Cellular Repair Processes
Cellular repair processes refer to the range of machinery that maintains and restores the integrity of the functional units of the cell, constitutively or in response to insult.
The cellular environment is an extremely dynamic one, and cells are continuously subject to a range of intrinsic and extrinsic stressors that threaten the integrity of their functional units, including organelles, proteins, DNA, and membranes and functional lipids. Accordingly, systems have evolved to reduce rate of generation of endogenous damage, detoxify damaging agents, and increase the intrinsic resistance of the cell’s macromolecular components to damage (Pamplona and Barja 2007).
When these preventive systems fail, however, it is essential to cell survival and function that the cell has machinery in place to repair and replace damaged functional units. The range of such machinery is vast but will be summarized briefly in this article. We will then review potential strategies toward the development of therapies to enhance or reinforce this machinery in order to intervene in the degenerative aging process and extend healthy lifespan.
Key Research Findings
In order to fulfill their biological functions, proteins must maintain precise three-dimensional folded structures. When proteins either fail to fold during synthesis or become misfolded in the course of their cellular lifetime, they can no longer execute their biological function; in addition, the aberrant conformation may be toxic to the cell, either directly or by protein aggregation (see entry for “Intracellular Aggregates”). Such misfolding can occur as a result of errors in protein biogenesis (including mRNA transcription and maturation as well as protein translation) or in the intrinsically error-prone process of protein folding itself (Klaips et al. 2018). Acute or chronic environmental or physiological stressors (including aging) can further increase the acute or steady-state levels of such misfolded proteins (Klaips et al. 2018).
Cells are equipped with several interacting systems to assure cellular protein structural integrity (sometimes termed “proteostasis”) (Klaips et al. 2018; Taylor and Dillin 2011) (see entry for “Proteostasis”). Defenses against protein misfolding exist at the levels of protein synthesis, protein folding and conformational maintenance, cytoprotective stress responses, and degradation of damaged and misfolded proteins (Klaips et al. 2018; Taylor and Dillin 2011).
At the level of protein synthesis, it is notable that several phylogenetically conserved interventions that retard aging reduce the rate of protein synthesis, which in addition to conserving energy also reduces the supply of proteins at risk of misfolding (Taylor and Dillin 2011; Tavernarakis 2008). These include calorie restriction (CR – see the entry “Diet and Caloric Restriction” in this volume), inhibition of the mechanistic target of rapamycin (mTOR) (see the entry for “Mechanistic Target of Rapamycin”), and reducing signaling through the insulin/insulin-like growth factor (IGF-1) pathway (see the entry for “Insulin/Insulin-Like Growth Factor Pathway”), although all of these interventions also affect other aspects of proteostasis. Molecular chaperones act on nascent polypeptide chains as they emerge from the ribosome, either “passively” stabilizing them into intermediates favorable to adoption of properly folded states or “actively” folding them via ATP-driven processes (Klaips et al. 2018; Taylor and Dillin 2011). Even after misfolded proteins have escaped initial conformational shaping, some chaperones can additionally protect the cell against toxic aggregates and aggregate-prone misfolded proteins by either disassembling aggregates into intermediates that can then be refolded or oppositely coordinating the aggregation of toxic aggregated or misfolded proteins into more inert assemblies (Evans et al. 2017; Taylor and Dillin 2011). Failure or insufficiency of this machinery can lead to the accumulation with age of recalcitrant aggregates that lead to cellular dysfunction.
A second layer of such protection is comprised of cellular stress responses, which upregulate components of protein quality control and/or inhibit protein synthesis when chronic or acute stressors cause the level of misfolded proteins to exceed the basal capacity of the protein maintenance machinery (Klaips et al. 2018; Taylor and Dillin 2011). Compartment-specific stress-response pathways include the heat-shock response in the cytosol and the mitochondrial and endoplasmic reticulum (ER) unfolded protein responses (UPR), all of which appear to engage in significant cross talk (Klaips et al. 2018; Kim et al. 2016). When properly functioning, these pathways act to re-establish the normal protein homeostasis of the cell, but when the chronic stress of aging and disease overwhelms the capacity of these pathways, their chronic activation can become dysfunctional and limit the dynamic range of the system to respond to new, additional stress (Klaips et al. 2018). Surprisingly, however, inhibitors of cellular stress responses can under some circumstances act to restore cellular homeostasis and function arising from the very misfolded protein stress that those pathways are engaged to relieve (Ma et al. 2013; Moreno et al. 2013; Sidrauski et al. 2013).
