Mitochondrial Targeted Interventions for Aging

Abstract

Changes in mitochondrial function play a critical role in the basic biology of aging and age-related disease. Mitochondria are typically thought of in the context of ATP production and oxidant production. However, it is clear that the mitochondria sit at a nexus of cell signaling where they affect metabolite, redox, and energy status, which influence many factors that contribute to the biology of aging, including stress responses, proteostasis, epigenetics, and inflammation. This has led to growing interest in identifying mitochondrial targeted interventions to delay or reverse age-related decline in function and promote healthy aging. In this review, we discuss the diverse roles of mitochondria in the cell. We then highlight some of the most promising strategies and compounds to target aging mitochondria in preclinical testing. Finally, we review the strategies and compounds that have advanced to clinical trials to test their ability to improve health in older adults.

MITOCHONDRIA IN AGING BIOLOGY

This is an exciting time to be studying mitochondria and aging, as there is now a well-established and important role for mitochondrial function in the biology of aging and age-related diseases. As a result, there is intensifying interest in developing strategies that target mitochondrial function to delay, slow, and reverse aging pathology and the decline in quality of life. Mitochondria play many roles in the cell and, despite this growing interest in mitochondrial targeted therapies, there is still a knowledge gap around the key aspects of mitochondrial function that drive aging and whether these are tissue specific or represent common mechanisms across multiple tissues. In this work, we will provide a brief overview of the roles that mitochondria play in the cell, their relationships to aging, and highlight some of the promising strategies being pursued to target mitochondria in preclinical studies and clinical trials. This is not meant to be a comprehensive review of the literature of mitochondria in aging biology, but instead we provide a brief background and highlight some of the most promising mitochondrial targeted interventions with clinical relevance for improving quality of life in our rapidly aging population (Fig. 1).

Figure 1.

Illustration of the multiples roles that mitochondria play in cell physiology that affect health and disease.

Mitochondria play a central role in cellular physiology and sit at the nexus integrating environmental and cell stressors with cell signaling and energetics that ultimately lead to adaptive or pathological responses. Mitochondria has traditionally been thought of in terms of cell ATP production or as the main source of oxidants in most cells. In fact, one of the earliest links between mitochondria and aging was proposed by Harman in 1972 (Harman 1972) when he implicated the mitochondria as the primary source of free radicals underlying the free radical theory of aging (Harman 1956). However, it is now clear that ATP production and redox homeostasis represent only a subset of the ways in which mitochondria communicate with the cytoplasm, nucleus, and the other cells to affect cellular health, and thus aging.

Mitochondrial Bioenergetics

Mitochondria are double-membrane organelles that originated evolutionarily through an endosymbiotic process where an alphaproteobacterium combined with a primitive eukaryotic cell (Sagan 1967). Most of the original bacterial genome has been transferred to the eukaryotic nucleus, but mitochondria maintain an independent circular genome that, in mammals, encodes for 13 protein subunits that are part of every complex of the electron transport system (ETS), except complex II, as well as mitochondrial tRNAs and ribosomal RNAs (Sanchez-Contreras et al. 2021). Mitochondria generate the majority of cellular ATP necessary to meet cell energy demands for growth, maintenance, stress response, and mechanical work through a process called oxidative phosphorylation. In the mitochondrial matrix carbohydrates, lipids, and amino acids are oxidized to generate NADH and FADH₂ through the tricarboxylic acid cycle (TCA) and β-oxidation. NADH and FADH₂ provide reducing equivalents (i.e., electrons) to the ETS located on the inner membrane of the mitochondria (Michal 1999) where they are used to pump protons from the matrix into the inner membrane space to generate a membrane potential that is used to drive ATP production as protons pass back into the matrix through the F1F0 ATP synthase (also known as complex V). ATP from the matrix is transported out of the matrix where it can be used in energy-consuming reactions by the cell in exchange for ADP, which is itself transported into the matrix by the adenine nucleotide transporter (ANT) (Nicholls and Ferguson 2002). The structure of the inner membrane, which is organized into tight folds called cristae that increase the membrane surface area for the inner membrane proteins that make up the complexes of the ETS, is critical for effective oxidative phosphorylation. In fact, swelling of the matrix and disorganized cristae is a common morphological sign of mitochondrial dysfunction (Taub et al. 2012).

Mitochondrial Redox Biology

Mitochondria are also a significant source of oxidants in most cells. During oxidative phosphorylation, electrons can leak from multiple sites in the ETS and react with molecular oxygen to form superoxide (Goncalves et al. 2015). Under typical conditions in most tissues, the primary sites of superoxide formation are from complex I and complex III of the ETS (Goncalves et al. 2015). Mitochondrial superoxide generation is membrane potential dependent, meaning that under conditions where membrane potential is high (e.g., normal respiratory function) but there is low ATP production, mitochondria generate more superoxide (Nicholls 2004). Inhibition or disruption of flux through the ETS can also lead to elevated superoxide production. In addition to the ETS, several matrix dehydrogenases can also generate superoxide, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glutamate dehydrogenase; but under most conditions, these contribute less than the ETS sources to mitochondria oxidant production (Goncalves et al. 2015). Because superoxide is highly reactive and can induce oxidative damage to macromolecules, the mitochondria contain antioxidant systems to detoxify superoxide. Superoxide dismutase (SOD2 in the matrix and SOD1 in the inner membrane space) is a highly efficient enzyme that rapidly converts superoxide into hydrogen peroxide (H₂O₂). H₂O₂ is less reactive than superoxide and can travel greater distances within the matrix and cell. In the matrix, H₂O₂ is converted to water either by the glutathione (GSH) peroxidase system using GSH as an intermediate or the peroxiredoxin–thioredoxin system. Both systems rely on NADPH, which is linked to NAD in the matrix and the cytoplasm. In addition to their important role in detoxifying superoxide, these redox intermediates, Prx, Trx, GSH:GSSG, and H₂O₂, play important roles in redox signaling underlying stress response pathways (Rohrbach et al. 2006; Garcia et al. 2010; Mailloux et al. 2012). Disrupted redox homeostasis linked to mitochondrial dysfunction with age is associated with many of the hallmarks of aging in worms, mice, and humans.

