Targetable Pathways for Alleviating Mitochondrial Dysfunction in Neurodegeneration of Metabolic and Non-Metabolic Diseases

Several lines of evidence have demonstrated that mitochondrial dysfunction has a primary role in the pathogenesis of PD, as many of the pathogenic mutations associated with PD are directly related to mitochondrial dysfunction () [,]. Parkin mutations are the most common cause of autosomal recessive PD, and are also the most sensitive to oxidative damage, and PINK1 mutations are the second most frequent cause of autosomal recessive early-onset PD []. In order to ensure cell survival, healthy neurons remove dysfunctional mitochondria efficiently via mitophagy as a quality control mechanism []. However, mutations resulting in a loss of function of both parkin and PINK1 result in the death of many cell types, including dopaminergic neurons, whose dysfunction is mainly responsible for the classical motor deficits of PD []. This is because these neurons have a reduced ability to remove dysfunctional mitochondria as a consequence of mutations in parkin and PINK1, preventing the recruitment of parkin to the mitochondria and therefore interfering with mitophagy efficiency []. This results in an accumulation of damaged mitochondria, making these cells vulnerable targets for OS and death []. Additionally, mitochondrial ROS production is significantly increased as a result of dysfunctional PINK1 and parkin []. A reduced ability to cleave PINK1 between Ala-103 and Phe-104 in order to generate the 53 kDA PINK1 protein, augments mitochondrial and cytoplasmic ROS production, in addition to significantly reducing basal ΔΨm []. Moreover, parkin has been recognised to conjugate both phosphorylated and unphosphorylated ubiquitin to the substrate located on the depolarized mitochondria []. However, if this mechanism is dysfunctional, an accumulation of abnormal mitochondria and an overproduction of ROS can occur. Figure 5 ^{22 35 56 37 57 37 58 59 60 60}

Studies infirst identified the role of parkin and PINK1 in the maintenance of the mitochondria []. PINK1 knockout (KO)were found to have defects in mitochondrial morphology, including fragmented mitochondrial cristae, hypersensitivity to OS, MRC defects, and degeneration of dopaminergic neurons, which are mechanisms that have been suggested as contributing early on in the pathogenesis of PD [,,]. In parkin KO, the same phenotype was observed as in the PINK1 KOas a result of these proteins operating in the same genetic pathway in order to maintain functional mitochondria []. Moreover, altered mitochondrial morphology and muscle degeneration in PINK1 KOwere salvaged via parkin overexpression, but this was not observed with PINK1 overexpression in parkin KO, indicating that PINK1 is an upstream regulator of parkin function []. Similar tomodels, PINK1 KO mice exhibit MRC defects within the striatum. A study carried out by Gispert et al. (2009) generated PINK1 deficient mice and identified characteristics similar to PD development in humans, including dopaminergic synapse dysfunction and protuberant mitochondrial dysfunction []. The study observed that PINK1 KO mice had impaired mitochondrial electron transport chain (ETC) complex I + III + IV activity within the brain, as well as reductions in ΔΨm and ATP generation, resulting in mitochondrial bioenergetic dysfunction. A reduction in ΔΨm below a certain threshold is often caused by uncouplers or OS, and is required for the activation of mitophagy, favouring the accumulation of PINK1 at the OMM [,]. However, PINK1 processing is incomplete in damaged mitochondria as a consequence of reduced ΔΨm. Consequently, a deficiency in PINK1 becomes more apparent under conditions of cellular stress, which may explain why dopaminergic neurons in PINK1-associated PD have an increased susceptibility to death, as these neurons are particularly vulnerable to increased levels of OS as a result of dopamine metabolism, and PINK1 KO causes increased ROS generation [,]. Dopamine and its oxidation by-products may contribute to increased ROS generation and mitochondrial respiration inhibition in PD, specifically the inactivation of mitochondrial electron transport chain (ETC) complex I, as these metabolites have been found to interact with the OXPHOS system [,,]. In isolated brain mitochondria from a rat model of PD, dopamine was found to inhibit mitochondrial respiration in a dose-dependent manner, as well as via interaction with mitochondrial toxins. PD patient studies have provided evidence for a 35% decrease in complex I activity, suggesting that dopamine may be a potential contributor to OS and mitochondrial dysfunction within dopaminergic neurons, leading to neuronal damage and degeneration. However, without stress conditions, PINK1 KO mice neurons also show reduced ATP levels, and isolated mitochondria from PINK1 KO cerebral tissue demonstrate a significant and progressive reduction in ΔΨm, although this may be associated with age. Drosophila melanogaster Drosophila Drosophila Drosophila Drosophila Drosophila Drosophila ^{37 56 61 62 63 56 61 64 65 38 61 66 67 68}

