ANTIOXIDANTS & REDOX SIGNALING Volume 21, Number 1, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2013.5777

FORUM REVIEW ARTICLE

Calcium Signaling Alterations, Oxidative Stress, and Autophagy in Aging Rodrigo Portes Ureshino, Katiucha Karolina Rocha, Guiomar Silva Lopes, Cla´udia Bincoletto, and Soraya Soubhi Smaili

Abstract

Significance: Aging is a multi-factorial process that may be associated with several functional and structural deficits which can evolve into degenerative diseases. In this review, we present data that may depict an expanded view of molecular aging theories, beginning with the idea that reactive oxygen species (ROS) are the major effectors in this process. In addition, we have correlated the importance of autophagy as a neuroprotective mechanism and discussed a link between age-related molecules, Ca2+ signaling, and oxidative stress. Recent Advances: There is evidence suggesting that alterations in Ca2+ homeostasis, including mitochondrial Ca2+ overload and alterations in electron transport chain (ETC) complexes, which increase cell vulnerability, are linked to oxidative stress in aging. As much as Ca2+ signaling is altered in aged cells, excess ROS can be produced due to an ineffective coupling of mitochondrial respiration. Damaged mitochondria might not be removed by the macroautophagic system, which is hampered in aging by lipofuscin accumulation, boosting ROS generation, damaging DNA, and, ultimately, leading to apoptosis. Critical Issues: This process can lead to altered protein expression (such as p53, Sirt1, and IGF-1) and progress to cell death. This cycle can lead to increased cell vulnerability in aging and contribute to an increased susceptibility to degenerative processes. Future Directions: A better understanding of Ca2+ signaling and molecular aging alterations is important for preventing apoptosis in age-related diseases. In addition, caloric restriction, resveratrol and autophagy modulation appear to be predominantly cytoprotective, and further studies of this process are promising in age-related disease therapeutics. Antioxid. Redox Signal. 21, 123–137.

Introduction

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he aging process is marked by a gradual loss in physical and mental capabilities due to a general decline in organ function, and aging can also be considered a risk factor for cancer, diabetes, and cardiovascular and neurodegenerative diseases. There are numerous aging theories, and most of them are likely compatible and may complement each other. The free radical theory of aging (65) predicts that there is an accumulation of molecules that are damaged by free radicals, mostly by oxidation, and it is extensively reported that there is an increase in oxidative stress in aging (49). In general, senescent cells in culture adopt a flattened, enlarged morphology and exhibit specific molecular markers, such as senescence-associated b-galactosidase, senescence-

associated heterochromatin foci, and the accumulation of lipofuscin granules (91). Although cellular senescence is an adaptive response to stress that contributes to extending the lifespan, this phenomenon might have a negative impact on the survival of the organism. During aging, senescent cells accumulate in proliferative tissues and release various degradative proteases, growth factors, and inflammatory cytokines that compromise the function of nonsenescent neighboring cells, creating a micro-environment which enables preneoplastic cells to develop into tumors, thus counterbalancing longevity with the risk of old-age cancer (183). In this review, we discuss the connection of molecular aging targets with cell physiology, starting from an overview of age-related alterations in Ca2 + signaling and oxidative stress, discussing the autophagy in this process, and developing a

Department of Pharmacology, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil.

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link between p53, Sirt1, and IGF-1, while focusing on agerelated alterations in Ca2 + signaling. Calcium, Oxidative Stress, and Apoptosis in Aging