Mitochondrial Metabolites in Aging

Mitochondria are also important regulators of intermediate metabolites that play an important role in regulating diverse cellular processes. In addition to their role in energy metabolism, TCA-cycle intermediates play key roles regulating gene expression and cell signaling that contribute to aging (Martínez-Reyes et al. 2016; Martínez-Reyes and Chandel 2020). Acetyl CoA is the common point of entry into the TCA for both pyruvate, following glycolysis, and fatty acids from β-oxidation and is also produced in the cytosol from citrate. It also contributes the acetyl group for acetylation of proteins and histones that drive epigenetic regulation of gene expression, both of which contribute to aging pathology. Elevating acetyl CoA in the brains of SAMP8 mice reduced brain aging (Currais et al. 2019). The concentration of α-ketoglutarate ([AKG] 2-oxoglutarate), the entry point for glutamate into the TCA cycle, also affects epigenetic regulation due to its role as a substrate for 2-oxoglutarate dioxygenases (Martínez-Reyes et al. 2016). AKG levels decrease in mammalian aging and alter epigenetics in multiple tissues. Elevating AKG levels reverses age-related pathology and cellular changes, including hearing loss, osteoporosis, and inflammation and extends lifespan in mouse models of aging (Asadi Shahmirzadi et al. 2020). Both fumarate and succinate, which are just upstream of AKG in the TCA, can also affect epigenetic regulation by inhibiting 2-oxoglutarate dehydrogenases (Ishii et al. 1998; Gallo et al. 2011; Edwards et al. 2013; Lima et al. 2022). NAD is a metabolic cofactor involved in multiple steps of the TCA cycle where it is reduced to NADH to provide electrons for complex I of the ETS. There is strong evidence that a decline in NAD levels with age is a driver of multiple age-related pathologies (Yoshino et al. 2018). This is due to its prolific role as a cofactor for enzymes involved in DNA damage response (PARP) and protein deacetylation (sirtuins). Defects in mitochondrial function, particularly in complex I activity, alter the NAD/NADH ratio and disrupt NAD homeostasis (Karamanlidis et al. 2013). Since defects in complex I function are common in aging tissues, this may contribute to reduced NAD with age (Karamanlidis et al. 2013). In a negative feedforward cycle, the decline in NAD further impairs mitochondrial function by leading to elevated acetylation though inhibition of sirtuin activity or increased pathological inflammation. This is supported by multiple reports showing that elevating NAD by supplementing with NAD precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), or inhibiting PARP activity to preserve NAD can prevent or reverse mitochondrial dysfunction and tissue degeneration (Ryu et al. 2016; Yoshino et al. 2018; Whitson et al. 2020; Romani et al. 2021). Given the systemic and prolific nature of the contribution of NAD and other TCA cycle metabolites to aging biology, maintaining metabolite homeostasis represents a potential mechanism by which preserved mitochondrial function could contribute to healthy aging.

Mitochondrial Calcium Homeostasis and Aging

Another important way in which mitochondria contribute to cell signaling is through their effect on calcium homeostasis. This effect is especially important in excitable cells like neurons and cardiac and skeletal muscle fibers. Mitochondria are typically in close physical association with the endoplasmic reticulum (ER)/sarcoplasmic reticulum, where they are poised to take up calcium upon release. This mitochondrial calcium uptake regulates mitochondrial ATP production by enhancing the activity of several dehydrogenases of the TCA cycle (Rossi et al. 2019). However, excessive calcium uptake also leads to elevated mitochondrial oxidant production and the opening of the mitochondrial permeability transition pore (mPTP) leading to the induction of cell death through apoptosis or necrosis. With age, mitochondria become more sensitized to calcium uptake resulting in a lower threshold for mPTP opening and cell death (Picard et al. 2010; Zhang et al. 2020a). Elevated mitochondrial redox stress can also contribute to calcium-induced stress through redox-dependent posttranslation of the ryanodine receptor that leads to increased calcium leak from the ER (Andersson et al. 2011; Umanskaya et al. 2014). This dysregulation of calcium homeostasis contributes to cardiac and skeletal muscle dysfunction in aging mice, which can be prevented by reducing mitochondrial oxidant production by expressing mitochondrial catalase (mCAT)

Mitochondrial Quality Control in Aging

The mitochondrial unfolded protein response (mUPR) is typically discussed in the context of proteotoxic stress, but can also be initiated by other aspects of mitochondrial dysfunction such as impaired oxidative phosphorylation or elevated redox stress (Feng et al. 2001; Liu et al. 2005; Dell'agnello et al. 2007). When the mUPR is activated, the transcription factors ATF4 (ATF1 in Caenorhabditis elegans), ATF5, and CHOP are transported to the nucleus where they initiate transcription of multiple chaperones and proteases that act to restore proteostasis (Shpilka and Haynes 2018). Elevated mUPR is a key component of the beneficial effects of transient mitochondrial stress (hormesis) on aging and longevity induced by disruption of the electron transport chain or elevated redox stress early in life. Impaired regulation of the mUPR is associated with the loss of proteostasis in sarcopenia, Parkinson's (PD), and Alzheimer's disease (AD) (Ji et al. 2020; Urbina-Varela et al. 2020; Lin et al. 2022).

