Autophagy is a major protein turnover pathway by which cellular components are delivered into the lysosomes for degradation and recycling. This intracellular process is able to maintain cellular homeostasis under stress conditions, and its dysregulation could lead to the development of physiological alterations. The autophagic activity has been found to decrease with age, likely contributing to the accumulation of damaged macromolecules and organelles during aging. Interestingly, failure of the autophagic process has been reported to worsen aging-associated diseases, such as neurodegeneration or cancer, among others. Likewise, it has been proposed in different organisms that maintenance of a proper autophagic activity contributes to extending longevity. In this review, we discuss recent papers showing the impact of autophagy on cell activity and age-associated diseases, highlighting the relevance of this process to the hallmarks of aging. Thus, understanding how autophagy plays an important role in aging opens new avenues for the discovery of biochemical and pharmacological targets and the development of novel anti-aging therapeutic approaches.

Several studies have demonstrated that CMA is a highly regulated and degradative event, involving HSC70 (heat shock protein 70 complex) and multimerization of the LAMP2A receptor (lysosome-associated membrane protein type 2A). Interestingly, not all proteins are able to be CMA substrates. To undergo CMA degradation, proteins must contain a KFERQ motif in their amino acid sequences, which is necessary to bind the chaperone HSC70 (10, 11). Substrate and the HSC70 complex can bind a 12-amino-acid cytosolic tail of LAMP2A for lysosomal docking. In addition, LAMP2A multimerization is necessary for substrate translocation into the lysosomal lumen. Cytosolic HSC70 is released from the multimeric complex, and then a chaperone HSP90 (located at the lumen of the lysosomal membrane) interacts with LAMP2A, stabilizing it during the substrate translocation. Finally, a luminal chaperone HSC70 is required to end the translocation process, and once inside the targeted protein is degraded by the lysosomal enzymes (12) (Figure 1). Autophagy can be induced by a variety of stressors, and nutrient restriction is one of the major stimuli, capable of rapidly activating the autophagic process with the concomitant inhibition of protein synthesis (1). Studies in both yeasts and mammals have characterized at least 40 autophagy-related genes (Atg), which encode proteins that participate in autophagy (13). In addition, the canonical autophagy pathway includes the inactivation of mammalian target of rapamycin complex 1 (mTORC1), allowing the phosphorylation and activation of the Unc-51-like kinase complex (Ulk1/2), with the subsequent cascade activation of the other ULK complex members such as FIP200 and ATG13 (14, 15). 

Another important complex that is activated is the BECLIN1, in which VPS34, one of its members, is translocated into the ER membranes and it produces high levels of phosphatidylinositol-3-phosphate, which is necessary for the recruitment of other effectors such as WIPI2b (16). Next, this effector interacts and recruits ATG16L, which binds ATG5-ATG12 conjugated to generate the ATG12-ATG5- ATG16L complex. This complex is required for the lipidation of LC3 (Microtubule-associated protein light chain 3), because it determines the site where LC3 will be conjugated and activated to LC3-II (17). Moreover, ATG3 (an E2-like protein) is associated with LC3-I and it binds to the complex through ATG12, allowing the conjugation of LC3-I with phosphatidylethanolamine to generate LC3-II. LC3-II, which is present in both inner and outer membranes of autophagosomal structures and is necessary for phagophore extension, cargo engulfment, and vesicle closure to form the autophagosome. Additionally, the targeted cargo is able to bind receptor/adaptor molecules like p62, NDP52, and NIX. These proteins contain a LC3 interacting region (LIR), which allows the recognition of elements to be engulfed by the phagophore and eliminated in an autophagic manner (18, 19). In addition to degradation, autophagy, or part of its machinery, can mediate a regulated cell death, named autophagydependent cell death (ADCD). Moreover, autophagy can participate in other cell death types [reviewed in reference (20)]. Interestingly, despite the fact that regulated cell death of malignant cells is a pro-survival mechanism at the whole organism level, it can also lead to tissue degeneration and function loss, and this can reduce the fitness of the aged individual (21).

