For many years, PINK1 and Parkin have been assigned cytoprotective roles in various organisms. Rana and colleagues demonstrated that the overexpression of Parkin in neurons promoted the lifespan of Drosphila. This was attributed to the efficient turnover of drosophila Mitofusin and the maintenance of normal mitochondrial morphology and respiratory activity by Parkin (A. Rana et al. 2013). Johnson and colleagues demonstrated in Parkin-expressing mouse embryonic stem cells that BAX is targeted for ubiquitylation and proteasomal degradation during activation of mitophagy, and subsequently prevents Cytochrome-C release (Johnson et al. 2012). Furthermore, Parkin has been shown to promote cell survival by stabilising MCL1 indirectly, via the degradation of FBXW7, an E3 ligase that ubiquitylates and promotes the degradation of MCL1 (Inuzuka et al. 2011, Ekholm-Reed et al. 2013).
While the concept of PINK1 and Parkin-induced cell death is contradicting our current understanding of the cytoprotective roles of PINK1 and Parkin, our observation is supported by Yoshii et al. (2011) who demonstrated that Parkin overexpression leads to disruption of mitochondrial membrane integrity (Yoshii et al. 2011). Yoshii and colleagues discovered that overexpression of Parkin in mouse embryonic fibroblasts (MEFs) promotes widespread ubiquitylation and proteasomal degradation of OMM proteins during mitophagy (e.g. TOM20, TOM40, TOM70a and OMP25). Using electron microscopy, they observed mitochondrial swelling, rupture of the OMM, and loss of cristae structure and suggested that the proteasomal degradation of these proteins may damage the integrity of the OMM and lead to the rupture to the OMM. They further showed that proteasomal inhibition could protect the integrity of the mitochondrial double membranes. Consistent with their observation, the cell death that I observed can also be inhibited by proteasomal inhibition whereas inhibition of the lysosome by vacuolar ATPase by folimycin did not rescue the cell death. While the authors did not detect cell death based on caspase 3 activation or nuclear
in our system is also caused by the rupture of OMM, causing cytochrome C release into the cytosol.
Immunofluorescence microscopy revealed that both proteasomal and lysosomal inhibition blocked the clearance of Parkin aggregates. However, proteasomal inhibition promoted the accumulation of large Parkin aggregates (2-3 aggregates per cell) while lysosomal inhibition accumulated numerous small Parkin aggregates. Further investigation of the effect of proteasomal inhibition on Parkin-mediated mitophagy revealed that p62, the adaptor for the recruitment of LC3, as well as LC3 itself were recruited to the mitochondria during both CCCP and CCCP plus epoxomicin treatments, suggesting that the initiation of autophagic process, including the ubiquitylation of mitochondrial proteins and the lipidation of LC3 were not affected by proteasomal inhibition. However, I observed that the Parkin aggregates were larger in epoxomicin co-treated cells, with LC3 punctae decorating the edges of the aggregates. In contrast, in the absence of epoxomicin, cells had smaller Parkin aggregates that were completely enveloped by LC3 (in a ring morphology). This suggests that smaller Parkin aggregates might be turned over more efficiently compared to larger Parkin aggregates. It is likely that the degradation of certain OMM proteins by the proteasome is required for the dissociation of large Parkin aggregate into smaller aggregates prior to lysosomal degradation. For example, the degradation of mitochondrial proteins that are involved in the fusion and fission machinery (e.g. MFN1/2) and the movement of mitochondria (e.g. MIRO1/2) might be impeded, affecting the dissociation of the Parkin- decorated mitochondrial aggregates (Fransson et al. 2006, Fransson et al. 2003, Chen et al. 2003). Both MFN1/2 and MIRO1/2 are known substrates of Parkin during mitophagy and the degradation of the two have been shown to promote mitophagy (Ziviani and Whitworth 2010, Gegg et al. 2010, Wang et al. 2011).
P97, an AAA-ATPase, has been shown to retrotranslocate MCL1 and MFN1 from the mitochondria OMM to the cytosol prior to proteasomal degradation (Xu et al. 2011, Tanaka et al. 2010). To further test if the physical action of
protein extraction from the mitochondria by p97 is responsible for the damage of the OMM, I used DBeQ, a p97 inhibitor. I observed that high concentrations of DBeQ treatment alone could induce OPA1 cleavage and Parkin recruitment, suggesting that mitophagy was induced upon p97 inhibition. Unlike proteasomal inhibition, p97 inhibition did not rescue the cells from cell death. Instead, simultaneous treatment of cells with CCCP and DBeQ exacerbated cell death. Since p97 is involved in a wide array of functions, ranging from chromatin remodelling, endocytic vesicle maturation, autophagosomal maturation to ER-associated degradation (ERAD), it is possible that the cell death observed is not a direct result of mitophagic cell death but a combined effect from multiple cellular stresses (Meyer et al. 2012, Chou et al. 2011). Indeed, Chou and colleagues reported that DBeQ is highly potent in activating caspase 3 and caspase 7 via a mechanism that is not yet understood (Chou et al. 2011). Therefore, no strong conclusion can be drawn from the effect of p97 on mitophagic cell death.
Cyt-C release is generally associated with apoptotic cell death (Jiang and Wang 2000). Indeed, I observed Cyt-C release during CCCP treatment using digitonin permeabilisation of the cell membrane. This was further supported by the observation of a small population of cells (20%) with partial cytosolic localization of Cyt-C.