In the presence of more extreme mitochondrial stress, the cell mitochondria are engulfed by autophagosomes in a process called mitophagy. Depending on cell type, there are multiple mitophagy pathways that have been documented (Fivenson et al. 2017). The most common mitophagy pathway in mammals is the PINK/Parkin pathway. The process can be induced by decreasing membrane potential in damaged mitochondria, which activates PINK1 and phosphorylation of the E3 ubiquitin ligase Parkin, further activating ubiquitin-binding domains of autophagy liquid chromatography (LC) adaptor and the formation of autophagosome. The autophagosome then fuses with the lysosome resulting in formation of autophagosomes for degradation (Lazarou et al. 2015). Mitophagy can also occur in a ubiquitin-independent pathway where, upon mitochondrial stress, autophagy receptors are recruited to the outer mitochondrial membrane (OMM), which then recruit LC3 and autophagosomes to the mitochondria (Iorio et al. 2022). In addition, several mitophagy proteins also work in the PINK1/Parkin independent pathway, such as Ambra1, Nix, a member of Bcl-2 family, and FUNDC1 (Iorio et al. 2022).

Reduced mitophagy with age has been identified in many tissues, including mouse heart, brain, and skeletal muscle and is a contributor to neurodegenerative disease. Enhanced mitophagy to maintain mitochondrial quality control is a key aspect of the beneficial effects of exercise on skeletal muscle function (Zhang et al. 2020b). Interventions that elevate mitophagy, such as spermidine and urolithin a (discussed more below), also have been demonstrated to have a positive effect on skeletal muscle function and cognitive impairment in mouse models (Ryu et al. 2016; Fan et al. 2017; Schroeder et al. 2021). Maintaining mitochondrial quality control through efficient mitophagy is not only important to maintain well-functioning mitochondria, but also to prevent the release of mitochondrial components into the cytoplasm and circulation as damage-associated molecular patterns (DAMPs). mtDNA and other macromolecules released into the cytosol from improperly degraded mitochondria stimulate the release of proinflammatory cytokines through the activation of the NLRP3 inflammasome or the stimulator of interferon gene (STING) pathways (Zhang et al. 2019; Chen et al. 2021; Masumoto et al. 2021; Lin et al. 2022; Qiu et al. 2022).

Mitochondrial-Derived Peptides in Aging

Mitochondrial-derived peptides (MDPs) provide another example of systemic mitochondrial signaling to affect organismal health (Kim et al. 2021). Mammalian mtDNA is known to encode 13 mRNAs for proteins of the ETS, 22 tRNAs, and 2 rRNAs. In the last several years, the presence of several open reading frames that encode small peptides has been demonstrated. The two most well described MDPs are humanin and MOTS-c, although several other MDPs have been recently described (D'Souza et al. 2020; Miller et al. 2022). Both humanin and MOTS-c are involved in multiple signaling pathways and decline with age in rodents and humans (Yen et al. 2018). Humanin has been found to promote cell survival by inhibiting cytochrome c release by interacting with proapoptotic proteins, localizing to the lysosomal membrane and promote chaperone-mediated autophagy, and interact with the IGF-1 pathway (Xiao et al. 2016; Qin et al. 2018; Kim et al. 2022). At the organ and organismal scale, humanin may provide protection from cardiovascular disease, cognitive decline, and AD pathology. MOTS-c has been referred to as an exercise mimetic for its ability to improve metabolic phenotype and activate AMPK and NRF2 signaling in skeletal muscle (Yang et al. 2021). High-intensity exercise increased levels of MOTS-c in both skeletal muscle and plasma of humans. Both humanin and MOTS-c administered to aged mice improve function and reverse some pathological effects of aging.

MITOCHONDRIAL TARGETED THERAPIES

Antioxidants are the most well-studied class of compounds and supplements related to mitochondrial function and aging. As has been well documented, the effect of an antioxidant strategy in clinical trials has been mixed at best (Peternelj and Coombes 2011). A recent randomized trial with 35,000 healthy women aged 45 and above showed no beneficial effect with 10 yr vitamin E supplementation for cardiovascular health and total mortality (Lee et al. 2005). Similarly, the HOPE trial indicated a potential increased risk of heart failure in the vitamin E daily supplementation group (Lonn et al. 2005). These large epidemiology studies have challenged the idea that supplementation with antioxidants is universally beneficial. However, more targeted strategies to alter mitochondrial oxidative stress have shown some promise. Other approaches now include natural and pharmaceutical interventions that directly interact with components of the mitochondria or enhance mitochondrial quality and turnover.

Mitoquinone (MitoQ)

One strategy for mitochondrial targeted antioxidants takes advantage of the negative charge across the inner mitochondrial membrane (IMM) to deliver antioxidant agents to the mitochondrial matrix using triphenylphosphonium ion (TPP⁺). The beneficial effects of these TPP⁺-conjugated antioxidants have been demonstrated in preclinical models of aging and age-related diseases as described below. One of these antioxidants is MitoQ, which is a TPP⁺-conjugated ubiquinone. MitoQ selectively concentrates in the mitochondria and prevents mitochondrial oxidative damage. It has been shown to prolong life span of SOD-deficient flies and improve pathology associated with antioxidant deficiency (Magwere et al. 2006). MitoQ shows beneficial effects in multiple models of neurodegeneration. In transgenic C. elegans with muscle-specific expression of human amyloid-β peptide (Aβ), a model of AD, MitoQ extends life span and improves health span of C. elegans while the ROS production, protein carbonyl content, and mtDNA damage burden remain unchanged (Ng et al. 2014). In in vitro models, MitoQ protects against Aβ-toxicity in primary neurons of amyloid precursor protein (APP) transgenic mice and in neuroblastoma cells treated with Aβ (Manczak et al. 2010). In vivo, 5-mo MitoQ treatment prevents cognitive decline and AD-like neuropathology in a widely used mouse model of AD (3xTg-AD mice), supporting the therapeutic benefits of MitoQ in AD (McManus et al. 2011). MitoQ treatment also shows neuroprotective effects in cell culture and mouse models of PD (Ghosh et al. 2010). In addition to its neuroprotective effects, MitoQ confers cardioprotection in multiple models of cardiovascular disease (Adlam et al. 2005; Graham et al. 2009; Supinski et al. 2009; Dare et al. 2015). MitoQ treatment reduces ischemia reperfusion (IR) injury in mouse and rat models of IR (Adlam et al. 2005; Mukhopadhyay et al. 2012; Dare et al. 2015). In spontaneous hypertensive rats, 8-wk MitoQ treatment reduced systolic blood pressure and attenuated cardiac hypertrophy (Graham et al. 2009). Despite its promise in animal models, MitoQ showed no effect on the progression of PD symptoms over 12 mo (Snow et al. 2010). This is currently under investigation for age-related vascular dysfunction (NCT04851288, NCT02597023).

