*4.3. Parkinson's Disease*

Parkinson's disease (PD) is very closely associated with mitochondrial dysfunction, owing to consistent evidence suggesting reductions in CI activity in PD patient brains and other tissues [288,289], in addition to genetic links between familial PD and mitochondrial dysfunction [290]. These well characterised mitochondrial abnormalities in PD and potential therapeutic strategies to target them have been reviewed extensively previously [291].

Lewy bodies, which form in the *SN,* are the main pathological hallmark of PD and are made up mainly of aggregated alpha-synuclein (α-syn), an abundant presynaptic molecule [292,293]. Alpha-synuclein is a 140 amino acid molecule, which is thought to play a role in neuronal plasticity and synaptic function [292,294,295]. The aggregation of α-syn is highly neurotoxic, and studies of transgenic mice overexpressing α-syn have shown that its accumulation can lead to a PD-like phenotype, consisting of the formation of prominent intraneuronal inclusion bodies, loss of dopamine neuron terminals, and motor deficits [296]. Intriguingly, much evidence has suggested that neuronal injury caused by α-syn may be mediated by mitochondrial dysfunction and degeneration [264,266,297–301].

Multiple studies have shown that α-syn localises to, and accumulates within, mitochondria (Table 2) [264,266,300,301]. This is thought to be mediated by a cryptic, noncanonical MTS within the N-terminal 32 amino acids of α-syn [266]. The transport of α-syn into mitochondria does not occur in the presence of oligomycin, which inhibits ATP synthase and thus depletes mitochondrial ATP, or, carbonyl cyanide-*m*-lorophenylhydrazone (CCCP), which disrupts the mitochondrial ∆ψ, highlighting that its import is dependent on both ATP and ∆ψ, consistent with the import requirements for known mitochondrial proteins [266]. The A53T point mutation that occurs in rare familial PD cases is also imported into mitochondria, but with significantly higher efficiency than the wildtype protein [266], which may account for the faster development of cellular abnormalities seen in cells expressing the A53T version of α-syn compared to the wildtype [265].

It has been previously shown by electron microscopy that the majority of mitochondrial α-syn accumulates at the IMM and that it interacts with CI [266]. This causes a significant reduction in CI activity, as well as an increase in ROS production, inducing oxidative stress [266], which may account for some of the toxic effects on dopaminergic neurons. Importantly, α-syn lacking the N-terminal MTS failed to localise to mitochondria and did not exhibit any of the mitochondrial dysfunctions seen in the wildtype [266].

A study carried out in cell models of PD showed that in vitro treatment with rotenone leads to an increase in S129 phosphorylation of α-syn [267]. The resulting post-translationally modified α-syn species were observed to bind with high affinity to TOM20 molecules, leading to a loss of the critical interaction between TOM20 and TOM22 (Table 2) [267]. Consequently, mitochondria have impaired protein import and widespread mitochondrial dysfunction, displayed by a loss of ∆ψ, reduced respiratory capacity, and increased oxidative stress in SH-SY5Y cells [267]. This α-syn/ TOM20 interaction and subsequent loss of import were also detected in the dopaminergic neurons from the *SN* of post-mortem brains of PD patients [267]. The authors highlighted mechanisms for rescuing this disorder, namely by in vivo knockdown of endogenous α-syn and by in vitro TOM20 overexpression, both of which preserve mitochondrial import and thus present potential therapeutic strategies for further investigation [267,302].

It has been shown that the core component of the TOM complex, TOM40, is downregulated in the midbrain of PD patients as well as in α-syn transgenic mice (Table 2) [268]. Importantly, levels of TOM20 remained the same, suggesting that this is a specific effect of TOM40, rather than a general reduction in mitochondrial proteins. Furthermore, this reduction in TOM40 levels corresponded with α-syn accumulation in PD brains, inferring a further functional link between α-syn aggregation and mitochondrial import dysfunction [268].

A recent study showed that, in addition to the key roles in mitochondrial quality control and biogenesis already established [303–308], Parkin, an E3 ubiquitin ligase, also plays a part in stimulating mitochondrial protein import, whilst stimulation of import is not achieved by disease-causing Parkin variants (Table 2) [27]. Furthermore, the results of this study showed that this effect relies on PINK1-mediated Parkin activation and results in ubiquitylation of TOM40 subunits, as well as an increase in K11 ubiquitin chains on mitochondria [27]. The importance of PINK1-Parkin regulation of mitochondrial import is highlighted by data showing excessively low levels of mitochondrial import in cells from PINK1- and PARK2-linked PD patients. This effect may be reversed by phosphomimetic ubiquitin in cells with residual Parkin activity, probably by bypassing the need for PINK1 dependent Parkin activation or by enhancing Parkin activity [27].

#### *4.4. Huntington's Disease*

Huntington's disease (HD) is an autosomal dominant neurological disorder characterised by neuronal loss in the striatal and cortical regions of the brain. The genetic cause of HD is an abnormal expansion of polyglutamine repeats (encoded by the CAG codon) in the huntingtin gene (HTT) [309].

N-terminal fragments of variant Huntingtin proteins, which form cytotoxic aggregates [310,311], have been shown to interact directly with mitochondria in cell and mouse models of HD (Table 2) [312,313]. Furthermore, a study showed that the variant Huntingtin localises to mitochondria from human HD brains isolated mitochondria, and that it directly interacts with the TIM23 complex, inhibiting import as a result (Table 2) [269]. These import defects were consistent in primary neurons expressing Huntingtin variant as well as in forebrain synaptosomal mitochondria in HD mice at early stages of the disease [269]. Notably, these import defects were not found in liver mitochondria from the same mice, suggesting that the import defects are specific to neurons [269]. Additionally, the inhibition of import preceded mitochondrial respiratory dysfunction and acted as a trigger for cell death, which was rescued upon augmentation of mitochondrial import by overexpression of TIM23 complex subunits, highlighting this pathway as a potential therapeutic strategy against HD [269].

Considering the early detection of impaired import in HD mice [269], it suggests that import defects precede the other mitochondrial insults described in HD models, namely decreased ∆ψ [314], reduced respiratory capacity and ATP levels [315,316], defective calcium buffering function [317], and altered mitochondrial morphology and number [318]. A plausible explanation is that inhibition of import would prevent key respiratory complex proteins from being imported and carrying out their functions, resulting in widespread mitochondrial damage.

Mutant Huntingtin has been linked to dysfunctions in the MIA pathway (Table 2) [270]. In neuronal cell lines, the expression of proteins of the MIA pathway were found to be significantly different to levels in control cells [270]. More specifically, ALR and CHCHD4 levels were reduced, and the ratio altered compared to control cells, whilst cytochrome *c* levels were increased, compared to the control group. Proteins that require the MIA pathway for import also displayed reduced expression levels, whilst CIV proteins not imported via this route, such as MTCO3, were unchanged, highlighting that this effect is specific to MIA substrates rather than a CIV effect [270]. In cells with a homozygous variant, however, levels of MTCO3 were also reduced [270], suggesting that there may be some CIV assembly defects. The observed effects on the MIA pathway were accompanied by deficient respiration, alterations in mtDNA, and changes in mitochondrial morphology [270]. These effects are consistent with what has been shown previously in both HD models and MIA deficient models [319–324].
