As mentioned above, the evolutionary theory of aging suggests that the proximate culprits in aging are likely to be any structural, bioenergetics, or functional constraints that prevent an organism from continuously maintaining (actively synthesizing, stabilizing, and renewing) its core macro- molecular complexes essential for biological function. An equally plausible culprit could be regu- latory mechanisms that respond to internal or external changes by causing a redistribution of resources away from somatic maintenance. These constraints typically depend on the ecological habitat of the organism, and other life history traits such as generation time, size, metabolic needs, availability of food, cellular and tissue organization, and interaction with its environment. How- ever, despite the remarkable variation in lifespans and life history traits, the proximate causes of aging of cells and tissues share common features.15As aging is the result of a complex interplay among various biological processes, the biological processes that are considered the drivers of aging are not only those that manifest during normal aging, but those whose experimental modulation can accelerate or decelerate the rates of aging in model organisms. These processes are briefly described below.
Genomic instability
Despite the many mechanisms dedicated to the repair of DNA damage, the accumulation of genetic damage is a common feature of aging seen in model organisms as well as humans.16 Somatic cells are continually exposed to environmental mutagens such as UV radiation, or toxic chemicals. Biochemical reactions and respiration themselves result in the formation of reactive oxy- gen species which damage DNA. Finally, chromosome segregation, DNA replication, and even DNA repair enzymes are error prone, guaranteeing that, with time, cells accumulate enough geno- mic alterations that could render them and the organism dysfunctional. Although it is difficult to unravel whether the accumulation of damaged DNA within cells over time is the cause or conse- quence of aging, increased DNA damage due to hereditary mutations in genes involved in DNA repair leads to premature aging syndromes such as ataxia-telangiectasia (A-T), Werner syndrome, Bloom’s syndrome, etc. Experimental detection of random low-abundance genomic alterations are difficult, and the first systematic studies to conclusively demonstrate an increase in DNA dam- age with age were conducted in experimental model systems such asDrosophila melanogaster, using an elegant method where alacZreporter gene integrated into specific loci of the organism served to read out mutational rate.17These experiments showed an age-dependent increase in mutations in thelacZ gene. They also revealed that the increase in mutations was dependent on environmental conditions such as temperature and were tissue specific. For instance, while mitotic tissue in the abdomen accumulated less than a twofold increase in mutations between
40 Veena Prahlad and Madhusudana Rao Chikka
young and old flies, cells in the thorax accumulated over three times more mutations in the same duration. The same tissue showed a higher amount of DNA damage when the organism was raised at a higher temperature. These studies suggest that cell-extrinsic features, and tissue-intrinsic fea- tures, such as different sensitivities of repair mechanisms or increased metabolic activity, collude to result in the amount of age-dependent DNA damage. Similar results were obtained in experiments on mammalian model systems.18
In all tissues, DNA damage in the form of single- or double-stranded breaks and DNA adducts initiate a molecular response to repair DNA, or in many cases, activate apoptosis. The DNA repair machinery present in all cells consists of highly conserved sets of enzymes that continuously mon- itor genomes for DNA damage and are responsible for the high integrity of the genome under physiological conditions. Repair mechanisms include nucleotide excision repair systems (NER) that remove bulky adducts that are incorporated into DNA; base excision repair (BER) enzymes, an error-prone ligation mechanism; nonhomologous end joining (NHEJ); and homologous recombination. Amongst these pathways homologous recombination is the least error prone and high fidelity mechanism as it uses a homologous sequence to template the repair of DNA.
Other repair pathways do not use homologous templates and are more error prone.
Within the nervous system, too, appropriate responses to DNA damage are required to maintain homeostasis and prevent disease. Human syndromes, such as A-T that results from defective responses to DNA damage, often feature overt neuropathology. As with all mitotically dividing cells, neural development is a critical period during which cells can accumulate mutations due to the errors in replication and repair of damaged DNA templates. The adult nervous system, how- ever, is also likely to continue to accumulate double-stranded DNA breaks given the brain’s large oxygen consumption, high metabolic rates, and the generation of reactive oxygen species (ROS).