Skq1

Another TPP⁺-conjugated antioxidant SkQ1 is a TPP⁺-conjugated plastoquinone. SkQ1 treatment extends the life span of Podospora, Ceriodaphnia, Drosophila, female outbred SHR mice, male BALB/c, and C57Bl/6 mice (Anisimov et al. 2008, 2011). OXYS rats are a model of accelerated aging and develop a wide range of accelerated aging phenotypes, including cataract, retinopathy, and high blood pressure (Solov'eva et al. 1975). In senescence-accelerated OXYS rats, dietary SkQ1 treatment ameliorates age-related cataract and retinopathy (Neroev et al. 2008). Eye drops containing SkQ1 reverse cataract and retinopathy in young to middle-aged OXYS rats and prevent the development of uveitis and glaucoma in rabbit models (Neroev et al. 2008). In both Wistar and OXYS rats, 4-mo dietary SkQ1 treatment prevents the age-related declines in age-related biomarkers like growth hormone (GH) and insulin-like growth factor 1 (IGF-1) (Kolosova et al. 2012). In addition, studies also suggest that SkQ1 is protective against cardiac, renal, and brain IR injuries (Bakeeva et al. 2008). SkQO1 has shown some promise in clinical trials of dry eye syndrome (NCT02121301) but has not been tested for aging or age-related pathologies in humans.

MitoTEMPO

MitoTEMPO is a TPP⁺-conjugated piperidine nitroxide (Trnka et al. 2008). In an ex vivo study, MitoTEMPO treatment enhances contractile function of hearts and aortic rings isolated from aged rats, normalizing the age-related impairments (Olgar et al. 2018). Ex vivo MitoTEMPO treatment also attenuates age-related changes in electrical activities of aged rat cardiomyocytes and is associated with an antiarrhythmic benefit (Olgar et al. 2020). Moreover, in vivo treatment with MitoTEMPO is protective in multiple preclinical models of age-related cardiovascular diseases, including pressure-overload-induced heart failure, diabetic cardiomyopathy, and hypertension (Dikalova et al. 2010; Ni et al. 2016; Dey et al. 2018). In a mouse model of hind limb ischemia, MitoTEMPO treatment enhances mitochondrial function and attenuates the age-related decline in blood flow recovery in aged skeletal muscles (Miura et al. 2017). In addition, MitoTEMPO also ameliorates muscle wasting in mice with chronic kidney disease (Liu et al. 2020).

Due to the dependence on mitochondrial membrane potential for mitochondrial targeting, the delivery of TPP⁺-conjugated antioxidants to mitochondria will be diminished when mitochondrial membrane potential is compromised in pathological conditions. Another limitation of these TPP⁺-conjugated antioxidants is that they can inhibit mitochondrial respiration and disrupt mitochondrial membrane potential at high concentrations (Kelso et al. 2001; Antonenko et al. 2008; Pokrzywinski et al. 2016). These antioxidants have been shown to process pro-oxidant properties at high concentrations; therefore, extra precautions are needed to be taken to determine the optimal dosages that exert antioxidant effects but not pro-oxidant activities.

Astaxanthin

Astaxanthin (AX) is one of the most powerful carotenoid compounds and has gained special interest during recent decades due to its strong antioxidant activity, anti-inflammatory effects and potential to preserve health span with age (Kidd 2011). Its unique structure spanning the membrane bilayer can scavenge ROS in both inner and outer layers of membrane unlike other antioxidants act either at the inner (vitamin E and β-carotene) or outer membrane (vitamin C) (Kidd 2011; Vrolijk et al. 2015; Sztretye et al. 2019). In comparison with several other carotenoids (zeaxanthin, lutein, and lycopene), AX demonstrated the greatest reduction in lipid peroxidation while preserving membrane structure. In addition, Kidd (2011) also reported AX-lowering plasma biomarkers of lipid peroxidation in humans. Furthermore, AX also exhibits unique benefits of improving fat oxidation, improving muscle fatigability, and enhancing exercise endurance in both aged mice and older adults. In preclinical studies, AX improved fat oxidation in mice through increased coimmunoprecipitation of fatty acyl transferase (FAT/CD36) with carnitine palmitoyltransferase I (CPTI), and AX reduced oxidative stress-induced modification of CPTI by hexanoyl-lysine adduct (HEL) (Ikeuchi et al. 2006; Aoi et al. 2008).