 Moreover, several KO mouse models have been shown to have extended lifespan, although the molecular mechanisms behind it and the connection with aging are not yet clear (35). Notably, another relationship between autophagy augmentation and extended lifespan has been reported in exceptionally healthy centenarian humans, who have increased levels of BECLIN1, compared to young people (36). We hope that in the next years these preliminary studies in humans will be more advanced, providing insights into our species longevity mechanisms from clinical case studies. The knockout for essential Atg genes is lethal in mice, and tissue-specific ablation has a less-dramatic phenotype, manifesting premature signs of aging (37). Specific-Atg5 or Atg7 KO leads to neurodegeneration or tissue abnormalities in most of the cases available in the literature [for a more detailed summary see reference (37)]. Finally, as we age, the incidence of cancer rises, probably because of the decline of homeostatic processes and the increase in the accumulation of potentially harmful molecules such as ROS and protein aggregates. Autophagy has been proposed to have a dual role in tumorigenesis, being important both in suppression as well as in tumor progression and surveillance (38, 39). ROS GENERATION AND AGING The ROS are considered metabolites of molecular oxygen during cellular respiration, being very reactive due to an unpaired electron (40). Mitochondria are the major ROS producers and perhaps the organelle most affected by them. In order to avoid detrimental effects of ROS, two important processes arise: Mitophagy and antioxidant system. 

Mitochondrial ROS can activate the autophagic pathway upon starvation by the activation of ATG4 (41), and this in turn leads to autophagic degradation of mitochondria (mitophagy) in order to reduce the ROS levels by limiting the number of mitochondria per cell (42). In addition, hypoxia and exercise can also trigger redox-dependent autophagy, suggesting that ROS might regulate the autophagic response to several stresses (43). Regarding the second process, the antioxidant system consists of several enzymes and molecules that react with ROS and neutralize them somehow, but the connection of antioxidants with lifespan is controversial (44). Notably, overexpression of a mitochondrial-targeted catalase in mice extends lifespan and reduces overall ROS, reinforcing the Free Radicals Theory of Aging in such model (45). Moreover, these mice showed a reduction in age-related pathologies (46). Additionally, Mn-superoxide dismutase (SOD2) heterozygous mice showed a life-long reduction, but surprisingly they did not have an accelerated aging phenotype. Nevertheless, this Sod2+/− mice showed a higher oxidative damage to DNA and had higher cancer incidence compared with wild type individuals (47). By contrast, knockout of 17 genes involved in the antioxidant system exhibited no effect in lifespan: Only the knockout for Cu/Znsuperoxide dismutase (Sod1) resulted in a decrease in longevity and premature aging as well (48). This mouse model showed an increase in senescent markers, suggesting that the oxidative stress that Sod1−/− mice suffer leads to DNA damage, promoting an irreversible state of quiescence (49).

 In addition, these Sod1−/− mice showed an accelerated sarcopenia, manifesting muscular mass loss and altered neuromuscular junctions (50). Despite these controversial and unexpected results in mice, the relationship between antioxidants and their role in healthy or pathologic aging needs to be deeply studied in the future. Finally, it is important to highlight that ROS have been proposed to be implicated in proliferation and survival signaling in certain conditions (42). A new concept has emerged recently in the aging field, termed “hormesis,” according to which low doses of a stressor can improve the cell response for a more detrimental condition in the future (32, 51). This could increase lifespan and cellular fitness (52). In this context, low levels of ROS can be beneficial due to the trigger of homeostatic responses, but its disproportional augmentation can lead to damage or aging (42, 53). From an autophagic perspective, an augmentation in ROS levels and a decline in mitophagy occur simultaneously, leading to aging (43, 52, 54). HALLMARKS OF AGING: AN AUTOPHAGIC VIEW In the last years, aging has begun to be seen as an active and highly regulated process (55). Age-related changes at cellular level include an increase in ROS, loss of proteostasis, genome instability, and telomere exhaustion, among others (23, 56, 57). These characteristic features of aging were termed “hallmarks of aging” by López-Otín et al. (52). In the following sections, we discuss how autophagy plays an important role in some of these hallmarks of aging, in both health and disease. 