Reactive oxygen species (ROS) are considered the most important oxidant molecules in cells and include superoxide (O2 - ), hydroxyl radical (OH - ), hydrogen peroxide (H2O2), and oxygen singlet (1O2) (158). The largest amounts of ROS are produced in mitochondria through the electron transport chain (ETC) via the leakage of electrons (particularly in complexes I and III) that react with free oxygen in the matrix, producing superoxide (14, 126). These organelles were predicted to participate at the leading edge of aging cellular alterations, because the increase in ROS and other harmful molecules could promote nuclear and mitochondrial DNA (mtDNA) mutations, contributing to a senescent phenotype (125). This outcome is a concern for genetic theories of aging, once DNA mutations cause toxic effects on biological systems (87, 184). Indeed, the accumulation of 8oxoguanosine (a DNA damage marker) in the promoters of genes with decreased expression was observed in the human brain, affecting proteins related to ATP synthesis and vesicle transport (105). Nuclear and mtDNA mutations can affect the transcription of ETC proteins, producing a mismatch in the coupling of mitochondrial respiration and increased ROS generation (66). To strengthen this current hypothesis, Trifunovic and coworkers (173) produced a mouse strain with a defective mtDNA polymerase (PolgA) that had an accumulation of mtDNA mutations associated with a decreased life expectancy and premature aging (displaying weight loss, alopecia, and osteoporosis). During aging, mtDNA mutations are estimated to be present in approximately 1% of the mitochondrial genetic material, which results in a decline in the function of this organelle (102), more frequently found in postmitotic cells (109). Navarro and coworkers (131) demonstrated that there is a 51% reduction in state 3 respiration (ADP-dependent) and 73% and 54% decreases in complex I and IV (ETC) activity, respectively, in the hippocampus of aged rats, in accordance with the previously reported reduction of the protein expression of these complexes (109). Besides, mitochondrial dynamics, particularly fusion and fission, are important to the maintenance of mtDNA integrity (11, 150). During these processes, mediated by proteins such as mitofusins (Mfs1 and Mfs2), Opa1 (fusion), and Drp1 (fission), there is a combination of mutated and normal mtDNA, known as mtDNA heteroplasmy. The excess of fission can cause respiratory impairment and an increase in oxidative stress (191); if fusion prevails, the presence of large and defective mitochondria can hamper mitochondrial autophagy (55) (described in section ‘‘Autophagy in Aging and Neurodegeneration’’), which may play an important role in cellular protection during senescence (95). Several authors have reported that there is reduced antioxidant capacity in aging (80, 154). Tian and coworkers (169) found a large amount of protein carbonyls but no change in lipid peroxidation in the heart, liver, kidney, and brain of aged rats; the latter also showed a reduction in the activity of catalase and no changes in superoxide dismutase (SOD) and glutathione peroxidase (GPx). The peroxisomes, single membrane-bound organelles containing anti-oxidants and

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oxidases (116, 178), play an important role in the aging process (137, 166). Several authors have reported alterations in catalase, acyl-CoA oxidase, and b-oxidation in the liver of Wistar rats (8), with an increase in b-oxidation in the aged brain (13). In fact, it was observed that systemic catalase overexpression in mitochondria extends the lifespan of mice (149). Ca2 + modulates the ETC and ATP production by its activity in dehydrogenases of the tricarboxylic acid cycle (59), and ATP synthase complex activation (167), in addition to its importance in maintaining the mitochondrial membrane potential (DJm) (38). Lu and coworkers (105) analyzed the expression of several genes in aged human brains and found, among other differences, alterations in the expression of Ca2 +-related genes. Accordingly, it has been proposed that a sudden imbalance in Ca2 + signaling can be considered an additional factor for cellular demise during aging (83), because it is mandatory for mitochondrial respiratory function. There are extensive data linking alterations in Ca2 + signaling, mitochondria, and apoptosis (39, 133). In particular, in the central nervous system (CNS), excessive glutamate in synaptic terminals can lead to Ca2 + overload (50, 132) via the overactivation of ionotropic NMDA (N-methyl-D-aspartic acid) receptors (140), which is called excitotoxicity. This phenomenon can contribute to cell death that occurs during brain aging (22, 177) and neurodegenerative processes (7, 30, 35). Despite excitotoxicity also occurring in glial cells, neurons are more susceptible to cell injury caused by glutamate. Astrocytes are known to support neurons (182) and to protect against oxidative stress and excitotoxicity (40). They are also pivotal in the neurotransmission process. Furthermore, GFAP, an astrocyte marker, is overexpressed in CNS of aged rats (48, 122); Castillo-Ruiz and coworkers observed an early astrogliosis after NMDA infusion in the cortex and striatum of aged rats, accompanied by increased degenerated neurons (25), showing that neurons are more vulnerable to injuries than glial cells. Mitochondria are particularly involved in the excitotoxicity process (for a review, see Smaili et al., 2011) (157). Ca2 + is taken up by mitochondria through a uniporter. This electrogenic channel has a low affinity for Ca2 + and forms ‘‘microdomains’’ of Ca2 + within the endoplasmic reticulum (ER), which releases Ca2 + through inositol trisphosphate receptor (IP3R) and ryanodine receptors (RyR) (143). The massive Ca2 + increase evoked by glutamate can cause mitochondrial Ca2 + overload (79, 136). Ca2 + accumulation in the mitochondria drives unregulated bioenergetics, which, in turn, can generate more ROS and eventually open the permeability transition pore (PTP) (5, 181), reducing cell viability and, ultimately, leading to cell death. This pathway is illustrated in Figure 1. Studies combining patch voltage clamping and fluorescence microscopy in neurons showed that intracellular Ca2 + buffering occurs in two steps: fast and slow (127). (i) The first stage, which appears to have high activity in aging, likely corresponds to a compensatory mechanism against a large influx of ions from voltage-operated Ca2 + channels (VOCC), and intracellular Ca2 + storage may have an important role in this process. Kirischuk and Verkhatsky (86) reported a decline in caffeine-sensitive ER storage in the cerebellar granule neurons of aged animals, and caffeine produced a plateau that was significantly higher in a cell senescence model (29). Indeed, it was demonstrated that the ER can store more Ca2 + in the striatum of aged rats (177). (ii) The second