Mitochondrial abnormalities in the dopaminergic neurons of PD patients and dopamine neuronal cultures were investigated by Chung et al. (2016), who identified mitochondrial defects and protein accumulation in parkin and PINK1 induced pluripotent stem cell (iPSC)-derived midbrain neuronal populations []. These neuronal populations revealed a considerable accumulation of abnormal mitochondria, in comparison with control iPSCs, which comprised of a smaller fraction of abnormal mitochondria after 75 days of differentiation. The abnormal mitochondria were also observed as being larger in size compared with the control neurons. This suggests that mutations in parkin and PINK1 are capable of triggering alterations in mitochondrial homeostasis, initiating multiple cellular pathogenic modifications. Another study with parkin mutant iPSC neurons showed increased OS and reduced GSH levels, accompanied by Nrf2 pathway activation []. Increases in OS were supported in post-mortem PD brains where increased levels of 4-hydroxyl-2-nonenal (HNE), a by-product of lipid peroxidation, and 8-hydroxy-guanosine and 8-hydroxy-deoxyguanosine, by-products of DNA and RNA oxidation, respectively, were identified []. Post-mortem PD brains were also found to have an increased expression of Nrf2 pathway proteins, including Nrf2 and NQO1, suggesting that the Nrf2 pathway is activated in iPSC-derived neurons in order to protect neurons from further oxidative damage. The Nrf2-ARE pathway is often downregulated in several neurodegenerative disorders, suggesting that parkin mutations are capable of impairing the Nrf2 antioxidant pathway []. Nevertheless, the cell is able to compensate by upregulating antioxidant protective pathways, and therefore provides an important therapeutic target. ^{58 69 70 38}

α-synuclein aggregation and accumulation is strongly associated with several neurodegenerative diseases, including PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MAS). Evidence of α-synuclein aggregates have been recognised in post-mortem PD brains to accumulate within mitochondria, despite its predominant localisation to the cytosol [,]. Overactivation of autophagy is a potential link between mitochondrial dysfunction and α-synuclein accumulation [], and intracellular accumulation has been shown to be capable of inducing OS and increasing ROS levels, as well as inducing mitochondrial fragmentation []. Although the mechanisms are not yet fully understood, evidence has shown that α-synuclein has direct effects on mitochondrial morphology due to mutations encoding α-synuclein increasing mitochondrial fragmentation as well as affecting mitochondrial biogenesis via the regulation of PGC1α [,]. A study using human dopaminergic neurons with overexpressed mutant A53T α-synuclein has provided evidence that basal and toxin-induced oxidative/nitrosative stress results in an inhibition of the MEF2C-PGC1α transcriptional network []. Myocyte enhancer factor 2 (MEF2C) is a transcription factor that binds to the PGC1α promoter activating it, and evidence has shown that the inhibition of this network contributes to mitochondrial dysfunction and apoptotic cell death in PD [,]. Moreover, this toxic protein has been found to interact with mitochondrial membranes, including in neuronal synapses, and has been associated with impaired complex I-dependent respiration and reduced ΔΨm, therefore impacting energy production and mitochondrial ROS production []. Recent evidence has identified an interaction between α-synuclein and MRC complex I, resulting in reduced mitochondrial activity and exacerbated ROS production []. The relevant molecular target has been found to be the-terminal domain of α-synuclein, which targets the aggregated protein towards MRC complex I. It has also been reported that soluble α-synuclein is capable of directly interacting with mitochondria-associated endoplasmic reticulum membranes (MAM), which can cause an overexpression of the proteins that alter mitochondrial morphology and cause mitochondrial fragmentation in cell models of PD []. Additionally, α-synuclein has been found to bind to OMM proteins, including the voltage-dependent anion-selective channel 1 (VDAC1), which has been implicated as a factor contributing to mitochondrial dysfunction in sporadic PD []. Levels of VDAC1 in nigral neurons of patients with sporadic PD were found to be reduced as a consequence of α-synuclein aggregation. ^{71 72 73 74 38 75 75 75 76 37 77 78 35} N

Thereby, parkin, PINK1, and α-synuclein represent important therapeutic targets in order to alleviate mitochondrial dysfunction and bioenergetic deficits, leading to neurodegeneration in both sporadic and familial PD.

