Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.     

How autophagy both activates and inhibits cellular senescence

Autophagy and cellular senescence are stress responses essential for homeostasis. While recent studies indicate a genetic relationship between autophagy and senescence, whether autophagy acts positively or negatively on senescence is still subject to debate. Although autophagy was originally recognized as a nonspecific lysosomal degradation pathway (general autophagy), increasing evidence supports a selective form of autophagy that mediates the degradation of specific targets (selective autophagy). Our recent study revealed distinctive roles of selective autophagy and general autophagy in the regulation of senescence, at least in part resolving apparently contradictory reports regarding the relationship between these 2 important homeostatic stress responses.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.     

Delivery of cytoplasmic proteins to autophagosomes

Autophagy represents a signaling-dependent regulated process that allows the degradation of some cellular proteins in autophagosomes, and plays a critical role in the management of cellular homeostasis under various stress conditions. In recent years, selective degradation of cytoplasmic proteins during stress has attracted considerable scientific interest. Here we examined the ability of resveratrol to induce autophagy in a variety of human cancer cell lines. We found that resveratrol-induced autophagy is accompanied by colocalization of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) with the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) in autophagosomes. In addition, we found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a previously shown PELP1-interacting protein, is co-recruited to autophagosomes in the presence of resveratrol. Although autophagy has been assumed to be a bulk and non-selective degradation process, in recent years, evidence of selective degradation of cytosolic proteins and organelles by autophagy is mounting. These observations suggest that the interaction of the target protein(s) with the delivery protein or proteins such as HRS facilitates the transport of certain cytoplasmic proteins to autophagosomes for their selective degradation, and thus, could influence the cytoplasmic as well as nuclear functions of nuclear receptor coregulators. Since PELP1 and, perhaps, other nuclear receptor coregulators are widely dysregulated in human cancers, these findings highlight the significance of the autophagic selective degradation of PELP1 following resveratrol (or other phytoestrogens) treatment in developing future strategies to use resveratrol under cancer prevention and therapeutic settings.     

Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-β-D-glucose-induced senescence-like growth arrest in human cancer cells

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.     

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle‐derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24^?/? (Z24^?/?) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin‐induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F‐actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei‐induced cGAS‐Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24^?/? mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H 2O 2-induced viability loss, which specifically evokes an autophagic response. Exposed to H 2O 2, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

PERK-ing up autophagy during MYC-induced tumorigenesis

Stress in the tumor microenvironment in the form of hypoxia and low glucose/amino acid levels activates the evolutionarily conserved cellular adaptation program called the unfolded protein response (UPR) promoting cell survival in such conditions. Our recent studies showed that cell autonomous stress such as activation of the proto-oncogene MYC/c-Myc, can also trigger the UPR and induce endoplasmic reticulum (ER) stress-mediated autophagy. Amelioration of ER stress or autophagy enhances cancer cell death in vitro and attenuates tumor growth in vivo. Here we will discuss the role of the UPR and autophagy in MYC-induced transformation. Our findings demonstrate that the EIF2AK3/PERK-EIF2S1/eIF2α-ATF4 arm of the UPR promotes tumorigenesis by activating autophagy and enhancing tumor formation. Therefore, the UPR is an attractive target in MYC-driven cancers.     

Research Article: Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-β-D-glucose-induced senescence-like growth arrest in human cancer cells

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.     

Purple sweet potato color attenuated NLRP3 inflammasome by inducing autophagy to delay endothelial senescence

Autophagy is a vital negative factor regulating cellular senescence. Purple sweet potato color (PSPC), one type of flavonoid, has been demonstrated to suppress endothelial senescence and restore endothelial function in diabetic mice by inhibiting the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome. However, the roles of autophagy in the inflammatory response during endothelial senescence are unknown. Here, we found that PSPC augmented autophagy to restrict high-glucose-induced premature endothelial senescence. In addition, PSPC administration impaired endothelium aging in diabetic mice by increasing autophagy. Inhibition of autophagy accelerated endothelial senescence, while enhancement of autophagy delayed senescence. Moreover, deactivation of the NLRP3 inflammasome triggered by PSPC was autophagy-dependent. Autophagy receptor microtubule-associated protein 1 light chain 3 and p62 interacted with the inflammasome component NLRP3, suggesting that autophagosomes target the NLRP3 inflammasome and deliver it to the lysosome for degradation. Altogether, PSPC amplified cellular autophagy, subsequently attenuated NLRP3 inflammasome activity and finally delayed endothelial senescence to ameliorate cardiovascular complication. These results suggest a potential therapeutic target in senescence-related cardiovascular diseases.     

TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS)

Autophagy plays an evolutionarily conserved role in host defense against pathogens. Autophagic protection

mechanisms against microbes range from regulating immune signaling responses to directly targeting the

pathogens for lysosomal degradation. Toll-like receptors (TLRs) that detect conserved molecular features shared by pathogens regulate several innate immune responses including autophagy. Our recent study demonstrates that autophagy reported in response to TLR4-stimulation in macrophages is selective

autophagy of aggresome-like induced structures (ALIS), and p62 (also known as SQSTM1) plays an essential role in this process. Treatment of macrophages with either Escherichia coli or lipopolysaccharide (LPS) results in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to an increase in the levels of p62 mRNA and protein, assembly of ALIS and their autophagic degradation. This study revealed a signaling

role for p62, distinct from its known function as a bacterial-targeting factor, which might be critical for cellular stress response during infection.