Mitochondrial therapeutics and mitochondrial transfer for neurodegenerative diseases and aging

Mitochondrial dysfunction and neurodegeneration: Progressive neurodegenerative diseases affect a significant proportion of the population; in a single year, there are as many as 276 million disabilities and 9 million deaths as a result of neurological diseases. Mitochondrial function, aging, and neurodegenerative processes appear to be intricately linked; central nervous system degeneration is a major feature of loss-of-function mitochondrial diseases, involving mutation of nuclear or mitochondrial DNA. Meanwhile, mitochondrial dysfunction occurs during healthy aging and is further associated with several neurological diseases, including Alzheimer’s disease (AD), Huntington’s disease, Friedreich’s ataxia, multiple sclerosis, motor neuron disease, Parkinson’s disease (PD), and vanishing white matter disease (VWMD) (Figure 1A). Aging increases neurodegenerative risk factors and processes, including progressively impaired cognitive and/or motor function due to cellular dysfunction, senescence, and/or neuronal death. Furthermore, impaired mitochondrial respiration, biogenesis, mitophagy, and axonal transport can be causative factors in dysfunctional protein synthesis, folding, aggregation, and trafficking, as well as inflammation, oxidative stress, and genomic instability.

Figure 1.

General mitochondrial etiology and therapeutic approaches to neurodegenerative diseases and aging.

(A) Schematic outlining mitochondrial dysfunction in neurodegenerative diseases and aging, and mitochondrial therapeutic strategies. Created with Microsoft Excel. (B) Cell-free mitochondria are stable in EX for several hours at 37°C but abolished by CM, and prolonged by presence of ethyl glycol tetraacetic acid (EGTA), as observed by accumulation of the cationic viable mitochondrial dye tetramethylrhodamine, ethyl ester (TMRE), which labels active mitochondria based on membrane potential. Loss of signal compared to the proton ionophore, FCCP treatment, which uncouples oxidative phosphorylation by transporting protons across the mitochondrial membrane and dissipating the membrane potential. Unpublished data. (C) Fluorescently labeled mitochondria (DsRed, orange) co-incubated with induced pluripotent stem cell-derived neurons (NE, eGFP, green) in neuronal cultures (NE), astrocyte cultures (AC, Rhodamine123, green), skeletal muscle cultures (SkM, mPlum, red), and neuronal and skeletal muscle co-cultures (MNSkM). Unpublished data. (D) Differentially abundant proteins include electron transport chain proteins and mitochondrial translational machinery. Created with Microsoft Excel. AC: Astrocyte; CM: cell culture media; eGFP: enhanced green fluorescent protein; EX: calcium-free extraction buffer; FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; MNSkM: motor neuron and skeletal muscle co-culture; mtDNA: mitochondrial DNA; NE: neuron; SkM: skeletal muscle; UPR: unfolded protein response.

Energy production by mitochondrial oxidative phosphorylation is a more efficient process compared to glycolysis (over 15 times more adenosine 5′-triphosphate [ATP] generated per glucose molecule). Accordingly, it is energetically favorable for the majority of eukaryotic cellular ATP to be generated by oxidative phosphorylation. Loss of cellular respiration is detrimental to cell viability, while upregulation of glycolysis and/or oxidative phosphorylation, leads to enhanced survival in slow-cycling, quiescent, or stem-like cancer cells, even under hypoxic conditions. Although a compensatory upregulation of glycolysis can be neuroprotective, neuronal differentiation typically involves a mitochondrial-meditated switch to oxidative phosphorylation. Accordingly, constitutive expression of glycolytic enzymes, hexokinase, and lactate dehydrogenase, inhibit neuronal differentiation. The generation of superoxide and reactive oxygen species (ROS), as a by-product of oxidative phosphorylation, can lead to oxidative damage to nucleic acids, proteins, and lipids. Neurons are amongst the longest-living cells, with the lowest turnover in the body; their function is highly dependent on supportive glial cell nutrient/antioxidant supplementation and the energy-intensive process of myelination, which requires high rates of protein synthesis. ROS can also uniquely impact neurons, such as through inhibition of trafficking kinesin complex Miro/Trak via p38 activation, and impairing mitochondrial motility, potentially causing synaptic dysfunction (Debattisti et al., 2017). Thus, targeting mitochondrial function offers the premise of mitigating cellular degeneration (Figure 1). Furthermore, the cumulative impact of oxidative damage is exacerbated by the inherent susceptibility of mitochondrial DNA to ROS-induced mutations. Consequently, neurons appear to have an inherent susceptibility to mitochondrial dysfunction.

