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

Effects of eight neuropsychiatric copy number variants on human brain structure

Many copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen’s d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions

N-terminal Huntingtin (Htt) phosphorylation is a molecular switch regulating Htt aggregation, helical conformation, internalization, and nuclear targeting

Huntington’s disease is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt). Phosphorylation of this protein region (Httex1) has been shown to play important roles in regulating the structure, toxicity, and cellular properties of N-terminal fragments and full-length Htt. However, increasing evidence suggests that phosphomimetic substitutions in Htt result in inconsistent findings and do not reproduce all aspects of true phosphorylation. Here, we investigated the effects of bona fide phosphorylation at Ser-13 or Ser-16 on the structure, aggregation, membrane binding, and subcellular properties of the Httex1-Q18A variant and compared these effects with those of phosphomimetic substitutions. We show that phosphorylation at either Ser-13 and/or Ser-16 or phosphomimetic substitutions at both these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic -helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates. In synthetic peptides, phosphorylation at Ser-13, Ser-16, or both residues strongly disrupted the amphipathic -helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Experiments with peptides bearing different combinations of phosphorylation sites within Nt17 revealed a phosphorylation-dependent switch that regulates the Httex1 structure, involving cross-talk between phosphorylation at Thr-3 and Ser-13 or Ser-16. Our results provide crucial insights into the role of phosphorylation in regulating Httex1 structure and function, and underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation, and their potential as therapeutic targets for managing Huntington’s disease

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

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

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

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 temperature-dependent 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 temperature- and 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

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