Loss of Proteostasis Proteostasis is one of the major functions of autophagy in normal tissues. Imbalance of proteostasis due to aging leads to protein aggregation, accumulation of misfolded proteins and in the end to cellular dysfunction, among others (23, 56, 57). Notably, carbonylation due to oxidative stress is one of the changes that leads to loss of proteostasis (44). To avoid cell death or dysfunction, numerous homeostatic mechanisms turn on, mainly autophagy (58) and the Ubiquitin-Proteasome-System (UPS). Because autophagy is considered one of the most important intracellular homeostatic processes, an alteration or deterioration of this pathway could modify the normal cell functioning, including a variety of diseases and normal cell physiology declination. Autophagosomes and lysosomes decline in an agedependent manner in muscles (59), heart (43), and several other tissues. Moreover, CMA has also been implicated in removing oxidized and potentially dangerous proteins by direct lysosomal degradation (60). The UPS is mostly implicated in the degradation of misfolded proteins, as well as short- and long-lived proteins by their ubiquitination. This process is achieved thanks to three major proteins that sequentially activate the ubiquitin tag (E1), transfer it to a second enzyme (E2), and finally ligate the ubiquitin tag to the target molecule (by E3 ligase), which eventually reaches the proteasome for degradation (61, 62).

 It is important to note that almost all regulatory proteins are substrates for this system (61, 63), and UPS declines with age [reviewed in (64)]. Interestingly, mTORC1 was found to regulate not only lysosomal protein degradation, but also proteasomal proteolysis of longlived proteins, independently of protein synthesis, suggesting a common regulation of both proteolytic systems by nutrientsensing (63). In addition, overexpression of a sole subunit of the proteasome enhanced its activity and the survival against several oxidants in two cell lines as well as primary culture of human fibroblasts (65). Moreover, proteasome activity decreased in an age-dependent manner (66). Overexpression of proteasome subunits in aged dermal human fibroblasts ameliorated the aged phenotype and restored the oxidized and ubiquitinated proteins to young levels (66). In the same way, transgenic mice with reduced proteasomal activity accumulated oxidized and ubiquitinated proteins, accelerating the aging phenotype and the age-related metabolic diseases (67). Besides, inhibition of proteasome activity impaired cell proliferation and shortened lifespan (68), reinforcing the importance of a correct proteostasis in healthy aging and longevity. Several studies have been done on neurodegenerative diseases related to aging and autophagy, including those most relevant for their high impact on human population. Most of them share the accumulation of ROS, misfolded proteins, and damaged organelles, aging being the main risk factor (69, 70). This accumulation interferes with proper axonal traffic, enhancing neurotoxicity. Both autophagy and CMA impairment hamper the correct protein-aggregates degradation and the remodeling of dendrites and axons, thus diminishing the nervous plasticity (71, 72).

