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Aristotle believed that we possess a finite amount of some “vital substance.” When that substance is consumed, we die. In other words, an organism's metabolic rate determines its lifespan.
As we age, the mitochondria become larger and less numerous, and sometimes develop abnormalities with their structure. Experiments performed on mice shows that increased levels of mitochondrial mutations are related to a variety of age related changes, such as osteoporosis, hair loss, and weight reduction. The “Mitochondrial Theory of Aging” posits that the accumulation of damage to the DNA of a mitochondria leads to aging in humans and animals.
Mitochondria are unique in that they are the only organelle in animal cells that possess their own DNA, referred to as mtDNA, which is separate from the DNA in the cell nucleus. When a cell divides, the mitochondria divide independently, and new mitochondria are passed to the new cells. New daughter created through mitosis are identical to the original cell but may contain mitochondria that have new mutations. Every new cell division has the possibility of resulted in mutations within the cell’s nucleus and within the mtDNA.
How are mitochondria and metabolic rate related?
Metabolic rate refers to the amount of energy that is used by an organism to maintain life processes. On a cellular level, the mitochondria use oxygen to convert food (glucose) to an energy storing molecule called adenosine triphosphate, or just ATP. This process is called cellular respiration. The ATP produced in this reaction is then used by the cell to maintain homeostasis and ensure that the cell and body function normally.
1. What claim is being made by the author? (This is the main point of the reading, also called a thesis and usually found in the first paragraph of an essay).
2. What evidence is provided to support the author’s claim? Write 1-2 sentences that summarize how the other supports her position.
3. Provide reasoning for the claim by connecting the evidence with the claim being made or connecting it to other scientific principles.
In pursuit of healthy aging
“Although previous work has shown how intermittent fasting can slow aging, we are only beginning to understand the underlying biology,” said William Mair, associate professor at Harvard Chan School. Mitochondrial networks in the muscle cells of C. elegans (pictured) have been key elements in the study.
Courtesy of Harvard Chan School
Mitchondria and muscle mass
Individuals 65 and older are the fastest growing segment of the American population. In 2016, about 11,000 people a day turned 70. What if, as we age, there were treatments to help us keep muscle mass and not lose functional ability as early. Clinicians at UCLA know that age is one of the most important contributing risk factors in disease. On the other hand, researchers at UCLA know that age is a modifiable risk factor in animal models with certain things like caloric restriction and drug therapies.
An important contributing investigator in this area is Jonathan Wanagat, MD, PhD., whose work has primarily been focused on why individuals lose muscle mass or muscle strength as they age.
"I&rsquom a basic science researcher, but I&rsquom also a geriatrician so I care for patients here at UCLA. By far and away in older individuals, their primary concern, and this goes across generations and across people of different backgrounds, is the fear of losing the ability to do what they are able to do now. "
- Dr. Jonathan Wanagat
Wanagat is a practicing Geriatrician at UCLA Health, and the concerns of his patients help to drive his passion for understanding why we lose muscle mass as we age, and how mitochondria and metabolism come into play.
One explanation he and his colleagues have identified are mitochondrial mutations, known as deletion mutations. Much of his previous work has been dedicated to demonstrating that these deletion mutations cause muscle fiber loss. As that work has progressed, they are also focusing on gaining a better understanding of the mutation process with the hope of finding a way to prevent the mutations from happening, accumulating, and ultimately killing the muscle fiber.
Wanagat has been focused on the mitochondria for 24 years and for him, &ldquomany roads that lead to the phenomena in aging go through the mitochondria. Whether it&rsquos mutations, autophagy, metabolism, ROS production, or fat metabolism - you name it - for me the part I&rsquom always interested in are the parts that touch mitochondrial biology.&rdquo
Mitochondrial Hormesis, Mitonuclear Protein Imbalance, and Metabolic/Stress Control of Lifespan
There are multiple overlaps and points of connection between the signaling events induced by CR, IGF-I inhibition, and mTOR inhibition. Interestingly, as revealed by recent findings, all of these appeared to elicit a heightened mitochondrial respiration that produces a mild to moderate elevation of ROS. Contrary to what might be expected from the free radical theory of aging, this elevation in ROS may underlie lifespan extension (136, 190). How could this be reconciled with current knowledge?
Before attempting to answer the above question, it is worth noting that manipulations having the opposite effect, namely a mild to moderate reduction in mitochondrial respiration, can also extend lifespan, although not necessarily through reduction of ROS (79). This has been demonstrated by ETC mutants of C. elegans (45, 95, 103, 161, 187, 188), Drosophila (31), and mice (110). In particular, the lifespan extension effect of clock 1 (clk-1)/coq-7 mutation, which encodes a mitochondrial hydroxylase required for the synthesis of coenzyme Q, is conserved from C. elegans to mice (110). clk-1 mutants are defective in mitochondrial respiration due to impaired electron transfer between complex I and complex III. This defect elevated the generation of ROS (127). The ROS thus generated apparently also did not shorten lifespan, but instead extended it. Intriguingly, therefore, ROS generation and lifespan extension can arise from dietary, pharmacological, or genetic manipulations that either increase or decrease mitochondrial respiration. Viewed generally, these are forms of metabolic and energetics stresses that might elicit cellular adaptive responses.