Several animal MMAemia models and patient studies have demonstrated that mitochondrial dysfunction is an important contributor to the development of neurodegeneration in MMAemia []. In patient studies, this has been supported by elevated levels of lactic acid—an indicator of impaired MRC function—in the CSF and globus pallidus of the brain, as well as elevated levels of the TCA cycle intermediate, citric acid, in the blood (). These elevated biomarkers are suggestive of an impairment of mitochondrial metabolism, such as mitochondrial abnormalities and MRC enzyme complex inhibition, ultimately leading to neuronal loss. In a study undertaken by Brusque et al. (2002), millimolar MMA concentrations consistent with those reported in MMAemia patients were found to inhibit the MRC in the cerebral cortex of young rats []. The MRC complex I activity was significantly inhibited by the presence of MMA, which was found to be a weak inhibitor of complex II (succinate dehydrogenase) activity, suggesting that MMA may be considered as a neurotoxin. Interestingly, inhibition of complex I activity has also been observed in the basal ganglia of PD patients. Therefore, it may be assumed that inhibition of the MRC complexes by MMA is perhaps very destructive to the brain, as OXPHOS is a highly active system in the cerebral tissue [,]. ⁸¹ Figure 7 ^{85 85 86}

Other studies have suggested that MMA may induce secondary excitotoxicity and OS, which are well-established mechanisms involved in neuronal damage []. Experimental evidence has found several OXPHOS defects in MMAemia patient and MMUT KO mice models, resulting in perturbations in antioxidant defence systems, which, as a consequence, cause oxidative damage, leading to MMA-induced deficient energy metabolism. Studies with MMAemia-patient fibroblasts have identified an increased expression of multiple proteins related to OS and apoptosis, including cytochromeand MnSOD, as well as significant increases in intracellular ROS levels []. Moreover, Atkuri et al. (2009) found that MMAemia patients expressed lower levels of GSH in multiple immune cells compared with healthy controls []. In cultured neurons, MMA accumulation was also found to reduce the ATP/ADP ratio and cause mitochondrial membrane depolarisation and ion gradient failure, leading to necrotic and apoptotic cell death [,,]. Additionally, secondary metabolites of MMA, such as MCA, MA, and propionyl-CoA, have been demonstrated to also inhibit the activity of the MRC and TCA cycle []. It has been suggested that neuronal damage in MMAemia is not caused by MMA alone, but by the synergistic contribution of alternate propionyl-CoA oxidation metabolites. These secondary metabolites are considered as neurotoxic agents, as they have been demonstrated to be capable of impairing OXPHOS via pyruvate dehydrogenase complex inhibition and inducing hyperammonaemia via-acetylglutamate synthetase inhibition. ^{87 85 88 89 90 91 87} c N

Although the mechanisms are not yet fully understood, recent evidence has demonstrated that a deficiency in the gene encoding methylmalonyl-CoA mutase,, affects the efficient functioning of the PINK1/parkin pathway in order to remove dysfunctional mitochondria via the autophagy-lysosomal system, a degradation system required for the removal of harmful cytosolic entities such as misfolded proteins, aged, and/or damaged organelles [,,]. This suggests that there may be a primary link between MMUT deficiency, mitochondrial dysfunction, and cellular stress in the development of neurodegeneration in MMAemia. MUT ^{20 81 92}

