*5.1. UPRmt*

The mitochondrial unfolded protein response (UPRmt) is known to be directly activated in response to impaired proteostasis in the mitochondrial matrix and has been extensively studied in *Caenorhabditis elegans*, where it was first identified [338]. The UPRmt is a transcriptional response pathway that eliminates proteotoxic stress and fine-tunes mitochondrial respiration [339,340].

The sensor for this pathway is stress activated transcription factor (ATFS-1, ATF5 in mammals), which contains both a weak N-terminal MTS and a strong C-terminal nuclear localisation sequence (NLS) [341]. Proteotoxic mitochondrial stress, caused by a variety of mitochondrial stressors including: impairment of the import machinery (*timm23* or *tomm40*(RNAi) or paraquat application via CI inhibition), loss of ETC quality control (*spg-7*(RNAi)), or mtDNA depletion (ethidium bromide application) [341], results in retargeting of ATFS-1 primarily to the nucleus. There, ATFS-1 acts with transcriptional regulators DVE-1 and UBL-5 to induce the production of mitochondrial chaperone proteins HSP-6 and HSP-60, as well as proteases CLPP-1, LONP-1, SPG-7, and YMEL-1, metabolic genes GPD-2 and SKN-1, and core component of the TIM23 complex, TIM17 [339,342,343]. ATFS-1 is also responsible for repressing the expression of ETC genes, thus shifting expression capacity to increase mitochondrial protein folding and reducing the proteotoxic stress from mistargeted proteins in the cytosol [344].

Importantly, the localisation of ATFS-1 is mediated by HAF-1, the previously identified UPRmt regulator and general attenuator of mitochondrial protein import during stress [345]. In the absence of HAF-1, ATFS-1 is unable to transition to the nucleus under stress conditions, thus failing to activate the UPRmt [341,345]. It is important to note that ATFS-1 has a relatively weak MTS, meaning that minor effects on mitochondrial protein import efficiency, such as partially depolarised mitochondria, can trigger the stress response pathway, even though some mitochondrial proteins with stronger targeting sequences may still be imported successfully under these conditions [346].

In mammalian cells, the UPRmt is thought to act in a similar way to that described above for *C. elegans*, where transcription factor ATF5 is regulated and triggers a stress response very similar to that described for the *C. elegans* homolog ATFS-1 [347]. However, studies have shown that integrated stress response (ISR) factor ATF4 is also involved in the transcriptional reprogramming of the mammalian UPRmt [348,349]. It is also thought that the heat shock response (HSR) is activated alongside the UPRmt in what is known as the mitochondrial to cytosolic stress response (MCSR) [338]. The HSR is activated by dysfunctional ETC activity or complex assembly and restores cytosolic proteostasis via transcription factor HSF-1 [350].

Given the vast mitochondrial dysfunction described in neurodegeneration, it is not surprising that there is an emerging body of evidence linking the UPRmt to neurodegeneration. In PD, variants of *C. elegans* PINK1 and Parkin orthologs PINK-1 and PDR-1 lead to increased activation of the UPRmt, which mitigates mitochondrial dysfunction caused by

the corresponding mutations, subsequently increasing dopaminergic neuron survival [351]. However, a study in *C. elegans* showed that prolonged UPRmt activation can in fact exacerbate mitochondrial dysfunction and dopaminergic cell death by favouring retention of dysfunctional mitochondria [352], which is important to note given the long-term and progressive nature of neurodegenerative diseases. Furthermore, the HSR has been shown to be activated in mouse and cell models of PD [353,354], and studies have also highlighted heat shock protein overexpression as an attenuator of α-syn aggregation and subsequent dopaminergic cell death [355].

In vivo studies also reveal that the accumulation of ALS SOD1 variant SOD1G93A in the IMS leads to activation of the UPRmt [356], consistent with other studies showing that activation of the UPRmt precedes disease onset and increases throughout disease progression in ALS mutant mice [357]. Similarly, in AD, accumulation of Aβ has been shown to activate the UPRmt [358], and there are high levels of UPRmt marker genes in postmortem brain samples from AD patients [359]. Interestingly, the inhibition of UPRmt by knockdown of genes coding for key UPRmt proteins HSP-6, HSP-60, and DVE-1 exacerbates AD phenotypes in *C. elegans* [360], suggesting that the UPRmt may play a protective role in AD progression.

Recently, evidence has shown that an earlier form of the UPRmt precedes the classical UPRmt, and is activated by the accumulation of unprocessed precursor proteins inside mitochondria, due to impaired processing by MPP [361]. In this case, yeast nuclear transcription factor Rox1 is relocalised to mitochondria, binding to mtDNA and regulating mtDNA transcription and translation, and maintenance of mitochondrial respiratory and import functions [361].