The conceptual theory put forth to explain how metabolic stress may in fact promote lifespan extension is referred to as mitochondrial hormesis, or “mitohormesis” (151). In brief, metabolically induced low-level mitochondrial stress elicits cellular responses that increase the stress tolerance of the cell. These changes collectively suppress acute and prolonged ROS production and facilitate cellular survival. For multicellular organisms, lifespan extension presumably occurs when the benefits of cellular mitohormesis translates to survival at the organismal level. A number of “retrograde signaling” pathways are now known that sense and transmit mitochondrial signals to effect changes in nuclear gene expression. One example of such signaling occurs as a consequence of inhibition of mitochondria respiration and the disruption of mitochondrial membrane potential (ΔΨm) (57, 80, 111). Originally characterized in yeast, a drop in ΔΨm initiates retrograde signaling through a set of retrograde (Rtg) regulation factors, which culminates in nuclear translocation of the heterodimer of two basic helix-loop-helix transcription factors, Rtg1 and Rtg3, which influences the expression of retrograde response target genes (80). A recent study using mammalian cybrid cells with mtDNA mutations identified 72 transcription factors that were potentially involved in mitochondrial retrograde signaling, including pathways not known to act in yeast (22). Activation of mitochondrial retrograde signaling extends the lifespan of yeast (93) and C. elegans (32), as well as human fibroblasts in culture (106). One form of ROS-mediated mitochondrial retrograde signaling is its activation of nuclear factor-κB (NF-κB) (51, 122), which heightens stress response and is survival promoting in many cell types (114). Mitochondrial ROS have also been shown to act through the mitogen-activated protein kinase p38 pertaining to hypoxia signaling in mammalian cells (41) and lifespan extension in C. elegans (161).
Stress and damage at the mitochondria also trigger the mitochondria-specific unfolded protein response (UPR mt ) (67, 138), a prominent mitochondria-nuclear signaling pathway that induces the expression of mitochondria protective molecular chaperones and other regulators of mitochondria homeostasis. The UPR mt is a potent transducer of ETC perturbation-based enhancement in lifespan (39). A recent report has shed light on how the UPR mt is initiated. Activating transcription factor associated with stress (ATFS)-1 is a sensor of mitochondrial stress and has both a nuclear localization signal and a mitochondrial targeting sequence. During mitochondrial stress, its mitochondrial import efficiency was reduced, allowing ATFS-1 to accumulate in the cytosol and translocate to the nucleus to enhance transcription of UPR mt genes (128).
Very recently, Houtkooper and colleagues have elaborated on the concept of “mitonuclear protein imbalance,” a stoichiometric imbalance between the expression of nuclear- and mitochondria-encoded proteins, as a conserved lifespan extension mechanism (74). Such an imbalance can be experimentally induced by knocking down mitochondrial ribosomal proteins. Mitonuclear protein imbalance, and consequentially UPR mt , is also generated by pharmacological perturbation of mTOR, as well as CR mimetics (74). Induction of mitonuclear protein imbalance appears to also underlie the lifespan-promoting effect of preserved NAD + levels and sirtuin activity in aged animals (123). Using a Drosophila model, Owusu-Ansah and colleagues showed that mild muscle mitochondrial distress caused by transgenic RNAi against complex I components preserved muscle function and extended lifespan (131). These muscle-restricted phenotypes apparently involved redox-dependent induction of genes that regulate UPR mt , as well as increased expression of the Drosophila gene ImpL2. The latter encodes an insulin-like growth factor-binding protein, which repressed insulin signaling and enhanced mitophagy. This is an important illustration that mitohormesis occurring in a major organ of energy expenditure could prolong lifespan.
It should also be emphasized that UPR mt is by no means a standalone pathway as far as aging is concerned, and it is not surprising that its connection with the classical endoplasmic reticulum (ER) stress-induced UPR (ER UPR) has been recognized, particularly in chronic inflammatory diseases (149). The ER and mitochondria are physically and functionally linked through the ER-mitochondrial contact sites (155), which regulate the key cellular process of cell death (49) and autophagy (61). In yeast, it was recently reported that the ER may be a major source of ROS in the event of mitochondrial dysfunction (99). Chronic low-grade inflammation is characteristic of aging organs such as the brain (141) and underlies many aging-associated disorders in humans (21). The process of autophagy has been deeply connected with cellular and organismal aging (156). In C. elegans, it has been shown that signaling from IGF-I and mTOR pathways converges on the regulation of autophagy, and this appears to be a key component of lifespan regulation (176).
Interestingly, recent findings provided clear evidence that signaling from mitochondria could modulate aging in a noncell autonomous manner. In investigating the lifespan extension effect of RNAi-mediated knockdown of nuclear-encoded cytochrome c oxidase-1 subunit Vb/COX4 (cco-1) in C. elegans, Durieux et al. (39) found that knocking down cco-1 in specific tissues like intestine and neurons significantly increased lifespan via upregulation of UPR mt , but this is not so for cco-1 knockdown in body-wall muscles. Intriguingly, mitochondrial perturbation in neurons resulted in upregulation of UPR mt in the intestine. It thus appears that mitochondrial respiratory stress in one tissue could activate the mitochondrial stress response pathway in not only surrounding cells within the same tissue but also those of a distal tissue. Indeed, mitochondrial ROS signaling through SKN-1/NRF2 from redox-sensitive neurons from the C. elegans head was shown to be sufficient for lifespan extension (161). The nature of this noncell autonomous signal, which Durieux et al. (39) termed a “mitokine,” is yet unclear. One possible candidate for a mitokine would be the ROS generated as a result of either mitochondrial respiratory impairment by genetic means or by CR and mTOR inhibition.
