Targeting The Mitochondrial Permeability Transition Pore To Prevent Age-Associated Cell Damage And Neurodegeneration Part 3

SIRT3 serves a protective role in inhibiting ROS production but it can also act to deacetylate CypD and inhibit mPTP opening [83]. Since SIRT3 is dependent upon NAD+ levels, CypD openings are therefore also dependent at least in part on NAD+ levels. 

In recent years, more and more studies have shown that there is a close relationship between NAD+ levels and memory. NAD+ is an essential coenzyme that can react with a variety of enzymes to participate in a variety of metabolic and regulatory processes in the body. In the body's metabolic process, NAD+ can also play an important role in anti-oxidation, anti-inflammatory, and energy metabolism.

Some of the latest studies have found that reduced NAD+ levels may be related to some neurological diseases, such as senile cognitive impairment and Alzheimer's disease. In these diseases, memory impairment is a very common symptom. Therefore, people began to explore the relationship between NAD+ and these diseases.

Many experiments have shown that increasing NAD+ levels can significantly improve memory, especially the memory of the elderly. In one experiment, researchers fed mice with food containing NAD+ and found that the mice's cognitive ability was significantly improved, much better than the control group.

In addition, studies have also shown that NAD+ also plays an important role in neuronal protection and repair. In some cases of neuronal damage, NAD+ can promote the autophagy process by activating SIRT1, thereby removing bacteriophages and garbage and promoting cell repair and regeneration. Neuronal damage is often closely related to memory loss, so NAD+ also has great potential in improving memory.

In general, the relationship between NAD+ levels and memory is inseparable. As people continue to deepen their research in this field, I believe that there will be more space and opportunities to explore this field in the future and make more contributions to improving the memory and anti-aging of the elderly. Let us look forward to future developments together! It can be seen that we need to improve memory. Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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Consequently, as NAD+ concentration declines as a byproduct of protection pathway activation, SIRT3 is unable to perform inhibition of CypD-induced mPTP opening [81, 83]. 

SIRT3's simultaneous effects on ROS production and CypD lead to an interplay between the two, which enables further mPTP openings. As discussed earlier, a decrease in SIRT3 activity leads to proapoptotic pathway activation through ROS-induced DDR [12, 77]. In particular, p53 binds to CypD to form a complex triggering mPTP openings [44]. 

Thus, ROS production and CypD activation are connected via SIRT3 inhibition. Pore openings are not limited to CypD's interaction with p53. Recently, a relationship was established between metformin, AMP kinase (AMPK), the peroxisome proliferator-activated receptor-α (PPARα)/mitochondria pathway, and CypD in cardiomyocytes [84]. 

It is known that activation of AMPK protects the heart from myocardial infarction and heart failure [84]. Thus, because it was found that metformin activates AMPK, metformin can be a potential impetus in driving myocardial protection. 

Metformin abolished oxidative stress-induced physical interactions between PPARα and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of mPTP formation [84]. Thus, the myocardial protective effects of metformin were found to converge at the mPTP.

1.4. Aging, Decreased Lifespan, and Neurodegenerative Diseases as Byproducts of mPTP Opening.

mPTP openings become more frequent and longer in duration as a byproduct of increased ROS production with age and subsequent ROS-induced ROS release [51, 53]. 

mPTP openings lead to the release of ROS which in turn stimulates proapoptotic pathways leading to further openings [12, 54–56]. Due to the protective pathway dependence on NAD+, depletion of NAD+ leads to the inhibition of protective pathways leaving the counter effects of proapoptotic signals to proceed unchallenged [81, 83]. 

Thus, it is necessary to address the effects of mPTP opening regarding the overall phenomenon of aging. It has long been held that ROS accumulation leads to oxidative stress and the subsequent observable phenomenon of aging [85]. 

Recently, ROS involvement in cellular senescence has received significant attention in organismal aging. Cellular senescence is thought to be initiated by genomic damage which activates DDR and subsequent pathways leading to growth arrest [86]. 

The accumulation of senescent cells in organismal tissue is commensurate with advancing age, and senescent cells reduce stem and progenitor cell numbers leading to impaired capacity for tissue regeneration [87–89]. 