Recent studies have shown that in rodents, natural behaviors such as exploration of a novel envi- ronment causes DNA double-strand breaks (DSBs) in multiple regions in the neurons of young adult mice.19In addition, being postmitotic, neurons depend on the more error-prone repair NHEJ mechanism for repair of double-strand breaks.20There is evidence that the DNA repair enzymes themselves do not function efficiently with age. For instance, cohesive end joining activity decreases with age of the animal, with expression of other DNA repair enzymes.21DNA damage response signaling itself is also reduced with age.22The lack of faithful DNA repair mechanisms seems to be particularly evident in the case of age-related neurodegenerative diseases, and several studies have shown that neurons accumulate more DNA damage in human neurologic disorders compared to age matched controls. Thus, DNA strand breakage is twofold higher in the Alzhei- mer’s disease (AD) cerebral cortex23 and in the substantia nigra of Parkinson’s disease (PD) patients,24compared with age-matched controls. However, despite these data, it is still unclear how and whether the random and stochastic accumulation of somatic mutations that might increase in neurons with age as a consequence of inefficient repair mechanisms contribute to the more stereotypic changes in neuronal function and phenotype that occur with age.
Telomere attrition
In contrast to the random nature of mutations, the more consistent shortening of telomeres, ribo- nucleoprotein complexes that cap eukaryotic chromosomes, has provided a powerful concept for the time-dependent senescence of somatic cells.25It has long been known that cells and unicellular organisms undergo replicative senescence, that is, they cease dividing after a certain number of cell cycles. This mitotic limit, or the Hayflick limit, is a consequence of the end replication problem faced by all mitotically active cells due to the fact that DNA polymerase can only synthesize a new strand of DNA in the 5’ 3’direction. Mammalian somatic cells do not express telomerase, the enzyme required to actively maintain the length of telomeres. Thus telomeres typically shorten with age in mitotically active somatic cells providing a limit to the number of cell divisions these 41 Aging and the Brain
cells can undergo. Stem cells and germ cells do express telomerase, and can replace the telomeres lost with every cell division, escaping replicative senescence.
Experimental manipulation of telomere length in mouse model systems has provided a causal link between telomere length and organismal aging. Mice with experimentally shortened or lengthened telomeres have decreased or increased lifespans respectively.26,27Remarkably, while telomerase-deficient mice show premature aging phenotypes and untimely tissue degeneration, these phenotypes can be reverted in aged mice by experimentally reactivating telomerase.28 Recently it was shown that chronic psychological stress, a condition that has frequently been cor- related with the earlier onset of age-related diseases can result in lower telomerase activity and shorter telomere length in adult women.29Telomere attrition also affects other cellular systems such as mitochondria. In telomerase-deficient mice the p53-mediated repression of PGC-1α and PGC-1βcauses decreased mitochondrial biogenesis.30This mitochondrial decline also occurs during physiological aging in wild-type mice and can be partially reversed by telomerase activation.31
While there are strong links between telomere attrition and organismal aging, given their post- mitotic status the aging of neuronal cells appears not to be directly affected by telomere length.
Studies in rats indicate that, while the percentage of short telomeres increased with age in the kid- ney, liver, pancreas, and lung of both males and females, telomere length did not change signif- icantly in brain tissue itself.32 Consistent with this, Werner syndrome patients that exhibit premature aging as a result of telomere dysfunction display an accelerated senescence phenotype in mesenchyme-derived tissues, but not in neural lineages. Remarkably, in recent studies while reprogramming Werner syndrome patient fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction, redifferentiating them to mesenchymal stem cells reasserted telomere dysfunction, whereas differentiating them to neural stem/progenitor cells did not.33 These studies suggest that instead of being the causal effector of aging, telomere length could serve more as a read out for aging. Indeed, although controversial, leucocyte telomere length (LTL) has been suggested as a surrogate marker of telomere length in other tissues/organs, and therefore a biomarker of aging, even providing a readout for neuronal health.34Shorter LTLs are evident in patients with age-related diseases such as diabetes, cardiovascular diseases, and neurodegenerative diseases.35In summary, these data suggest that telomere attrition is typically thought to affect neu- ronal tissue by affecting the proliferative capacity of adult neuronal stem cells, and telomerase- deficient mice show deficits both in proliferation in the subependymal zone (SEZ) and neurogen- esis in the olfactory bulb yielding reduced numbers of neurons and also underdeveloped dendritic arbors.36
Mitochondrial damage
Numerous lines of arguments link mitochondrial dysfunction with aging. Mitochondrial DNA (mtDNA) is compact, and encodes critical subsets of gene products of the electron transport chain (ETC) required for oxidative phosphorylation. Thus, most mutations that affect mtDNA have consequences for the respiratory function of cells. Respiratory function of the mitochondria is, in turn, linked with the production of ROS, the abnormal increase of oxidative stress, metabolic imbalances that can be expected to lead to cellular damage accumulation, respiratory deficiency, and decreasing the amount of adenosine triphosphate (ATP) required for functioning of the tissue.