If ROS is one form of mitochondrial retrograde signaling that contributes to extension of lifespan, what is the nature of the species that is responsible for ROS-based signaling? The superoxide anion O2 − is extremely reactive and has a very short half-life. It is readily dismutated into H2O2 by both mitochondrial and cytoplasmic SODs and reacts with Fe-S clusters within mitochondrial proteins. The hydroxyl radical is even more short-lived and reactive. On the other hand, H2O2 is biochemically less toxic and has a longer half-life, and being uncharged allows it to passively diffuse across membranes, or through facilitated diffusion mediated by aquaporin channels (8, 120). Other than SOD-based reactions, H2O2 may also be generated by electron transfer processes involving molecules like the longevity gene product and signaling adaptor p66Shc (119). In response to oxidative stress, a fraction of the cytoplasmic p66Shc translocates to mitochondria, where it binds cytochrome c and acts as an oxidoreductase, transferring electrons from cytochrome c to molecular oxygen (50). Interestingly, H2O2 is also formed in the ER as a by-product of oxidative protein folding (178), or through the activity of ER NADPH oxidase-4 (186). Should ROS act as a signal, the exact operative chemical species of ROS involved may well be H2O2. H2O2 generated from 1-methylnicotinamide, a metabolite of nicotinamide generated by the activity of sirtuins on NAD + , was recently proposed to underlie the lifespan-prolonging effect of sir-2.1 in C. elegans (160). However, at the moment it is unclear if effective concentrations of H2O2 could be induced and sustained as a signal that could act in not just intercellular but interorgan signaling. Of course, it is equally likely that the mitokine is a protein or a small molecule. Possible candidates include the mitochondrial-derived peptides such as humanin, which is a known mediator of stress response (100, 189), or a metabolism-regulating cytokine, such as FGF21 (16, 109).
EXAMPLES OF PROJECTS IN THE LAB:
Is aging loss of digital or analog information? Is epigenetic noise the reason we age?
We have developed “The Relocalization of Chromatin Modifiers (RCM) Hypothesis,” which proposes that epigenetic changes due to relocalization of chromatin factors in response to DNA damage may be a chief cause of aging. This is true for yeast cells and it appears to be true for all eukaryotes. In response to a DNA break, proteins that regulate gene expression move to the break to help repair, resulting in gene expression changes. In young cells, this process is reset upon completion of DNA repair. However, not all proteins make it back to where they came from, which leads to gene expression changes and a loss of cell identity. We have developed the “ICE” mouse (for inducible changes in the epigenome), which allows us to induce DNA breaks and drive epigenetic changes that accelerate aging. Work is now centered on reversing this aging process.
C57BL/6 Siblings: Control vs ICE at 16 months of age
Reprogramming cells to be young again
Our work has led us to the conclusion that the loss of epigenetic information is likely the root cause of aging. By analogy, if DNA is the digital information on a compact disc, then aging is due to scratches. We are searching for the polish. Our work has led us to identify reprogramming factors that we believe will enable us to reset a cell’s epigenetic status and reverse its age. We have developed human-compatible viral vectors to deliver the reprogramming genes to specific tissues or the entire body, thereby causing cells to act younger and wounds to heal faster. Our current focus is on nerve regeneration and the reversal of other symptoms of aging. We see treatments being possible for companion animals and humans to dramatically improve their health and lifespan.
Can we develop drugs that slow aging?
Our work on SIRT1 led us to an exciting finding that the level of nicotinamide adenine dinucleotide (NAD+), cofactor of SIRT1, declines with age. We study the mechanisms by which the NAD+ level affects DNA repair and look for therapeutic targets to improve this process. In particular, we focus on delineating the biology of NAD+-depleting and producing enzymes as direct tools to control the NAD+ level in the cells toward increased health-span and improved physiological resilience.
The discovery of longevity genes showed that it is possible to greatly slow the pace of aging and disease by manipulating just one central pathway. This raises the possibility that we can find small molecules that can treat multiple, seemingly unrelated diseases, with a single medicine. Our lab has been highly active in this area, starting with the discovery of sirtuin activating compounds (STACs) in 2003. Since then, potent activators have been discovered and some of these are now in clinical trials, producing positive results. We have active studies to understand how STACs work at the molecular and the physiological levels using cutting-edge enzymological and structural methodologies and mouse genetic models in which we can delete genes at any time throughout the lifespan of the animal, and in specific organs. We published, for example, that the ability of resveratrol and a STAC called SRT1720 to increase mitochondrial function, require the SIRT1 gene in vivo. We have an active program to develop novel molecules that raise NAD levels. We are testing them for their effects on aging and age-related diseases. Human clinical trials with NAD-boosting molecules are ongoing.
Improving Health Through NAD+ Boosting
Understanding the role of mitochondria in aging and disease
The study of mitochondria has experienced a renaissance in recent years. A large body of evidence indicates that common aging-related diseases have a mitochondrial component. Yet, surprisingly little is known about what leads to the progressive loss of mitochondrial fitness during aging. We investigate the cellular mechanisms that could be employed to maintain the mitochondrial homeostasis and ultimately prolong health-span.
One of our research avenues led to the discovery of ongoing asynchrony between the nuclear and mitochondrial genomes during aging. Utilizing novel genetic and pharmacological approaches, we are using this knowledge to restore metabolic function in aged mice back to youthful levels. We established that mitochondrial NAD+ levels dictate cell survival, which we refer to as the “Mitochondrial Oasis Hypothesis.” Following up on this work, we have formulated an exciting hypothesis that leakage of NAD+ from mitochondria is a cause of aging and memory loss.