Astaxanthin has been consumed by humans as a supplement for 30 yr. There is growing evidence showing AX improves athletic performance and exercise capacity and reduces injury markers (Earnest et al. 2011; Djordjevic et al. 2012). Due to its benefits on muscle and cardiovascular health, AX as supplementation for more vulnerable populations such as older adults has gained more interest. A recent study that paired AX treatment with exercise training in older adults reported that adding AX supplementation improved muscle strength and muscle endurance in the tibialis anterior muscle compared with exercise alone (Liu et al. 2018). The improved muscle function in this group was associated with metabolic benefits of the AX supplementation, including improved fat oxidation during lower intensity exercise (Liu et al. 2021). These results point to a beneficial effect of AX supplementation to improve exercise-induced metabolic adaptation under low-intensity stimulus. Unfortunately, this study did not collect tissues to specifically measure differences in adaptive signaling associated with the functional improvements. The same study also reported sex-dependent increases in exercise efficiency and reduced CHO oxidation in older males, but not females. This study points to the value of clinical trials that combine exercise training with mitochondrial-targeted treatment, especially with nutraceutical supplements, to test strategies to enhance a healthy lifestyle in older populations.

There are many other natural products currently being investigated for their beneficial effects on mitochondrial and aging recently reviewed in detail (Liang et al. 2021). For example, a recent review of pipeline drugs for AD over the past 5 yr indicated 121 agents in 136 trials of AD therapies. Among those in phase 3: tricaprilin, ginkgo biloba, and ANAVEX2-73 (blarcamesine) were targeted for mitochondria dysfunction (Cummings et al. 2020). The mechanisms of action of these natural products are typically not straightforward and their functions are frequently defined by their effect on specific mitochondrial or cellular processes such as oxidative stress, as in the carotenoid astaxanthin discussed above, mitochondrial membrane potential, mitochondrial mitophagy, as in urolithin a discussed below, or biogenesis. One example of a compound derived from a plant-based natural product is J147. J147 is a derivative of curcumin, a plant-based compound with antioxidant and anti-inflammatory activities. J147 improves on the bioavailability of curcumin and possesses neurotrophic activity (Peterson and Popkin 1980). Due to its neurotrophic activity, studies with J147 have focused on neurodegenerative diseases. Several preclinical reports suggest a beneficial effect of J147 treatment in rodent models of AD (Kepchia et al. 2021). Recently, J147 has been demonstrated to target the ATP5A subunit of the mitochondrial ATP synthase (complex V) (Goldberg et al. 2018). Recently, J147 has been shown to inhibit the mitochondrial ATPsynthase, elevate mitochondrial membrane potential and ROS production, and modulate AMPK/mTOR signaling. The effect on membrane potential and ROS signaling appears to contribute to an effect of J147 treatment on calcium homeostasis that leads to altered downstream signaling (Kepchia et al. 2021). Like many of the natural-derived compounds the details of the mechanism of action of J147 on AD and aging remain to be worked out. However, based on data from rodent models of AD and the SAMP8 accelerated aging model, there is clear potential for J147 as a mitochondrial targeted strategy for extending health span (Prior et al. 2016; Kepchia et al. 2022). As a shared target for both aging and dementia, J147 is currently in phase I clinical trial (NCT03838185). This phase I clinical trial is intended to test the biosafety of this compound with a secondary goal of testing for a clinically meaningful effect on cognitive function among both young and old adults (Table 1).

Table 1.

Rodent model ischemia reperfusion (IR) injury

Preclinical	Intervention	Model	Physiology/phenotype	References
	MitoQ	Transgenic Caenorhabditis elegans mode of Alzheimer's disease (AD)	Extends life span, delays Aβ-induced paralysis, but no change in ROS, protein carbonyl content, or mtDNA damage	Ng et al. 2014
		Wild-type and SOD-deficient flies	Prolongs life span only in SOD-deficient flies	Magwere et al. 2006
		AD mouse model	Protects against Aβ-toxicity	Manczak et al. 2010
		AD mouse model	Prevents cognitive decline	McManus et al. 2011
		Mouse model of Parkinson's disease (PD)	Neuroprotection effects	Ghosh et al. 2010
		Hypertension rat	Reduces systolic blood pressure and attenuates cardiac hypertrophy	Graham et al. 2009
	SkQ1	Accelerates aging mouse model	Accelerates aging mouse model	Neroev et al. 2008
		Accelerates aging rabbit model	Prevents the development of uveitis and glaucoma	Neroev et al. 2008
		Accelerates aging rat model	Prevents age-related decline in growth hormone (GH) and insulin-like growth factor 1 (IGF-1)	Kolosova et al. 2012
	MitoTEMPO	Aged rat	Enhances contractile function of hearts and aortic rings	Olgar et al. 2018
		Aged rat	Attenuates age-related changes in electrical activities; antiarrhythmic benefit	Olgar et al. 2020
		Aged mice under limb ischemia	Improves mitochondria function and blood flow in skeletal muscle	Miura et al. 2017
		Mice with chronic kidney disease (CKD)	Reduces muscle wasting	Liu et al. 2020
	Astaxanthin	Aged mice	Improves muscle fatigability, endurance, and fatty acid utilization	Ikeuchi et al. 2006
	J147	Rodent model of AD	Inhibits mitochondria ATP synthase; elevates membrane potential and ROS	Pior et al. 2016; Kepchia et al. 2018
	Elamipretide	Rodent model of heart failure (HF)	Reduces cardiac hypertrophy and improves cardiac function	Dai et al. 2011, 2012, 2013
		Canine model of HF	Ejection fraction, stroke volume, cardiac output, and left ventricle (LV) contractility	Sabbah et al. 2016
		Rodent model of IR injury	Preserves cardiac function after IR injury	Petri et al. 2006; Szeto et al. 2008
		Aged mice	8-wk reverse cardiac aging phonotype: hypertrophy, diastolic	Chiao et al. 2020
		Rodent aging model	ATP production and fatigue resistance after 1 h single injection	Siegel et al. 2013
		Aged rodent	8-wk improvement in mitochondrial function, redox homeostasis, and exercise tolerance	Campbell et al. 2019
	Spermidine	Rodent aging model	4-wk supplementation improved autophagy; reduced of LV	Eisenberg et al. 2016
		Rodent stem cell	2-wk treatment maintain regeneration and reduced protein	Eisenberg et al. 2009
	Urolithin A (UA)	C. elegans	Extends life span and increases expression of mitogene and respiration capacity in aged	Ryu et al. 2016
		Rodent	Improves exercise tolerance in old mice and rat