Considering that ROS plays a vital role in cellular senescence and the mPTP plays a vital role in the release of ROS, it is therefore likely that the mPTP contributes to the progression of senescence. 

Despite knowledge of the interaction between ROS and the mPTP, little work has been done about the relation between the mPTP and cellular senescence. Hofer and colleagues investigated rat ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial susceptibility to Ca2+-induced mPTP openings with aging and calorie restriction [90]. 

They found that IFM exhibited an increased susceptibility towards mPTP openings during senescence. A decline in Ca2+ retention was observed with aging, particularly during senescence [90]. It is important to note that SSM did not exhibit these same results, and mPTP's association with senescence may be dependent on the tissue type. 

SSM aside, these results would suggest that the mPTP plays a role in the induction of cellular senescence and thus tissue aging, as evidenced by the decline in Ca2+ retention. 

As described above, genomic damage initiates the cellular pathway inducing senescence. Since it is known that ROS release through the mPTP is capable of inducing DDR, it is clear that a relation between mPTP opening, induction of cellular senescence, and cell and tissue aging exists. Another proposed mechanism by which mPTP opening leads to cellular aging is through increased levels of autophagy. 

While autophagy is commonly thought to increase longevity due to its ability to clear damaged proteins and dysfunctional organelles, it can be detrimental at very high levels [91]. Elevated autophagy shortened lifespan in C. 

elegans lacking serum/glucocorticoid-regulated kinase-1 (sgk1) because of increased mitochondrial permeability [91]. Furthermore, mice maintaining sgk-1 displayed lower levels of mitochondrial permeability, normal levels of autophagy, and normal lifespan. Based on these results, sgk-1 is suggested to modulate mPTP opening, which in turn mediates mitochondrial permeability, autophagy, and lifespan [91]. 

Since mitochondrial permeability is enhanced in the absence of sgk-1, it can be concluded that lifespan reduction as a byproduct of elevated autophagy is likely due to increased mPTP activity. Research on neurodegenerative diseases, particularly on PD, has uncovered significant findings regarding mPTP openings and aging. 

PD is characterized by two phenomena including loss of dopaminergic neurons in the substantia nigra [92] and accumulation of highly insoluble fibrillar aggregates of the protein alpha-synuclein [93]. 

Recently, Ludtmann and colleagues [64] investigated the relationship between monomeric and oligomeric α-synuclein encoded by the gene SNCA and their subsequent effects on mPTP openings and cellular death. 

While α-synuclein in its monomeric form improves ATP synthase efficiency, upon protein aggregation and subsequent formation of the oligomeric form, a toxic gain of function is observed. 

Specifically, as it relates to the mPTP, the oligomers induce selective oxidation of the ATP synthase beta subunit resulting in an increased probability of mPTP opening. 

This finding is significant as induced pluripotent stem cell- (iPSC-) derived neurons bearing SNCA triplication generate α-synuclein aggregates that interact with ATP synthase and induce mPTP opening, leading to neuronal death [94].

PD is, however, not fully characterized by neuronal death alone. Loss of the antioxidant protein (protein-disulfide reductase) glutathione (GSH), a reduction in mitochondrial complex I activity, increased oxidative damage of DNA, and elevated free iron levels in the substania nigra have all been documented in patients suffering from PD [95, 96]. 

As mentioned earlier, ROS production increases with age, specifically in complexes I and III with the inhibition of electron transport [51, 52]. Furthermore, ROS accumulation within the mitochondria can lead to ROS-induced ROS release via the mPTP [53]. 

ROS released from the mitochondria can damage nuclear DNA and lead to proapoptotic signals which stimulate further mPTP openings [54–56]. Thus, the mPTP links two key processes associated with PD: a reduction in mitochondrial complex I activity leading to increased mitochondrial ROS, which in turn prompts mPTP openings and subsequent ROS release inducing increased DNA damage [51–56]. 

It is also important to note that neuroinflammation is observed in PD [64], and inflammation leads to extracellular acidification [33] which in turn leads to increased ROS production in the cell driving further mROS release from the matrix of the mitochondria via the mPTP [5, 34, 35]. 

