Ultrastructural studies of PINK1-deficient cells often display increased lysosomal content (26
), shown to be due to an increase in mitochondrial autophagy [(26
), Fig. ]. Knockdown of essential autophagy proteins exacerbates cell death in stable PINK1 shRNA lines, indicating that autophagy plays a compensatory role in PINK1-deficient cells (26
). Mitochondrial autophagy induced by PINK1 deficiency is regulated by canonical Beclin 1-dependent mechanisms (77
). Moreover, endogenous Parkin levels increase in some stable PINK1-deficient lines (78
), suggesting a role for Parkin-mediated mitophagy as a compensatory response. Although it remains to be determined whether or not Parkin is necessary for mitophagy in PINK1-deficient cells, cell survival is enhanced by transient overexpression of Parkin (26
). As Parkin may show multiple neuroprotective mechanisms, we employed siRNA to Atg7, the E1-like activating protein for Atg12 and LC3, to study the contribution of autophagy. Inhibition of autophagy substantially reduced the ability of Parkin to confer protection (78
), indicating that a major neuroprotective effect of Parkin in this context involves autophagy.
Figure 2. Selective mitophagy in PINK1-deficient cells. (A) The majority of SH-SY5Y cells stably expressing PINK1 shRNA exhibit autophagic vacuoles (AVs; arrowheads) and at least some morphologically preserved mitochondria. As observed in other pathological states (more ...)
In our ultrastructural studies, a minor fraction of PINK1-deficient cells exhibit swollen, pale mitochondria reminiscent of those observed in toxin-treated cells (Fig. A, right cell). Quantitative analysis indicates that these cells do not show upregulation of lysosomes/late autophagic vacuoles (Fig. B), and most probably account for the low level of basal cell death observed in the PINK1 shRNA cells (26
). In contrast, cells with relatively intact mitochondrial morphologies show the highest content of late autophagic vacuoles (Fig. B), further supporting a role for autophagy in mitochondrial quality control and cytoprotection among individual cells in these stable lines.
As steady-state levels of autophagic vacuoles are not necessarily reflective of autophagic flux or activity, we performed several studies to verify enhanced mitochondrial autophagy. PINK1 knockdown cells show reduced mitochondrial mass as assessed through western blot analysis for either matrix or membrane proteins, or by analysis of the cytoplasmic area occupied by mitochondria (26
). Flux studies using the autophagosome–lysosome fusion inhibitor bafilomycin demonstrate intact lysosomal turnover of both early and total LC3 puncta, as well as lysosome-dependent loss of GFP-tagged mitochondria (26
). Further ultrastructural studies revealed that a 2 h pulse of bafilomycin trapped approximately three times as many early autophagosomes in PINK1 shRNA cells compared with control cells (Fig. C), indicating an increased rate of autophagosome formation. In contrast to control cells that showed a random mixture of cytoplasmic constituents, mitochondria were enriched within early autophagosomes of PINK1-deficient cells (Fig. D), consistent with selective mitophagy.
We also found that overexpression of PINK1 is capable of suppressing toxin-induced increases in autophagic vacuoles (26
), and the ability of RNAi-resistant PINK1 to reverse the autophagic phenotype in stable PINK1 shRNA lines was not dependent upon the N-terminal mitochondrial-targeting sequence. We propose that one effect of PINK1 in maintaining stable mitochondrial networks includes suppression of autophagy. This could be through either direct or indirect mechanisms, such as reducing redox signals for autophagy induction (79
). The possibility that PINK1 overexpression could promote maturation/clearance of toxin-induced autophagosomes also remains to be determined.
Interestingly, several recent papers indicate that PINK1 promotes Parkin-associated mitophagy (48
), potentially with direct effects on Beclin 1 (80
). As depolarized mitochondria are less capable of processing PINK1 to the proteasomally degraded Δ1 form, it is proposed to accumulate on the surface of mitochondria to recruit Parkin (75
). Another study showed that overexpression of PINK1 and Parkin caused trafficking of mitochondria to perinuclear aggresome-assembly areas (73
), although this effect was only observed when both proteins were concurrently overexpressed. The trafficking effect could relate to the association of PINK1 with the Miro/Milton-trafficking adaptors (54
), as Miro is implicated in both anterograde and retrograde trafficking. As Milton mediates kinesin-dependent anterograde transport, it is interesting to note that the Miro/Milton complex can associate with truncated PINK1, while the effects of overexpressed PINK1 on Parkin recruitment and retrograde mitochondrial aggregation are dependent upon its full-length sequence (75
). Overexpressed PINK1 has been reported to exhibit a cytosolic orientation of its kinase domain (69
), and arrested import/processing of overexpressed PINK1 by depolarized mitochondria appears to be sufficient to recruit Parkin to mitochondria (75
In these studies, either acute PINK1 knockdown or PINK1−/− MEFs showed impaired ability to recruit Parkin to chemically depolarized mitochondria. On the other hand, increased Parkin promotes compensatory mitophagy in PINK1-deficient SH-SY5Y cells (26
). Thus, it is unclear whether or not stable physical association of Parkin with mitochondria is necessary for mitophagy or if transient enzymatic interaction is sufficient. Although Parkin recruitment to mitochondria is associated with its ability to cause loss of fluorescently labeled mitochondria, Parkin recruitment and mitochondrial clearance can be experimentally dissociated (75
). Furthermore, studies in neurons may reveal additional regulatory mechanisms, as chemical uncouplers appear to induce complete loss of mitochondrial fluorescence from the affected cells. While glycolysis-competent cell types can survive mitochondrial depletion, excessive mitochondrial degradation is detrimental in neuronal cells (41
Another caveat to consider is whether or not loss of mitochondrial fluorescence could reflect other mechanisms. As discussed above, there are several proteases within mitochondria that could contribute to loss of mitochondrial constituents or quenching of mitochondrial fluorescence in chemically depolarized cells. Mitochondrially targeted proteins are subject to proteasome degradation under depolarizing conditions where import is impaired (84
), and mitochondrial depolarization or permeability transition dissipates intermembrane space proteins. Quantitative ultrastructural analysis to demonstrate increased mitochondria in autophagosomes at early time points, with EM confirmation of absent mitochondrial structures at later time points, would help resolve these possibilities. Alterations in translational regulation, biosynthesis and import/assembly of mitochondrial constituents could also contribute to depletion of mitochondrial content within cells, affecting quality control and the outcome of autophagic responses to injury. As autophagic recycling represents the final tier of mitochondrial quality control in the presence or absence of sufficient PINK1 function, further strategies to enhance selective mitophagy (38
) while promoting mitochondrial biogenesis (85
) may prove effective for multiple forms of Parkinson's disease.