Clinical	Intervention	Model	Physiology response	References	Clinical trial #
	Humanin	Blood	Higher circulation level in children of centenarians compared to control	Yen et al. 2020	NCT03431844
	Elamipretide	Skeletal muscle	Improves mitochondria function (ATPmax) 2 h postinjection	Roshanravan et al. 2021	NCT02245620
		HF	Single infusion is safe	Daubert et al. 2017	NCT02388464
			High-dose ELAM-positive changes in LV volume
		Barth syndrome	Improves 6 min walk and knee extensor strength at 36 wk	Reid et al. 2021	NCT03098797
		Atherosclerotic renal artery stenosis	Attenuates postprocedural hypoxia, increases renal blood flow, and improves kidney function	Saad et al. 2017	NCT01755858
		Mitochondrial myopathy	Increases 6 MWT after 5 d treatment, the improvement appears to be dose-dependent	Karaa et al. 2018, 2020	NCT03323749
		Reperfusion injury	Safe treatment is not associated with a decrease in myocardial infarct size	Chakrabarti et al. 2013; Gibson et al. 2016	NCT01572909
	AX	Skeletal muscle, metabolism	Improves fat oxidation at lower intensity exercise	Liu et al. 2021	NCT03368872
			Improves TA muscle strength and endurance	Liu et al. 2018	NCT03368872
			Improves strength, as well as improves endurance	Earnest et al. 2011	NCT01241877
			Astaxanthin (AX) improves athletic performance and exercise capacity, reduces injury markers	Djordjevic et al. 2012	NA
		Human blood	Lowers plasma biomarker of lipid peroxidation	Kidd et al. 2011	NCT01167205
	MitoQ	Early-onset AD mild cognitive impairment	Carotid artery, endothelial function, and brain blood flow	NA	NCT03514875
		PD	No effect on progression over 12 mo	Snow et al. 2010	NCT00329056
		Aging-related vascular dysfunction	Determines 3 mo mitoQ for endothelial function in older adults and the mechanisms		NCT04851288
	Tricaprilin	AD	Targets mitochondria dysfunction	NA	NCT05809908
	Ginkgo biloba	AD	Targets mitochondria dysfunction	NA	NCT03090516
	ANAVEX2-73	Mild cognitive impairment, due to AD or early-stage mild dementia	Target mitochondria dysfunction	NA	NCT03790709
	S-equol	AD	Positive results but not clinically significant (p < 0.06) that S-equol does not influence platelet mitochondria COX activity	Stancu et al. 2016	NCT02142777
					NCT03101085
	J147	Aging and dementia	Phase I safety trial, completed but results not published	NA	NCT03838185
	UA	Aging skeletal muscle	Improves muscle endurance after 2 mo	Liu et al. 2022	NCT03283462
			Beneficial for systemic metabolism and reduces inflammation after 4 mo
		Aging skeletal muscle	4 wk supplementation elevates mitophage, mitochondrial biogenesis, and fatty acid oxidation gene expression	Andreux et al. 2019	NCT04160312
	Spermidine	Blood	Level reduced in older, but nonagenarian and centenarians remain the same level as middle-aged	Pucciarelli et al. 2012; Kiechi et al. 2018	NCT03378843
			Higher spermidine linked to increased survival

Elamipretide

The Szeto–Schiller (SS) peptides are tetrapeptides with an alternating aromatic–cationic amino acids motif that were serendipitously found to preferentially concentrate in the IMM (Zhao et al. 2004; Doughan and Dikalov 2007; Bakeeva et al. 2008). Elamipretide, also referred to as SS-31 peptide, is the most studied member of the SS peptide family. It was later shown that the mitochondrial localization of elamipretide was conferred by its strong affinity with cardiolipin (CL), a phospholipid exclusively found in the mitochondrial membranes (Birk et al. 2014). The mitochondrial uptake of elamipretide is independent of the mitochondrial membrane potential, and, therefore, elamipretide can be taken up by depolarized mitochondria (Zhao et al. 2004; Doughan and Dikalov 2007). Originally thought to act as an ROS scavenger in the mitochondria, it is now clear that elamipretide reduces production of oxidants by the ETS (Szeto 2014; Birk et al. 2014). In vitro studies show that elamipretide increases oxygen consumption and ATP production in mitochondria (Birk et al. 2014). Its interaction with CL stabilizes cristae structure (Brown et al. 2014; Szeto 2014) and alters the charge distribution around the IMM (Mitchell et al. 2020). Recently it has been shown that elamipretide directly interacts with several proteins in the IMM, including the ANT, F1FO ATP synthase, and complexes I and III of the ETC (Chavez et al. 2020).

Studies have demonstrated the protective effects of elamipretide in cardiac aging and age-related cardiovascular disease. In mouse models of pressure-overload induced heart failure, elamipretide attenuates cardiac hypertrophy and improves cardiac function (Dai et al. 2011, 2012, 2013). The protective effects of elamipretide have also been demonstrated in a canine model of heart failure (Sabbah et al. 2012, 2016). In the canine model, 2-h elamipretide treatment improves ejection fraction, stroke volume, cardiac output, and left ventricle (LV) contractility index (Sabbah et al. 2012) and 3-mo elamipretide treatment enhances ejection fraction and reduces LV end-diastolic pressure (Sabbah et al. 2016). Elamipretide also reduces cardiac IR injury and preserve cardiac function in various IR models (Petri et al. 2006; Szeto 2008). Recently, we have demonstrated that 8-wk elamipretide treatment reverses preexisting cardiac aging phenotypes, including cardiac hypertrophy, diastolic, and systolic dysfunction, in old C57/BL6J mice (Chiao et al. 2020; Pharaoh et al. 2023). Importantly, the improved cardiac function in old mice is accompanied by improved mitochondrial respiration, reduced oxidative damage, and normalized myofilament phosphorylation in the heart (Chiao et al. 2020). Elamipretide treatment is shown to improve mitochondrial respiration by reducing the age-related increase mitochondrial proton leak (Chiao et al. 2020), via a mechanism dependent on its interaction with ANT1 (Zhang et al. 2020a). A later study also found that combining elamipretide with NMN synergistically enhances the cardiac NAD⁺ pool and improves cardiac function in old mice (Whitson et al. 2020).

