The role of protein degradation in neuronal cell death
thesisposted on 2009-12-09, 10:50 authored by Joanna Marie Norman
In neuronal systems, the degradation of intracellular proteins, controlled by the ubiquitin-proteasome system and autophagy, is of paramount importance for normal cellular homeostasis. The dysfunction of either of these pathways leads to the accumulation of protein aggregates, as seen in neurodegenerative conditions, culminating in neuronal cell death. In the current study I investigated the cleavage of the proteasome subunits, S1, S6´ and S5a in cerebellar granule neurons induced to undergo apoptosis through the withdrawal of potassium. The cleavage of S1 and S6´ and the loss of proteasomal activity corresponded with the activation of caspase-3; however the role of the proteasome was shown to be limited in this model as cells had passed the death commitment point. In addition, I developed a multiubiquitinated fluorescent sensor for the analysis of the proteasomal function on a single cell level, and characterised its use in SH-SY5Y cells. I have also constructed epitope-tagged plasmids encoding the autophagy-related proteins and examined their potential regulation by cell death proteases in an in vitro cleavage assay. Most of the autophagy-related proteins were cleaved in the in vitro model and the potential cleavage sites were identified for mutagenesis. The cleavage of Beclin 1 was also observed in apoptotic cerebellar granule neuron lysates. Finally, I investigated the mechanisms by which the HDACi, TSA, exerts a neuroprotective effect in cerebellar granule neurons. I have demonstrated that it increases the expression of a number of BCL2 family proteins, in particular MCL1, which was hypothesised to contribute to the neuroprotection observed. Taken together, I have demonstrated in this thesis that there are multiple levels of control during cell death; defining their importance is essential for the development of future drug targets.