Toxicity of mitochondrial dysfunction: The neurodegenerative consequence of mitochondrial dysfunction is exemplified by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), a chemical precursor to the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) that was identified as an unexpected by-product of a synthetic heroin variant, causing PD-like symptoms in recreational users. MPTP is metabolized by monoamine oxidase-B to MPP⁺ once diffused through the blood-brain barrier. The binding of MPP⁺ to respiratory chain complex I (NADH-ubiquinone oxidoreductase) results in a decrease in ATP generation efficiency, increased free radical generation, and an upregulation of PD-related genes, such as SNCA, LRRK2, and PRKN, compromising dopaminergic neuron survival. At acute dosages (3 × 20 mg/kg), MPTP causes loss of dopaminergic neurons (~75% nigral neurons) but can also cause spinal motor neuron dysmorphia and death (~30%) in vivo (Vivacqua et al., 2012). The resulting oxidative stress also leads to upregulation of mitochondrial fragmentation, and mitophagy failure, leading to the accumulation of degraded mitochondria, and induction of the unfolded protein response. This was observed by increased expression of endoplasmic reticulum stress markers, such as immunoglobulin heavy chain binding protein (BiP, also known as 78-kDa glucose-regulated protein, GRP78), and the transcription factor, CCAAT enhancer binding protein (C/EBP) homologous protein. MPTP has also been observed to induce neuroinflammation with astrocyte and microglial activation, abnormal tau phosphorylation, and cognitive defects. Hence, mitochondrial dysfunction, by way of compromised ATP generation and free radical production can result in potentially causative neuroinflammation and proteotoxicity that is not limited to PD but also other neurodegenerative diseases and aging. In turn, α-synuclein and tau aggregates have been shown to inhibit mitochondrial Complex I activity in rodent models of PD and AD. Early studies demonstrating protection against MPTP-induced toxicity by ROS scavengers, including N-acetyl-cysteine, alpha-tocopherol, beta-carotene, and ascorbic acid, suggest that the effects of acute mitochondrial dysfunction can be mitigated by antioxidant treatment. However, targeting the early free radical theory of aging is contested by the necessity of ROS produced by normal mitochondrial function, serving as regulators of cell signaling and cell cycle progression. In particular, ROS inactivation of the tumor suppressor, phosphatase, and tensin homolog (PTEN), is thought to support neural stem cell growth and neurogenesis. There is little consistent evidence to suggest that overexpression of antioxidant proteins (e.g., catalase, superoxide dismutase 1 [SOD1]) or treatment with antioxidant pharmaceuticals can increase the life span of cells in the laboratory, and in fact may even compromise it (Bratic and Larsson, 2013). Additionally, it has been argued that progeroid phenotypes can be induced by mitochondrial DNA mutations, and accumulate with age, without necessarily inducing oxidative stress (Bratic and Larsson, 2013). However, laboratory observations investigating antioxidant usage and aging have been limited in their ability to account for environmental stress factors encountered during daily living (e.g., sunlight, alcohol consumption, smoking, and psychological distress). Furthermore, the sensitivity of telomeric repeats to oxidative stress leaves telomeres particularly vulnerable to dysfunctional mitochondrial ROS production. This is potentially due to iron binding propensity (leading to a greater rate of Fenton chemistry conversion of hydrogen peroxide to superoxide) or high guanine content (susceptible to the formation of irreparable 8-oxoG lesions in G-quadruplex structures). Downstream hydrogen peroxide generated by electron transport chain (ETC) dysfunction has been demonstrated to diffuse into the nucleus and cause telomere attrition but not necessarily nuclear DNA damage. It remains plausible that mitochondrial DNA and telomeric DNA damage could be induced by oxidative stress from non-laboratory environmental factors, and thus that antioxidant supplements may compensate for decreased antioxidant protein activity with age, such as glutathione peroxidases, catalase, and SODs.

