The mitochondrial disease associated protein Ndufaf2 is dispensable for Complex-1 assembly but critical for the regulation of oxidative stress.

Deficiency in human mitochondrial Complex-1 has been linked to a wide variety of neurological disorders. Homozygous deletion of the Complex-1 associated protein, Ndufaf2, leads to a severe juvenile onset encephalopathy involving degeneration of the substantia nigra and other sub-cortical regions resulting in adolescent lethality. To understand the precise role of Ndufaf2 in Complex-1 function and its links to neurologic disease, we studied the effects on Complex-1 assembly and function, as well as pathological consequences at the cellular level, in multiple in vitro models of Ndufaf2 deficiency. Using both Ndufaf2-deficient human neuroblastoma cells and primary fibroblasts cultured from Ndufaf2 knock-out mice we found that Ndufaf2-deficiency selectively reduces Complex-1 activity. While Ndufaf2 is traditionally referred to as an assembly factor of Complex-1, surprisingly, however, Ndufaf2-deficient cells were able to assemble a fully mature Complex-1 enzyme, albeit with reduced kinetics. Importantly, no evidence of intermediate or incomplete assembly was observed. Ndufaf2 deficiency resulted in significant increases in oxidative stress and mitochondrial DNA deletion, consistent with contemporary hypotheses regarding the pathophysiology of inherited mutations in Complex-1 disorders. These data suggest that Ndufaf2, unlike other Complex-1 assembly factors, may be more accurately described as a chaperone involved in proper folding during Complex-1 assembly, since it is dispensable for Complex-1 maturation but not its proper function.

Parkin selectively alters the intrinsic threshold for mitochondrial cytochrome c release.

Autosomal-recessive mutations in the Parkin gene are the second most common cause of familial Parkinson's disease (PD). Parkin deficiency leads to the premature demise of the catecholaminergic neurons of the ventral midbrain in familial PD. Thus, a better understanding of parkin function may elucidate molecular aspects of their selective vulnerability in idiopathic PD. Numerous lines of evidence suggest a mitochondrial function for parkin and a protective effect of ectopic parkin expression. Since mitochondria play a critical role in cell survival/cell death through regulated cytochrome c release and control of apoptosis, we sought direct evidence of parkin function in this pathway. Mitochondria were isolated from cells expressing either excess levels of human parkin or shRNA directed against endogenous parkin and then treated with peptides corresponding to the active Bcl-2 homology 3 (BH3) domains of pro-apoptotic proteins and the threshold for cytochrome c release was analyzed. Data obtained from both rodent and human neuroblastoma cell lines showed that the expression levels of parkin were inversely correlated with cytochrome c release. Parkin was found associated with isolated mitochondria, but its binding per se was not sufficient to inhibit cytochrome c release. In addition, pathogenic parkin mutants failed to influence cytochrome c release. Furthermore, PINK1 expression had no effect on cytochrome c release, suggesting a divergent function for this autosomal recessive PD-linked gene. In summary, these data demonstrate a specific autonomous effect of parkin on mitochondrial mechanisms governing cytochrome c release and apoptosis, which may be relevant to the selective vulnerability of certain neuronal populations in PD.