Finally, when other systems fail to prevent protein misfolding and aggregation, protein degradation pathways (most notably the ubiquitin-proteasomal system (UPS) and the autophagy-lysosomal system) allow the cell to degrade irretrievably damaged proteins and recycle their components (Taylor and Dillin 2011). Smaller and short-lived proteins are primarily degraded by the UPS, but larger proteins and aggregates are unable to pass through the relatively narrow proteolytic chamber of the 20S proteasome and may impede the entrance of subsequent substrates if so targeted, leading to dysfunction and protein toxicity. Such substrates are accordingly targeted to the lysosome for degradation (Liebl and Hoppe 2016; Taylor and Dillin 2011). Specific delivery of proteins to the proteasome or the lysosome is regulated by the activity of E3 ubiquitin ligases, which tag damaged proteins with ubiquitin chains that direct them to their fate (Liebl and Hoppe 2016).
Autophagy delivers damaged or no longer needed proteins (as well as organelles, parts of the ER, and intracellular pathogens) to the lysosome for degradation. It can be divided into bulk autophagy (macroautophagy) and forms of selective autophagy (Evans et al. 2017). In macroautophagy, an isolation membrane is extended over a region of cytosol, nonselectively engulfing damaged proteins and other substrates to form an autophagophore that then fuses directly with the lysosome or late endosomes/multivesicular bodies. Selective autophagy, by contrast, involves a more orchestrated process of tagging specific proteins and organelles for degradation using the ubiquitin system, followed by selective targeting to either an autophagosome or directly to the lysosome (depending on the pathway) via selective autophagy receptors.
Among the modes of selective autophagy are chaperone-mediated autophagy (CMA) and microautophagy (Evans et al. 2017; Taylor and Dillin 2011). The CMA machinery recognizes substrates bearing a KFERQ-type motif and delivers them to the lysosome, where they are trafficked across the lysosomal membrane by a protein complex featuring lysosomal-associated membrane protein 2A (LAMP2A) (Kaushik and Cuervo 2018). Microautophagy embraces several processes with distinctive membrane structures and machinery, but all feature the selective surrounding of a small region of the cytoplasm for delivery to the lysosome (Oku and Sakai 2018). (Mitophagy – the selective degradation of damaged mitochondria – and the selective autophagy of peroxisomes, inflammasomes, and certain classes of aggregated proteins (Evans et al. 2017) will not be treated here.)
Dysfunction of the cell’s proteolytic machinery and associated cellular dysfunction is observed in aging and particular diseases of aging, with affected cells accumulating autophagic vacuoles that fail to fuse with the lysosome, as well as abnormal proteins and toxic lysosomal hydrolases (Friedman et al. 2015; Scrivo et al. 2018).
DNA is subject to a wide range of damaging agents of metabolic, lifestyle, environmental, and chemical origin. It is thought that the genetic code is subject to thousands of random alterations in each cell every day, yet the activity of the DNA repair machinery ensures that fewer than 0.1% of base changes continue on to be fixed as a mutation (Alberts et al. 2002). Mutations can lead to least three deleterious effects on the cell (and the organism): cell death, cellular senescence, and cancer (Hoeijmakers 2009). In addition, mutations in cells that survive in tissues without suffering these fates are widely suspected of causing cellular and (collectively) tissue dysfunction, contributing to age-related tissue decline and disease (Szilard 1959; Gorbunova and Seluanov 2016). This hypothesis has been given additional impetus by recent reports of clonal expansion of mutation-bearing cells in aging tissues in association with increased risk of diseases of aging (Risques and Kennedy 2018; Martincorena et al. 2018; Martincorena and Roshan 2015), including in some cell types that are postmitotic under normative physiologic conditions (Fischer and Stringer 2008).
The cell has a range of specialized machinery devoted to the specialized repair of specific forms of DNA damage (Ciccia and Elledge 2010; Jackson and Bartek 2009; Hoeijmakers 2009). Single-strand breaks are repaired by single-strand break repair (SSBR), with mechanisms that partially overlap with base excision repair (BER – vide infra), whereas DSBs are alternatively routed through nonhomologous end joining (NHEJ) or homologous recombination (HR).
