SUMMARY The primary cause of Huntington’s disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into non-human primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Objectives To assess to what extent educational differences in total life expectancy (TLE) and disability-free life expectancy (DFLE) could be reduced by improving fruit and vegetable consumption in ten European countries. Methods Data from national census or registries with mortality follow-up, EU-SILC, and ESS were used in two scenarios to calculate the impact: the upward levelling scenario (exposure in low educated equals exposure in high educated) and the elimination scenario (no exposure in both groups). Results are estimated for men and women between ages 35 and 79 years. Results Varying by country, upward levelling reduced inequalities in DFLE by 0.1-1.1 years (1-10%) in males, and by 0.0-1.3 years (0-18%) in females. Eliminating exposure reduced inequalities in DFLE between 0.6 and 1.7 years for males (6-15%), and between 0.1 years and 1.8 years for females (3-20%). Conclusions Upward levelling of fruit and vegetable consumption would have a small, positive effect on both TLE and DFLE, and could potentially reduce inequalities in TLE and DFLE.

Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the fulllength protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches. Huntington disease (HD)2 is an inherited neurodegenerative disorder with onset in midlife for which symptoms include motor disturbances, personality changes, and cognitive decline (1). The mutation is a CAG/polyglutamine (polyQ) repeat expansion located at the N terminus of huntingtin (Htt), a protein of many diverse functions (2, 3). Individuals with (CAG) 35 or less remain unaffected, those with (CAG) 40 and above will develop HD within a normal lifespan, whereas repeats above (CAG) 70 will invariably cause childhood onset.Mouse models of HD include transgenic mice that express either N-terminal fragments of, or full-length human Htt, and the more genetically precise knock-in models, in which mutant CAG repeats have been inserted into the mouse Hd gene (Hdh) (1). R6/2 mice expressing mutant exon 1 Htt (4) exhibit an early onset and rapid progression of HD-related phenotypes, which have allowed extensive complementary analyses and enabled this model to be used as a screening tool. In our colony, nuclear inclusions can be readily detected by immunohistochemistry in the cerebral cortex, striatum, and hippocampus by 3 weeks of age (5, 6), Rotarod impairment is apparent by 6 weeks, and end-stage disease occurs at 15 weeks. The HdhQ150 knock-in mouse carries ϳ150Q (7). In our homozygous HdhQ150 (Hdh Q150/Q150 ) colony, nuclear inclusions were detected by immunohistochemistry in the striatum and hippocampus by 6 months and the cortex by 8 months, an impaired Rotarod performance was apparent by 18 months of age, and end stage disease occurs at around 22 months (8). Our recent comparison of R6/2 and Hdh Q150/Q150 mice at late stage disease (standardized for strain background and CAG repeat size) found that both models exhibit widespread and comparable phenotypes (8 -10). Nuclear inclusions and neuropil aggregates were distributed throughout all regions of the...

BACKGROUND:Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. METHODS:We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. RESULTS:This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. CONCLUSIONS:We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin–proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.

Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

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

Huntington disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. Previous studies have proposed that activation of the heat shock response (HSR) via the transcription factor heat shock factor 1 (HSF1) may be of therapeutic benefit. However, the effect of disease progression on the HSR and the therapeutic potential of this pathway are currently unknown. Here, we used a brain-penetrating HSP90 inhibitor and physiological, molecular, and behavioral readouts to demonstrate that pharmacological activation of HSF1 improves huntingtin aggregate load, motor performance, and other HD-related phenotypes in the R6/2 mouse model of HD. However, the beneficial effects of this treatment were transient and diminished with disease progression. Molecular analyses to understand the transient nature of these effects revealed altered chromatin architecture, reduced HSF1 binding, and impaired HSR accompanied disease progression in both the R6/2 transgenic and HdhQ150 knockin mouse models of HD. Taken together, our findings reveal that the HSR, a major inducible regulator of protein homeostasis and longevity, is disrupted in HD. Consequently, pharmacological induction of HSF1 as a therapeutic approach to HD is more complex than was previously anticipated.