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Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.
Parkinson disease (PD) is characterized by the degeneration of dopaminergic and other monoaminergic neurons, leading to deficits in motor, autonomic and eventually cognitive systems. Although PD is predominantly sporadic, there are familial mutations that segregate with disease. Mutations in parkin, an E3 ubiquitin ligase, and in PTEN-induced kinase 1 (PINK1), a mitochondrially-targeted serine/threonine kinase, are associated with autosomal recessive forms of PD. While the pathogenesis of PD remains unclear, certain observations are common between PD patients and toxin or genetic models of PD. Mitochondrial dysfunction, reactive oxygen species (ROS), protein aggregation and autophagosomes have each been observed in PD tissues and models.
Some PINK1 mutants have reduced kinase activity and others show reduced protein stability; these observations suggest that PINK1 normally functions to protect neurons. Likewise, parkin overexpression is protective in several models of neuronal injury, including PINK1 deficiency. Recent data indicate that the neuroprotective effects of parkin and of PINK1 may converge at regulating mitochondrial homeostasis, although their specific effects on mitochondrial dynamics and autophagic turnover appear dissimilar.
We recently found that RNA interference-mediated knockdown of PINK1 expression leads to mitochondrial fragmentation, mitochondrial ROS production, decreased mitochondrial cristae density, and cell death in multiple clonal lines derived using two distinct shRNA sequences. Autophagy played a protective role in clearing damaged mitochondria, a process that is facilitated by Drp1-dependent fission and parkin overexpression. The loss of mitochondrial content, as assessed using fluorescence image analysis and western blot, is inhibited by bafilomycin or RNAi knockdown of Atg7 or Atg8 (Supplement to Dagda et al. 2009). Use of antioxidants reveals a role for ROS upstream of fission, autophagy and cell death. Interestingly, MnTBAP but not catalase suppresses the increase in GFP-LC3 puncta levels in PINK1-deficient cells. This suggests a role for intracellular superoxide or peroxynitrite in signaling mitophagy, consistent with prior observations that ROS are necessary for induction of canonical autophagy.
Electron microscopy studies of the PINK1-deficient lines reveal structural disruption and loss of inner membrane cristae folds, whereas overexpression of PINK1 increases cristae density. Consistent with previous studies, we found that PINK1 knockdown led to decreased transmembrane potential, which was ameliorated by MnTBAP treatment (our unpublished data), suggesting that ROS production is upstream of mitochondrial depolarization. Given that other groups have shown that membrane depolarization can inactivate the mitochondrial fusion protein Opa1, promote calcineurin-mediated activation of the fission protein Drp1, and initiate parkin-enhanced mitophagy, increased ROS/respiratory chain dysfunction likely represents an early effect of mitochondrial PINK1 loss of function.
Although mitochondrial injury elicits autophagic degradation of mitochondria in both the 1-methyl-4-phenylpyridinium (MPP+) and the PINK1-deficient model, the role of autophagy is quite different in these two injury models. Interestingly, reports in several systems that suggest a pro-death or detrimental role for autophagy involve increased autophagic/mitophagic flux that is regulated through a Beclin 1/PI3K-independent mechanism, including MPP+ toxicity, resveratrol-treated cancer cells, mutant LRRK2 mediated neurite shortening, and during PUMA-induced mitochondrial stress. In contrast, the current study indicates that canonically regulated autophagy induced in the context of PINK1 deficiency contributes to neuroprotective clearance of damaged mitochondria. Canonical autophagy has also been shown by other groups to mediate clearance of alpha-synuclein oligomers/aggregates. One possible explanation is that mitophagy is beneficial in cases of chronic or mild mitochondrial injury. On the other hand, toxin treatment may cause more severe and widespread damage to a greater percentage of mitochondria, resulting in excessive mitophagy.
In PINK1-deficient cells, inhibition of either autophagy or mitochondrial fission increases cell death, but parkin overexpression confers protection. The increased parkin expression correlates with further enhancement of autophagy in PINK1-deficient cells, suggesting that one mechanism by which parkin confers protection may be through enhanced autophagic recycling of dysfunctional mitochondria. In contrast, we found that restoring PINK1 expression using an RNAi-resistant plasmid results in suppression of autophagy in the shRNA lines. These data support the concept that PINK1 and parkin may serve to maintain mitochondrial morphology and promote cell survival through different mechanisms, with the effects of parkin mediated through autophagy induction, as reported in other models of mitochondrial dysfunction.
It is also interesting to note that PINK1 knockout mice exhibit much more subtle pathology than observed in cell culture or in Drosophila models. While there are many possible reasons for this, reports of increased parkin levels in PINK1-deficient cells, which we have also observed (our unpublished data), and the role of parkin in promoting clearance of depolarized mitochondria suggest another possibility. Whether or not there is significant parkin upregulation in PINK1 knockout mice remains to be reported; however, this could theoretically contribute to developmental compensations.
To summarize, PINK1-deficient cells exhibit alterations in mitochondrial function, morphology and autophagic turnover. Respiratory chain dysfunction involving increased superoxide production appears to function as an upstream signal to trigger fission, autophagy and depolarization-related recruitment of parkin to mitochondria. Each of these responses would serve to facilitate compensatory autophagic clearance of damaged mitochondria. Based on these data, we propose that PINK1 functions to prevent mitochondrial damage, whereas parkin facilitates recycling of damaged mitochondria in a pathway that converges downstream of PINK1 to maintain a healthy mitochondrial complement.
Supported by funding from the National Institutes of Health (AG026389, R01 supplement AG026389-03W1 and NS053777). S.J.C. was supported in part by F31 NS064728 and R.K.D. by F32 AG030821.
Previously published online: www.landesbioscience.com/journals/autophagy/article/10050
Punctum to: Dagda RK, Cherra SJ, III, Kulich SM, Tandon A, Park D, Chu CT. Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem 2009; 284:13843–55; PMID: 19279012; DOI 10.1074/jbc.M808515200.