We are also interested in identifying new genes and signaling cascades in the human genome that control mitochondrial function. We are developing novel genome mining algorithms, using advanced sequencing and proteomics tools, and high-throughput screening methods to map the most complete network of mitochondrial regulators. This work will provide new insights into fundamental aspects of mitochondrial biology and how mitochondrial defects may be prevented or corrected. We are also interested in finding novel secreted factors that increase mitochondrial function and are candidates for signaling factors that have recently been implicated in the systemic control of aging in simple organisms.
Mitochondrial network surrounding nucleus (mitoTimer)
Delaying menopause and reversing female infertility
Ovarian stem cells are a recently discovered type of cell than can give rise to oocytes in culture and produce healthy oocytes in vivo. This work may overturn the dogma that a female is born with a set number of eggs that are simply lost over time due to damage and genomic instability. We are using our knowledge gained from studying aging and metabolism to understand how female infertility may be delayed or reversed. Our goals are to identify genes and small molecules that can reactivate ovarian stem cells in vivo to treat premature ovarian failure, chemotherapeutic ovarian failure (in cancer patients) and extending the healthy and fertile period for women.
Two cell embryos
Can we slow down or even reverse neurodegenerative diseases?
Neurodegenerative diseases strike primarily in mid to late life, and thus their incidence rises in aging populations. We are actively working on identifying the molecular drivers of neuronal degeneration such as novel genes, epigenetic changes and metabolic imbalance by applying diverse experimental approaches including classical studies of genes and gene function, advanced omics and novel transgenic mouse models, including the NICE mouse (for neuronal inducible changes in the epigenome) which allows us to study the effects of epigenetic changes in the aging brain. Through our studies we aim to develop therapeutic interventions that can prevent the onset or slow the progression of disease, and possibly even reverse it by regenerating the damaged tissues.
Healthy brain vs brain with A-beta plaques – photo courtesy of Jaime Ross
Uncovering the human secretome
Peptide hormones regulate embryonic development and most physiological processes by acting as endocrine or paracrine signals. They also hold great therapeutic potential either as medicines or targets for treating both common and rare diseases. Yet identifying peptide-coding genes below
300 base pairs is inherently difficult because they exist within the “genomic noise”. Our goal is to uncover the human secretome and use newly discovered hormones to improve the human condition. Over the past few years, we have developed a unique pipeline of technologies that combines breakthroughs in math, computer hardware and software, proteomics, mass spectrometry, and high-throughput screening, each of which has been optimized and integrated. Using this platform, for which we have been awarded an NIH Director’s Pioneer Award, we have discovered thousands of putative peptide-coding genes. Our aim is to screen these peptides for activities to determine their biological roles and potential therapeutic application in biology and disease settings.
Mitochondrial Dysfunction in Age-Related Metabolic Disorders
Aging is a natural biological process in living organisms characterized by receding bioenergetics. Mitochondria are crucial for cellular bioenergetics and thus an important contributor to age-related energetics deterioration. In addition, mitochondria play a major role in calcium signaling, redox homeostasis, and thermogenesis making this organelle a major cellular component that dictates the fate of a cell. To maintain its quantity and quality, mitochondria undergo multiple processes such as fission, fusion, and mitophagy to eliminate or replace damaged mitochondria. While this bioenergetics machinery is properly protected, the functional decline associated with age and age-related metabolic diseases is mostly a result of failure in such protective mechanisms. In addition, metabolic by-products like reactive oxygen species also aid in this destructive pathway. Mitochondrial dysfunction has always been thought to be associated with diseases. Moreover, studies in recent years have pointed out that aging contributes to the decay of mitochondrial health by promoting imbalances in key mitochondrial-regulated pathways. Hence, it is crucial to understand the nexus of mitochondrial dysfunction in age-related diseases. This review focuses on various aspects of basic mitochondrial biology and its status in aging and age-related metabolic diseases.
The bioenergetic system in the aging brain is complex, adaptive, and dynamic. Chronological aging and endocrinological aging both drive critical aspects of the bioenergetic system in brain. Coupling between brain and peripheral metabolism is dynamic and has implications for therapeutic and nutritional interventions to address brain metabolic distress. Given the parallel metabolic phenotype between aging female brain and prodromal AD, our observations provide insights into preventative and therapeutic windows of opportunity to sustain brain metabolic health and reduce risk of AD.
Regulatory Role of MERCs in Mitophagy
Autophagy exerts a protective role against cellular senescence through the elimination of damaged organelles and intracellular protein aggregates (Rubinsztein et al., 2011 Ranieri et al., 2018). Interestingly, MERCs have been proposed recently as platforms for autophagy initiation and function (Hamasaki et al., 2013 Bockler and Westermann, 2014) on the basis of their role in modulating lipid composition of ER–MT interface. In particular, the artificial increase of PE was found to regulate positively the autophagic flux and, thus, to extend significantly the lifespan in yeast, mammalian cells, and flies (Rockenfeller et al., 2015). In addition, MAM lipid-rafts microdomains and the GD3 ganglioside were reported to participate in the initial organelle scrambling activity that finally leads to the formation of autophagosome (Garofalo et al., 2016).