In healthy cells, phospholipids are assymetrically-distributed between the outer and inner membranes. The outer membrane is composed mostly of cholinephospholipids, such as phosphatidylcholine and sphingomyelin whereas the inner membrane is composed mostly of aminophospholipids, including phosphatidylserine (PS) and phosphatidylethanolamide (PE). The distribution of PS and PE is actively maintained by the enzyme aminophospholipid translocase (‘flippase’) (Diaz and Schroit 1996). During apoptosis, an enzyme known as the phospholipid scramblase is cleaved and activated by effector caspases, which promotes bidirectional movement of phospholipids, thereby exposing PS and PE to the cell surface (Zhou et al. 1997, Martin et al. 1995). The externalized PS and PE can be recognized
affinity to the plasma membrane serves as an alternative read out for apoptosis. I treated the cells with CCCP in the presence of annexin V conjugated with a fluorescent dye and a membrane impermeable dye, DRAQ7. During CCCP treatment, annexin V was seen to localize to the plasma membrane (at 8hr) and this event precedes the incorporation of DRAQ7 (at 12hr).
The apoptotic nature of this cell death is also confirmed by its sensitivity of Z- VAD-FMK, a broad spectrum caspase inhibitor (Slee et al. 1996). However, Z-VAD-FMK does not differentiate between the intrinsic and extrinsic pathway. The current view is that caspase 8 is cleaved and activated during extrinsic apoptotic activation, which could either cleave caspase 3 to directly trigger apoptosis, or cleave Bid into tBid to activate Bak and Bax oligomerisation on the OMM, resulting in Cyt-C release into the cytosol. Cyt- C then triggers the assembly of the apoptosome that subsequently activates Caspase 9. Caspase 9 cleaves Caspase 3 to promote apoptosis. On the other hand, intrinsic apoptosis is triggered directly via the mitochondria where internal insults trigger the release of Cyt-C from the mitochondria, sharing the same downstream mitochondria-mediated pathway as described above for extrinsic apoptosis (Mukhopadhyay et al. 2014).
In our setting, it seems that both caspase 8 and caspase 9 are required for the activation of apoptosis. We postulate two explanations to the observation (Figure 4.11). In the first scenario, cell death might take place via the extrinsic pathway but activation of caspase 8 alone is insufficient to activate caspase 3. Instead, the activation of caspase 9 via Bid cleavage and Cyt-C release might be required to amplify the caspase cascade. This is less likely to be the case as the extrinsic triggers are usually Fas, TNF or TRAIL ligands that bind to their respective receptors to activate the extrinsic pathway. In the alternative, and more likely scenario, cell death might take place via the intrinsic pathway. In a situation where a small subset of damaged mitochondria is not efficiently engulfed by the lysosomes, only a small amount of Cyt-C is released into the cytosol. This low level of Cyt-C might not be sufficient to activate apoptosis. Instead, a positive feedback loop via
the activation of caspase 3, which has been reported to cleave caspase 8 might be required to further activate tBid and pemeabilise the OMM to amplify the caspase cascade (Figure 4.11)(Sohn et al. 2005, Viswanath et al. 2001). Viswanath and colleagues demonstrated in rat adrenal medulla cell line (PC12 cells) that 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) treatment (150µM) induces caspase 9 activation within 2hr of treatment, followed by caspase 3 activation (2-4hr). Caspase 8 activation and Bid cleavage did not occur until late stages (4-6hr). They further demonstrated that inhibition of caspase 9 using LEHD-FMK, a cell-permeable caspase-9 specific inhibitor, prior to MPTP treatment did not block Cyt-C release but inhibited caspase 8 activation and the subsequent cell death (Viswanath et al. 2001). Therefore, it is possible that the cell death observed in our cell line follows a similar intrinsic apoptotic pathway as PC12 cells.
Very recently, Zhang and colleagues reported that HeLa cells expressing Parkin underwent apoptosis after valinomycin and mitophagy after CCCP treatment (Zhang et al. 2014). They demonstrated that the differential response was due to the selective ubiquitylation and degradation of MCL1 by Parkin during valinomycin but not CCCP treatment, which dictate whether cells undergo mitophagy or apoptosis. While I have not tested valinomycin in hTERT-RPE1-YFP-Parkin cells, I propose that the trigger and mechanism for our cell death observation is most likely different from that described by Zhang and colleagues. MCL1 degradation induces apoptosis by allowing BAK and BAX oligomerisation on the OMM for Cytochrome-C release. In chapter 6, I will demonstrate that BAK and BAX knockdown does not affect CCCP-induced cell death, which argues against the upstream effect of MCL1 degradation.
Figure 4.11 Proposed model for the involvement of caspase 8 and caspase 9 in Parkin-mediated mitophagic cell death.
Mitochondrial depolarisation triggers PINK1 and Parkin recruitment to the outer mitochondrial membrane (OMM). High expression of Parkin causes aberrant ubiquitylation of OMM proteins, resulting in their extraction by p97 for proteasomal degradation, which affects the integrity of the outer mitochondrial membrane. Cytochrome-C is release from the mitochondrial intermembrane space, triggering the intrinsic apoptotic pathway. The assembly of the apoptosome activates caspase- 9, which then activates caspase 3. Caspase 3 and caspase 8 can inter-activate each other, further amplifying the caspase cascade to trigger apoptosis.
PINK1 Casp9 Casp8 Parkin Mitochondrial depolarisation extraction for proteasomal degradation apoptosome amplification Apoptosis OMM substrate p97 ubiquitin Cyt-C autophagosomal engulfment Casp3