In skeletal muscle, a single treatment with elamipretide increases the efficiency and magnitude of in vivo ATP production in old skeletal muscles and improves in vivo fatigue resistance after 1 h (Siegel et al. 2013), while 8-wk elamipretide treatment improves in vivo mitochondrial function, redox homeostasis, and exercise tolerance (Campbell et al. 2019) in aged mice. However, 4-mo daily treatment with elamipretide did not alter fatigue resistance, atrophy, and contractile properties of ex vivo extensor digitorum longus muscles, despite reduced mitochondrial redox stress (Sakellariou et al. 2016). These different results suggest a complex role for mitochondrial redox stress in aging muscle that may be dependent on the intact system integrating circulatory and nervous inputs into the muscle. In addition to the effects in aging heart and skeletal muscle noted above, elamipretide is protective in other models of age-associated diseases, including PD (Plecitá-Hlavatá et al. 2009), AD (Howitz et al. 2003), insulin resistance (Anderson et al. 2009), age-related vision decline (Alam et al. 2022), and kidney pathology (Sweetwyne et al. 2017) in preclinical models. Due to its promising results in preclinical studies, elamipretide has been the subject of multiple clinical trials.

In a clinical trial in older adults with low mitochondrial function (ATP_max < 0.7 mM/sec and P/O < 1.9), a single elamipretide treatment improved in vivo maximum mitochondrial ATP production (ATP_max) in a hand muscle compared to placebo (Roshanravan et al. 2021). This positive effect on ATP_max returned to placebo level by day 7 following treatment, consistent with 16 h half-time of elamipretide in human blood (Daubert et al. 2017). This acute response parallels that observed in aged mice and is consistent with the ability of elamipretide to reversibly bind to CL and restore mitochondrial ETS function. (Birk et al. 2013; Brown et al. 2014). A post hoc analysis revealed an elevation in first dorsal interosseous (FDI) muscle fatigue resistance 7 d after treatment. This separation between the effects on ATPmax and muscle endurance suggests that mitochondrial ATP production may not be the most important driver of muscle function and exercise tolerance with aging. This supports a complex role for the mitochondria involved in many different aspects of cell physiology outlined in the introduction of this review. In addition to the trial mentioned above for aging skeletal muscle, elamipretide is being tested in clinical phase I and phase II studies focused on mitochondrial myopathy, macular degeneration, cardiovascular, and renal disease population (Chakrabarti et al. 2013; Gibson et al. 2016; Daubert et al. 2017; Saad et al. 2017; Karaa et al. 2018, 2020; Hortmann et al. 2019; Butler et al. 2020; Reid Thompson et al. 2021).

Mitochondrial Quality Control

Mitophagy is impaired with increasing age- and age-related diseases. The decline in mitochondrial quality control and associated loss of mitochondrial function has been linked to slow walking speed and reduced muscle strength in older individuals (Picca et al. 2023). Improving mitochondrial biogenesis by restoring the level of mitophagy is associated with delaying the age-related decline in muscle health (Madeo et al. 2018).

Spermidine

Spermidine is a metabolic polyamine that is naturally present in food sources that acts as an autophagy inducer among other cellular activities (Pietrocola et al. 2016). Four weeks of spermidine treatment improved autophagic flux in aging cardiomyocytes and reduced pathology by promoting autophagosome turnover (Eisenberg et al. 2016). Support for induction of mitophagy as the mechanism of action of spermidine comes from results demonstrating that genetic inhibition of autophagy abolishes the life span extension in flies and worms (Eisenberg et al. 2009). Spermidine also has been reported to maintain regenerative function in aging muscle stem cells and reduced protein aggregates with 2 wk of treatment (García-Prat et al. 2016). Interest in spermidine as an aging intervention is supported by the observation that aging is associated with decline in tissue spermidine concentration in model organisms as well as humans (Madeo et al. 2018). Interestingly, a cross-sectional observation reported whole blood spermidine content decreased in older adults but remained at the level of young (middle-aged) among healthy nonagenarians and centenarians (Pucciarelli et al. 2012). Additional survey-based studies also reported reduced prevalence of heart failure related to consumption of spermidine intake linked to lower mortality (Kiechl et al. 2018) and that spermidine dietary intake was linked to a reduced risk for cognitive impairment in humans (Picca et al. 2023). These results support a potential role for spermidine in healthy aging and have led to more rigorous clinical trials testing spermidine in the context of healthy aging. A small pilot study suggested spermidine supplementation may have protective effects on cognitive function in older adults at risk for dementia (Wirth et al. 2018). A follow-up phase IIb study is ongoing (Wirth et al. 2022; NCT03094546).

