Regulation of the redox homeostasis during polyglutamine misfolding in Huntington’s Disease
Sajjad, Muhammad Umar (2010) Regulation of the redox homeostasis during polyglutamine misfolding in Huntington’s Disease. University of Southampton, School of Biological Sciences, Doctoral Thesis , 278pp.
Huntington’s Disease (HD) is one of many neurodegenerative diseases that are associated with protein misfolding, aggregation and oxidative stress. While several changes in the redox homeostasis have been shown to occur in HD animal models and HD brains, the formal relationships between intracellular protein misfolding that occurs in HD, redox dysregulation and cellular toxicity are unknown. Therefore, several cellular models of intracellular polyglutamine (polyQ) protein misfolding were established for mechanistic studies.
Various in vitro transient and stable cell expression systems expressing an N-terminal fragment of huntingtin (htt) (httExon 1, httEx1) with/or without a polyQ expansion and fused to fluorescent proteins were characterized. Mutant httEx1 (mhttEx1) constructs expressed in both neuronal and non-neuronal cell lines produced early polyQ aggregates and intracellular inclusion bodies (IBs) followed by cell toxicity that increased over time in time-course experiments. Using oxidation-sensitive probes, reactive oxygen species (ROS) were measured in polyQ-expressing cells using single, live-cell imaging analysis by confocal microscopy or population assays in order to explore the relationship between polyQ aggregation, ROS production and cellular toxicity. This study highlighted an early increase in ROS due to the expression of aggregation-prone mhttEx1 in both transient and stable cellular systems that coincided with polyQ aggregation, but preceded cell death. Suppression of ROS and toxicity was achieved by two antioxidant compounds (L-NAC and Trolox). Moreover, the use of MitoQ (Coenzyme Q10 covalently attached to triphenylphosphonium cation (TPP+)) at nanomolar concentrations abrogated the increased ROS due to mhttEx1 suggesting a mitochondrial origin of ROS.
Given that molecular chaperones regulate the folding/misfolding of proteins and are involved in the regulation of the cellular redox homeostasis, the role of the redoxactivatable chaperone DJ-1 in HD was investigated. Protein expression analysis in HD cell models, a rodent model of HD and human HD brain samples showed an up-regulation of DJ-1 protein expression compared to control samples. Oxidation of DJ-1 was also elevated
in the human HD cortex. To test for a functional role of DJ-1 elevation and oxidation in HD, DJ-1 was overexpressed with wild-type or mhttEx1 in cell lines and mouse primary astrocytes. Overexpression of DJ-1 accelerated mhttEx1 aggregation and toxicity both of which could be suppressed by exposure of cells to mild oxidants suggesting that DJ-1, when redox-activated to a chaperone, modulates polyQ aggregation and toxicity. This hypothesis was tested by overexpression of mhttEx1 with a DJ-1 mutant lacking a critical redox activatable cysteine (Cys106). The C106S-DJ-1 mutant lost its ability to reduce polyQ aggregation and toxicity under oxidising conditions upon co-expression with mhttEx1 suggesting that DJ-1 indeed functions as a modulator of polyQ misfolding and toxicity.
Together this work suggests that ROS may be produced during polyQ aggregation and is involved in cellular toxicity. This study also shows that DJ-1 regulates both, polyQ aggregation and toxicity in cell models and given the increased DJ-1 expression in vitro and in vivo (human HD), this protein could be a potential target for HD therapy.
|Item Type:||Thesis (Doctoral)|
|Subjects:||R Medicine > RB Pathology
R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
Q Science > QH Natural history > QH301 Biology
|Divisions:||University Structure - Pre August 2011 > School of Biological Sciences
|Date Deposited:||03 Dec 2010 16:43|
|Last Modified:||08 Jun 2012 13:00|
|Contributors:||Sajjad, Muhammad Umar (Author)
Wyttenbach, Andreas (Thesis advisor)
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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