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FIG. 1. Excitotoxicity, ROS, and apoptosis. On excessive glutamate (Glu) stimulation, there is a massive Ca2 + increase due to an influx by ionotropic (iGluR; e.g., NMDA) and metabotropic receptors (mGluR group I), which activates Gq protein, stimulates phosphatidylinositol 4,5-bisphosphate cleavage, resulting in IP3 formation and activation of IP3R in the ER. This release of Ca2 + from the ER can be enhanced by the activation of Ca2 + -induced Ca2 + release via RyR. In mitochondria, Ca2 + primarily enters through the uniporter and activates the tricarboxylic acid cycle (TCA) and ATP synthase (ATPs), prompting the ETC and elevating DJm. This process enhances the probability of electron leakage, particularly via complexes I and III; these electrons then react with free oxygen in the matrix and produce superoxide. This process is the origin of ROS, which can damage cell membranes and DNA, evoking DDR (DNA damage response) and enhancing and activating p53 through its phosphorylation (p) and acetylation (a). p53 directly (by transactivation in the cytosol) and indirectly (by transcription in the nucleus) activates Bax and other pro-apoptotic proteins. Bax can oligomerize at the mitochondrial outer membrane, releasing cytochrome c and APAF-1; in the presence of pro-caspase 9 and dATP, the apoptosome is formed, which activates the intrinsic apoptosis pathway. p53 also modulates oxidative stress by inducing the expression of several antioxidants (e.g., Sesn, Gpx, and MnSod) and mitochondrial respiration modulators (e.g., Tigar, Sco2). ([: stimulation) (>: inhibition). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars stage, corresponding to slow Ca2 + buffering, appears to be more dependent on mitochondria (68). Lopes and coworkers (104) found that there was a greater release of mitochondrial Ca2 + after protonophore (FCCP - carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone) addition in the colon of aged rats. In addition, it was demonstrated that the aged striatum had increased mitochondrial Ca2 + storage in the CNS, which was accompanied by an increase in ROS production and a reduction in DJm (177). In summary, mitochondrial Ca2 + accumulation might promote an increase in ROS and alter bioenergetics, leading to PTP

opening and a loss of DJm (64), which can cause apoptosis (155, 156). The link between apoptotic proteins of the Bcl-2 family, which comprises pro- (e.g., Bax, Bak) and antiapoptotic (e.g., Bcl-2, Bcl-xl) members, and Ca2 + signaling has been extensively studied. In the ER, Bcl-2 was reported to interact with IP3R and reduce the release of Ca2 + (145), though some studies have indicated that Bcl-2 could increase (56), reduce (46, 70, 138), or have no effect (187) on the ER Ca2 + content. In mitochondria, Bcl-2 overexpression increases the concentration or accumulation capacity of Ca2 + (70, 90, 199),

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FIG. 2. PC12 cells overexpressing Bcl-2 (Bcl-21) display alterations in intracellular Ca21 stores. In the left panel, Bcl-2 + cells show a reduced ER Ca2 + content, which was evaluated after the incubation with the ER Ca2 + mobilizer agent Tapsigargin (Tap). On the other hand, the overexpression of Bcl-2 increases mitochondrial Ca2 +, which was analyzed after the addition of the mitochondrial Ca2 + mobilizer agent FCCP. *Significant difference (p £ 0.05, Student’s t test). Adapted from Hirata et al., 2012 (70). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

which is associated with the elevation of DJm and the mitochondrial volume (90) (Fig. 2). The proapoptotic protein Bax otherwise induces ER and mitochondrial Ca2 + release and the loss of DJm, which might be related to ETC inhibition and the partial release of cytochrome c (24, 155) (Fig. 3). Alterations in the expression of Bcl-2 can be directly related to oxidative stress, because Bcl-2 modulates the intracellular levels of ROS (90). Conversely, an increase in ROS can lead to a reduction in Bcl-2 levels (69). Indeed, greater ROS production was accompanied by a reduction in Bcl-2 expression in the striatum of aged rats (177), but with an enhanced activity of the antioxidant enzyme GPx, resulting in no change in lipid peroxidation (Fig. 4).