Besides its role as tethering factor, Mfn2 has emerged as an important regulator of mitophagy, the selective degradation of MT during autophagy activation (Chen and Dorn, 2013 Bockler and Westermann, 2014). In particular, during mitophagy PINK1-phosphorylated Mfn2 functions as a receptor for parkin that, in turn, mediates MFN2 ubiquitination, as a signal to mark damaged MT recruitment and ubiquitination leading to mitophagy initiation. Thus, giving that Mfn2 localizes at MERCs, it is conceivable to speculate that MERCs is primarily involved and participate to mitophagy, rather than to fission and fusion processes.
Studies performed in yeast have provided an interesting model for the role of MERCs in the removal of aged MT from the cell. Indeed, during yeast cells mitosis, tethering activity of MERCs is essential to segregate maternal MT and accumulate toxic protein aggregates, separating those from the MT acquired by the bud, which are largely free of aggregates (Mogk and Bukau, 2014 Zhou et al., 2014). Interestingly, this mechanism, which account for a strategy to rejuvenate cellular environment, involves MERCs and is gradually lost by cells with advanced replicative age, suggesting the participation of MERCs in the mitochondrial quality control (Zhou et al., 2014). More interestingly, in human mammary epithelial cells, a similar event was observed following cellular division. In mammary cells, fine-tuned fission events allow daughter cells, which must maintain stemness properties, to receive newly synthetized MT, while the daughter cells, undergoing to differentiation, receive aged MT (Katajisto et al., 2015). As a consequence, this mechanism is settled to preserve the regenerative capacity of the tissue and prevent senescence. Similarly, recent works have revealed how MERCs were spatially linked to the mitochondrial DNA synthesis both in human and yeast (Murley et al., 2013 Lewis et al., 2016). In other words, it was found that MERCs coordinate replication of mitochondrial nucleoids with mitochondrial fission in order to distribute the proper nucleoids into the daughter MT. Altogether, these evidences confirm the involvement of MERCs in the modulation of mitochondrial fission as a strategy to cope with the establishment of cellular aging.
Giving the crucial role of Mfn2 in ER–MT tethering, mitochondrial fusion, and mitophagy, it is not surprising the increase of experimental evidences and studies linking Mfn2 to aging and age-related diseases. Notably, all the processes related to mitochondrial dynamics ascribed to Mfn2 are important to optimize mitochondrial function and avoid senescence and degeneration. Indeed, Mfn2-knockout in MEFs, cardiomyocyte, and neurons, impairs mitophagy leading to damaged MT accumulation, cell death, and tissue degeneration. Remarkably, these pathological mechanisms involving Mfn2 dysfunction are found in numerous age-related diseases, such as Alzheimer, Parkinson, diabetes, and cardiomyopathies (Filadi et al., 2018).
Not by coincidence, a progressive reduction of Mfn2 that caused impaired mitophagy and accumulation of dysfunctional MT has been reported in the skeletal muscles of aging mice, linked to sarcopenia (Sebastian et al., 2016). In the PolG mice model of premature aging, obtained by expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG mice), mouse cells displayed higher level of the mitochondrial fission protein Fis1, in parallel with increased mitophagy, which likely contributes to the sarcopenic phenotype observed in premature aging (Joseph et al., 2013). In contrast, wild-type aged mice were characterized by higher level of Mfn2 and Mfn1 and reduced levels of Fis1, suggesting increased mitochondrial fusion and reduced mitophagy, probably in response to the physiological accumulation of mitochondrial DNA mutations in the aged muscles. Similarly, increased mitochondrial fusion was also found in senescent mesenchymal stromal stem cells that showed higher levels of Mfn1 and OPA1, together with increased mitochondrial mass and ROS, compared to younger cells at lower passages (Stab et al., 2016).
To summarize, stimulating autophagy in animal models can certainly ameliorate several aging-associated phenotypes. Collectively, these data indicate that the beneficial effects derived from lifespan extension regimens can (at least in part) be explained by the induction of mitophagy. Future studies should provide further insights into how these mechanisms intersect with the mitophagy pathway in order to maintain mitochondrial fitness in vivo.
MAM in aging and senescence: a proteomic perspective
The MAM proteome was comprehensively analyzed for the first time by Zhang et al. 19 , who identified 991 proteins in the “heavy” MAM fraction (which can be isolated at lower centrifugal forces compared to standard MAM isolation procedures). Later on, Poston et al. 20 reported 1212 candidates, including weak soluble proteins, present at the MAM. Among them were commonly recognized MAM proteins: ACAT1, BiP/GRP78, calnexin, calreticulin, Erlin-1, Erlin-2, ERP44, HSPA9, MFN1, PDIA3, VDAC1, VDAC2, and VDAC3. The MS analysis enabled the characterization and classification of proteins identified in MAM into three groups: (1) those localized only in MAM (“MAM-resident proteins”) (2) those localized in MAM but present in other cellular compartments (“MAM-enriched proteins”) and (3) those temporarily present in MAM (“MAM-associated proteins”) 20 . Up to date, increasing number of reports has been published describing importance of the MAM proteome in regulation of cellular biology and senescence 17,18,19,20,21,22,23 .