Accumulating evidence indicates a critical role for mitophagy in neurodegeneration and aging, in maintaining the functional cellular mitochondrial pool, and in the induction of protein degradation and mitophagy pathways that remove degraded or depolarised mitochondria (D’Amico et al., 2021). Additionally, mitophagy marker upregulation has been observed in calorie restriction experiments, including neurodegeneration-relevant proteins, such as PTEN-induced kinase 1 (PINK1), Parkin, ubiquitin, p62, and LC3-II, along-side mitochondrial biogenesis transcriptional regulators, proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and sirtuins. The frequently studied PINK1/Parkin-dependent pathway of mitophagy has been observed to be dysregulated in PD, as well as AD, amyotrophic lateral sclerosis (ALS), and Huntington’s disease. PINK1 and parkin knockout mice typically demonstrate Parkinsonism with age (> 1 year), with an accumulation of fragmented mitochondria and loss of dopaminergic neurons demonstrating the age-related nature of cell death induced by mitophagy failure. Small molecule activation of PINK1 may provide a route to attenuate neurodegeneration. For example, kinetin triphosphate enhances PINK1 activity, and rescues mitochondrial morphology and motor symptoms in cultured neurons and PINK1 knockdown flies, however has not demonstrated improvement in commonly explored rodent models. Meanwhile, the premise of niclosamide, as a PINK1 mitophagy activator and its beneficial effects on neuronal differentiation and TDP-43 mislocalization, is largely limited to cell culture studies. However, the upregulation of autophagy by urolithin A has demonstrated comparably greater efficacy in several aging worm, neurodegenerative, and injury mouse models (D’Amico et al., 2021).

Recent studies have aimed to further characterize the pathological formation of the mitochondrial permeability transition pore (mPTP), and its relevance to neurodegeneration and aging. Pore formation, under high matrix Ca²⁺ levels and oxidative stress, is thought to be favored by Ca²⁺ binding to either the ADP/ATP translocase (ANT) or the ATP synthase F1 β-subunit and Cyclophilin D (CypD) binding to oligomycin-sensitivity conferring protein, followed by induction of a c-ring leak channel (Frigo et al., 2023). The mPTP has been associated with neuronal hyperexcitability, or interaction with protein aggregates, e.g., α-synuclein, amyloid-β, and triggering the mitochondrial unfolded protein response in aging. Genetic depletion of CypD, or inhibition by cyclosporin, can mitigate mPTP formation, with CypD ablation restoring oligomycin-sensitivity conferring protein function, synaptic oxygen consumption, ATP synthesis, and cognition in AD mouse models. These recent studies suggest CypD and ANT inhibition as potential therapeutic targets, although the inhibition of CypD by cyclosporin A is limited as a therapeutic due to its inherent toxicity.

Therapeutics to enhance mitochondrial function: There are several promising potential therapeutic strategies to target mitochondrial dysfunction.

(i) ETC or tricarboxylic acid (TCA) cycle modulating supplements (e.g., nicotinamide adenine dinucleotide precursors, idebenone, pyruvate, succinate) support efficient transfer of electrons throughout complex I–V subunits of the ETC. Supplementation of nicotinamide, nicotinamide riboside, or nicotinamide mononucleotide, as a precursor to NAD⁺, can support the availability of NADH for the ETC. In addition, as a ubiquitous cellular cofactor, in particular, for the sirtuin family proteins, nicotinamide mononucleotide has also been observed to support a range of cellular and physiological functions (Fang, 2019). Sirtuins play key roles in DNA repair and genomic stability, and clinical studies have found nicotinamide mononucleotide can improve cellular mitochondrial biogenesis and increase telomere length. However, while supplementation of NAD⁺ precursors in humans has demonstrated increases in circulating NAD⁺ levels and improvements in hippocampal blood flow and frailty measures, critically they have not shown benefits in cognitive function in AD patients. Similarly, nicotinamide riboside treatment increased blood NAD⁺ but led to no changes in cognition in people with mild cognitive impairment. Nicotinamide riboside supplementation demonstrated improvements in mitochondrial membrane potential (ΔΨM), oxygen consumption rate, ATP levels, and delayed senescence in neural and muscle stem cells. Therefore, although age-associated physical decline may be prevented by improved mitochondrial respiration, NAD⁺ precursor supplements do not appear to lead to a change in survival.