NHEJ is the dominant mechanism for DSB repair in human cells and directly religates the broken strand of DNA. This entails the recognition, capture, and stabilization of the two broken ends of DNA to prevent nonspecific processing, the assembly of the NHEJ machinery at the site, bridging the two sections of broken DNA molecule, processing the DNA ends (if necessary) to allow them to be ligated, and finally, ligation of the broken ends and resolution of the NHEJ complex (Davis and Chen 2013). NHEJ is highly versatile, as it can religate any type of broken DNA strand and does not require a homologous stretch of DNA to act as a template for repair; this latter feature also allows it to operate in any phase of the cell cycle, as the sister chromatid is only available as a template during S and G2 (Davis and Chen 2013). The lack of a template for repair in favor of direct processing and ligation of the broken ends, however, carries the risk that genetic material may be lost.
By contrast, HR is a highly accurate if more restricted mechanism of DSB repair, using the sister chromatids as homologous templates to restore the original sequence of the broken DNA ends. Through several subpathways, the HR machinery recognizes the broken DNA ends; processes them into single-stranded DNA tails; guides them to invade the intact strand for use as a template, forming a D-loop; synthesizes the replacement sequence; and finally religates the broken DNA strands, following which the intercrossed DNA molecules are severed and restored to their normal double-stranded configuration. (Li and Heyer 2008). (As noted below, the HR machinery is also involved in interstrand crosslink (ICL) repair.)
Mismatch repair (MMR) corrects errors introduced during DNA replication or by a range of mutagenic agents, such as base mismatches and insertions or deletions.
BER is responsible for repairing minor chemical alterations of DNA bases such as spontaneous deamidation of cytosine to uracil, as well as abasic sites (which are intermediates in its repair mechanism). Distinct subpathways exist for repair of single nucleotides (single-nucleotide BER) and for the replacement of two or more nucleotides in the same damaged strand (long-patch BER). The damaged base is first recognized by a DNA glycosylase, which then removes the base through cleavage of the N-glycosidic bond. This leaves an abasic site, which is next cleaved by an AP endonuclease and then repaired via DNA polymerase and ligase proteins (Robertson et al. 2009).
Many animals and other eukaryotes can correct the more complex lesions (such as pyrimidine dimers) induced by ultraviolet (UV) light via photoreactivation of the enzyme photolyase; however, this enzyme is inactive in humans (Lucas-Lledó and Lynch 2009). Instead, these and other helix-distorting lesions are repaired via the more cumbersome nucleotide excision repair (NER) system. There are two NER pathways, which differ in the mechanism by which damage is initially recognized: global genome NER (GG-NER) scans the entire genome for bulky DNA lesions, including regions that are not transcribed, whereas transcription-coupled NER (TC-NER) exclusively detects lesions that interfere with transcription by stalling RNA polymerase II (Hoeijmakers 2009). Once detected, DNA damage is excised as part of a 22–30-base oligonucleotide; DNA polymerases are then recruited to repair the remaining single-stranded DNA, and DNA ligase seals the remaining nick (Jackson and Bartek 2009; Hoeijmakers 2009).
Interstrand crosslinks (ICLs) are adventitious covalent bonds between opposing strands of DNA. Because they prevent the unwinding of the DNA duplex by helicases, they block both transcription and DNA replication, leading to chromosome instability, breakage, or rearrangements and ultimately cell death; DNA crosslinking agents are accordingly used as cytotoxic chemotherapy. Because they involve both strands of DNA and must be repaired before cell replication, ICLs are challenging lesions for repair, necessitating an initial “unhooking” of the DNA via incisions made by nucleases on either side of the ICL, followed by the coordinated action of other canonical DNA repair pathways – a complex set of mechanisms that remain poorly understood (Hashimoto et al. 2016; Muniandy et al. 2010).