All these impairments are chronic, and the consequences can accumulate over time, plausibly caus- ing age-dependent tissue degeneration and dysfunction. Since, mtDNA lacks protective histones and it is thought that mtDNA repair enzymes are limited, the frequency of mtDNA mutations are thought to be higher than that of the nuclear genome.37However, while mtDNA is particularly susceptible to mutations due to the oxidative microenvironment of the mitochondria, mtDNA cir- cles divide and assort independently and cells can contain numerous mitochondria each with a
42 Veena Prahlad and Madhusudana Rao Chikka
multiplicity of heterogeneous genomes, allowing them to exist in a state of“heteroplasmy,”where mutant and wild-type mitochondria co-exist in the same cell, and different cells have different pro- portions of functional and dysfunctional mitochondria.38Therefore, dysfunction in a subset of mitochondria within a cell can be compensated by the working of others. This ability of mitochon- dria to replicate, and to break apart in cycles of fission and fusion, subsequently getting rid of por- tions of the mitochondrion that are dysfunctional through a process called mitophagy,39 complicates our ability to draw a direct connection between mitochondrial dysfunction and aging.
Despite this, single-cell analyses have revealed that mutant mitochondria do accumulate within individual aging cells and may even attain a state of homoplasmy in which one mutant genome prevails.40In somatic cells defective mitochondria causes respiratory chain deficiency that accumu- lates in mosaic patterns in heart, skeletal muscle and brain.41Mitochondrial dysfunction as such is particularly highly associated with age-related neurodegenerative diseases. The brain is a highly metabolic tissue and neurons depend solely on oxidative phosphorylation for ATP. Mitochondrial genomic dysfunction has been seen in brain samples from Alzheimer’s disease (AD) patients where a higher percentage of Cytochrome c oxidase-deficient neurons, neurons with dysfunctional elec- tron transport chains are found concomitant with higher levels of mtDNA mutations and degraded mtDNA, compared with age-matched controls.42,43 Furthermore, one of the few DNA repair pathways known to be functional in mitochondria, the BER pathway, seems to be defective in AD postmortem brain whole tissue lysates.44,45
Mitochondrial dysfunction also appears to have a causal link to Parkinson’s disease (PD), where hereditary mutations in mitochondrial complex I of the ETC complex, PTEN-induced putative kinase 1 (PINK1), as well in Parkin, an E3-ubiquitin ligase required to clear dysfunctional mito- chondria through mitophagy cause familial PD.46Chemical inhibitors of Complex I, including the common pesticides rotenone and paraquat, are strongly associated with PD and remarkably cause selective dopaminergic neuron loss in invertebrate as well as vertebrate animal models of the dis- ease.47ETC deficiency in dopaminergic neurons leads to excessive mitochondrial fragmentation and an impaired supply of fresh mitochondria to dopaminergic nerve terminals in striatum. In line with these observations, a high level of mtDNA deletion is associated with ETC deficiency in sub- stantia nigra neurons from PD patients.48Similarly, mitochondrial dysfunction is implicated in both amyotrophic lateral sclerosis (ALS) and Huntington’s disease. In ALS, mutant SOD-1, a mitochondrial enzyme, which contributes to the largest proportion of the known familial ALS cases, is thought to inhibit VDAC1 (voltage-dependent anion-selective channel protein (1) directly, leading to reduction of energy production in mitochondria.49In addition, oxidative dam- age is seen in mitochondrial DNA in spinal motor neurons of transgenic ALS mice.50Similarly, in Huntington’s disease the interaction of mutant Huntington’s protein with mitochondria is thought to cause decreased mitochondrial enzyme activity resulting in mitochondrial metabolic deficits.51
Mutations and deletions in mitochondrial DNA52as well as mutations in nuclear genes encod- ing gene products required for mitochondrial function are hereditary causes of age-related degen- erative diseases, besides neurodegeneration. Mice that are deficient in the proof-reading-enzyme PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase,53display a mtDNA mutator phenotype and accumulate mitochondria with random point mutations and deletions. This is accompanied by numerous premature aging phenotypes, such as reduced subcutaneous fat, alo- pecia (hair loss), and kyphosis (curvature of the spine). Similarly in other model systems mitochon- drial DNA mutations cause respiratory deficiency in stem cells,54while not showing significant increases in ROS production. These studies, taken together, strongly link mutations in mitochon- drial DNA with aging. However, while the premature aging phenotypes of the mutator mouse have proven fairly persuasive, experiments measuring thein vivorate of mutations in mice dem- onstrate that thein vivorate of change of the mitochondrial genome at a single–base pair level is more than 10 times lower than expected, although an 11-fold increase in mitochondrial point mutations was observed with age.55This mutation rate was several orders of magnitude less than 43 Aging and the Brain
what even the mutator mouse could sustain without any obvious features of rapidly accelerated aging, suggesting that as with nuclear DNA mutations, mitochondrial DNA mutations could be merely correlated with aging and not the cause.