The etiology of PD is complex and multifactorial involving environmental factors, genetic susceptibility, and aging that together promote disease progression [97]. 

The findings reviewed above suggest that the mPTP is also likely to have a potential role in the pathogenesis of PD. Age-related dysfunctions of the mPTP extend to and are prevalent in age-related pathologies mediated by various factors such as inflammation. Inflammation is an early step in the pathogenesis of AD [98], and neuroinflammation is a process that occurs in PD [99]. 

As was noted earlier, extracellular acidification can increase ROS production, which leads to increased PT via the opening of the mPTP [59]. mPTP dysfunction may also be involved in the progression of AD. 

In its later stages, AD is characterized by massive amyloid-beta (Aβ) deposition in the parenchyma and the cerebrovascular walls [100, 101]. Recent findings show that mitochondrial damage in AD is linked to Aβ toxicity [102–105]. 

Some examples include decreased mitochondrial respiratory chain function [105, 106], increased mitochondrial ROS generation [105, 107], and changes in mitochondrial structure [108]. 

The interaction of Aβ species with certain regulators of the mPTP is likely responsible for the aforementioned damage. Specifically, the interaction of Aβ species with CypD and the upregulation of CypD expression was found to decrease the threshold of mPTP activation [109]. 

An AD mouse model overexpressing a mutant human form of amyloid precursor protein (mAPP) has also been shown to demonstrate increased CypD levels [109]. Thus, Aβ appears to be an important mediator connecting AD to the mPTP. 

CypD is considered a crucial component for mitochondrial permeability transition pore (mPTP) formation [4, 110]. Du et al. found that mitochondrial function and learning/memory were significantly improved in CypDdeficient mice [109, 111]. 

These results suggest that pore formation is a necessary step in the pathogenesis of AD and that the ablation of CypD in mice gives lifelong protection against Aβ-induced mitochondrial and behavioral dysfunction [111]. 

Other studies have shown that Aβ oligomers induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload and mitochondrial PT [112]. 

This is significant because, as mentioned earlier, Ca2+ overload can lead to mPTP activation [49, 50]. Nonsteroidal anti-inflammatory drugs (NSAIDs), including salicylate and sulindac sulfide, were able to inhibit mitochondrial Ca2+ overload through mitochondrial depolarization. These studies highlight the role of mPTP dysfunction in neurodegenerative disease.

1.5. Potential Therapies to Mitigate mPTP Opening.

Previous work suggests that mPTP opening plays a role in both injury and aging, thus targeted therapies to inhibit continued and frequent opening of the mPTP may serve to promote longevity and healthspan (Table 1). 

As discussed earlier, PD, AD, and other age-related disorders are thought to be byproducts of mPTP openings. Research targeting the mPTP whether directly or indirectly is divided into two areas. 

The first area involves therapeutics that require some form of interaction with CypD, and the second area involves therapeutics that require no interaction with CypD [113]. 

Of the therapies that inhibit CypD, cyclosporin A (CsA) has evoked great interest as it has shown cytoprotective properties in cellular models due to its ability to interfere with the interaction of CypD with the mPTP [114]. Specifically, CsA has been shown to block mitochondrial Ca2+ efflux and allow mitochondria to accumulate large amounts of Ca2+ [115]. The mechanism that is responsible for increased Ca2+ retention is indicative of mPTP closure [45]. 

This point is supported by Crompton and colleagues, who found that the ability of mitochondria to retain Ca2+ in the presence of CsA was due to CsA inhibition of the mPTP [116]. CypD in particular was shown to be the target of CsA [117]. 

mPTP openings were studied in ischemic reperfusion injury in rat hearts to determine the efficacy of CsA in cardioprotection. Cardioprotection was observed in a narrow range, between 0.2 and 0.4 μM, as benefits were lost at concentrations above 0.4 μM [118]. 