Idebenone (structurally similar to coenzyme Q10 and Vitamin E/alpha-tocopherol) can transfer electrons directly to complex III, while also serving as a free radical scavenger. Idebenone is approved in Europe for preventing vision impairment and promoting vision recovery in Leber’s hereditary optic neuropathy. Although it has shown less promise in a number of human studies, including for AD, Huntington’s disease, and multiple sclerosis, there is evidence for reduced dopaminergic neuronal damage, and improved motor dysfunction in PD rodent models, with a human clinical trial for PD underway. There are a few examples of supplementation of TCA substrates in clinical studies. For example, pyruvate supplementation, as a precursor of acetyl-CoA, can support mitochondrial respiration by increasing NADH generation, preventing cellular senescence, reducing oxidative stress and hyperexcitability, and preventing the development of cognitive deficiencies in AD mice. Succinate supplementation can support the TCA cycle as an intermediate substrate of succinyl-CoA, but also directly supports the ETC as a substrate of complex II, to promote production of FADH₂. Dimethyl succinate supplementation could overcome AD-associated α-ketoglutarate dehydrogenase and succinyl-CoA synthetase TCA deficiency in an induced pluripotent stem cell-derived neuronal model, increasing oxygen consumption, NADH concentration, and synaptic density. Moreover, succinate supplementation has been demonstrated to support TCA cycle function with restoration of oxygen consumption rate levels in rotenone (mitochondrial complex I inhibitor)-treated human glial cultures and exhibiting an increased lactate/pyruvate ratio in people with traumatic brain injury. Cell-membrane permeable pro-drugs of succinate have been recently developed to target complex II and bypass complex I deficiency disease and may be applicable to neurodegenerative diseases in the future.

(ii) ΔΨM supportive drugs (e.g., SS-31, SBT-272, ursodiol) stabilize the inner mitochondrial membrane and reduce electron leakage. A restorative effect in primary mitochondrial diseases can be observed with the cardiolipin-binding peptide elamipretide/SS-31. This approach led to the regeneration of axons, myelin sheath thickness, and motor function in a mouse model of Friedreich’s ataxia (Liu et al., 2022). Mitochondrial protein leakage is thought to be associated with the aging cardiomyocyte mitochondria, with SS-31 demonstrating reversal of the mPTP, mitigating diastolic dysfunction in primary rodent cells. Meanwhile, SBT-272 has demonstrated improved bioavailability and central nervous system uptake over SS-31 in rodent models of ALS and PD. SBT-272 has been reported to reduce dopaminergic neuron loss, α-synuclein aggregation, and neuroinflammation markers, ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein, in A53T α-synuclein PD mice. SBT-272 can also enhance ΔΨM, and mitochondrial axonal motility, and decrease neuroinflammatory markers in hTDP-43A315T ALS/frontotemporal dementia mice. Future studies are needed to assess the impact of SS-272 on survival and cognitive and motor dysfunction in neurodegenerative diseases. We recently identified ursodiol (ursodeoxycholic acid, UDCA) in a cytoprotective endoplasmic reticulum stress drug screen in a VWMD patient-derived stem cell astrocyte model, and found that UDCA increased ΔΨM and reduced ROS generation (Ng et al., 2020). Mitochondrial dysfunction has been well characterized in VWMD, due to mutations in eIF2B causing dysfunctional protein synthesis regulation. Interestingly, increases in ΔΨM and reduction activity (used as a proxy for mitochondrial activity or cell viability) by UDCA were observed without an increase in bulk ATP generation (Ng et al., 2023), possibly owing to tight regulation of ATP levels. UDCA can block the mPTP, and recent studies suggest UDCA can activate 5′ AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), PINK/Parkin, reduce dopaminergic cell death and improve movement dysfunction in a PD mouse model. UDCA has also been trialed in PD patients with minor improvements in cognitive and movement scores and with possible increases in ATP generation. The ALS therapeutic, AMX0035 (Relyvrio), which includes sodium phenylbutyrate and the UDCA taurine-conjugated derivative (tauroursodeoxycholic acid, TUDCA) was controversially approved by the U.S. Food and Drug Administration on the basis of neurofilament and SOD1 biomarker decrease, but not clinical endpoints in human studies. Unfortunately, AMX0035 recently failed in Phase III clinical trials for ALS. Evidently, further studies are required to identify whether ΔΨM modifying agents have the capacity to ameliorate disease progression.