An increase in the Bax/Bcl-2 protein ratio may have a positive correlation with apoptosis, and an increase in the bax/bcl-2 mRNA ratio may also indicate an increase in the number of apoptotic cells in aging brains (37, 168, 177). Proteins such as Bcl-2 and Bax share a common C-terminal hydrophobic domain that enables intimate contact with organelles, including the ER, mitochondria, and nucleus (196). Briefly, pro-apoptotic proteins bind to the membranes of organelles (such as mitochondria) and promote the release of proapoptotic factors, such as cytochrome c (73, 155). In the cytoplasm, cytochrome c forms a complex (apoptosome) with APAF-1/caspase-9 (157), and caspase-9 cleaves procaspase-3, activating it and subsequently cleaving inhibitor

FIG. 3. Bax induces Ca21 mobilization from intracellular stores and releases cytochrome c, in primary culture astrocytes. (A) Recombinant Bax evokes Ca2 + rise even after the addition of Tapsigargin (Tap) or FCCP. (B) In addition, the recombinant Bax induces the release of cytochrome c from mitochondria to the cytosol. *Significant difference (p £ 0.05, Student’s t test). Adapted from Carvalho et al., 2004 (24). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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FIG. 4. In aging, the increased generation of oxidant molecules (e.g., ROS) can be balanced by the increase of antioxidant enzymes (e.g., Gpx), which may result in no changes in lipid peroxidation in aged striatum. *Significant difference (p £ 0.05, Student’s t test). Adapted from Ureshino et al., 2010 (177).

of caspase-activated DNase (iCAD), which releases CAD to fragment the DNA (42). There is also a concomitant release of endonuclease G (EndoG), which also participates in DNA fragmentation (162), a hallmark of apoptosis (82). Autophagy in Aging and Neurodegeneration

Autophagy, which literally means ‘‘self-eating’’ and was coined by Nobel Laureate Christian de Duve in 1963, enables cells to digest cytosolic components via lysosomal degradation. In addition to scavenging the cytosol for macromolecules and damaged organelles, the process of autophagy also provides a way to supply cells with amino acids and energy due to recycling (88). With regard to the turnover of longlived proteins and the removal of damaged organelles and cellular debris, autophagy is believed to constitute an antiaging process (9). During aging, particularly in tissues populated by postmitotic cells, there is an accumulation of insoluble particles

FIG. 5. Autophagic process in aged cells. During aging, particularly in post mitotic cells, there is a progressive accumulation of lipofuscin, which impairs the autophagy of toxic protein aggregates and mitophagy. Then, damaged mitochondria are not removed in aged cells, which increase oxidative stress. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

called lipofuscins, which can aggregate in autophagosomes and hamper the autophagic system (18, 91) (Fig. 5), a valuable protective mechanism for the cell (110). Since the autophagic system is impaired in aging, damaged mitochondria produce a large amount of ROS and cannot be recycled, which increases cell oxidative stress (18). In addition, analyses of brain gene expression (i.e., Atg5, Atg7, and Beclin 1) in the elderly revealed a down-regulation of autophagic genes compared with young subjects; conversely, an up-regulation was observed in patients with Alzheimer’s disease (47). In 1935, McCay and coworkers reported that reducing caloric intake in rats by 20%–40% extends the lifespan (111, 117), and caloric restriction (CR) was also found to extend the lifespan in yeast, worms, rodents, and monkeys (153). In addition, it is well described that autophagy may be important for increasing longevity (67, 77). Moreover, CR modulates multiple genetic pathways that are involved in aging, including protein homeostasis, which is primarily maintained by removing damaged proteins through the ubiquitin– proteasome system, and autophagy, a process that protects cells from undesirable effects produced by damaged proteins (101), as previously mentioned. From another perspective, a reduction in mitophagy (mitochondrial autophagy) may also be involved in aging-related diseases, such as Parkinson’s disease. The etiology focuses on genetic and environmental factors (51) and is associated with aging, though there are many cases of the early onset of this disease (54). In this process, the protein aggregation of filamentous intracytoplasmic inclusions called Lewy bodies cannot be removed by autophagy (183). Approximately 5% of Parkinson’s disease cases are caused by genetic changes that lead to alterations in the expression of proteins such as Pten-induced novel kinase 1 (PINK1) and parkin (52, 179), which are related to mitophagy (194). When mitochondria are damaged, PINK1 accumulates in the mitochondrial outer membrane for the recruitment of parkin and subsequent mitochondrial degradation (130), and one of the most common stimuli used to sensitize the PINK1-parkin system is the dissipation of DJm (114). It is interesting that there is a reduction in DJm in the cells of many tissues during aging (60, 177, 190). Taken together, the intersection between oxidative stress, Ca2 + , and autophagy should be clarified with regard to molecular alterations in the aging process. Molecular Aging Targets

As mentioned earlier, some proteins are potential molecular targets for improving lifespan or at least reducing the age-associated risk for degenerative diseases. Proteins such