Mitochondrial structure and MERCs
Mitochondrial malfunctioning and structural variations have been linked with aging and age-associated disorders 21,22 . Mitochondrial morphology is very dynamic and can vary from a fragmented to a filamentous network as an effect of competition between the processes of fusion and fission, which are the key determinants of the mitochondrial quality control 23 . In particular, the levels of mitochondrial fusion proteins Mfn1 and Mfn2 were shown to be increased in aging skeletal muscle, indicating for upregulated fusion, likely in response to the accumulated mutations in the mitochondrial DNA 24,25 . The increased fusion was accompanied by reduced levels of the fission protein Fis1. Interestingly, mitochondrial network rearrangements are regulated by MERCs, which have been shown to mark the sites of mitochondrial fission 26 . Furthermore, senescent human adipose-derived mesenchymal stromal/stem cells exhibited increased levels of mitochondrial mass, superoxide and mitochondrial fusion proteins as mitofusin 1 (Mfn1) and dynamin-related GTPase (OPA1) compared with young cells at low passages 27 . These observations indicate that changes in mitochondrial morphology observed in aging cells can be linked to the misregulated processes of fission and fusion.
Misfolded protein aggregates present in MERCs
The loss of proteostasis, which is manifested by the decreased protein degradation ability of a cell, is one of the hallmarks of aging. Consequently, aggregates of damaged or misfolded proteins accumulate, leading to cell degeneration, and many pathologies. It has been recently reported that mitochondria are involved in the asymmetric segregation of the toxic aggregates during cell division in yeast 28,29,30 , which provides a mechanism for rejuvenation of the bud. In this process, the cellular debris is retained in the older mother cell, while the younger bud is essentially free of toxic protein waste. The protein aggregates have been shown to associate with the ER surface and localize at MERCs, indicating the possible role of MERCs in the protein quality control system 28 . A similar process was observed in immortalized human mammary epithelial stem-like cells undergoing asymmetric division, where newly synthesized mitochondria segregated preferentially to the daughter cell maintaining stemness properties, while daughter cells which received older mitochondria gave rise to differentiated cells 31 . Further studies using the split-GFP system in human RPE1 cells and in yeast revealed that cytosolic proteins prone to aggregation are imported into mitochondria in order to undergo degradation by mitochondrial proteases, such as Pim1 29 . This indicates that mitochondria play a role in both segregation and degradation of protein aggregates.
Cooperation of mitochondria, the ER and MAM in ROS production
Reactive oxygen species (ROS) and aging
Increased intracellular levels of ROS and consequential oxidative damage to proteins, lipids, and DNA have been reported in many models of aging 32,33,34 . Although it is now clear that aging process is far too complex to be explained by one mechanism, the evidence that accumulation of oxidative damage is among the events contributing to aging phenomenon is quite extensive. Proteins responsible for intracellular ROS generation are located nearly in all subcellular compartments including mitochondria and the ER 34,35 . ROS present at moderate levels participate in intracellular signaling however, excessive amount of these highly reactive molecules is harmful. Since MAM are dynamic structures enhancing communication between mitochondria and ER, they may play role in regulation of ROS production by ER and mitochondria.
ROS sources in mitochondria and ER
Mitochondrial respiratory chain has long been recognized as the main source of deleterious free radicals such as superoxide radical anion (O2 .•− ), which are responsible for age-related oxidative stress 36,37 . In recent years this view has been challenged and other intracellular ROS sources are gaining increased attention 38 . Depending on the tissue type, physiological state or pathological conditions, various enzymes localized in different subcellular compartments may be the dominant ROS producers. However, the significance of mitochondrial ROS in the aging process is supported by the marked overrepresentation of the mitochondrial proteome among the proteins subjected to oxidative damage throughout a lifespan 39 . The main ROS produced in mitochondria is superoxide radical anion O2 •− , which is dismutated to H2O2. In turn, H2O2 gives rise to highly reactive OH in the reaction catalyzed by transition metals. There are several sites in mitochondria where ROS can be formed, including the respiratory chain complexes I and III. The rate of superoxide generation by these sites depends strictly on the redox state of the respiratory chain 33 . Other known mitochondrial ROS sources, releasing either O2 •− or H2O2, include the following: mitochondrial cytochrome b5 reductase 40 and monoamine oxidases 41 (associated with outer mitochondrial membrane), dihydroorotate dehydrogenase 42 , and glycerol-3-phosphate dehydrogenase (located at the outer surface of the inner mitochondrial membrane) 43 , electron transfer flavoprotein-ubiquinone oxidoreductase (localized on the matrix face of the inner mitochondrial membrane), and two mitochondrial matrix enzyme complexes: α-ketoglutarate dehydrogenase 44,45 , and pyruvate dehydrogenase 35 . Interestingly, most of the abovementioned proteins and protein complexes have been found to be increasingly carbonylated during aging and senescence 39 .
When compared with mitochondria, ROS production in the ER is less studied, partly due to the limited choices of appropriate tools for measuring the ROS levels in this compartment. In the ER, proteins from the cytochrome P450 family 46 , NADPH oxidase 4 (Nox4) 47 , and endoplasmic reticulum oxireductin (Ero1) 48 are the well-known ROS producers. Ero1 exists in two isoforms: Ero1-α and Ero1-β 49,50,51 . Interestingly, Ero1-Lα binds to the ER membrane especially in regions involved in MAM formation, and approximately 75% of Ero1-Lα is localized in the MAM fraction 52 . There is still missing evidence regarding ROS levels in the ER at different stages of life however, aging appears to be accompanied by increased oxidative damage and the dysfunction of specific ER proteins, such as the ryanodine receptor (RyR) 53, the chaperones protein disulfide isomerase (PDI) and immunoglobulin heavy chain binding protein (BiP) 54,55 .
