A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum

Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH₄Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.     

Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.     

Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.     

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

脑缺血后N-乙基马来酰亚胺敏感因子ATP酶失活致神经元自噬流障碍的病理机制

缺血性脑卒中是由脑血管梗塞引起的急性脑血管病,具有较高的发病率、致残率和致死率。研究发现,过度自噬或自噬不足均可导致细胞损伤。自噬包括自噬体的形成和成熟、自噬体与溶酶体融合、自噬底物在自噬溶酶体内的降解和清除,这些过程呈连续状态则称为自噬流。研究发现,脑缺血可导致自噬体与溶酶体间发生融合障碍,从而引发自噬流损伤。细胞内膜融合由3种核心组分介导,即N-乙基马来酰亚胺敏感因子（N-ethylmaleimide sensitive factor,NSF） ATP酶、可溶性NSF黏附蛋白（soluble NSF attachment protein,SNAP）及可溶性NSF黏附蛋白受体（soluble NSF attachment protein receptors,SNAREs）。当SNAREs介导自噬体与溶酶体融合后以非活性的复合体形式存留于自噬溶酶体膜,须被NSF再激活为单体后方可发挥新一轮的膜融合介导作用,而NSF是唯一可再激活SNAREs的ATP酶。新近研究表明,脑缺血可显著抑制NSF ATP酶活性,导致其对SNAREs再激活减少,这可能是自噬体与溶酶体间发生融合障碍并导致神经元自噬...     

Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.     

TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells

Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA⁻, tipB⁻, tipC⁻, and tipD⁻). We found a clear autophagic dysfunction in tipC⁻ and tipD⁻ while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.     

Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells interferes with their maintenance

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K⁺/H⁺ ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH⁺ population in HMLER cells than in the ALDH⁻ population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH⁺ population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH⁺ population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.     

Regulation of the breakdown rates of biotin-containing proteins in Swiss 3T3-L1 cells.

1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis.     

The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation

Metabolic stress induces autophagy as an alternative source of energy and metabolites. Insufficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. Here, we performed a functional screen in search of novel autophagy regulators from natural products. We showed that oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, is a potent autophagic flux inhibitor. Exposure to OC results in an increased number of autophagosomes and impaired degradation of SQSTM1/p62. Costaining of GFP-LC3B with LysoTracker Red or LAMP1 antibody demonstrates that autophagosome-lysosome fusion is blocked by OC treatment. Furthermore, OC inhibits lysosomal proteolytic activity by altering lysosomal acidification and downregulating the expression of lysosomal cathepsins. Importantly, OC can eliminate the tolerance of cancer cells to nutrient starvation. Starvation dramatically increases the susceptibility of cancer cells to OC-induced CASP3-dependent apoptosis in vitro. Subsequent studies in xenograft mouse model showed that OC has anticancer potency as revealed by increased staining of cleaved CASP3, LC3 puncta, and SQSTM1, as well as reduced expression of lysosomal cathepsins. Combined treatment with OC and caloric restriction potentiates anticancer efficacy of OC in vivo. Collectively, these data demonstrated that OC is a novel autophagic flux inhibitor and might be useful in anticancer therapy.     

Increased autophagy accelerates colchicine-induced muscle toxicity

Colchicine treatment is associated with an autophagic vacuolar myopathy in human patients. The presumed mechanism of colchicine-induced myotoxicity is the destabilization of the microtubule system that leads to impaired autophagosome-lysosome fusion and the accumulation of autophagic vacuoles. Using the MTOR inhibitor rapamycin we augmented colchicine’s myotoxic effect by increasing the autophagic flux; this resulted in an acute myopathy with muscle necrosis. In contrast to myonecrosis induced by cardiotoxin, myonecrosis induced by a combination of rapamycin and colchicine was associated with accumulation of autophagic substrates such as LC3-II and SQSTM1; as a result, autophagic vacuoles accumulated in the center of myofibers, where LC3-positive autophagosomes failed to colocalize with the lysosomal protein marker LAMP2. A similar pattern of central LC3 accumulation and myonecrosis is seen in human patients with colchicine myopathy, many of whom have been treated with statins (HMGCR/HMG-CoA reductase inhibitors) in addition to colchicine. In mice, cotreatment with colchicine and simvastatin also led to muscle necrosis and LC3 accumulation, suggesting that, like rapamycin, simvastatin activates autophagy. Consistent with this, treatment of mice with four different statin medications enhanced autophagic flux in skeletal muscle in vivo. Polypharmacy is a known risk factor for toxic myopathies; our data suggest that some medication combinations may simultaneously activate upstream autophagy signaling pathways while inhibiting the degradation of these newly synthesized autophagosomes, resulting in myotoxicity.     

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP₃R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP₃R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP₃R-dependent Ca²⁺ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca²⁺ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP₃Rs may play a role in the delayed apoptotic response observed in these cells.     

Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

Autophagy is an important proteolytic pathway in skeletal muscles. The roles of muscle fiber type composition and oxidative capacity remain unknown in relation to autophagy. The diaphragm (DIA) is a fast-twitch muscle fiber with high oxidative capacity, the tibialis anterior (TA) muscle is a fast-twitch muscle fiber with low oxidative capacity, and the soleus muscle (SOL) is a slow-twitch muscle with high oxidative capacity. We hypothesized that oxidative capacity is a major determinant of autophagy in skeletal muscles. Following acute (24 h) starvation of adult C57/Bl6 mice, each muscle was assessed for autophagy and compared with controls. Autophagy was measured by monitoring autophagic flux following leupeptin (20 mg/kg) or colchicine (0.4 mg/kg/day) injection. Oxidative capacity was measured by monitoring citrate synthase activity. In control mice, autophagic flux values were significantly greater in the TA than in the DIA and SOL. In acutely starved mice, autophagic flux increased, most markedly in the TA, and several key autophagy-related genes were significantly induced. In both control and starved mice, there was a negative linear correlation of autophagic flux with citrate synthase activity. Starvation significantly induced AMPK phosphorylation and inhibited AKT and RPS6KB1 phosphorylation, again most markedly in the TA. Starvation induced Foxo1, Foxo3, and Foxo4 expression and attenuated the phosphorylation of their gene products. We conclude that both basal and starvation-induced autophagic flux are greater in skeletal muscles with low oxidative capacity as compared with those with high oxidative capacity and that this difference is mediated through selective activation of the AMPK pathway and inhibition of the AKT-MTOR pathways.     

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.     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

基于自噬溶酶体形成机制调控缺血性脑卒中后神经元自噬流

脑卒中是由脑血管阻塞或出血引发的急性脑血管病，约84%的临床脑卒中患者由脑缺血引起。研究表明，自噬广泛参与并显著影响脑卒中病理生理进程。自噬是一个将陈旧蛋白质、损伤细胞器及多余胞质组分等呈递给溶酶体进行降解的代谢过程，其包括自噬的激活、自噬体的形成和成熟、自噬体与溶酶体融合、自噬产物在自噬溶酶体内消化和降解等过程。自噬流通常被定义为自噬/溶酶体信号机制。最近发现，自噬流障碍是导致缺血性脑卒中后神经元损伤的重要原因，而在自噬过程中任一步骤发生障碍均可导致自噬流损伤。本文重点对自噬体-溶酶体融合的机制，以及该机制在缺血性脑卒中后发生障碍的致病机理进行详细阐述，以期基于自噬体-溶酶体融合机制对神经元自噬流进行调节，进而诱导缺血性脑卒中后的神经保护。本文可为脑卒中病理机制研究指明方向，为脑卒中治疗探寻新的线索。