Urolithin A

Urolithin A (UA) belongs to a family of urolithins produced in the colon following microbiota-mediated transformation of natural polyphenols ellagitannins (ETs) and ellagic acid (EA) (D'Amico et al. 2021). Natural compounds abundant in foods such as pomegranates, berries, and nuts. However, only 40% of older adults can convert the natural food source to UA due to variation in the gut microbiomes with age, health status, and dietary intake (D'Amico et al. 2021). Recent studies have demonstrated a mechanism where UA supplementation activates mitophagy pathways and induces mitochondrial biogenesis. In worms, UA supplementation extended life span dependent on autophagy genes bec-1, sqst-1, and vps-34 and the mitophagy genes pink-1, dct-1, and skn-1 expression (Ryu et al. 2016). In young worms, UA treatment unexpectedly reduced basal respiration, while maintaining maximum respiratory capacity, despite reduced mitochondrial content indicated by the lower mtDNA to nuclear DNA ratio. However, in aged worms, UA treatment led to increased expression of mitochondrial genes and preserved reserve respiratory capacity. This same study also demonstrated induction mitophagy-related genes, greater ubiquitination of mitochondrial proteins, and improved exercise tolerance in aged mice and rat models supporting a link between improved mitochondrial quality control through mitophagy and aging skeletal muscle function (Ryu et al. 2016).

A phase I study of safety and tolerability of UA supplementation showed 4 wk of UA supplementation in healthy sedentary older adults decreased plasma acylcarnitine level (Andreux et al. 2019). The study also demonstrated a direct impact of UA on elevated gene expression in skeletal muscle for genes associated with mitophagy, mitochondrial biogenesis, and fatty acid oxidation (Ryu et al. 2016). This study concluded UA supplementation is safe, bioavailable, and has a positive impact on mitochondria health. A follow-up, small-phase II clinical trial demonstrated that UA supplementation significantly improved the secondary end point of muscle endurance in two disparate muscles after 2 mo of treatment (FDI, primarily type II fiber; TA, primarily type I fiber) (Liu et al. 2022). Despite the absence of improvement in the primary endpoint, 6 min walk test, with UA, this study suggests that UW supplementation may directly improve aged skeletal muscle performance, even in the absence of exercise. Plasma levels of acylcarnitines, ceramides, and C-reactive protein were also decreased by UA at 4 mo, indicating beneficial effects of UA treatment on systemic metabolism and inflammation, despite the absence of an effect on muscle mitochondrial ATP production. These findings are consistent with the aforementioned phase I clinical study after 4-wk UA supplementation (Andreux et al. 2019) and indicate that UA warrants further study as a mitochondrial-targeted strategy to preserve function with age.

Mitochondrial Biogenesis

AMPK acts as a cellular energy sensor that works upstream to regulate signaling pathways that activate mitochondrial biogenesis, autophagy, and cellular stress. Aging-related decline of AMPK sensitivity activates cellular stress, impairs metabolic regulation, increases oxidative stress, and reduces autophagy, all of which contribute to age-related decline (Stancu 2015). AMPK can up-regulate both mitochondrial biogenesis and mitophagy to induce fragmentation of damaged mitochondria and recycling of damaged mitochondria to improve the mitochondrial pool. Indirect activators include any modulator that causes AMP or calcium accumulation, such as metformin, resveratrol, and curcumin. Metformin inhibits complex I of mitochondrial respiration leading to increased AMP/ATP ratio. Another category of AMPK activator was first identified by Abbott laboratories in 2006 (AICAR), which generates AMP mimetic compound AICAR monophosphate (ZMP), an allosteric activator of AMPK. Additionally, salicylate is a pro-drug form of aspirin. Aspirin is a derivative rapidly broken down to salicylate upon entering circulation, also resulting in activation of AMPK.

MITOCHONDRIAL-DERIVED PEPTIDES

One exciting class of compounds are the MDPs described above. Humanin was the first discovered MDP and has been shown to have strong neuroprotective effects against AD. It was initially cloned from the resilient occipital lobe of the brain of an AD patient and found that the peptide protected against amyloid-β toxicity in neuronal cells (Yen et al. 2020). In humans, the cerebrospinal fluid level of humanin is reduced in AD patients (Yen et al. 2020). The circulating level in children of centenarians also showed higher levels of humanin compared with age-matched controls (Yen et al. 2020). The clinical trial registry clinicaltrials.gov indicates that at least one study has been completed to investigate the level of humanin in myocardium tissue and blood in relation to early complication occurrence and frequency after cardiac operation (NCT03431844).

After the initial discovery of humanin, seven additional MDPs, SHLPs1-6, and MOTS-c have been discovered (Miller et al. 2022). SHLPs, small humanin-like peptides, are encoded from the 16S rRNA region, and, at least for SHLP2-3, share some biological function with humanin. MOTS-c has also been characterized as an aging modulator and has found to decline in the circulation of middle-aged and older adults compared to younger controls. Importantly, the same study also reported MOTS-c was highest in aged human skeletal muscle suggesting it was tissue specific (D'Souza et al. 2020). As noted above MOTS-c treatment reproduces some of the benefical effects of exercise, and both humanin and MOTS-c have promise as potential interventions to preserve health span.

CONCLUSION

As stated at the beginning of this review, this is an exciting time to be working at the intersection between mitochondrial biology and aging due to the rapid development of new strategies and increasing interest and ability to conduct clinical trials focused on aspects of healthy aging. The aforementioned compounds, while not a comprehensive review of every mitochondrial-targeted compound in the literature, highlight promising new targets and strategies to capitalize on the important role for mitochondria in healthy aging. The greater appreciation of the diverse and complex roles of mitochondria in regulating cell health in aging is leading to a growth in strategies focused on improving mitochondrial health rather than the more typical targeting of specific enzymes or pathways. Given the multiple points of intersection between mitochondria and aging biology, these more wholistic approaches to mitochondrial health present new opportunities to identify new interventions, but also create a cycle where these new interventions provide new insights into the mitochondrial mechanisms of aging. These factors combined with growing interest from industry in aging is creating an environment likely to lead to breakthroughs in mitochondrial-targeted interventions for healthy aging in the next several years.