Mitochondria-ER contact sites as modulators of ROS synthesis and targets of oxidative damage
The MAM structure facilitates mitochondrial calcium uptake upon its release from the ER by coupling IP3R with a voltage-dependent anion channel (VDAC) 56 . The influx of Ca 2+ to the mitochondrial matrix affects multiple aspects of mitochondrial function, such as Krebs cycle enzyme activity, ATP synthesis, mitochondrial permeability transition pore (PTP) opening, the mitochondrial membrane potential and respiration, and in consequence, mitochondrial ROS production 57,58,59,60,61 . Mutual dependencies between ER function and mitochondrial ROS production have also been demonstrated upon the aging-dependent deterioration of RyR function 53,59 . In the skeletal muscle of aged mice, increased carbonylation and cysteine nitrosylation of RyR1 was accompanied by channel “leakiness,” reduced Ca 2+ transients upon electric stimulation of the muscle fibers, increased ROS levels and impaired muscle force production. The mitochondrially targeted overexpression of catalase diminished the oxidative modifications of RyR 59 . On the other hand, RyR1 destabilization by rapamycin treatment resulted in increased Ca 2+ levels in the mitochondrial matrix, a decreased mitochondrial membrane potential and enhanced mitochondrial superoxide production 59 . Furthermore, increased mitochondrial lipid peroxidation in the skeletal muscle of mice with the Y522S mutation in RyR1 was associated with increased Ca 2+ leakage through the channel 62 . Interestingly, mitochondrial damage, as well as accompanying muscle dysfunction, could be diminished by treatment with the antioxidant N-acetylcysteine, indicating involvement of ROS 62 .
The translocation and enrichment of the MAM fraction with the Ero1-Lα isoform is regulated by the oxidoreductive status of the ER environment. In fact, hypoxic conditions lead to the complete relocation of Ero1-Lα from MAM 52 . Ero1-Lα is a FAD-dependent oxidase that together with PDI plays an essential role in protein folding 63,64 . PDI directly interacts with newly synthesized and folded proteins and catalyzes disulfide bond formation by accepting electrons. In turn, Ero1 restores the oxidized state of PDI and transfers the accepted electrons from PDI to molecular oxygen, leading to H2O2 synthesis 64,65,66 . In addition, Ero1-Lα is crucial in the regulation of calcium release via MAM and IP3R1. During ER stress, Ero1-Lα oxidizes IP3R1, which potentiates the release of Ca 2+ from the ER 49 . Next, ERp44 (ER luminal chaperone protein), which can also be found in MAM, binds to IP3R1, resulting in the inhibition of Ca 2+ transfer to mitochondria at MERCs 67 . Interestingly, IP3R1 oxidation by Ero1-Lα causes the dissociation of ERp44 from IP3R1, thus promoting the activation of calcium release via IP3R1 49,68 .
Proteins present in MAM and involved in ROS generation are presented in Fig. 2.
Schematic representation of ER, mitochondria, and MAMs with major mechanism of ROS production and Ca 2+ cross-talk. 2OGDH Oxoglutarate dehydrogenase, CYB5R3 NADH:cytochrome b5 reductase, cyt. c cytochrome c, DHODH dihydroorotate dehydrogenase, Ero1 endoplasmic reticulum oxireductin, ETF electron transfer flavoprotein-ubiquinone oxidoreductase, Ero1α endoplasmic reticulum oxidoreductin, GPDH glycerol-3-phosphate dehydrogenase, GRP75 75 kDa glucose-regulated protein, NADH:ubiquinone oxidoreductase (I), CoQH2-cytochrome c reductase (III), IMM inner mitochondrial membrane, IP3R inositol triphosphate receptor, KGDHC α-ketoglutarate dehydrogenase complex, MAO monoamine oxidases A/B, Nox4 NADPH oxidase 4, OMM outer mitochondrial membrane, p66Shc p66Shc protein, PDI protein disulfide isomerase, PDH pyruvate dehydrogenase, VDAC voltage-dependent anion channel
P66Shc and its involvement in ROS production and aging
Among the many proteins found in the MAM, the 66-kilodalton isoform of the growth factor adapter Shc (p66Shc) protein has been reported to stimulate ROS synthesis and be tightly connected with the oxidative challenge, age-derived diseases and the aging process 69,70,71 . P66Shc together with p52Shc and p46Shc belongs to the ShcA family, and plays the role of a dominant negative regulator in the signal transduction from the growth factor receptor via the Ras-mediated signaling 72,73 . Furthermore, it has been demonstrated that p66Shc knockout mice are less sensitive to oxidative and hypoxic stress and live approximately 30% longer than wild-type animals 69 .