Despite these promising results, a recent clinical trial showed that CsA failed to improve clinical outcomes and prevent adverse left ventricular remodeling in patients with an acute anterior ST-segment elevation myocardial infarction (STEMI) [117]. This raises questions regarding the viability of targeting CypD to promote cardioprotective. 

It is possible that these seemingly conflicting studies on the cardioprotection offered by CSA could be explained by the means of drug administration. Since cardioprotection was observed in a narrow range in rat hearts, it is possible that the dosage administered in the clinical trial, 2.5 mg/kg body weight, was too low/high of a concentration [117]. 

Further research supporting CypD as a viable cardioprotective target was conducted by Parodi-Rullman et al. on induced myocardial infarction in rats [119]. They found that CypD inhibition exerts cardioprotective effects in reperfused but not in non reperfused infarcted hearts of female rats, and the effects are observed only during acute postinfarction injury. 

CypD remains a viable target for age-related pathologies, although the timing and dosage of drug administration should be refined and optimized to demonstrate clear benefits for the patient. 

The CypD inhibitor, CsA derivative Nmethyl-isoleucine-4-cyclosporin (NIM811), has been investigated as a therapeutic alternative to CsA alone. In a study conducted to determine the efficacy of NIM811 in inhibiting the mPTP, it was found that both mitochondrial permeability transition onset and apoptosis were prevented when NIM811 was added to rat hepatocytes [114]. 

The potential of NIM811 for reducing mitochondrial permeability and improving cell survival has also been shown in animal models of spinal cord injury [120], traumatic brain injury [121], and hindlimb ischemia-reperfusion injury [122]. 

CypD-independent therapeutics have received attention (Table 1). Melatonin in particular has been studied as a potential inhibitor to the mPTP that does not require CypD interaction. 

Melatonin has been shown to inhibit mPTP activation as evidenced by reduced mitochondrial swelling and increased Ca2+ capacity [123]. This is further supported by Andrabi et. al who studied the effects of melatonin on mPTP openings in rat brain models [124]. The release of cytochrome c was used to assess pore opening, and rats treated with melatonin displayed a marked decrease in cytochrome c release [124]. 

These results would support the assertion that melatonin does indeed inhibit mPTP activation. Postmortem analyses of cerebrospinal fluid show a marked decrease in melatonin concentration with age [125], which could in theory contribute to increased mPTP opening with aging. mPTP openings lead to swelling of the mitochondria, rupture of the outer mitochondrial membrane, and subsequent release of intermembranous proteins [126]. 

Melatonin supplementation may therefore represent one option to suppress mPTP opening in older adults who are likely to have relatively low endogenous levels of melatonin.

In addition to melatonin, other CypD-independent therapeutics include mitotargeted compounds (Table 1). Mitotargeted therapeutics acting in the absence of CypD interaction include electron scavengers, cinnamic anilides, N-phenylbenzamides, and isoxazoles. 

One small molecule that directly targets the mPTP is (E)-3-(4-fluoro-3- hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide (compound 22), a cinnamic anilide that inhibits oxidative stress and chemical crosslinker-induced mPTP opening [127]. 

Other novel CypD-independent therapies exist, classified in the same cinnamic anilide series and performing similar functions as compound 22. One example is GNX-4728 which was administered in a mouse model of amyotrophic lateral sclerosis (ALS). GNX-4728 was found to slow disease progression, improve motor function, and extend lifespan by nearly twofold. 

Furthermore, Ca2+ retention was established, which is again indicative of mPTP closure [128]. Regarding N-phenylbenzamides, the most prominent therapeutic candidate is compound 4, (3-(benzyloxy)-5-chloro-N-(4-(piperidin-1-ylmethyl)phenyl)benzamide). Compound 4 induced a concentration-dependent increase in the calcium retention capacity (CRC) of permeabilized HeLa cells suggesting mPTP inhibition [129]. 

The isoxazole, compound 1, 5-(3- hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3- carboxamide, produced similar results in an isolated mouse liver mitochondria model. Compound 1 was shown to inhibit mitochondrial swelling without interfering with the inner mitochondrial membrane potential [130]. Electron scavengers are microtargeted therapeutics acting in the absence of CypD interaction. 

Some of the most studied drug therapies in this category include SS-31, XJB-5-131, MitoQ, EUK-8, and MitoTEMPO. SS-31 can increase cell survival and reduce intracellular ROS in neuronal N2A cells treated with t-butyl hydroperoxide (tBHP) [131]. XJB-5-131 can improve postischemic recovery of aged hearts, reduce Ca2+-induced swelling in mitochondria, and reduce total mROS levels in cardiomyocytes [132]. 

It was also observed that XJB-5-131 improved motor skills and cognitive functions in rats with traumatic brain injury. These results are seen as a byproduct of decreased levels of mROS and subsequent prevention of cardiolipin oxidation [133]. Both MitoQ and EUK-8 employ the same electron-scavenging mechanisms as the therapies above. 

MitoQ's therapeutic effects were examined in rat cardiac ischemia-reperfusion injury and MitoQ decreased cell death and decreased mitochondrial damage [134]. EUK-8's effects were examined in presymptomatic heart/muscle-specific manganese-superoxide dismutase- (Mn-SOD-) deficient mice. It was observed that EUK-8 suppressed the progression of cardiac dysfunction and diminished ROS production and oxidative damage [135]. 

Again, while the above therapies do not interact directly with the mPTP, they do reduce ROS levels or production within the mitochondria, which leads to inhibition of mPTP opening.

MitoTEMPO has been investigated as a potential therapeutic in the treatment of AD. A recent study was performed in which Aβ toxicity, a hallmark of AD, was measured in primary cultured mouse neurons. Upon treatment with MitoTEMPO, it was found that Aβ-induced mitochondrial superoxide production and neuronal lipid oxidation were significantly decreased. 

Furthermore, a protective effect on mitochondrial bioenergetics was observed as evidenced by preserved mitochondrial membrane potential [136]. These results would indicate that MitoTEMPO has the potential to protect neuronal function in AD. While the previous therapies are in the developing stages, one therapy has been approved for treating acute ischemic stroke in Japan. 

Edaravone is a free radical scavenger that produces neuroprotective effects. The mechanism in which this is accomplished is similar to the other scavengers in that edaravone captures and reduces excessive ROS [137]. Similarly, as with the other scavengers, the therapy acts on the relationship between ROS and mPTP activation. Thus, as a byproduct of edaravone administration, a decrease in ROS is observed and a decrease in mPTP activation occurs.

2. Summary and Conclusions

Mitochondrial dysfunction is now thought to play a significant role in tissue degeneration and loss of function that occurs in multiple organ systems with advanced age. 

A key factor in this process is the generation of reactive oxygen species in the mitochondria of aged cells, which is in turn associated with cell death, senescence, and tissue damage. The mitochondrial permeability transition pore appears to play a significant role in ROS production with aging. For example, the continued opening of mPTP and release of mROS lead to DNA damage. Cytoprotective pathways are activated to counter oxidative damage; however, the continued activation of these pathways leads to a depletion of NAD+. 

Since both the PARP1 and sirtuin protective pathways are dependent on NAD+, they lose their ability to suppress mPTP opening and inhibit mROS release and production. These findings point to mPTP inhibition as a potential therapeutic target for age-related disorders. Mitotargeted compounds and small molecules such as NIM811 have, at least in animal models, demonstrated success in promoting cell survival in settings associated with significant cellular damage such as spinal cord injury, traumatic brain injury, and ischemic stroke. 

Despite this, the application of mPTP-targeted drugs in a medical setting remains elusive. This is evidenced by CsA, which failed to improve clinical outcomes and prevent adverse left ventricular remodeling in patients with an acute myocardial infarction. The electron scavenger edaravone remains one of the only mPTP-targeted drugs approved for clinical use as a neuroprotective agent. 

Future studies should be directed at exploring more long-term use of these small molecules in older animals to determine their effects on the development and progression of chronic age-associated disorders of the brain, musculoskeletal, and cardiovascular systems.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

Funding for this research was provided by the National Institute on Aging, US National Institutes of Health (AG036675), and US Army Medical Research and Materiel Command CDMRP Program Grant DM160252.

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