Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine-and temperaturedependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperatureand polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients

Phosphorylation of huntingtin at residue T3 is decreased in Huntington's disease and modulates mutant huntingtin protein conformation.

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease

MGL recognises CD45RA on human Tregs and modulates their suppressive capacity.

<p>(A) Flow cytometry analysis of purified Treg stained with anti- CD4, CD25, FOXP3 and CD45RA antibodies. (B) Competition studies of rhMGL-Fc binding to CD45RA expressed by Tregs using GalNAc polymer. Filled histograms represent the untreated Tregs, while the open histograms show Tregs treated with rhMGL-Fc or rhMGL-Fc+GalNAc. (C) Western blot analysis of Treg lysate immunoprecipitated with rhMGL-Fc. Samples were run in 4–12% SDS-PAGE gel and were analysed with anti-CD45RA and rhMGL-Fc. These results are representative of one donor out of three. (D) Immunosuppression capacity of Tregs alone and Tregs treated with rhMGL-Fc after four days of co-culture with CD4⁺ CD25^- T cells (ratio 1:5, Treg:CD4⁺CD25^- T cells), in presence of anti-CD3 and anti-CD28. (E) IFNγ spots produced by CD8+ T cells (5×10⁴/well) stimulated with mDCs in presence or not of Tregs ± rhMGL. The results correspond with the mean of three independent experiments ± standard deviation (SD). * corresponds to <i>p</i><0.05 and ** to p<0.01.</p

Phospho-S129 Alpha-Synuclein Is Present in Human Plasma but Not in Cerebrospinal Fluid as Determined by an Ultrasensitive Immunoassay

Accumulation and aggregation of misfolded alpha-synuclein is believed to be a cause of Parkinson's disease (PD). Phosphorylation of alpha-synuclein at S129 is known to be associated with the pathological misfolding process, but efforts to investigate the relevance of this post-translational modification for pathology have been frustrated by difficulties in detecting and quantifying it in relevant samples. We report novel, ultrasensitive immunoassays based on single-molecule counting technology, useful for detecting alpha-synuclein and its S129 phosphorylated form in clinical samples in the low pg/ml range. Using human CSF and plasma samples, we find levels of alpha-synuclein comparable to those previously reported. However, while alpha-synuclein phosphorylated on S129 could easily be detected in human plasma, where its detection is extremely sensitive to protein phosphatases, its levels in CSF were undetectable, with a possible influence of a matrix effect. In plasma samples from a small test cohort comprising 5 PD individuals and five age-matched control individuals we find that pS129 alpha-synuclein levels are increased in PD plasma samples, in line with previous reports. We conclude that pS129 alpha-synuclein is not detectable in CSF and recommend the addition of phosphatase inhibitors to plasma samples at the time of collection. Moreover, the findings obtained on the small cohort of clinical plasma samples point to plasma pS129 alpha-synuclein levels as a candidate diagnostic biomarker in PD

Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD. (C) 2019 Published by Elsevier Inc

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

Phosphorylation of the N‐terminal domain of the huntingtin (HTT ) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD ) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK ‐binding kinase 1 (TBK 1), that efficiently phosphorylates full‐length and N‐terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo . TBK 1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans ) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK 1‐mediated neuroprotective effects are due to phosphorylation‐dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT . These findings suggest that upregulation and/or activation of TBK 1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation