeat expansion in exon 1 of the HTT gene that translates to a polyglutamine tract in the huntingtin protein (HTT). The formation of HTT fragments has been implicated as an essential step in the molecular pathogenesis of HD and several proteases that cleave HTT have been identified. However, the importance of smaller N-terminal fragments has been highlighted by their presence in HD postmortem brains and by the fact that nuclear inclusions are only detected by antibodies to the N terminus of HTT. Despite an intense research effort, the precise length of these fragments and the mechanism by which they are generated remains unknown. Here we show that CAG repeat length–dependent aberrant splicing of exon 1 HTT results in a short polyadenylated mRNA that is translated into an exon 1 HTT protein. Given that mutant exon 1 HTT proteins have consistently been shown to be highly pathogenic in HD mouse models, the aberrant splicing of HTT mRNA provides a mechanistic basis for the molecular pathogenesis of HD. RNA-targeted therapeutic strategies designed to lower the levels of HTT are under development. Many of these approaches would not prevent the production of exon 1 HTT and should be reviewed in light of our findings.

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington's Disease Mice

Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies.

Erratum: SIRT1 Activity is linked to its brain region-specific phosphorylation and is impaired in huntington's disease mice (PLoS ONE (2016) 11:1 (e0145425) DOI: 10.1371/journal.pone.0145425)

[This corrects the article DOI: 10.1371/journal.pone.0145425.].

Disruption to schizophrenia-associated gene Fez1 in the hippocampus of HDAC11 knockout mice

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1–9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.

In vivo neutralization of the protagonist role of macrophages during the chronic inflammatory stage of Huntington’s disease

Neurodegenerative diseases, characterised by the progressive and selective neuronal death in the central nervous system, are frequently accompanied by an activated immune system. In Huntington’s disease (HD), clinical and animal studies show evidence of immune activity, along with hyper-reactive monocyte/macrophage responses, while application of immunosuppressive regimens have imparted beneficial effects to HD mice. These findings suggest a contributory role of the immune system in HD pathology, with immune-based interventions offering a potential therapeutic strategy. Herein, we show that peripheral and CNS immune system activity increased with disease progression in HD mouse models and defined the phenotype of the immune response. Additionally, the depletion of monocytes and macrophages in vivo, via clodronate liposome treatment, revealed a major contributory role of these innate immune cells to the chronic inflammatory milieu observed during the course of the disease. This suggests that peripheral immunomodulatory strategies targeting monocytes and macrophages could be relevant for HD.

Regulatory mechanisms of incomplete huntingtin mRNA splicing

Huntington’s disease is caused by a CAG repeat expansion in exon 1 of the HTT gene. We have previously shown that exon 1 HTT does not always splice to exon 2 producing a small transcript (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The mechanisms by which this incomplete splicing occurs are unknown. Here, we have generated a minigene system that recapitulates the CAG repeat-length dependence of HTTexon1 production, and has allowed us to define the regions of intron 1 necessary for incomplete splicing. We show that manipulation of the expression levels of the splicing factor SRSF6, predicted to bind CAG repeats, modulates this aberrant splicing event and also demonstrate that RNA polymerase II transcription speed regulates the levels of HTTexon1 production. Understanding the mechanisms by which this pathogenic exon 1 HTT is generated may provide the basis for the development of strategies to prevent its production.Incomplete splicing of HTT results in the production of the highly pathogenic exon 1 HTT protein. Here the authors identify the necessary intronic regions and the underlying mechanisms that contribute to this process.

A03 Regulatory mechanisms of incomplete HTT MRNA splicing in huntington’s disease

Huntington’s disease is caused by a CAG repeat expansion in exon 1 of the HTT gene. We have previously shown that exon 1 HTT does not always splice to exon 2 producing a small transcript (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The mechanisms by which this incomplete splicing occurs are unknown. Here, we have generated a novel minigene system that recapitulates the CAG repeat-length dependence of HTTexon1 production, and has allowed us to define the regions of intron 1 necessary for incomplete splicing. We show that manipulation of the expression levels of the splicing factor SRSF6, predicted to bind CAG repeats, modulates this aberrant splicing event and also demonstrate that RNA polymerase II transcription speed regulates the levels of HTTexon1 production. Understanding the mechanisms by which this pathogenic exon 1 HTT is generated will provide the basis for the development of strategies to prevent its production.

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

B4 Detection of the aberrantly spliced exon 1 – intron 1 htt mRNA in HD patient post mortem brain tissue and fibroblast lines

Background We have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and present in all knock-in mouse models of HD (with CAG ≥ 50), utilising cryptic polyadenylation sites located at 677 bp and 1145 bp into mouse intron 1. The read-through product is also readily detected in the YAC128 and BACHD mouse models which both use a cryptic polyadenylation site that is located 7327 bp into human intron 1. However, the presence of this small exon 1 – intron 1 mRNA was not easy to detect in HD patient tissues using the assays that we initially developed. Aims To develop novel quantitative RT-PCR assays to detect the exon 1 – intron 1 HTT mRNA in tissues from HD patients. Methods We have now established a set of qPCR assays that quantify sequences located close to the cryptic polyadenylation site in human HTT intron 1. These have been applied to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD. Results The exon 1 – intron 1 HTT mRNA can be readily detected in the somatosensory cortex, hippocampus and cerebellum of post mortem brains from individuals with HD, particularly in those with early onset disease. These human HTT intronic sequences are also present in fibroblasts from juvenile HD patients. We shall also present data on changes in the relative abundance of the exon 1 – intron 1 Htt mRNA in a knock-in mouse model with disease progression. Conclusion The highly pathogenic exon 1 HTT protein is generated in the tissues of people with HD through the aberrant splicing of HTT. Funding Medical Research Council and CHDI Foundation