Inactivating mutations in thegene can result in a loss-of-function of the MMUT enzyme activity, and these mutations can either be a complete () or partial () loss-of-function, leading to the accumulation of toxic organic acids within the mitochondrial matrix, initiating mitochondrial network structural and functional abnormalities. MMUT is expressed in various tissues, including the basal ganglia of the brain [], however it is highly expressed within the mitochondria of kidney tubular cells, therefore the consequences of MMUT deficiency on mitochondrial network function and homeostasis have been investigated using these cells []. In MMAemia-patient kidney tubular cells, an MMUT deficiency can result in the accumulation of damaged and dysfunctional mitochondria as a consequence of deficient MMUT inactivating PINK1/parkin-mediated mitophagy, therefore producing excessive levels of ROS and causing cellular distress []. In this same study (Luciani et al., 2020), a link between MMUT deficiency and autophagy induction was reported, suggesting that an MMUT deficiency may impair mitochondrial homeostasis and function via compromised PINK1/parkin-directed priming of MMA-stressed mitochondria to autophagic-lysosomal degradation []. Both MMA and control cells were treated with the mitochondrial complex I inhibitor [], rotenone, in order to induce mitochondrial damage. This study observed, under normal and stress-induced conditions, a reduced amount of parkin clusters and reduced translocation of parkin to damaged mitochondria in MMA cells, in addition to mitochondrial abnormalities such as mitochondrial fragmentation, reduced ΔΨm, and impaired mitochondrial respiration and ATP generation []. As a consequence of the defective PINK1/parkin priming mechanisms, mutantcells fail to transport their damaged mitochondria to the autophagy-lysosomal degradation systems, therefore triggering an accumulation of MMA-diseased mitochondria within the cell []. Thus, the disrupted clearance of MMA-stressed mitochondria triggers a level of mitochondrial dysfunction that causes cellular distress and damage. MUT MUT 0 MUT - MUT ^{93 92 20 20 94 20 90}

In order to demonstrate the central role of MMUT in maintaining mitochondrial network function and homeostasis, Chen et al. (2020) analysed a KOzebrafish model that displayed an abnormal mitochondrial morphology characterised by perturbed mitochondrial cristae organisation, in addition to impaired mitochondrial bioenergetics, contributing to increased mitochondrial OS []. This was also observed in an earlier experimental study on rats, where chronic exposure to MMA resulted in mitochondrial swelling and disorganised cristae of the tubulum epithelium []. Moreover, studies with mutant kidney cells have demonstrated that mitochondrial homeostasis and function can be improved by therapeutic interventions that target the PINK1/parkin pathway by activating mitophagy-mediated degradation in order to avoid mitochondrial-derived cellular stress and damage, as well as maintaining efficient mitochondrial quality control mechanisms []. MUT ^{92 95 20}

Impaired mitochondrial function has been suggested as a collective pathogenic feature of LSDs, as the majority of LSDs frequently present with primary lysosomal impairment in addition to mitochondrial dysfunction []. Experimental studies have reported mitochondrial morphological abnormalities, such as mitochondrial fragmentation and/or elongation, as well as dysfunctional mitochondrial bioenergetics, as a consequence of impaired mitochondrial respiration, reduced MRC activity, and/or decreased ΔΨm (). In mouse models of neuropathic GD, the most common LSD caused by mutations in the() gene, which encodes for the lysosomal enzyme, glucocerebrosidase (GCase), neurons, and astrocytes that do not expresswere found to have defective autophagic and proteasomal machinery, causing a primary lysosomal defect and leading to an accumulation of dysfunctional mitochondria within the cell []. Furthermore, Osellame et al. (2013) reported evidence of impaired mitochondrial function in neurons and astrocytes of type II GD, as morphological abnormalities were indicated by small and fragmented mitochondria []. The resting ΔΨm was measured inKO () mice models and was found to be significantly lower in comparison with mice neurons and astrocytes derived fromhomozygous () andheterozygous () mutations.andneurons were incubated with oligomycin—an ATPase inhibitor —in order to assess the function of the MRC, demonstrating that the ΔΨm inneurons was not affected by incubation with oligomycin, but was significantly reduced inneurons, suggesting that the reduced ΔΨm is maintained by ATP synthase (complex V) working in reverse, which is a mechanism implicated in PINK1 KO-associated PD. Moreover, confocal imaging of neurons and astrocytes located within the midbran indicated significant mitochondrial fragmentation incells, in comparison withandcells, suggesting that GD can cause increased mitochondrial fragmentation and MRC impairments, including reduced complex I and complex II + III activity, leading to mitochondrial dysfunction. Interestingly, evidence has also demonstrated an association between an absence or loss-of-function ofand an increased risk of PD in GD patients, as the genetic causes of familial PD are thought to induce the same biochemical and molecular events that underlie the pathogenesis of several LSDs. ¹⁰¹ Figure 8 ^{102 102} glucocerebrosidase gba gba, gba gba −/− gba gba +/+ gba gba +/− gba +/+ gba −/− gba +/+ gba −/− gba −/− gba +/+ gba +/− gba