(iii) Compounds that support regulation of mitochondrial turnover (e.g., NAD⁺ precursors, rapamycin, urolithin A) promote increases in healthy mitochondria. Caffeine can increase mitochondrial biogenesis and oxidative respiration, and protect against rotenone-mediated neurotoxicity in a PD rodent model and has been shown to reduce the risk and/or slow the progression of AD and PD. Supplementation of NAD⁺ is also able to increase the expression and activity of key regulators of mitochondrial genes, including NAD-dependent protein deacetylase sirtuin-1 activity, which can stimulate mitochondrial biogenesis via peroxisome PGC1α. The overexpression of PGC1α increased mitochondrial number, motility, Complex I and Complex IV expression in C9ORF72 ALS motor neurons, and improved cognitive impairment of APP23 AD mice. Rapamycin treatment, which inhibits mTOR, upregulated mitophagy and mitophagosome/lysosome fusion in amyloid precursor protein/presenilin 1 (APP/PS1) AD mice, while also enhancing learning, memory, and synaptic plasticity (Wang et al., 2021). Furthermore, urolithin A extended life span in worm models, dependent on mitophagy gene expression and independent of an effect on ROS levels. Urolithin also enhanced AMPK activation, decreased inflammatory pathways mediated by nuclear factor kappa light chain enhancer of activated B cells and P38 mitogen-activated protein kinase, reduced amyloid-β (Aβ) deposition, astrogliosis and microgliosis and cognitive impairment in APP/PS1 AD mice. Evidently, urolithin A possesses a range of pharmacological properties that are not limited to mitophagy; its effect in reducing activation of inflammatory dendritic and microglial cells and infiltrating peripheral Th17 cells has been correlated to aryl hydrocarbon receptor activation, rather than autophagy or mitophagy (D’Amico et al., 2021).

(iv) Antioxidant treatment and pathway activation (e.g., coenzyme Q10, MitoQ, N-acetylcysteine, edaravone, EPI-589, idebenone, nuclear factor erythroid 2-related factor 2 activators) ameliorates oxidative damage from mitochondrial-produced ROS, and increases ETC efficiency. The ROS scavenger, edaravone, has demonstrated a cytoprotective effect against endoplasmic reticulum stress, and increased antioxidant metallothionine protein expression (MTM1, MT1X, MT2A) that were downregulated in VWMD astrocytes (Ng et al., 2020, 2023). In this endoplasmic reticulum stress screen, many glucocorticosteroids were also found to be cytoprotective. The protective mechanism could be through (i) amelioration of the maladaptive protein synthesis stress response that is inherent to eIF2B mutations in VWMD or (ii) glucocorticosteroid upregulation of antioxidant metallothionine proteins. Glucocorticoids have been shown to induce glucocorticoid receptor translocation to mitochondria and increase ΔΨM at low doses, although they can induce the opposite effect, along with apoptosis, at higher concentrations. In ALS, free radical scavengers, such as EPI-589, have been demonstrated to delay disease progression in vivo. Additionally, edaravone was approved by the U.S. Food and Drug Administration in 2017 to treat ALS, although clinical trials failed to reach significance between treatment groups. Well-marketed antioxidant supplements, such as coenzyme Q10 or the conjugated mitochondrial-targeting triphenylphosphonium cation (TPP⁺) variant, mitoquinone, have been frequently studied but not found to provide clinical benefit in PD. Recently, activation of the antioxidant nuclear factor erythroid 2-related factor 2 pathway by omaveloxolone has been demonstrated to significantly reduce hippocampal and cortical neuronal loss and Aβ plaque load, as well as cognitive dysfunction in AD mice. As a natural compound, sulforaphane has also been observed to activate the nuclear factor erythroid 2-related factor 2 pathway. This was correlated with anti-inflammatory effects and mitigation of protein aggregation, including reduction of tumor necrosis factor-α and interleukin-1β, reduction of Aβ plaques, as well as reduction of tau pathology in AD mouse models. Aside from the premise of small molecules to modulate mitochondrial function, ROS accumulation and/or antioxidant gene expression, there has been emerging evidence supporting the efficacy of whole organelle mitochondrial transfer, as a means to improve overall mitochondrial capacity in cells/tissues.

