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Mutant Huntingtin toxicity modifiers revealed by a spatiotemporal proteomic profiling
Mutant Huntingtin toxicity modifiers revealed by a spatiotemporal proteomic profiling
Huntington's disease (HD) is a genetic hereditary disorder characterized by aggregation of polyQ-expanded mutant Huntingtin (mHTT) protein and progressive neurodegeneration within different brain regions, but specially in cortex and striatum. The pathology is associated with motor, cognitive and psychiatric symptoms. A hallmark of HD is the aggregation of polyglutamine-expanded (polyQ) huntingtin from soluble oligomers to inclusion bodies. Still nowadays, the character of these aggregates and the transition to the neuronal functional disorder, is poorly understood. In this thesis, the progression of the disease was assessed in a spatiotemporal manner in the R6/2 mice, a HD model, in order to find molecular signatures that could lead first, to a more detailed description of the disorder and second, to the elucidation of possible protein candidates that eventually have the ability of modify HD-related toxicity. Initially, it was approached by mass spectrometry-based quantitative proteomics to break down the spatiotemporal mechanisms of degeneration in HD. The formation of insoluble inclusion bodies throughout the disease progression correlated with the profound remodeling of the soluble proteome. The complexity in protein numbers of the aggregates was detailed through a quantitative characterization. This deep analysis unraveled the dependency of the aggregates' protein sequestration on specific biophysical features and sequence domains. Based on the proteomic data and applying different criteria, a follow-up study of some proteins was carried out. Overexpression of a selected group of the sequestered proteins improved the cellular viability in a cell line model of HD and reduced in most cases the inclusion body size. The effect of most of those proteins was specific to a mHTT-toxicity induced context. These results suggest that widespread loss of function contributes to aggregate-mediated toxicity. The strong effect of one of the protein candidates in the viability assays, Hepatome-derived grow factor (HDGF), lead to a closer examination. The effect of the protein was confirmed in primary neurons, in both transient transfection and in long-term viral transduction. Overexpression of HDGF in the striatum of R6/2 mice significantly rescued their exploratory behavior and ameliorated their clasping phenotype. In summary, the thesis represents a multi-disciplinary study in the R6/2 mouse model, spanning from proteomics to in vivo overexpression of a validated sequestered protein, which appears to be a potential therapeutic mHTT-toxicity modifier. Collectively, the study provides an integrative approach to solve HD molecular mechanisms and contributes to fill in the gap between identification of disease-associated pathways and their corresponding phenotypes.
Huntington, neurodegeneration, aggregation, mice, proteomics
Gutiérrez Ángel, Sara
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Gutiérrez Ángel, Sara (2019): Mutant Huntingtin toxicity modifiers revealed by a spatiotemporal proteomic profiling. Dissertation, LMU München: Faculty of Biology
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Abstract

Huntington's disease (HD) is a genetic hereditary disorder characterized by aggregation of polyQ-expanded mutant Huntingtin (mHTT) protein and progressive neurodegeneration within different brain regions, but specially in cortex and striatum. The pathology is associated with motor, cognitive and psychiatric symptoms. A hallmark of HD is the aggregation of polyglutamine-expanded (polyQ) huntingtin from soluble oligomers to inclusion bodies. Still nowadays, the character of these aggregates and the transition to the neuronal functional disorder, is poorly understood. In this thesis, the progression of the disease was assessed in a spatiotemporal manner in the R6/2 mice, a HD model, in order to find molecular signatures that could lead first, to a more detailed description of the disorder and second, to the elucidation of possible protein candidates that eventually have the ability of modify HD-related toxicity. Initially, it was approached by mass spectrometry-based quantitative proteomics to break down the spatiotemporal mechanisms of degeneration in HD. The formation of insoluble inclusion bodies throughout the disease progression correlated with the profound remodeling of the soluble proteome. The complexity in protein numbers of the aggregates was detailed through a quantitative characterization. This deep analysis unraveled the dependency of the aggregates' protein sequestration on specific biophysical features and sequence domains. Based on the proteomic data and applying different criteria, a follow-up study of some proteins was carried out. Overexpression of a selected group of the sequestered proteins improved the cellular viability in a cell line model of HD and reduced in most cases the inclusion body size. The effect of most of those proteins was specific to a mHTT-toxicity induced context. These results suggest that widespread loss of function contributes to aggregate-mediated toxicity. The strong effect of one of the protein candidates in the viability assays, Hepatome-derived grow factor (HDGF), lead to a closer examination. The effect of the protein was confirmed in primary neurons, in both transient transfection and in long-term viral transduction. Overexpression of HDGF in the striatum of R6/2 mice significantly rescued their exploratory behavior and ameliorated their clasping phenotype. In summary, the thesis represents a multi-disciplinary study in the R6/2 mouse model, spanning from proteomics to in vivo overexpression of a validated sequestered protein, which appears to be a potential therapeutic mHTT-toxicity modifier. Collectively, the study provides an integrative approach to solve HD molecular mechanisms and contributes to fill in the gap between identification of disease-associated pathways and their corresponding phenotypes.