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FIG. 6. Relationship between altered age-related molecules. IGF-1 signaling inhibits the transcriptional activity of FoxO3, which modulates p53 through the up-regulation of p19Arf. Sirt1 participates in the deacetylation of FoxO3 and p53, modulating their transcriptional functions. ([: stimulation) (>: inhibition).

as the transcription factor forkhead box protein O3 (FoxO3), whose polymorphisms have been associated with extreme longevity in humans (3, 189), and other molecules remain the focus of orthomolecular therapeutics for aging-associated diseases. FoxO3, a transcriptional activator that modulates DNA repair and apoptosis (16, 141, 192), plays a role in oxidative stress by causing a reduction in the expression of mitochondrial respiratory complexes and, thus, in mitochondrial respiratory activity (45). In addition, FoxO3 acts in the modulation of SOD2 (89), catalase (164), peroxiredoxin III (28), and sestrin 3 (26, 61), though the role of FoxO3 in Ca2 + signaling still needs to be established. The following molecules have been associated with FoxO3. (i) p53: FoxO3 leads to the activation of p53-dependent apoptosis (193) and up-regulates p19Arf, an upstream regulator of p53 (12). (ii) Sirt1: FoxO3 and p53 are deacetylated by the Sirt1 deacetylase (17, 124). (iii) IGF-1: insulin and growth factors inhibit the transcriptional activity of FoxO3 by phosphorylating its nuclear export sequence (16) (Fig. 6). These proteins are very important modulators of oxidative stress and autophagy and

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play a considerable role in Ca2 + signaling. Next, we discuss the relationship between age-related molecules and the critical alterations in cell physiology. p53 is a tumor suppressor protein that is known to induce apoptosis via the up-regulation of proteins at the transcriptional level or via cytosolic action (57). The most important activity of p53 is an increase in Bax expression (121) and the transactivation of Bax in the cytosol (27), leading to the oligomerization of Bax at the mitochondrial outer membrane to release pro-apoptotic factors. The p53 DNA-binding domain is responsible for its transcriptional activity and is also important in the cytosol, where it interacts with Bcl-2, acting similar to a BH3-only protein (119), potentially switching the cell fate to apoptosis. With regard to the aging process, several groups have used genetically modified animal models by mutating or overexpressing p53, and such mice showed an increased capacity for tumor resistance but a reduced (174), unaltered (53), or increased lifespan (112), depending on p53 activation. In the striatum of aged rats, there is an increase of phosphorylated p53, which can increase bax gene expression (Fig. 7). The cellular alterations that underlie the activation of p53 remain under discussion and may be associated with oxidative stress and Ca2 + homeostasis. Jorda´n and coworkers (78) showed that p53 is important for the hippocampal neuronal cell death promoted by the depolarization agent veratridine, which induces Ca2 + overload (Fig. 8). On exposure to UV light, a classic p53 inducer (93), there is a differential modulation of mitochondrial uniporter activity in young and aged striatum slices, which may be dependent on p53 only in aged rats (Ureshino and coworkers, unpublished data). Notably, the Ca2 + -binding protein S100B is capable of down-regulating p53 in melanoma cells, thereby preventing apoptosis (100). However, the exact relationship between p53 and Ca2 + remains to be established. With regard to oxidative stress, it has been shown that apoptosis caused by p53 activation is dependent on the previous elevation of ROS, leading to the activation of the intrinsic pathway of apoptosis (139). Similarly, it has been demonstrated that the activation of p53 requires an exogenous source of ROS to cause apoptosis; in fact, cell death by oxidative stress is prevented in the absence of p53 (106). As previously reported, oxidative stress in aging promotes the accumulation of DNA damage (approximately two times higher compared with young animals) (2). Dorszewka and Adamczewska-Goncerzewicz (36) found an increase in p53

FIG. 7. Increase in phosphorilated p53 (p.p53) expression is related to a rise in bax gene expression in aged striatum, which may cause an increase in cell vulnerability to apoptosis. *Significant difference (p £ 0.05, Student’s t test).

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FIG. 8. Interrelationship between Ca21 signaling, ROS generation, autophagy, and some important molecules in the aging process. Molecules such as Sirt1, p53, and IGF-1 can modulate autophagy and cross-talk with Ca2 + signaling and oxidative stress. Sirt1 up-regulates autophagy by deacetilating ATG5 and ATG7, helps in LC3 II conversion, and activates AMPK, which inhibits mTOR. Caloric restriction (CR) also stimulates AMPK through an increase in the AMP/ATP ratio or NAD + /NADH and promotes Sirt1 expression. Resveratrol acts directly and increases Sirt1 and ROS scavenger. Sirt1 modulates Ca2 + signaling, acting on SERCA and CaM. p53 increases sestrin (Sesn) levels, which participates in the peroxiredoxin cycle, reducing ROS levels, in addition to the stimulation of AMPK. Although the mechanism is not clear, cytosolic p53 acts by inhibiting autophagy and also modulates Ca2 + by increasing VOCC function. In contrast, the IGF-1 pathway has a pro-aging activity, enhancing ROS production and reducing GPx. IGF-1/IGFR binding increases PI3K activity through insulin receptor substrate 1 (IRS-1), elevating IP3 levels (releasing Ca2 + from the ER) and activating AKT, resulting in the activation of mTOR (through the inhibition of TSC2 and Rheb) and the inhibition of autophagy. Finally, the elevation of ROS can contribute to the p53-mediated cell death ([: stimulation) (>: inhibition). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