The premise of exogenous horizontal mitochondrial transfer: approach, potential and limitations: Mitochondrial transfer is a promising organelle therapy and has demonstrated amelioration of cognitive and motor deficits when delivered to PD 6-hydroxydopamine mice (Chang et al., 2021) and Aβ-ICV AD mice (Nitzan et al., 2019). Furthermore, mitochondrial replacement may be harnessed to deliver an exogenous genetic or protein payload. This could be introduced as a DNA plasmid into mitochondria by electroporation, or by means of mitochondrial targeting sequence fusion proteins. We have found that the mitochondria extracted from donor cells and transferred to recipient disease cells retain their ΔΨM, following electroporation (Ng et al., 2023). Furthermore, a cytoplasmic reporter payload can be delivered with low efficiency in vitro, while dsRed labeled mitochondria can be transferred into recipient cells. However, this did not result in observable increases in ATP production (Ng et al., 2023). Additionally, in the absence of ethyl glycol tetraacetic acid-mediated calcium chelation, we found that ΔΨM was lost upon addition to the cell culture medium (physiological free Ca²⁺ ~1.2–2 mM cf. cytosolic Ca²⁺ 0.1 µM). Furthermore, after transfer from calcium-free extraction buffer, cell-free mitochondria appeared to lose membrane potential within ~6 hours at 37°C (Ng et al., 2023) (Figure 1B). It is possible that susceptibility of cell-free mitochondria to high calcium environments with loss of membrane potential could emerge from mPTP formation. As such, further research is needed to establish the respiration potential of transferred mitochondria in vivo, and whether mitochondria can functionally integrate with the existing mitochondrial population for a sustained period. However, cell-free mitochondria were able to be preserved for at least 48 hours in sucrose ethyl glycol tetraacetic acid buffer at 4°C (Ng et al., 2023), and are able to be functionally cryopreserved with trehalose, supporting their application as an inventoriable therapeutic. We have found that mitochondrial transfer increases the abundance of ETC proteins, and mitochondrial translational proteins in a number of cell types in vitro in neuronal, astrocyte, and skeletal muscle cells (Figure 1C). Patient-derived induced pluripotent stem cells offer a solution for a scalable, continuous source of autologous or allogenic and inventoriable organelle-replacement products for neurodegenerative diseases. Furthermore, mitochondria can be genetically modified with overexpression constructs in host cells, driven by a mitochondrial targeting sequence. Upregulation of mPTP stabilizing proteins (e.g., oligomycin sensitivity-conferring protein) may stabilize the mPTP, or calcium transport proteins (e.g., the mitochondrial sodium-calcium exchanger, NCLX) may prevent mitochondrial calcium overload. Trafficking kinesin TRAK and Miro proteins, overexpressed in transferred mitochondria, could enhance axonal transport of transferred mitochondria to support neurite regeneration with a directional bias towards synapse formation in central nervous system or peripheral neurons. Nonetheless, the utility of stem-cell-derived mitochondrial transfer is overshadowed by its relative complexity. Unlike neurons, pluripotent stem cells represent a cell stage that relies extensively on glycolysis rather than oxidative phosphorylation. Thus, it may be necessary to differentiate stem cells, to avoid extraction of relatively immature mitochondria. In addition, maintaining clinical standards in the extraction and transfer process, and possible contraindications of systemic administration, will require careful consideration and optimization.