Mitochondria have a separate genome from the nucleus, which is subject to much higher levels of mutation because of its proximity to the cell’s major source of reactive oxygen species (the electron transport chain) and its more error-prone replicative machinery (de Grey 1999). The most prominent lesions of mitochondrial DNA in aging – and the most closely associated with diseases of aging – are large (1.1–10-kb) deletions, for which no true repair mechanism exists; these clonally expand to occupy all mitochondrial genomes in a small fraction of (primarily postmitotic) cells in aging human tissues (de Grey 1999) (<1% in the heart, skeletal muscle, and much of the brain (de Grey 1999), but perhaps as high as 12% in the putamen (Corral-Debrinski et al. 1992)). The mitochondrial DNA repair machinery is more limited and less well-understood than that of the nucleus but includes forms of BER and MMR and possibly HR as well (Ma and O’Farrell 2015; Chen 2013).
The DNA Damage Response
While the DNA repair machinery is specifically recruited to repair individual lesions as they arise, a threshold level of cellular DNA damage activates the DNA damage response (DDR) – a coordinated network of signaling pathways that regulate DNA repair, cell cycle progression, and ultimately cell fate (return to normal cell cycling, apoptosis, or senescence) (Mirzayans et al. 2017; Maréchal and Zou 2013; Jackson and Bartek 2009). The kinases ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) (and some would add DNA-dependent protein kinase (DNA-PKcs) Maréchal and Zou 2013) are, respectively, recruited primarily by DSBs and by replication stress or other broad types of DNA damage. They orchestrate the phosphorylation of hundreds of protein targets directly, as well as additional secondary targets through phosphorylation of CHK1, CHK2, and p38 MAPK (Maréchal and Zou 2013; Jackson and Bartek 2009).
A key nexus of the DDR machinery is the transcription factor p53, whose specific phosphorylation mediates its stabilization and cellular localization, and thereby its activity (Mirzayans et al. 2017). Upon activation, p53 activates cell cycle checkpoints to allow an opportunity for DNA repair and determines cell fate based on the level and type of genotoxic stress signaling it receives from ATM and ATR, as well as cell type and additional intrinsic and extrinsic properties of the cell (Mirzayans et al. 2017). Signaling from ATR to withdraw from the cell cycle is responsive to the ongoing progress versus stalling of DNA repair during G2 (Feringa et al. 2018). Once activated, p53 activates both pro-growth-arrest and pro-apoptotic mediators in response to genotoxic stress, but the threshold for execution of apoptosis is higher due to the restraining influence of antiapoptotic proteins, such that the level of DNA damage dictates which fate will prevail (Kracikova et al. 2013). Otherwise, the cell can proceed into a permanent state of growth arrest (senescence) or return to status quo ante after DNA repair is complete – the latter function carried out by the serine/threonine phosphatase WIP1 (wild-type p53-induced phosphatase 1) (Mirzayans et al. 2017).
The integrity of the cell membrane is essential to cell survival and function, maintaining an intracellular environment favorable to cell function and selectively controlling the entry and exit of needed substrates and wastes, as well as helping anchor the cell in its anatomical position externally and the cytoskeleton internally. Under normal physiologic conditions, different cells are exposed to cyclic, periodic, or constant mechanical sheer and stretch, in addition to membrane trafficking and remodeling processes, depending on the cell and tissue type, which may puncture or otherwise damage it. Additionally, membranes can be damaged by extrinsic or metabolic insults and acute attack by pathogens or lymphocytes. Mechanisms for membrane repair are therefore essential to cellular integrity.
The simplest mechanism of membrane repair is a thought to be a spontaneous self-sealing action of membrane tension, which can repair small (<1 μm) breaches via physical forces that oppose the “line tension” created by the phospholipids surrounding the membrane breach (McNeil and Terasaki 2001; Tang and Marshall 2017). In nucleated cells, it has been proposed that larger breaches of the membrane elicit a Ca2+-regulated exocytotic response, forming a “patch vesicle” that fuses with the edges of the membrane breach and seals it (McNeil and Terasaki 2001; Tang and Marshall 2017). Alternatively, membrane-patching materials can be derived from the fusion of membrane materials derived from organelles and existing intracellular vesicles that “explode” outward to the breach, in an “explodosis” mechanism (Tang and Marshall 2017).
Muscle sarcolemma exhibits what may be either one variant of this broad conceptual model, a distinct repair mechanism, or an alternative explanation for the observations underlying the patch model in other systems. Dysferlin, the protein whose mutation is responsible for Miyoshi muscular dystrophy 1, regulates vesicle trafficking and fusion, and mutant muscle cells fail to properly repair after injury, including notably deficits in the repair of cell membranes (Han 2011). Injured muscle cells were shown to be repaired by individually exocytosed lysosomes that were tethered to the cell membrane by a mechanism requiring dysferlin (Defour et al. 2014).