Moreover, a study carried out by Cleeter et al. (2013) identified a significant reduction in ATP synthesis involving both MRC complex I and complex II/III-linked ADP phosphorylation, as a result of a 20-day incubation with a selective inhibitor of GCase activity, conduritol-β-epoxide (CβE), with inhibited levels of GCase activity consistent with those reported in GD patients []. Despite an observed significant reduction in ATP synthesis, individual MRC protein activities were unaltered, suggesting that defective ATP synthesis may be due to dysfunctional electron transfer in the MRC and/or defective mitochondrial membrane. Inhibition of GCase with CβE caused mitochondrial function abnormalities and increased OS, which are defects frequently observed in the PD brain and mitochondrial disease [,]. Additionally, following CβE treatment, a significant and progressive elevation in free radical generation was observed, which may have contributed to the observed mitochondrial fragmentation as a result of increased OS []. ^{103 22 103 103}

Evidence of elevated OS has been reported in patient GD fibroblasts through the presence of an increased generation of superoxide ions from non-phagocytic NADPH oxidase together with protein carbonyls from augmented protein oxidation []. It is possible that OS may also result from an accumulation of damaged mitochondria whose aberrant MRC function may become a major source of ROS generation, contributing to OS-induced cellular damage []. Furthermore, evidence of OS has also been reported in NPC human fibroblasts; post-mortem cerebellum; and MPS IV patient urine, plasma, and leukocytes, where increased protein oxidation, lipid peroxidation, and DNA damage have been described []. In NPC human fibroblasts, a reduced SOD2 and/or CAT activity was observed, as well as diminished GSH levels, in addition to MPS IV patient erythrocytes showing decreased GSH activity, suggesting the presence of increased OS in other LSDs. Furthermore, mitochondrial-associated OS events are also known to be dysfunctional in the PD brain, which may result from a loss of cerebral GSH status, which has been reported in post-mortem studies []. ^{104 101 99 35}

The accumulation of defective mitochondria may possibly be due to dysregulated mitochondrial biogenesis and mitophagy pathways, which have been reported in animal and cellular models of several LSDs []. Impaired mitophagy through impaired PINK1/parkin recruitment and/or function has recently been investigated in mouse models of GD, which have shown reduced colocalisation of autophagosomes and mitochondria, therefore leading to reduced recycling of damaged mitochondria in mice primary neurons []. This was demonstrated in a study undertaken by Osellame et al. (2013), whereneurons failed to recruit parkin despite a reduction in ΔΨm. In order to assess the failed recruitment of parkin, cells were treated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)—an uncoupler of mitochondrial OXPHOS leading to the collapse of ΔΨm—in order to promote PINK1 accumulation at the OMM []. However, once the mitochondria were depolarised, theneurons showed no difference in the levels of PINK1 in comparison to the controls, confirming that a reduced ΔΨm inneurons is not sufficiently low enough for parkin recruitment, suggesting that ΔΨm must be near to complete dissipation in order to be selected for turnover. As a consequence, damaged mitochondria remain unmarked for turnover via mitophagy, resulting in impaired and defective mitochondria accumulating inneurons, therefore compromising neuronal cell function. In another study by Moren et al. (2019), GD cellular models derived from neural crest stem cells containingandmutations were found to increase PGC1α levels under carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP)-induced mitophagy—another mitochondrial OXPHOS uncoupler promoting cell death []—therefore increasing the mitochondrial content via the activation of mitochondrial biogenesis []. As a result of treatment with CCCP, mitochondrial depolarisation led to a significant elevation in PGC1α incell lines in comparison with control andcell lines, which is possibly a compensatory mechanism for the underlying mitochondrial defects and GCase deficiency in GD. This study identified that impaired turnover of depolarised mitochondria and GCase-deficient neurons in GD are possible mechanisms that may be associated with early mitochondrial impairments and dysfunction in this disorder. ^{105 99 102 106 107} gba -/- gba -/- gba -/- gba -/- gba +/+ gba +/− gba +/+ gba +/−