mRNA in the cortex, cerebellum, and spinal cord of 24month-old rats, with only the cortex exhibiting a significant increase in p53 protein levels, which was associated with a higher Bax/Bcl-2 ratio and DNA fragmentation. It should be noted that p53 can regulate redox signaling (148) via the expression of antioxidant enzymes such as GPx (163) and manganese superoxide dismutase (MnSOD) (75). p53 was recently shown to be intimately involved in oxidative metabolism, as it up-regulates the expression of proteins called sestrins (sestrin 1 and sestrin 2) (20), which participate in the ROS depuration cycle of peroxiredoxin, reducing this enzyme from its SO2H to its SOH form (Fig. 8). In addition, some authors have reported that p53 can modulate biochemical processes, including glycolysis and mitochondrial oxidative phosphorylation, through the expression of genes encoding the glycolysis regulatory protein TIGAR (TP53induced glycolysis regulator) (107) or complex IV protein of the ETC, such as SCO2 (synthesis of cytochrome c oxidase 2) (113), thereby controlling the oxidative stress status. Moreover, p53 is also related to the modulation of autophagy, as it can transactivate several proteins (for a review, see Maiuri et al., 2010) (108). Damage-regulated autophagy modulator, a phylogenetical lysosomal protein (31), the b1 and b2 subunits of AMPK (adenosine monophosphateactivated protein kinase) (44), and sestrin 1 and sestrin 2 (19), which also activate AMPK, are direct targets of p53. AMPK activation is responsible for inhibiting the mammalian target of rapamycin (mTOR), promoting autophagy (146). De-

spite an unclear mechanism, it was reported that p53 can also inhibit autophagy when it is present in the cytosol (165) (Fig. 8). Sirtuins (Sirt) are nicotinamide adenine dinucleotide (NAD + )-dependent class III histone deacetylases that function in both the nucleus and cytoplasm (63) and are involved in several biological processes, such as oxidative metabolism, inflammation, DNA transcription, insulin secretion, cellular senescence, apoptosis, and DNA repair (62, 72). Several studies support the importance of Sirt1 in the aging process, and it has been noted that Sirt1 and its yeast homolog Sir2 play an important role in lifespan (72). According to Michain and Sinclair (118), the overexpression of Sir2 prolonged longevity in yeast, flies, and worms. Sirt1 is the most studied member of the family, because it has been shown to prevent oxidative stress and DNA damage (62). Haigis and Sinclair (63) discussed that the anti-aging effects of Sirt1 could be related to increased stress resistance in mammalian cells in vivo, as Sirt1 accumulation has been associated with stress responses (185, 195). It was demonstrated that an Sirt1 agonist restored the age-related loss of cardioprotection (171), and the overexpression of this protein in pancreatic b cells in mice caused an enhancement of glucosestimulated insulin secretion (10). Despite the abundant evidence of the anti-aging role of this protein, some authors have provided data regarding neuroprotection on Sirt1 inhibition (99) and have reported that Sirt1 in Caenorhabditis elegans and Sir2 can also accelerate cellular aging and death (43,103),

