Baldo, Barbara. Innovative approaches to monitor mutant huntingtin and to facilitate its degradation in Huntington's disease models. 2011, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9707
The mechanisms leading to the toxicity induced by mutant huntingtin are not well understood. However the acquisition of a misfolded conformation and the formation of intracellular inclusions constituted by shorter fragments of the mutant protein are considered important in the neurodegenerative process.
In my thesis project I have investigated mechanisms to enhance the cellular degradation of mutant huntingtin. A second focus was on the development of an immunoassay to detect and quantify aggregates in HD models.
I analyzed the data obtained form a high through-put screen aimed to identify small molecular weight compounds decreasing mutant huntingtin levels in cells. Among all compounds screened, only inhibitors of heat shock protein 90 (Hsp90) showed a significant effect on mutant huntingtin clearance. I therefore investigated the mechanisms of Hsp90 chaperone inhibition and the reduction of soluble mutant huntigtin levels. Data from biochemical assays demonstrated that mutant huntingtin degradation is enhanced upon compound treatment and that the protein is cleared through the ubiquitin-proteasome system. This was independent from the heat shock response induced after pharmacological Hsp90 inhibition. Co-immunoprecipitation experiments suggested that mutant huntingtin is a client protein of Hsp90. The results were replicated in different cellular models including full length mutant huntingtin expressed from the endogenous locus, thus highlighting the importance of Hsp90 in stabilizing soluble mutant huntingtin and suggesting the possible application of Hsp90 inhibitors as therapies in HD.
In the second project I developed a sensitive method to detect mutant protein aggregates in HD models. To this purpose I implemented the already established time resolved fluorescence resonance energy transfer (TR-FRET) based immunoassay for the detection of soluble mutant and wild-type huntingtin. A mixture of either donor or acceptor fluorophore labeled single monoclonal antibody directed against an epitope exposed on the huntingtin aggregate surface was used. This strategy allowed for energy transfer and therefore a measurable TR-FRET signal, only in presence of mutant aggregated protein. I could demonstrate the sensitivity of the bioassay on a microtiter set up both as a single assay and in a duplex combination with the previously developed TR-FRET assay for soluble huntingtin.
I applied the TR-FRET for aggregated huntingtin to samples from R6/2 and HdhQ150 mice, expressing exon 1 and full length mutant huntingtin, respectively. In brain homogenates from both models there was an age-dependent, inverse correlation between soluble and aggregated mutant huntingtin. These findings supported the importance of the relation between aggregated and soluble protein in disease progression. Furthermore, I detected the inverse correlation also in peripheral tissues of R6/2 mice where the presence of aggregates was previously demonstrated with other methods. An in-depth analysis of R6/2 samples in a combination of TR-FRET and size exclusion chromatography suggested a differential specificity of the two antibody combinations used for different aggregate populations. The TR-FRET method provides a new means to characterize the aggregation process as well as to test the efficacy of possible disease modifying treatments for HD.
|Committee Members:||Rüegg, Markus|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Neurobiology > Pharmacology/Neurobiology (Rüegg)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||120 S.|
|Last Modified:||30 Jun 2016 10:42|
|Deposited On:||22 Dec 2011 09:54|
Repository Staff Only: item control page