Protein homeostasis (proteostasis) is maintained by multiple processes, including protein degradation, which regulate proteins levels in cells, including neurons. Loss of proteostasis is often associated with defects in protein degradation and can lead to protein aggregation, a hallmark of ageing diseases including neurodegeneration. The ubiquitin-proteasome system (UPS) is the major selective protein degradation pathway in cells. It involves a number of enzymes that act in a sequential manner to post-translationally attach a small protein modifier, ubiquitin, onto lysine residues of a protein target. E3 ubiquitin ligases function in the last step of this cascade by mediating the transfer of ubiquitin onto the protein substrate. Protein ubiquitination is well characterised in dividing cells and different types of enzymes and ubiquitin chains have been shown to play various roles from DNA repair to regulation of mitosis. In contrast, the roles and functions of ubiquitin signalling are less well delineated for terminally differentiated neurons. In this thesis, I investigated the expression, localisation and mode of regulation of the E3 ubiquitin ligase HECTD1 in mouse-derived cortical neurons. I show that HECTD1 is expressed in adult mouse brain and mouse embryo-derived cortical neurons where it is cleaved in response to bath application of NMDA. This cleavage is mediated through extrasynaptic NMDA receptors in mature (12 DIV) neurons whereas in young (7 DIV) neurons it requires the activation of both synaptic and extrasynaptic pools of the NMDA receptors. HECTD1 proteolytic cleavage is calcium-dependent and requires calpain and caspase activity in a sequential process and is independent on apoptosis. It was also shown that HECTD1 cleavage can be induced using FCCP (a mitochondrial uncoupler), antimycin A (mitochondrial complex III inhibitor) and thapsigargin (ER stress inducer). Those drugs are known to induce an increase in intracellular calcium levels. Furthermore, using biochemical fractionation and immunofluorescence, a pool of HECTD1 was identified at the mitochondria. A more refined fractionation protocol in fact revealed that this E3 ligase might indeed be found at ER-MAMs (ER-mitochondria associated membranes). HECTD1 localisation at mitochondria and potentially ER-MAM, where much of calcium trafficking occurs, is of particular interest especially since HECTD1 cleavage is calcium-dependent. These results open new questions regarding the mode of regulation of E3 ubiquitin ligases by calcium as well as the role of ubiquitin signalling at ER-MAMs. These findings will likely give valuable insight for human disorders, in particular neurodegeneration and heart diseases where unbalanced calcium and dysfunctional ER-MAMs drive many of the damages associated with these pathologies.
Regulation and subcellular localisation of a novel E3 ubiquitin ligase in mouse primary cortical neurons
Jasem, S. (Author). 19 Feb 2020
Student thesis: Doctoral Thesis › PhD