Thus, the effect of mitochondrial dysfunction on aging may be the result of the complex role of mitochondria in cellular physiology, where it is not only required to maintain metabolic homeo- stasis and respiratory balance, but also interacts with numerous aspects of cellular function. Mito- chondria communicate with other organelles in a cell, such as the nucleus and endoplasmic reticulum, and mutations in mtDNA that result in stable perturbations of mammalian mitochon- drial function can elicit a dramatic alteration in nuclear gene expression. Abnormal oxidative stress caused by mitochondrial impairment also leads to the dysregulation of ROS-dependent cellular signaling pathways, mitochondrial surveillance, and defense mechanisms, which in turn can cause metabolic alterations in the cell and activation of the Toll like receptors and innate immune response. Additionally, mitochondrial dysfunction can elicit excessive activation of the fission and fusion machinery, resulting in the recruitment of the NLRP3 inflammasome, and cause chronic inflammatory responses.56Experimental studies on model organisms show that preventing mtDNA dysfunction can delay the progress of neurodegenerative disease.
Protein damage
In addition to the gradual loss of genomic integrity through somatic mutations, dysfunction of DNA repair, mtDNA deletions and mutations, and telomere attrition, aging is also associated with dramatic changes in protein structure. Most protein-based biological processes proceed within a narrow range of optimal conditions (temperatures, redox states, etc.) outside of which efficiency steeply declines and damage can occur. Proteins suffer continual damage from fluxes in their envi- ronment including temperature-induced denaturation, oxidative damage, denaturation by changes in ionic conditions, UV/photodamage, etc. Despite this, ectothermic animals are able to sustain very similar rates of metabolic activity at widely different temperatures of adaptation or clines, and metabolic adaptation proceeds independent of what may be predicted from the Q10- of enzymatic reactions at the two temperatures (temperature compensation). Such remark- able function and stability in protein-based function is maintained through the presence of highly conserved and elaborate systems of protein synthesis, protein maintenance, and stabilization. In particular, cellular quality control mechanisms and protein degradation systems are geared to maintain protein homeostasis or proteostasis.
With age protein dysfunction increases, there occur alterations in the ability of protein com- plexes to remain stable, and the diverse protein aggregates can be seen in varied tissues of a met- azoan. Indeed, almost all age-related tissue dysfunction is associated with protein aggregates, although the best known are the Lewy bodies in PD and the plaques and tangles in AD. While a single-point mutation or deletion in DNA can arguably cause a permanent loss of function, the heterogeneity of the proteome and the renewability of most of the protein components of a cell, coupled with their presence in more than one copy, makes it unclear how damage in a subset of proteins can lead to degenerative phenotypes. However, although the loss of protein function due to aggregation does affect cells, in the majority of age-related protein aggregation diseases, the aggregates are thought to cause a gain of toxic function that leads to degeneration.57,58One reason for the toxicity of misfolded proteins is thought to be the imbalance between the aberrant inter- actions that promote the misfolding of other proteins and their ability to sequester members of the protein quality control system such as molecular chaperones needed to maintain protein function.
In addition, there is evidence from numerous organisms that stress-induced synthesis of cytosolic and organelle-specific chaperones and other protein quality control components is significantly impaired in aging.59,60Mutant mice deficient in a co-chaperone of the heat-shock family exhibit accelerated aging phenotypes.61 Conversely, animal models overexpressing chaperones are
44 Veena Prahlad and Madhusudana Rao Chikka