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thus constituting an issue under continuous discussion in the aging process. Sirt1 regulates the cellular metabolic state through the phosphorylation of AMPK (92), which is activated by an elevation in the AMP/ATP ratio or certain kinases, such as liver kinase B1 (LKB1) and Ca2 + /calmodulin-dependent protein kinase kinase b (CaMKKb) (71). Sirt1 activation and NAD + increases are associated with an increase in the intracellular Ca2 + concentration (134) and can also activate Ca + 2 channels, promote a vasorelaxant effect (176), and improve SERCA2a (sarco-ER Ca2 + -ATPase, an ER Ca + 2reuptake channel) expression and Ca + 2 uptake in the myocardium (161) (Fig. 8). CR has been reported to increase Sirt1 expression and promote autophagy through the AMPK pathway (23), which inhibits mTOR (146). Furthermore, Sirt1 is involved in the autophagy process through the deacetylation of ATG 5 and ATG 7; aids in autophagosome formation (94); and is also associated with the conversion of LC3I to LC3II via proteolytic cleavage and lipidation (123) (Fig. 8). CR also promotes the stimulation of neurogenesis and increases synaptic plasticity, resulting in the resistance to cognitive decline (115). Resveratrol (3-5-4¢ trihydroxystilbene), a polyphenol present in various vegetables and fruits, has been reported to be a powerful antioxidant and protector of mitochondria against oxidative stress (120) through its activity as a free radical scavenger (1). Resveratrol was also demonstrated to protect mitochondria via an increase in citrate synthase activity and to improve mitochondrial respiration in the presence of fatty acid substrates (170). It has been reported that resveratrol acts directly or indirectly on the activation of Sirt1, enhancing its expression (63); similar to CR, resveratrol has been associated with increased autophagy (123). Insulin-like growth factors (IGFs) are anabolic and mitogenic hormones that regulate cell growth and differentiation, cell metabolism, and apoptosis (96, 142). IGF-1 stimulates the IGF-1 receptor (IGFR) and activates insulin receptor substrate 1 (IRS-1), the major cytosolic substrate (129, 147), leading to the activation of phosphoinositide 3-kinase (PI3K) (128, 147), which targets AKT (198). In the autophagic pathway, AKT inhibits tuberous sclerosis protein 2 (TSC2); the heterodimer TSC1/2 antagonizes the mTOR signaling pathway via the inhibition of Rheb (Ras homologue enriched in the brain). Thus, AKT activation leads to the activation of mTOR and blocks autophagy (98). In addition, active PI3K increases the levels of IP3 (76, 147), which causes Ca2 + release from the ER; this Ca2 + is, consequently, taken up by mitochondria, thereby elevating mitochondrial respiration in cardiomyocytes (21, 76). This pathway is shown in Figure 8. Moreover, IGF-1 was also correlated with increased L-type Ca2 + channel gene expression in skeletal muscle (186), enhancing the amplitude of the Ca2 + current but not modifying the voltage dependence of Ca2 + channel currents in the cortex of young and aged rats (151). Since IGF-1 activates cell growth and survival, its synthesis is down-regulated in the aging process (74), and there is evidence that associates the reduction in circulating growth hormone (GH) and IGF-1 with age-related functional deficits in many organs (84, 159). This situation is evident in the cardiac system where IGF-1 acts as an important protector in the heart (175), as the activation of IGFR promotes the

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conservation of telomere length and the growth of cardiac progenitor cells (172). Furthermore, the overexpression of IGF-1 has been related to cardiomyocyte contraction improvement in aged mice (97), and the loss of IGF-1 contributes to an age-related decline in cardiovascular function (34). Dardevet and coworkers (33) reported a reduction in the IGF-1 response in aged rats due to the diminished expression (*80%) of IGFR and consequent reduced glucose transport. IGF-1 may modulate oxidative metabolism either by enhancing ROS production (144) or by reducing certain antioxidant enzymes, such as GPx (15) (Fig. 8). Guevara-Aguirre and coworkers (58) monitored individuals with GH receptor mutations, which are related to IGF-1 deficiency, extracted their serum and treated cells, and observed a reduction in the expression of RAS, PKA (protein kinase A), and TOR and the up-regulation of SOD2. Under physiological conditions, Sirt1 can increase IGF-1 signaling, promote ROS production, and alter redox signaling (99). In addition, resveratrol, an Sirt1 activator, have been associated with a reduction in insulin/IGF-1 signaling and increased mitochondrial biogenesis (6, 197), thus promoting protection against age-related alterations. Although a reduction in IGF-1 signaling appears to be crucial for increasing longevity, the association between IGF-1 and sirtuins remains controversial (43, 72, 99, 103, 197). The conserved insulin/IGF-1 signaling pathway regulates autophagy through mTOR by activating AKT (98, 146), and this interaction controls cell metabolism and longevity in various species (146). In mammals and worms, a loss of function in the insulin/IGF-1 signaling pathway inhibits tumor growth by activating the p53-mediated cell death pathway (180). IGF-1 induces premature cellular senescence by inhibiting sirtuins and activating p53 (85), and it is also related to the activation of mTOR (188). Thus, a reduction in insulin/IGF-1 signaling increases the lifespan of C. elegans (81) and mice (4). This repression can be related to the activation of AMPK (41), which can also reverse age-related alterations (32). Interestingly, mutations that reduce the activity of the IGF-1 receptor (160) and also in IGF-1 pathway genes (135) were found in centenarians. The mouse strains Ames dwarf, Snell dwarf, GHRKO, and Little mice are known for their reduced body size and increased longevity. These strains share a GH deficiency, and the proposed mechanism of delayed aging included reduced systemic IGF1 levels (for a review, see Bartke and Brown-Borg, 2004) (4). It is interesting that Ames dwarf mice display mTOR downregulation, additional evidence that autophagy increases longevity (152). Figure 8 summarizes the relationship mentioned earlier between molecular aging targets (p53, Sirt1, and IGF-1) and the critical age-related alterations in cell physiology, including Ca2 + signaling, oxidative stress, and autophagy. Concluding Remarks