The accumulation of protein aggregates, such as α-synuclein, may further deteriorate mitochondrial function and have been reported in post-mortem cerebral samples of LSD patients []. Although α-synuclein accumulation is strongly associated with both sporadic and familial forms of PD, the assessment of numerous LSD patient samples has revealed the presence of Lewy bodies and neurotoxic intracellular accumulation of α-synuclein in cerebral tissue, including in the cortex, hippocampus, and substantia nigra [,]. Interestingly, Lewy bodies containing aggregated α-synuclein have been associated with mitochondrial fragmentation and dysfunction in several cellular models of LSDs []. This was investigated by Choi et al. (2012), where brain homogenates from the cerebral cortex of the post-mortem brain samples of patients withmutations were found to contain aggregated α-synuclein []. This study analysed the presence of intracellular α-synuclein inclusions using SDS-PAGE and Western blot, demonstrating that most patients withmutations exhibited bands of insoluble oligomeric forms of α-synuclein, in comparison with the controls and GD patients withoutmutations. Cerebral tissue from animal models with neuronal variants of GD revealed disrupted mitochondrial cristae and the presence of rounded, fragmented mitochondria associated with aggregated α-synuclein accumulation, as well as reduced oxygen consumption and reduced ATP levels []. These protein aggregates have been found to colocalise with mitochondria, suggesting that α-synuclein may cause a direct impairment to mitochondrial function, although it has not yet been fully elucidated whether α-synuclein accumulation is a primary cause of mitochondrial damage in LSDs or whether it is a secondary consequence caused by increased generation of ROS and/or impaired autophagic systems []. ^{99 99 108 99 109 108 99} gba gba gba

In a study undertaken by Luth et al. (2014), the accumulation of α-synuclein was demonstrated to directly inhibit MRC complex I activity, as well as reduce ΔΨm and increasing ROS generation and alter calcium homeostasis []. Therefore, these studies suggest that the accumulation of protein aggregates, including α-synuclein, are risk factors for the development of mitochondrial dysfunction in LSDs. Moreover, the presence of Lewy bodies has also been reported in other LSDs, including the presence of phosphorylated α-synuclein in MPS III patient cerebral tissue samples, in addition to samples of cerebral tissue from NPC patients, which were positive for the presence of aggregated α-synuclein [,]. The plasma of GD and NPC patients, in addition to NPC lymphoblasts, were also reported to contain α-synuclein oligomers [,]. LSDs are complex disorders, therefore therapeutic strategies that target several different mitochondrial-associated molecular pathways may be beneficial for improving mitochondrial function and quality control mechanisms, particularly in neuronal cells, in order to alleviate mitochondrial dysfunction and neurodegeneration in LSDs. ^{110 111 112 113 114}

Natural polyphenols, including resveratrol (3,5,4′-trihydroxystilbene) and curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), are produced in plants in response to injury and stress, and are found in several food sources []. These redox-active compounds have become an interesting mitochondria-targeting therapeutic in order to preserve the structure and function of the mitochondria and neurons []. Polyphenols exhibit antioxidant and neuroprotective properties including the ability to activate transcription factors such as Nrf2, which are involved in the expression of antioxidant enzymes, and can directly regulate the mitochondrial apoptosis system and induce mitochondrial biogenesis in order to improve mitochondrial mass and function [,,]. These compounds have shown many beneficial effects in the mitochondria, however they often exhibit very low bioavailability, poor absorption, and rapid metabolism, notably in the brain [,], which may compromise the effectiveness of these compounds in neurometabolic and neurodegenerative diseases. ^{120 121 121 122 123 120 124}

