We have shown that lack of the recessive parkinsonism protein DJ-1 causes several defects in mitochondria related to oxidative stress. Specifically, mitochondria in DJ-1-deficient cells are depolarized and have an increased tendency to fragment, which agrees with other studies (39
). Furthermore, we defined a common phenotype and relationship between DJ-1 and two other genes for recessive parkinsonism, PINK1 and parkin.
Numerous reports demonstrate that the lack of DJ-1 increases sensitivity to reactive oxygen species (ROS) and mitochondrial complex I inhibitors (5
). In response to oxidative stress, DJ-1 can upregulate glutathione synthesis (30
), which may be related to the ability of DJ-1 to interact with mRNA transcripts related to glutathione metabolism (24
). Supporting the concept that ROS metabolism is important in mediating loss of DJ-1 phenotypes, increasing intracellular glutathione fully rescued the mitochondrial phenotypes. We have previously shown that the ability of DJ-1 protein to respond to oxidative stress is critical for maintenance of mitochondrial morphology (35
). These results suggest that DJ-1 influences mitochondrial function and morphology through an oxidative stress pathway.
The two principal mitochondrial phenotypes seen in these DJ-1-deficient cells, namely loss of membrane potential and an increased tendency to fragment, are similar to observations made for PINK1 knockdown (7
). For both PINK1 and DJ-1, the morphological effects can be rescued by blocking mitochondrial fission. However, the fusion effects may be secondary to other mechanisms. For example, in PINK1-deficient cells, calcineurin inhibitors can block the tendency for mitochondrial fragmentation, whereas loss of Δψm
), and thus fragmentation is a secondary event.
In a previous report in mammalian cells, PINK1 deficiency triggers increased fragmentation of mitochondria, which can be rescued by parkin but not by DJ-1 (19
). Because parkin also rescues the DJ-1 deficiency phenotype in this study, we might conclude that DJ-1 is upstream of PINK1/parkin. However, because we see that DJ-1 is still active at preventing loss of mitochondrial connectivity induced by rotenone in the absence of PINK1, we suggest instead that DJ-1 and PINK1/parkin are parallel pathways. We do not find evidence for a native co-complex of the three proteins (36
), which indirectly supports a parallel relationship.
DJ-1 now shares an additional similarity to PINK1 and parkin, an association with autophagy. Autophagy has been extensively addressed in the field of recessive parkinsonism. Reductions in mitochondrial membrane potential initiate parkin-mediated autophagy (4
), which is inhibited in PINK1-deficient cells (21
). However, our study does not find a complex, whereby PINK1 and parkin physically interact before or after mitochondrial depolarization despite other reports showing such evidence (45
The association of DJ-1 with autophagy is varied. DJ-1 silencing has resulted in the upregulation (39
) and downregulation (40
) of autophagy. Our results suggest that the loss of DJ-1 promotes an increase in autophagy, as an increase in LC3-GFP-positive punctatae and LC3-II protein levels are seen. These data indicate that DJ-1 function may either contribute to the regulation of autophagy or mitigate the downstream effects of ROS, as studies have shown that ROS can upregulate autophagy (49
Future studies should address the role of autophagy in mediating damage caused by ROS. It has been suggested that ERK1/2 regulates lysosomal degradation and more specifically, ERK2 kinase activation is necessary for autophagy (25
). Recently, decreased ERK2 phosphorylation was noted in the mitochondrial fraction of DJ-1-deficient cells (40
) and it was inferred that DJ-1-mediated ERK2 phosphorylation could putatively control autophagic and lysosomal functions. Our data differ from those of that study as we used affinity chromatography to separate phosphorylated proteins from the unphosphorylated whole cellular fraction. Although we did not observe a change in ERK1/2 phosphorylation in DJ-1-deficient cells, it would be worthwhile to examine the phosphorylation status of the mitochondrial pool of ERK1/2.
Additional studies are needed to identify the underlying molecular mechanisms relating PINK1/parkin and DJ-1. We suggest that oxidative stress is a likely common factor as it is important here for DJ-1 function, triggers phenotypes associated with loss of PINK1 (25
) and is sufficient to activate parkin (4
). If correct, this suggests that mitochondrial function under oxidative conditions is a determinant of neuronal survival in recessive parkinsonism.