The underlying causes of aging and the molecular mechanisms of longevity might help in preventing age-associated diseases and/or improving the quality of life. Although previous studies of aging were primarily observational, describing changes in the morphology and function of tissues, organs, and organisms, more recent in vitro and in vivo studies involving age models have identified relevant molecules and their respective signaling pathways.

CALCIUM, ROS, AUTOPHAGY, AND AGING

Within the scope of this review, Ca2 + signaling may be associated with certain apoptotic pathways; however, despite being the focus of many studies as an important element for cell death, the exact role of Ca2 + signaling in this process is unknown. Nonetheless, it is known that Ca2 + actively participates in cell death after excitotoxic stimulation. In this case, the increased influx of Ca2 + primarily through NMDAR leads to the accumulation of Ca2 + in mitochondria, which might accelerate oxidative phosphorylation and modify mitochondrial bioenergetics, contributing to an increase in ROS production. This event may contribute to the occurrence of PTP opening and the release of proapoptotic factors. In fact, alterations in some proteins such as p53, Sirt1, and IGF-1, which may change signaling, can be related to an increased susceptibility to Ca2 + -mediated cell death. Meanwhile, a direct link between the molecules, Ca2 +signaling, and aging remains to be clarified. In addition, since the role of autophagy within many cellular contexts appears to be predominantly cytoprotective and the induction of autophagy by pharmacological or genetic methods has life-extending effects, further studies of this fundamental cellular process are necessary to better address its role in the prevention of aging effects and/or treatment of age-related degenerative diseases. This study highlights the complexity of oxidative stress, Ca2 + signaling, and autophagy interaction with age-related interest molecules and underscores the importance of understanding the molecular mechanisms through which these processes of autophagy function in aging. This relation is not fully established, and there are some controversial studies that keep this issue under discussion. Finally, further investigations between molecular aging theories and cellular metabolism may promote advances in therapeutics and increases in healthspan. Acknowledgments

This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES). References

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Address correspondence to: Prof. Soraya Soubhi Smaili Department of Pharmacology Federal University of Sa˜o Paulo Rua Treˆs de Maio, n.100 Sa˜o Paulo/SP 04044-020 Brazil E-mail: [email protected]

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Date of first submission to ARS Central, December 11, 2013; date of final revised submission, January 24, 2014; date of acceptance, February 8, 2014. Abbreviations Used Dwm ¼ mitochondrial membrane potential AMPK ¼ adenosine monophosphate-activated protein kinase ATPs ¼ ATP synthase CAD ¼ caspase-activated DNase CaMKKb ¼ Ca2+ /calmodulin-dependent protein kinase kinase b CNS ¼ central nervous system CR ¼ caloric restriction DDR ¼ DNA damage response ER ¼ endoplasmic reticulum ETC ¼ electron transport chain FCCP ¼ carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone FoxO3 ¼ forkhead box protein O3 GH ¼ growth hormone GPx ¼ glutathione peroxidase iCAD ¼ inhibitor of caspase-activated DNase IGF ¼ insulin-like growth factor IP3 ¼ inositol trisphosphate IP3 R ¼ inositol trisphosphate receptor IRS-1 ¼ insulin receptor substrate 1 LKB1 ¼ liver kinase B1 MnSOD ¼ manganese superoxide dismutase mtDNA ¼ mitochondrial DNA mTOR ¼ mammalian target of rapamycin NAD ¼ nicotinamide adenine dinucleotide NMDA ¼ N-methyl-D-aspartic acid p.p53 ¼ phosphorilated p53 PI3K ¼ phosphoinositide 3-kinase PINK1 ¼ Pten-induced novel kinase 1 PTP ¼ permeability transition pore Rheb ¼ Ras homologue enriched in brain ROS ¼ reactive oxygen species RyR ¼ ryanodine receptors SCO2 ¼ synthesis of cytochrome c oxidase 2 SERCA ¼ sarco-endoplasmic reticulum Ca2+ -ATPase Sesn ¼ sestrin SOD ¼ superoxide dismutase Tap ¼ Tapsigargin TCA ¼ tricarboxylic acid cycle TIGAR ¼ TP53-induced glycolysis regulator TSC2 ¼ tuberous sclerosis protein 2 VOCC ¼ voltage-operated Ca2+ channels