Mitochondrial Involvement in Neuronal Cell Death

Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. In this thesis, I demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the Nterminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress. Protein aggregation is a common pathogenic mechanism in diverse neurodegenerative disorders. TAR DNA-binding protein-43 (TDP-43) cytoplasmic inclusions are pathological hallmarks of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Furthermore, TDP-43 mutations have been identified in both familial and sporadic forms of ALS. Despite evidence linking dysfunction of TDP-43 to neuronal cell death, it is currently unknown whether TDP-43-induced toxicity occurs via a gain of function or loss of function mechanism. In the present study, I initially examined the effects of either overexpression or knockdown of TDP-43 on neuronal survival in vitro. Overt neuronal cell death was not induced by either experimental paradigm. However, it was noted that both endogenous and overexpressed TDP-43 partially localized to mitochondria within diverse neuronal and astrocytic cell types. Furthermore, a significant decrease in the function of mitochondrial ETC Complex I occurred following TDP-43 knockdown in NSC34 motor neuron/neuroblastoma hybrid cells. These data suggest a novel link between TDP-43 localization and function and mitochondrial physiology. Despite the preponderance of evidence linking TDP-43 dysfunction to neuronal cell death, current investigations indicate that the mechanism by which this protein exerts its toxicity is very complex. Furthermore, little insight has been given into the regulation of TDP-43 nuclear export and cytoplasmic localization. Previously, we described that TDP-43 consistently localizes to the mitochondria in diverse neuronal cell types. In addition, siRNA-mediated TDP-43 depletion was associated with a deficit in mitochondrial ETC complex I. In the present study, I report that TDP-43 nuclear export is enhanced by reductions in mitochondrial glutathione (GSH) levels. Accordingly, redistribution of TDP-43 from the nucleus to the cytoplasm is induced by ethacrynic acid (EA) depletion of cellular glutathione levels, and this effect is inhibited by overexpression of the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), which specifically increases the mitochondrial GSH pool. Furthermore, inhibition of cJun N-terminal kinase (JNK) but not p38 MAP kinase effectively ameliorates EA induced TDP-43 nuclear export. These data suggest novel regulation of TDP-43 cellular distribution and highlights a relationship between mitochondrial physiology, TDP-43 dysfunction, and neuronal cell death