 In Parkinson’s disease (PD), the cytoplasmic aggregates are formed by α-synuclein and ubiquitin (or Lewy bodies) in dopaminergic neurons of substantia nigra, leading to their death (32). Alterations in UPS (71) and also in CMA can develop the disease too. It is worth noting that overexpression of Lamp2A improved CMA performance and decreased αsynuclein cytoplasmic levels (73). In the same way, Alzheimer’s disease (AD) is characterized by intracellular accumulation of tau protein as well as β-amyloids (Aβ), derived from the amyloid precursor protein (APP). This aggregate formation impairs normal cell function, finally leading to cell death (32). Also, extracellular Aβ-plaques secreted by autophagosomes can interrupt intercellular communication (72), another hallmark of aging (52). Besides, Apolipoprotein E4 (ApoE4) is the main genetic risk for sporadic AD and was found to promote the disruption of the lysosomal membrane together with Aβ, leading to neuronal degeneration (72, 74). Mutations in Presenilin1 or 2 (PS1 and PS2, two transmembrane subunits of gamma-secretase), as well as in tau protein or in APP are common causes of the familial AD (72). Other neurodegenerative diseases implicate alterations in the autophagic process, i.e., SENDA, Huntington, Amyotrophic Lateral Sclerosis, and Frontotemporal Dementia disorders [reviewed in (70, 75, 76)]. In all these cases, the lack of proper degradation by autophagy promotes the aggregation of several proteins and the consequent malfunctioning of axonal transport. Regarding metabolic diseases and autophagy, it is well known that islet amyloid deposition leads to type 2 diabetes in humans due to the amyloidogenic property of human islet amyloid polypeptide (hIAPP).

 It is important to note that mice do not develop such aggregation. In order to bypass this model difference, Kim et al. developed transgenic mice expressing hIAPP specifically in β-cells and bred them with Atg71β−cell mice. Male mice had premature diabetes, while females had hyperglycemia but never developed the disease, suggesting a synergism between autophagy deficiency and human amyloid overexpression. Moreover, primary culture of monkey islet cells overexpressing precursors of hIAPP showed that autophagic inhibition by 3-methyladenine (3-MA) increased pro-hIAPP dimer or trimer accumulation, blocking the autophagic activity in these pancreatic cells (77). It is also important to highlight that diabetes or glucose handling deficiencies are risk factors for the AD, as the amyloids properties of proteins implicated in metabolic diseases and AD are similar and probably interconnected (78). More studies regarding the connection between metabolic and neurodegenerative diseases are required for a better understanding of the molecular basis of such relationships at systemic level. Finally, sarcopenia is characterized by a progressive loss of muscle mass and strength thanks to an imbalance between production and degradation of proteins (79). Aged-related declination of autophagy (both mitophagy as well as CMA) promotes sarcopenia by protein accumulation interference with normal myofibers functioning, but an exacerbation of autophagy can also result in cellular stress and finally death (79). Thus, an age-related imbalance of proteostasis could drive a variety of diseases involving both protein accumulation and degradation. Mitochondrial Dysfunction Mitophagy is a basal process involved in the autophagic degradation of mitochondria (76, 80, 81). 

It is necessary in normal differentiation of certain cell types such as red blood cells (82), in embryogenesis, immune response, cell programming, and cell death (80). Mitophagy is required not only to remove damaged mitochondria, but also to promote the biosynthesis of new ones, supporting the mitochondrial quality control (76, 80). Given that mitochondria are implicated in bioenergetics and ROS production, the mitophagy plays an important role in cell homeostasis. Additionally, a decrease in mitophagy is observed in aged animals and this contributes to aging phenotype (81). Canonically, mitophagy is triggered by the cytosolic exposition of mitochondrial outer membrane (MOM) proteins, which have a LIR domain. The mitophagy is tightly regulated by several molecules, NIX and BNIP being two of the most widely characterized mitochondrial adaptors for autophagic machinery (83). NIX activation is associated with an increment in mitochondrial degradation in HeLa cells, protecting them against cellular stress (84). Interestingly, NIX has a LIR domain which binds LC3 once it is activated by phosphorylation (84). Additionally, PINK1 and PARKIN have been involved in the regulation of mitophagy when the mitochondrion loses its membrane potential (80). These proteins have been considered as key components in controlling the activation of mitophagy (85) and also as participants of mitophagy-associated cancer resistance. PINK1 and PARKIN are activated in response to an increment of intracellular ROS levels, which stimulate the MAPK and ERK1/2 signaling cascades, triggering parapoptosis in non-malignant cells, which bypass the caspases activation and, thus, the apoptosis (86) (Figure 2).