The critical processes of mitochondrial fission and fusion are regulated by members of the dynamin family of GTPases. Imbalances in mitochondrial fission and fusion contribute to neuronal cell death. For example, increased fission mediated by the dynamin-related GTPase, Drp1, or decreased fusion resulting from inactivating mutations in the OPA1 GTPase, cause neuronal apoptosis and/or neurodegeneration. Recent studies indicate that post-translational processing regulates OPA1 function in non-neuronal cells and moreover, aberrant processing of OPA1 is induced during apoptosis. To date, the post-translational processing of OPA1 during neuronal apoptosis has not been examined. Here, we show that cerebellar granule neurons (CGNs) or neuroblastoma cells exposed to pro-apoptotic stressors display a novel N-terminal cleavage of OPA1 which is blocked by either pan-caspase or caspase-8 selective inhibitors. OPA1 cleavage occurs concurrently with mitochondrial fragmentation and cytochrome c release in CGNs deprived of depolarizing potassium (5K condition). Although a caspase-8 selective inhibitor prevents both 5K-induced OPA1 cleavage and mitochondrial fragmentation, recombinant caspase-8 fails to cleave OPA1 in vitro. In marked contrast, either caspase-8 or caspase-3 stimulates OPA1 cleavage in digitonin-permeabilized rat brain mitochondria, suggesting that OPA1 is cleaved by an intermembrane space protease which is regulated by active caspases. Finally, the N-terminal truncation of OPA1 induced during neuronal apoptosis removes an essential residue (K301) within the GTPase domain. These data are the first to demonstrate OPA1 cleavage during neuronal apoptosis and they implicate caspases as indirect regulators of OPA1 processing in degenerating neurons.

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. Here, we 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 N-terminal 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.