Regulation of Neuronal Apoptosis by the Mitochondria

It is well accepted that many pathological neurodegenerative diseases are mediated, at least in part, by programmed cell death or apoptosis. A variety of mechanisms that lead to apoptosis have been extensively characterized in a wide range of neuronal types. Mitochondria are a critical starting point for a multitude of apoptotic insults initiating a process described as the intrinsic death pathway. Conflicting evidence has been presented as to what particular steps are essential for initiation of intrinsic neuronal apoptosis i.e. what signals are required and in what chronological order do they occur. Cultured cerebellar granule neurons (CGNs) are an established model to study neuronal apoptosis. CGNs require depolarization and serum for their survival in culture. When deprived of these stimuli, CGNs die via an intrinsic apoptotic cascade characterized by Bim induction, Bax translocation, cytochrome c release, caspase-9 and caspase-3 activation. Mitochondrial events upstream of cytochrome c release are still unclear. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis, but the precise role of mPTP opening in neuronal apoptosis is controversial. This thesis tested the hypothesis that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria in CGNs undergoing apoptosis. During the course of these experiments, we further postulated that other normal mitochondrial functions, such as fission and fusion, regulate the progression of intrinsic apoptosis. To test these hypotheses, we utilized a variety of biochemical assays, most notably western blots, immunocytochemistry, and cell imaging procedures in cultured CGNs. More specifically, we expressed tagged pro- and anti-apoptotic proteins in CGNs, allowing us to alter the apoptotic signals working upstream and downstream of the mitochondria. Results showed that expression of both, a wild type Bax-alpha and a novel point mutant of Bax-alpha (T182A Bax-alpha) that constitutively localized to the mitochondria induced intrinsic apoptosis. Apoptosis induced by expression of the BaxT182A mutant, but not WT Bax, was independent of mPTP opening. More importantly, addition of Cyclosporin A, an mPTP inhibitor, blocked wild type Bax translocation and cytochrome c release, but did not prevent T182A Bax translocation, subsequent cytochrome c release and cell death. These results illustrate a signaling role of the mPTP in translocating Bax to the mitochondria. The requirement of mPTP opening can be subverted by a constitutively active mutant of Bax. This thesis also demonstrates a role for fission proteins in the progression of intrinsic, neuronal apoptosis. Mitochondrial fission and fusion are natural processes of the cell that maintain a healthy network of energy producing mitochondria. Recent evidence has implicated defects of the mitochondrial fusion and fission machinery in human disease. In these pathologies rampant mitochondrial fission leads to spontaneous apoptosis or a heightened sensitivity to apoptotic insults. To investigate the role of mitochondrial fission in intrinsic apoptosis, we utilized hfis1WT and DN constructs to enhance and block mitochondrial fission in cultured CGNs, respectively. Results showed a direct role for the scaffold protein, hfisl, in regulating mitochondrial apoptotic events leading to cytochrome c release and caspase activation. Collectively, the results presented in this thesis broaden our knowledge of the myriad ways in which neuronal apoptosis can be regulated at the level of the mitochondria and provide new potential targets for the prevention of neurodegeneration. The form and content of this abstract are approved.