Recombinant adeno associated viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches

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

The 5-Hydroxymethylcytosine Landscape of Prostate Cancer

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.publishedVersionPeer reviewe

Comparative Analysis of Total Alpha-Synuclein (αSYN) Immunoassays Reveals That They Do Not Capture the Diversity of Modified αSYN Proteoforms

Background: The development of therapeutics for Parkinson’s disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression, and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the α-synuclein (αSYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. Objective: To determine whether the three commercial kits that have been extensively used for total αSYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant αSYN proteoforms. Methods: We investigated and compared the ability of the different assays to detect the diversity of αSYN proteoforms using a library of αSYN proteins that comprise the majority of disease-relevant αSYN variants and post-translational modifications (PTMs). Results: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant αSYN species, and that these assays are unable to recognize most disease-associated C-terminally truncated variants of αSYN. Moreover, several N-terminal truncations and phosphorylation/nitration PTMs differentially modify the level of αSYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the αSYN proteoforms analyzed by the three immunoassays. Conclusion: Our results show that the tested immunoassays do not capture the totality of the relevant αSYN species and therefore may not be appropriate tools to provide an accurate measure of total αSYN levels in samples containing modified forms of the protein. This highlights the need for next generation αSYN immunoassays that capture the diversity of αSYN proteoforms.</jats:p

Comparative Analysis of Total Alpha-Synuclein (alpha SYN) Immunoassays Reveals That They Do Not Capture the Diversity of Modified alpha SYN Proteoforms

Background: The development of therapeutics for Parkinson's disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression, and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the alpha-synuclein (alpha SYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results.Objective: To determine whether the three commercial kits that have been extensively used for total alpha SYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant alpha SYN proteoforms.Methods: We investigated and compared the ability of the different assays to detect the diversity of alpha SYN proteoforms using a library of alpha SYN proteins that comprise the majority of disease-relevant alpha SYN variants and post-translational modifications (PTMs).Results: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant alpha SYN species, and that these assays are unable to recognize most disease-associated C-terminally truncated variants of alpha SYN. Moreover, several N-terminal truncations and phosphorylationinitration PTMs differentially modify the level of alpha SYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the alpha SYN proteoforms analyzed by the three immunoassays.Conclusion: Our results show that the tested immunoassays do not capture the totality of the relevant alpha SYN species and therefore may not be appropriate tools to provide an accurate measure of total alpha SYN levels in samples containing modified forms of the protein. This highlights the need for next generation alpha SYN immunoassays that capture the diversity of alpha SYN proteoforms

Comparative analysis of total alpha-synuclein (αSYN) immunoassays reveals that they do not capture the diversity of modified αSYN proteoforms

AbstractBackgroundThe development of therapeutics for Parkinson’s disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the α-synuclein (αSYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results.ObjectiveTo determine whether the three commercial kits that have been extensively used for total αSYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant αSYN proteoforms.MethodsWe investigated and compared the ability of the different assays to detect the diversity of αSYN proteoform using a library of αSYN proteins that compromise the majority of disease-relevant αSYN variants and post-translational modification.ResultsOur findings showed that none of the three tested immunoassays accurately capture the totality of relevant αSYN species and are unable to recognize most disease-associated C-terminally truncated variants of αSYN. Moreover, several N-terminal truncations and phosphorylation/nitration differentially modify the level of αSYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the αSYN proteoforms analyzed by the three immunoassays.ConclusionsOur results showed that these immunoassays do not capture the totality of the relevant αSYN species and therefore may not be appropriate tools to provide an accurate measure of total αSYN levels in samples containing modified forms of the protein. This highlights the need for next-generation αSYN immunoassays that capture the diversity of αSYN proteoforms.</jats:sec

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