SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1.

Objective Brown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase.Methods We carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the β3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins.Results Our findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII.Conclusions Our data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1

A randomized, double-blind, placebo-controlled trial evaluating cysteamine in Huntington's disease

BACKGROUND: Cysteamine has been demonstrated as potentially effective in numerous animal models of Huntington\u27s disease. METHODS: Ninety-six patients with early-stage Huntington\u27s disease were randomized to 1200 mg delayed-release cysteamine bitartrate or placebo daily for 18 months. The primary end point was the change from baseline in the UHDRS Total Motor Score. A linear mixed-effects model for repeated measures was used to assess treatment effect, expressed as the least-squares mean difference of cysteamine minus placebo, with negative values indicating less deterioration relative to placebo. RESULTS: At 18 months, the treatment effect was not statistically significant - least-squares mean difference, -1.5 ± 1.71 (P = 0.385) - although this did represent less mean deterioration from baseline for the treated group relative to placebo. Treatment with cysteamine was safe and well tolerated. CONCLUSIONS: Efficacy of cysteamine was not demonstrated in this study population of patients with Huntington\u27s disease. Post hoc analyses indicate the need for definitive future studies. © 2017 International Parkinson and Movement Disorder Society

Human Mesenchymal Stem Cells Prolong Survival and Ameliorate Motor Deficit through Trophic Support in Huntington's Disease Mouse Models

We investigated the therapeutic potential of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in Huntington's disease (HD) mouse models. Ten weeks after intrastriatal injection of quinolinic acid (QA), mice that received hBM-MSC transplantation showed a significant reduction in motor function impairment and increased survival rate. Transplanted hBM-MSCs were capable of survival, and inducing neural proliferation and differentiation in the QA-lesioned striatum. In addition, the transplanted hBM-MSCs induced microglia, neuroblasts and bone marrow-derived cells to migrate into the QA-lesioned region. Similar results were obtained in R6/2-J2, a genetically-modified animal model of HD, except for the improvement of motor function. After hBM-MSC transplantation, the transplanted hBM-MSCs may integrate with the host cells and increase the levels of laminin, Von Willebrand Factor (VWF), stromal cell-derived factor-1 (SDF-1), and the SDF-1 receptor Cxcr4. The p-Erk1/2 expression was increased while Bax and caspase-3 levels were decreased after hBM-MSC transplantation suggesting that the reduced level of apoptosis after hBM-MSC transplantation was of benefit to the QA-lesioned mice. Our data suggest that hBM-MSCs have neural differentiation improvement potential, neurotrophic support capability and an anti-apoptotic effect, and may be a feasible candidate for HD therapy

The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients

The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Proteome-scale mapping of binding sites in the unstructured regions of the human proteome

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of similar to 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered &gt;2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteomewide discovery of SLiM-based interactions.De tre första författarna delar förstaförfattarskapet.</p

Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery

Neocortical morphometry in Huntington's disease: Indication of the coexistence of abnormal neurodevelopmental and neurodegenerative processes

Huntington's disease (HD) is an inherited, autosomal dominant disorder that is characteristically thought of as a degenerative disorder. Despite cellular and molecular grounds suggesting HD could also impact normal development, there has been scarce systems-level data obtained from in vivo human studies supporting this hypothesis. Sulcus-specific morphometry analysis may help disentangle the contribution of coexisting neurodegenerative and neurodevelopmental processes, but such an approach has never been used in HD. Here, we investigated cortical sulcal depth, related to degenerative process, as well as cortical sulcal length, related to developmental process, in early-stage HD and age-matched healthy controls. This morphometric analysis revealed significant differences in the HD participants compared with the healthy controls bilaterally in the central and intra-parietal sulcus, but also in the left intermediate frontal sulcus and calcarine fissure. As the primary visual cortex is not connected to the striatum, the latter result adds to the increasing in vivo evidence for primary cortical degeneration in HD. Those sulcal measures that differed between HD and healthy populations were mainly atrophy-related, showing shallower sulci in HD. Conversely, the sulcal morphometry also revealed a crucial difference in the imprint of the Sylvian fissure that could not be related to loss of grey matter volume: an absence of asymmetry in the length of this fissure in HD. Strong asymmetry in that cortical region is typically observed in healthy development. As the formation of the Sylvian fissure appears early in utero, and marked asymmetry is specifically found in this area of the neocortex in newborns, this novel finding likely indicates the foetal timing of a disease-specific, genetic interplay with neurodevelopment