Mitochondrial stress response

Mitochondria are involved in apoptotic cell death and as such their response to cellular stress is essential in deciding whether a cell survives and recovers or

undergoes apoptosis. Mitochondria respond to cellular stresses in a number of ways including changes in morphology, release of damage associated mitochondrial proteins (DAMPs) and possibly activation of the mitochondrial unfolded protein response (UPRmt_{). Each of these responses will be specific to dealing with the stress} to which the cell has been exposed and each is described in more detail below.

1.5.1 Morphology and dynamics

It has been demonstrated that under low levels of cellular stress, mitochondria fuse to become a connected network (Tondera et al., 2009; Shutt and McBride, 2013; Picard et al., 2014; Leduc-Gaudet et al., 2015), likely allowing functional

complementation and increasing the ability of mitochondria to cope with the stress. However, at higher levels of mitochondrial stress the mitochondrial network

fragments and mitochondria swell. Mitochondrial swelling appears to occur due to an influx of water caused by higher ion concentrations in the matrix over the cytosol as a result of mitochondrial permeability transition pore (PTP) opening (Di Lisa et al., 2001) due to calcium overload (Halestrap et al., 1986) and depolarisation of the mitochondria (Minamikawa et al., 1999).

An alternative to mitochondrial swelling, is the formation of donut or toroid shaped mitochondria (Long et al., 2015). Toroid mitochondria have an increase in

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return to normal mitochondria is easier upon removal of mitochondrial stress (Liu and Hajnoczky, 2011).

1.5.2 Mitochondrial derived vesicles

Mitochondrial derived vesicles have been found to be an early response to oxidative stress occurring prior to mitochondrial depolarisation (Soubannier et al., 2012a; Soubannier et al., 2012b) and are thus form an essential housekeeping and quality control mechanism. Targeting of HeLa cells with a subtoxic dose of ROS, produced MDVs detectable by electron microscopy and confocal microscopy, prior to

mitochondrial fragmentation which are targeted to lysosomes (Soubannier et al., 2012a). There are two species of MDVs which differ based on their subcellular destination and protein composition, with peroxisome targeted MDVs containing MAPL and lysosomal targeted MDVs containing TOM20 (Neuspiel et al., 2008; Soubannier et al., 2012a). Electron microscopy has demonstrated MDVs to be of a regular spherical shape around 80 to 120 nm in size (Neuspiel et al., 2008).

Mechanistic studies suggest GTP hydrolysis is not essential for MDV formation suggesting the process to be independent of DRP1(Soubannier et al., 2012b). More recently, treatment of HeLa cells transfected with GFP-parkin with antimycin A lead to parkin recruitment to the OMM (McLelland et al., 2014). Furthermore, parkin mutants did not generate MDVs and MDV formation was found to require PINK1 similar to mitophagy (McLelland et al., 2014). The involvement of PINK1 and parkin, in both quality control processes, has led to the suggestion that when mitochondrial damage exceeds a threshold level, the membrane potential dissipates and PINK1 detects this change and orchestrates the switch from MDV formation to mitophagy (McLelland et

al., 2014).

1.5.3 The mitochondrial unfolded protein response

The mitochondrial unfolded protein response is a mechanism for dealing with proteotoxic stress specifically in the mitochondria. Although originally identified in cultured rat hepatoma cells (Martinus et al., 1996), the molecular mechanisms involved in the UPRmt _{have mainly been investigated in Caenorhabditis elegans (C.}

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elegans) (Pellegrino et al., 2013), as such the mammalian UPRmt _{has not yet been} fully elucidated.

The mitochondrion, as a double membrane organelle, presents a proteostasis risk for the cell since they require import of numerous proteins to specific locations within the mitochondrion. Thus, the mitochondria are equipped with numerous chaperone proteins that aid in the correct folding and localisation of the imported mitochondrial proteins (Bukau et al., 2006). In addition to this, a number of proteases (e.g. Lon protease, ClpXP, YME1L and paraplegin) are also found within the mitochondria and degrade those proteins that are misfolded (Tatsuta and Langer, 2009).

To date, the established signalling pathway (Figure 1.12), for the induction of the UPRmt _{in C. elegans, is initiated by ClpP which degrades misfolded proteins to} peptides, which are pumped across the IMM and detected by ATFS-1 (Pellegrino et

al., 2013). ATFS-1 is a transcription factor and upon activation localises to the

nucleus activating UPRmt _{genes including chaperone proteins mtHsp70 and Hsp60.} In comparison, the mammalian UPRmt _{(Figure 1.13) is not completely characterised} but appears to have two signalling pathways one for the matrix and one for the IMS. The matrix UPRmt _{functions by activating the JNK2 signalling pathway which}

activates the transcription factor CHOP, which upregulates UPRmt _{genes including} Hsp60, mtDnaJ, ClpP and CHOP (Zhao et al., 2002). The IMS UPRmt _{functions via} the AKT signalling pathway which activates ERα increasing expression of Htra2, NRF1 and activating proteasome activity (Papa and Germain, 2011). No mammalian homologue of ATFS-1 has been identified.

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Figure 1.12 The mitochondrial unfolded protein response (UPRmt_{) signalling pathway}

in C. elegans. Unfolded proteins are degraded by ClpP protease and undergo HAF-1 mediated export from the mitochondria. HAF-1 mediated peptide release leads to the nuclear accumulation of ATFS-1, UBL-5 and DVE-1 in the nucleus. This leads to the transcriptional activation of Hsp60 and mtHsp70 to restore correct protein folding. Figure adapted from. Figure adapted from Pellegrino et al. (2013).

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Figure 1.13 The mitochondrial unfolded protein response (UPRmt) signalling

pathway in mammals. The pathway has not been fully elucidated, however it is thought that there are two branches a matrix stress specific and an intermyofibrillar space (IMF) specific. Accumulation of unfolded proteins in the matrix signals the upregulation of CHOP via JNK and c-Jun. CHOP transcriptionally activates the

expression of ClpP protease, Hsp60 chaperone protein and mtDnaJ. Accumulation of unfolded protein in the IMS leads to AKT dependent phosphorylation of ERα. Which transcriptionally activates Htra2 protease and Nrf1 transcription factor expression. Figure adapted from Pellegrino et al. (2013).

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