An additional Ca2+-regulated mechanism involves the “purse-string” contraction of actomyosin around the breach, drawing the remaining membrane and associated cortical actin cytoskeleton together to close the gap (Tang and Marshall 2017).
The initial repair of cell membranes is followed by a more protracted period of repair and reorganization that ultimately restores full membrane function (McNeil and Terasaki 2001).
Examples of Application
Biomedical gerontology has long been focused on modulating the repair machinery of the cell in hopes of better maintaining the molecular fidelity, function, and survival of cells (and therefore organisms) with age. To some extent, this has been evident in successful antiaging interventions in model organisms. Genetic or pharmacological inhibition of the mTOR pathway, for instance, extends total and healthy lifespan in numerous species, and attenuates many gross anatomical aging phenotypes at necropsy, presumably in large part by decreasing the accumulation of a variety of damaged cellular components, though evidence for the latter is mostly confined to transgenic models bearing disease-associated mutations (Johnson et al. 2013; Wilkinson et al. 2012) (see entry for “Mechanistic Target of Rapamycin”).
In another notable study, the age-related decline in the abundance of the lysosomal receptor for CMA (LAMP-2A) was opposed via a repressible LAMP-2A transgene whose expression was restricted to the liver, in which organ its age-related decline is best characterized (Zhang and Cuervo 2008). In young (6-month-old) animals, withdrawal of the suppressing agent prevented the age-related decline of degradation of a typical CMA substrate (GADPH), while the age-related decline in UPS, overall proteolytic activity, and possibly macroautophagy were ameliorated. In parallel, the ultrastructural and gross morphological features of the aging liver were better preserved, along with liver function and mitochondrial morphology and function. Similar benefits were obtained in late-onset (22-month-old) animals, albeit at substantially reduced efficacy (Zhang and Cuervo 2008). However, CMA continued to decline with age despite the additional LAMP-2A protein, in part due to a rise in its instability at the lysosomal membrane; moreover, accumulation of lipofuscin (a persistent, recalcitrant intracellular aggregate (see entry for “Intracellular Aggregates”)) was attenuated but not arrested or reversed (Zhang and Cuervo 2008).
Calorie restriction (CR) – the most well-characterized of antiaging interventions – clearly attenuates many of the cellular aberrations of aging, but in many cases in ways that seem to involve reduced generation of aging lesions rather than upregulation of repair, and it cannot undo forms of aging for which there is no intrinsic repair mechanism. For instance, while CR reduces the rate of accumulation of nuclear DNA mutations in several tissues with age (Garcia et al. 2008), it does not appear in most cases to proactively upregulate most elements of the nuclear DNA repair machinery, though it does retard their age-related decline (e.g., Wikinson et al. 2012; Rao 2003; Licastro et al. 1988 – but contrast Cabelof et al. 2003 and others for BER). Meanwhile, CR lowers BER activity in muscle and brain mitochondria while elevating it in the liver (Stuart et al. 2004). Again, CR appears to retard the rate of generation of focal electron transport chain abnormalities in skeletal muscle associated with large deletions in mitochondrial DNA (most likely by reducing the rate of stochastic ROS generation at Complex I and by increasing the resistance of mitochondrial membranes to oxidation (Pamplona and Barja 2007)), yet does not retard their spread once initiated (Bua et al. 2004).
Metabolic pathways are inherently fraught targets for intervention, inasmuch as they are the very processes that maintain the life of the organism, and are poised to balance competing risks by millennia of natural selection. For instance, pathways involved in cell proliferation, apoptosis, and senescence balance the risk of tissue denudation versus cancer; the regulation of the autophagy machinery and protein synthesis via mTOR and other regulators must balance the benefits of rapid growth against loss of protein and organellar fidelity; and robustly engaging the antimutagenic activity of DNA-PK during aging comes at the cost of metabolic disease (Park et al. 2017).