Mitochondrial-derived peptides as a potential mode of action: The mode of action of exogenously delivered mitochondria may not be due to bioenergetic supplementation but the expression and release of mitochondrial-derived peptides. This may explain why even in studies where intravenously administered mitochondria are not detected in the central nervous system, alterations in brain cellular processes are detected. One of the first mitochondrial-derived peptides discovered, humanin, can phosphorylate STAT3 and prevent Bax mitochondrial translocation, inhibiting caspase-3 mediated cell death, and is able to improve learning and memory accompanied by reduction of amyloid plaques in 3×Tg-AD mice (Niikura, 2022). Intranasal delivery of humanin induced mitochondrial biogenesis via PI3K/AKT, further intracellular humanin expression, and mitigated loss of dopaminergic neurons in a PD mouse model. Meanwhile, a potent derivative of humanin, colivelin, mitigated cholinergic neuronal cell loss, delayed the onset and time to death of SOD1 transgenic ALS mice, and prevented learning and memory deficits in AD mouse models. Although in vitro and animal studies have shown promise, there remains an absence of clinical studies with humanin, its derivatives, and other mitochondrial-derived peptides.

Conclusions: The interrelated nature of mitochondrial dysfunction and the inherent impact of energy dysregulation in cellular stress, proteotoxicity, and cell death implies that mitochondrial therapeutics may be beneficial for multiple neurodegenerative diseases and aging, i.e., to treat degeneration as a secondary mitochondrial disease. Nuclear or mitochondrial genetic abnormalities owing to hereditary or environmental toxins can play key roles in neurodegeneration and aging. Although antioxidants have demonstrated convincing evidence of mitigating stress factors that induce mitochondrial dysfunction, the therapeutic effect of antioxidant supplementation remains controversial in efficacy as a regenerative treatment. Given the current evidence regarding supplementation of antioxidants and NAD⁺ precursors, it is likely that their potential benefit in neurodegeneration and aging involves protection against disease processes or environmental stress factors, without necessarily extending lifespan. Alternatively, it may be the case that improving mitochondrial function would benefit some people and not others and the lack of patient stratification in clinical trials obscures any potential benefits. In this case, improved pre-clinical screening and personalized medicine approaches would be beneficial. Thus, there remains a need to discover and validate therapeutic options targeting ETC/TCA function, ΔΨM, mitochondrial biogenesis, mitophagy, mitochondrial transfer, and mitochondrial-derived peptides. Patient-derived stem cell and transdifferentiation models provide a convenient translational platform for disease-relevant drug screening (Ng et al., 2022a) at the drug discovery stage. We have investigated rapid, scalable, and cost-effective methods to generate patient-derived stem cell cultures, assay oxidative stress, ΔΨM, and mitochondrial function-relevant gene expression assays (Ng et al., 2022b, 2023) that can be employed for high-throughput screening of experimental, off-label, and complementary medicine compounds. Additional human studies complemented with 31P magnetic resonance spectroscopy of ATP and NAD and cognitive and movement disorder outcomes are necessary to thoroughly screen and support mitochondrial therapeutic development. In an age of emerging gene and cell-based therapies, further research is warranted to explore the most effective mitochondrial-based strategies to slow neurodegenerative disease progression and aging.

This article is dedicated to Dr. Simon Myers (Late, Western Sydney University, Australia). We thank Prof. Martina Sanderson-Smith (University of Wolllongong, Australia) for usage of equipment.

This work was supported by a MAWA Project Grant (to NN, MN, LO). The aim of MAWA is to advance medical science and improve human health and therapeutic interventions without the use of animals or animal products.