Doctor of Philosophy
School of Biological Sciences
Whiten, Daniel R., The Role of clusterin in motor neuron disease and the molecular mechanism of its action, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2016. http://ro.uow.edu.au/theses/4727
Amyotrophic lateral sclerosis (ALS) is a currently untreatable disease characterised by the progressive loss of motor neurons. Death typically occurs as a result of respiratory failure within just 3 years of diagnosis. Aberrant protein aggregation is a hallmark of ALS – in particular, the aggregation of transactive response DNA binding protein 43 kDa (TDP-43) to form cytosolic inclusions is observed in >95% of ALS cases. Mutations in the gene encoding TDP-43 itself are believed to underpin the pathology in approximately 5-10% of all cases. Clusterin (CLU) was the first secreted mammalian chaperone identified. In recent years, it has been established that under endoplasmic reticulum (ER) stress conditions the secretion of CLU is reduced, and instead the protein is retrotranslocated to the cytosol. Much of the work described in this thesis was directed towards testing the hypothesis that (under ER stress) CLU may interact with TDP-43 in the cytosol to influence its aggregation and/or the clearance of inclusions.
The first aim of this project was to develop a robust technique that could be used to quantify the effect of intracellular CLU (or any protein, drug or treatment) on the number of intracellular inclusions formed by a target protein, such as TDP-43. In Chapter 3, a flow cytometric method (termed flow cytometric characterisation of inclusions and trafficking; FloIT) is described that involves the analysis of detergentlysates of cells. By measuring both fluorescent protein inclusions and fluorescentlystained nuclei, the number of inclusions originating from a known number of cells can be measured. FloIT was shown to accurately measure inclusions formed by a variety of proteins and can be adapted for use in a variety of applications, such as measuring the co-aggregation of proteins into inclusion bodies and the nuclear trafficking of fluorescent molecules.