Several experimental studies have identified mitochondria as the key targets of resveratrol, as it has been shown to be a regulator of MRC function, capable of inducing mitochondrial biogenesis and reducing ROS production via its interaction with SIRT1 and AMPK via PGC1α [,]. Gueguen et al. (2015) identified MRC complex I as a direct target of resveratrol action (), resulting in an increase in its enzymatic activity following treatment with low doses of the compound (1–5 μM) []. This interaction between MRC complex I and resveratrol results in the stimulation of key signalling pathways, including the SIRT1-dependent upregulation of several antioxidant enzymes in the brain, such as SOD1 and SOD2, GPx, and CAT [,,]. Resveratrol has been found to promote neuronal survival via inducing apoptotic cell death, in addition to the regulation of secondary consequences of disease pathogenesis, including inhibiting OS and preventing neuro-inflammation [,]. In primary fibroblast cultures from patients with early-onset PD, resveratrol treatment was found to induce apoptosis in order to efficiently remove damaged mitochondria and misfolded proteins []. Increased mRNA expression of several PGC1α target genes was also identified in parkin-mutated fibroblasts following resveratrol treatment, which was then demonstrated to improve mitochondrial oxidative function, while simultaneously reducing OS and increasing mitochondrial biogenesis []. Interestingly, resveratrol treatment resulted in increased MRC complex I and citrate synthase (CS) activity—a biomarker for mitochondrial enrichment—increased ATP production and reduced lactate concentrations, suggesting an induction of mitochondrial biogenesis. Resveratrol is able to rapidly cross the BBB, however it should be noted that resveratrol is rapidly metabolised and has both a low solubility and bioavailability, which may reduce the efficacy of this potential therapeutic compound, although this may be improved with the addition of bioenhancers or drug-delivery carriers [,]. In vitro animal studies with bioenhancers have shown increased plasma concentrations of resveratrol through the use of combination therapies, for example, combining resveratrol and hydroxypropyl-β-cyclodextrin, which increased the solubility and absorption of this compound, in addition to nanoformulations of resveratrol, which increased its surface area for absorption []. However, the effects of bioenhancers have not yet been widely studied in humans. ^{116 125} Figure 9 ^{125 125 126 127 120 128 129 130 120 131 120}

Curcumin has a wide range of health promoting functions, including cytoprotective and neuroprotective properties, in addition to its favorable absorption, bioavailability, and long half-life in the CNS []. The long half-life of curcumin in the CNS (6–7 h in plasma) is thought to be due to its amphiphilic nature and a high concentration of lipids within the brain, meaning that this compound may be beneficial in patients who present neurologically [,]. Several experimental studies have suggested that curcumin can interact simultaneously with multiple molecular targets, resulting in a cascade of biochemical and molecular events and activating multiple signalling molecules, such as AMPK, SIRT1, and Nrf2, and therefore protecting the cells from oxidative injury by increasing mitochondrial mass and protein expression elicited by the induced mitochondrial biogenesis [,,]. Curcumin has also been shown to exhibit a protective effect against mitochondrial dysfunction and apoptosis, in addition to improving oxygen consumption rates, increasing cell viability, and ΔΨm in PINK1-deficient cells []. Models of PD have also demonstrated the potential neuroprotective effects of curcumin, as rat SNpc neurons were shown to be protected from apoptosis following curcumin treatment, leading to increased dopamine levels within the striatum, and reduced cytotoxicity and cell viability [,,]. Curcumin has been shown to prevent cytochrome c release and inhibit α-synuclein-induced neuronal toxicity and death in a PC12 cell model of PD [,,]. Moreover, curcumin treatment was demonstrated to be beneficial in an NPC1 mice model, where triple combination therapy involving curcumin, miglustat (targets sphingolipid synthesis and storage in GD and NPC), and ibuprofen (reduces CNS inflammation) was found to enhance neuroprotection and cell survival, in addition to ameliorating the autophagic flux function []. ^{132 132 133 119 120 134 135 135 136 137 138 139 140 141}