“Strategies for Engineered Negligible Senescence” (SENS) (see entry of this title) is proposed as an alternative and potentially more powerful approach to the repair and maintenance of molecular, cellular, and organismal integrity with age, and thereby health and survival with age, in part because it conceptually offers the potential to bypass these metabolic dilemmas (de Grey et al. 2001; de Grey and Rae 2007). In this “damage-repair” heuristic, the cellular and molecular lesions that accumulate in aging tissues are targeted directly, rather than targeting the metabolic determinants of damage generation or repair. Depending on the nature of the lesion, these “rejuvenation biotechnologies” remove, repair, replace, or render harmless particular forms of aging damage through their own action, with minimal perturbation of metabolism (de Grey et al. 2001; de Grey and Rae 2007). Examples include the delivery to the lysosome of engineered hydrolases to degrade intracellular aggregates implicated in age-related disease (de Grey 2002; de Grey et al. 2005) (vs. modulating autophagy and other proteostatic machinery) (see entry for “Intracellular Aggregates”), senolytic agents to remove senescent cells (vs. “senomorphic” agents, which modulate the senescence-associated secretory phenotype or inhibit or reverse the conversion to senescence) (Kim and Kim 2019) (see entries for “Cell Senescence” and “Senolytic Drugs”), and allotopic expression of mitochondrially encoded proteins, in order to obviate the effects on cell function of acquiring a homoplasmic mitochondrial population bearing large, irreparable deletions in the mitochondrial DNA, as occurs in a minority of cells with age (Boominathan et al. 2016; de Grey 1999; de Grey and Rae 2007). An additional argument in favor of this approach is that it allows for the targeting of recalcitrant forms of damage that the organism may not have the intrinsic capacity to remove or repair, such as large mitochondrial DNA deletions or advanced lipofuscin (de Grey et al. 2001; de Grey and Rae 2007; contrast Zhang and Cuervo 2008).
Future Directions for Research
Although there are many putative “accelerated aging” models (Miller 2004) based on mutations in elements of the DNA repair machinery, there is no unconfounded evidence that upregulation of DNA repair retards “normative” aging (Gorbunova and Seluanov 2016). A convincing test of this general hypothesis would be a valuable contribution to the field.
Additionally, as noted, the nature and extent of mitochondrial DNA repair mechanisms remain poorly understood and should be further explored. Moreover, the dysfunction of the autophagic/lysosomal pathway observed in affected cells in aging and specific diseases of aging presents several potentially attractive targets for therapies to reverse these age-related defects, though with the potential for perverse effects of imbalancing the integrated system as a result of selectively inducing steps in the pathway (Friedman et al. 2015). A proposed alternative following the direct “damage-repair” approach of SENS is the introduction of novel engineered lysosomal hydrolases to target specific damaged proteins that accumulate in aging cells (de Grey 2002; de Grey et al. 2005). Preliminary evidence supports this approach (Mathieu et al. 2012; Schloendorn et al. 2009), including the recent demonstration that recombinant manganese peroxidase reduces the burden of the main toxic bisretinoid implicated in age-related macular degeneration in model Stargardt’s macular degeneration mice (Moody et al. 2018), and two candidate enzymes that have emerged from this work have been licensed to biotechnology startups for therapeutic development. More challenging are targets that accumulate as cytosolic inclusion bodies, with uncertain capacity for lysosomal targeting or degradation. Further work in both areas is needed.
Brain neuron and other cell membranes exhibit a number of changes with age, including disordering of lipid rafts, a shift to more unsaturated fatty acyl groups in phospholipids, and alterations in cholesterol and sphingomyelin content and distribution (Colin et al. 2016; Fülop et al. 2012). In rodents, CR is known to oppose several of these changes (Babenko and Storozhenko 2016; Hernández-Corbacho et al. 2011; Tacconi et al. 1991). Discovery of whether these changes are adaptive changes in response to primary aging damage or are themselves a form of primary age change, and means to oppose these changes, would be useful contributions.
Cells resist the accumulation of damage through intrinsic molecular stability, detoxification of damaging agents, tuning the metabolic generators of such damage, and also by mechanisms of repair for proteins, DNA, and membranes. Nonetheless, a significant amount of damage eludes these mechanisms; degenerative aging is driven by the accumulation of unrepaired damage in the tissues, leading to loss of functional capacity and ensuing specific diseases of aging along with nonspecific loss of homeostasis and resilience. Augmenting these repair mechanisms – either by modulating the endogenous damage-repair machinery or through direct removal, repair, and replacement through exogenous biotechnology – is a promising strategy for extending life- and healthspan.
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