While p66Shc is considered a cytosolic protein, it has also been found in the following locations: (a) the mitochondrial matrix 74 (b) the mitochondrial intermembrane space 70 (c) associated with the OMM from its cytosolic side 71 and finally (d) in the MAM fraction. Exogenous or endogenous oxidative stress can stimulate the critical phosphorylation of p66Shc at the Ser36 residue 69 and enhance its translocation to or association with mitochondria 75 . The p66Shc is phosphorylated at Ser36, and subsequently isomerized, dephosphorylated, and finally translocated to the mitochondrial intermembrane space (MIMS) and/or the MAM fraction, where it participates in ROS production 70,75,76,77,78,79,80 . The p66Shc catalyzes the reduction of O2 to H2O2 in the mitochondrial intermembrane space at the cost of cytochrome c oxidation, which appears to be an important step in the induction of apoptosis through the mitochondrial pathway 70 . Unfortunately, whether p66Shc is translocated to the MIMS in mitochondria 70 or binds to the OMM (from the cytosolic side) involved in MAM formation 71 remains a matter of debate. Yet, regardless in which cellular compartment p66Shc contributes to ROS production 81 , its participation in the feedback loop of ROS-induced p66Shc ROS production indicates that p66Shc could be involved in mammalian lifespan regulation. Thus, by translating oxidative stress damage into cell death, p66Shc becomes an apoptotic inducer shortening the lifespan 75 . The p66Shc mRNA and p66Shc protein were highly expressed in fibroblasts from centenarians compared with fibroblasts from young and elderly individuals 82 . In contrast, the primary cultures of skin fibroblasts derived from newborn and 18-month-old mice expressed similar levels of p66Shc 71 . However, the expression of p66Shc was significantly higher in the liver, heart, lungs, skin, and diaphragm of adult mice than in newborn littermates 69 . Higher levels of p66Shc in the MAM isolated from the livers of old mice and increased ROS production by crude mitochondria (containing MAM) argue in favor of the translocation of p66Shc to the MAM in the cellular response to age-related oxidative stress 71,83 . Moreover, p66Shc is also present in plasma membrane-associated membranes (PAM). Interestingly, the level of p66Shc changes reciprocally in PAM and MAM, depending on the age of the animal 71 .
It has been demonstrated that an extracellular agonist-stimulated Ca 2+ uptake by mitochondria in mouse embryonic fibroblasts (MEFs) is gradually decreased with culture time (see Fig. 3) 75 . Interestingly, such dependency was not reported in p66Shc-deficient MEFs 75 . After oxidative challenge, a reduction in the mitochondrial Ca 2+ response and fragmentation of the three-dimensional mitochondrial network was observed in wild-type MEFs, but only minor changes in the Ca 2+ response and morphology were detected in p66 Shc–/– cells 75 . Moreover, the inhibition of p66Shc phosphorylation at Ser36 with the use of hispidin, a specific blocker of the PKCβ isoform, preserved the mitochondrial morphology in wild-type MEFs. Similarly, no alterations in the passage-dependent decrease in mitochondrial calcium were observed in these cells after treatment with hispidin 75 .
Mitochondrial Ca 2+ responses ([Ca 2+ ]mit) in MEFs during ATP challenge as a function of a passage number. The pseudocolor scale (on the right) indicates the approximate changes of mitochondrial calcium level, where light pink represents low Ca 2+ and red high (physiological) Ca 2+ levels. Blue dots—represent senescence marker, β-galactosidase activity
MAM, the link between mitochondria and the ER in mitochondrial Ca 2+ uptake in senescent cells
Studies of a neuronal aging model revealed increased Ca 2+ transfer from the ER to mitochondria in long-term cultured neurons, whereas no functional coupling was observed between the ER and mitochondria during short-term culturing 84 . The increased Ca 2+ uptake by mitochondria is considered to be responsible for the downregulation of store-operated calcium entry, which in turn causes the impaired stability of mushroom spines, leading to aging-associated cognitive decline 84 . The increased ER-mitochondria Ca 2+ transfer was accompanied by the upregulation of the mitochondrial calcium uniporter (MCU) 85 , which suggests the involvement of MERCs in the process, since they are hotspots for Ca 2+ signaling 86,87 . Increased Ca 2+ transfer to mitochondria could serve as a regulatory mechanism to counterbalance the loss of mitochondrial potential in aging cells. The proposed mechanism of the Ca 2+ flux through MERCs involves control over the calcium channel expression level as well as the number and structure of MERCs. Indeed, the number of contact sites is a well-known determinant of the extent of Ca 2+ transferred between mitochondria and ER 88,89 . The mechanism of such regulation relies on the laws of diffusion, according to which doubling the distance causes a fourfold increase in the travel time required, thus reducing the efficiency of diffusional transport at larger distances 18 . Recently, it was demonstrated that ultrastructure of the MERCs itself, in particular the thickness of MERCs, is a crucial factor regulating the efficiency of Ca 2+ transport 18 . Interestingly, knockdown of MCU and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), both involved in the accumulation of calcium in mitochondria, resulted in senescence escape, indicating the role of mitochondrial calcium accumulation in senescence induction 90 . Similarly, lower number of contacts between mitochondria and the ER in senescent human fibroblasts could be also responsible for the compromised mitochondrial calcium uptake in senescent cells. Notwithstanding this, additional studies are needed to identify which factors have the highest influence of the regulation of Ca 2+ fluxes through MERCs in aging cells.
Lifespan varies considerably among even closely related species, as exemplified by rodents and primates. Despite these disparities in lifespan, most studies have focused on intra-specific aging pathologies, primarily within a select few systems. While mice have provided much insight into aging biology, it is unclear if such a short-lived species lack defences against senescence that may have evolved in related longevous species. Many age-related diseases have been linked to mitochondrial dysfunction that are measured by decreased energy generation, structural damage to cellular components, and even cell death. Post translational modifications (PTMs) orchestrate many of the pathways associated with cellular metabolism, and are thought to be a key regulator in biological senescence. We propose hyperacylation as one such modification that may be implicated in numerous mitochondrial impairments affecting energy metabolism. Keywords: Comparative biology, Sirtuin 3, Acylation, Mitochondria, Aging