CoQis a potent antioxidant and essential cofactor carrying electrons from complexes I and II to complex III of the MRC, and is a well-known activator of PGC1α [,]. CoQhas been suggested to have a role as a neuroprotective agent, as this lipophilic antioxidant has been shown to improve cognitive function, facilitate ATP synthesis, and upregulate mitochondrial function in neurodegeneration []. A study carried out by Somayajulu et al. (2005) investigated the role of CoQas a neuroprotective agent when neuronal cells were exposed to hydrogen peroxide (HO)-induced OS []. In human neuroblastoma (SH-SY5Y) cells pre-treated with water-soluble CoQfollowing HOtreatment, a significant reduction in mitochondrial ROS generation, preserved ΔΨm, increased mitochondrial ATP production, and the prevention of mitochondrial membrane collapse were observed in comparison with the control mitochondria. This suggests that CoQcan act as a potent antioxidant at the mitochondrial level, as CoQhas been demonstrated to stabilise the mitochondrial membrane and increase mitochondrial numbers as a consequence of HO-induced OS, which may be due to the ability of CoQto increase PGC1α signalling [,,]. Studies with senescence-accelerated mouse (SAM) models have revealed that a diet supplemented with CoQcan induce pathways involving SIRT1, SIRT3, and PGC1α, preventing mitochondrial deterioration by increasing OS resistance []. In this study, Western blot analyses demonstrated that CoQinduced the deacetylation and activation of PGC1α and SOD2 via SIRT1 and SIRT3, therefore increasing mitochondrial mass and function via PGC1α-activated mitochondrial biogenesis. This study also identified that increased levels of cyclic adenosine monophosphate (cAMP) can mediate the metabolic effects of PGC1α on mitochondrial function and biogenesis via the increased expression of PGC1α. These findings provide biochemical evidence that CoQis capable of regulating mitochondrial activity by enhancing the activity of the cAMP-AMPK-SIRT1-PGC1α-pathway. Furthermore, at the inner mitochondrial membrane level, CoQhas been acknowledged as a modulator of mPTP, and has been demonstrated to prevent the opening of the mPTP in order to prevent ΔΨm collapse, therefore inhibiting mitochondrial-mediated apoptosis [,,]. A summary of the neuroprotective effects of the bioactive quinones are outlined in. 10 10 10 2 2 10 2 2 10 10 2 2 10 10 10 10 10 ^{142 143 144 145 142 145 146 143 147 148 149} Figure 10

PQQ, a natural redox cofactor, has been reported to stimulate mitochondrial biogenesis and has been associated with several health benefits due to its antioxidant properties, including improved mitochondrial function, energy utilization, and longevity, in addition to inhibiting apoptosis, providing protection from ROS, and improving neurologic functions in animal and cellular models [,,]. A study carried out in mouse hepatocytes (Hepa1-6 cells) by Chowanadisai (2013) demonstrated that PQQ treatment was capable of increasing the mitochondrial content and stimulating mitochondrial biogenesis, indicated by an increased CS and cytochromeactivity, mtDNA content, and cellular oxygen respiration []. PQQ exposure was shown to promote phosphorylation of cAMP response element-binding protein (CREB), activating the PGC1α promoter, therefore increasing PGC1α mRNA transcription and protein expression, in comparison with the post-translation regulation of PGC1α by resveratrol. In addition, PQQ exposure to the Hepa1-6 cells was also found to increase the activation of NRF1 and 2. Moreover, the neuroprotective effects of PQQ were studied in a mouse rotenone-induced PD model, where PQQ was found to protect SH-SY5Y cells and dopaminergic neurons from neurotoxicity and to prevent mitochondrial dysfunction through the promotion of mitochondrial biogenesis, in addition to preventing motor deficits and reducing dopaminergic neuronal loss in the SNpc and dopamine depletion in the striatum []. PQQ has been shown to be a promising therapeutic candidate in animal models and cell cultures in order to ameliorate mitochondrial dysfunction through mitochondrial biogenesis induction, and this compound has therapeutic potential, as PQQ is water-soluble and low dietary concentrations are easily absorbed in the intestines, in comparison with natural polyphenols, which are water-insoluble and have poor absorption []. ^{150 151 152 151 153 151} c

Idebenone (2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone) is a synthetic analogue of CoQthat is active in the CNS and is capable of crossing the BBB [,,,]. It is an important antioxidant that has demonstrated a neuroprotective role in several neurological disorders, including PD. The pharmacological function of idebenone is centred on the activation of the electron transfer system, in addition to the reduction of non-respiratory oxygen consumption after conversion to its reduced, ubiquinol form by the MRC complex I []. Idebenone has also been found to be successful at inhibiting oxidative processes, such as lipid peroxidation. In a study carried out by Yan et al. (2019), idebenone treatment (100 mg/kg) in mice was shown to improve MPTP-induced dopaminergic neuronal death and the neurological deficits associated with PD, in addition to diminishing the inflammatory response by reducing the expression of several proinflammatory cytokines in the striatum and substantia nigra []. Moreover, in vitro studies have also demonstrated that treatment with idebenone can protect against MPTP-induced motor dysfunction and reduce neuronal death in the substantia nigra and striatum. 10 ^{154 155 156 157 154 156}