AMP-Activated Protein Kinase: A Universal Regulator of Autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca²⁺ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca²⁺-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca²⁺ ([Ca²⁺]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.

Addendum to:

Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2

M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä

Mol Cell 2007; 25:193-205     

AMP-dependent kinase and autophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/day, ip) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK, and the downstream signal mTOR. Ethanol challenge altered cardiac geometry and function as evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca²⁺ rise, prolonged relengthening and intracellular Ca²⁺ decay, as well as reduced SERCA Ca²⁺ uptake, which effects were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7, and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C, whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D, and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggest that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux.     

The regulation of autophagy by calcium signals: Do we have a consensus?

Macroautophagy (hereafter called ‘autophagy’) is a cellular process for degrading and recycling cellular constituents, and for maintenance of cell function. Autophagy initiates via vesicular engulfment of cellular materials and culminates in their degradation via lysosomal hydrolases, with the whole process often being termed ‘autophagic flux’. Autophagy is a multi-step pathway requiring the interplay of numerous scaffolding and signalling molecules. In particular, orthologs of the family of ∼30 autophagy-regulating (Atg) proteins that were first characterised in yeast play essential roles in the initiation and processing of autophagic vesicles in mammalian cells. The serine/threonine kinase mTOR (mechanistic target of rapamycin) is a master regulator of the canonical autophagic response of cells to nutrient starvation. In addition, AMP-activated protein kinase (AMPK), which is a key sensor of cellular energy status, can trigger autophagy by inhibiting mTOR, or by phosphorylating other downstream targets. Calcium (Ca²⁺) has been implicated in autophagic signalling pathways encompassing both mTOR and AMPK, as well as in autophagy seemingly not involving these kinases. Numerous studies have shown that cytosolic Ca²⁺ signals can trigger autophagy. Moreover, introduction of an exogenous chelator to prevent cytosolic Ca²⁺ signals inhibits autophagy in response to many different stimuli, with suggestions that buffering Ca²⁺ affects not only the triggering of autophagy, but also proximal and distal steps during autophagic flux. Observations such as these indicate that Ca²⁺ plays an essential role as a pro-autophagic signal. However, cellular Ca²⁺ signals can exert anti-autophagic actions too. For example, Ca²⁺ channel blockers induce autophagy due to the loss of autophagy-suppressing Ca²⁺ signals. In addition, the sequestration of Ca²⁺ by mitochondria during physiological signalling appears necessary to maintain cellular bio-energetics, thereby suppressing AMPK-dependent autophagy. This article attempts to provide an integrated overview of the evidence for the proposed roles of various Ca²⁺ signals, Ca²⁺ channels and Ca²⁺ sources in controlling autophagic flux.     

Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ^~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.     

Saikosaponin-d inhibits proliferation by up-regulating autophagy via the CaMKKβ–AMPK–mTOR pathway in ADPKD cells

Autosomal dominant polycystic kidney disease (ADPKD) is a common heritable human disease. Recently, the role of repressed autophagy in ADPKD has drawn increasing attention. Here, we investigate the mechanism underlying the effect of Saikosaponin-d (SSd), a sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump (SERCA) inhibitor. We show that SSd suppresses proliferation in ADPKD cells by up-regulating autophagy. We found that treatment with SSd results in the accumulation of intracellular calcium, which in turn activates the CaMKKβ–AMPK signalling cascade, inhibits mTOR signalling and induces autophagy. Conversely, we also found that treatment with an autophagy inhibitor (3-methyladenine), AMPK inhibitor (Compound C), CaMKKβ inhibitor (STO-609) and intracellular calcium chelator (BAPTA/AM) could reduce autophagy puncta formation mediated by SSd. Our results demonstrated that SSd induces autophagy through the CaMKKβ–AMPK–mTOR signalling pathway in ADPKD cells, indicating that SSd might be a potential therapy for ADPKD and that SERCA might be a new target for ADPKD treatment.

     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins

Autophagy is an intracellular degradation process, through which cytosolic materials are delivered to the lysosome. Despite recent identification of many autophagy-related genes, how autophagosomes are generated remains unclear. Here, we examined the hierarchical relationships among mammalian Atg proteins. Under starvation conditions, ULK1, Atg14, WIPI-1, LC3 and Atg16L1 target to the same compartment, whereas DFCP1 localizes adjacently to these Atg proteins. In terms of puncta formation, the protein complex including ULK1 and FIP200 is the most upstream unit and is required for puncta formation of the Atg14-containing PI3-kinase complex. Puncta formation of both DFCP1 and WIPI-1 requires FIP200 and Atg14. The Atg12-Atg5-Atg16L1 complex and LC3 are downstream units among these factors. The punctate structures containing upstream Atg proteins such as ULK1 and Atg14 tightly associate with the ER, where the ER protein vacuole membrane protein 1 (VMP1) also transiently localizes. These structures are formed even when cells are treated with wortmannin to suppress autophagosome formation. These hierarchical analyses suggest that ULK1, Atg14 and VMP1 localize to the ER-associated autophagosome formation sites in a PI3-kinase activity-independent manner.Key words: autophagosome, PI3-kinase, isolation membrane, endoplasmic reticulum, ULK     

mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

Autophagy is a lysosomal degradation pathway important for cellular homeostasis and survival. Inhibition of the mammalian target of rapamycin (mTOR) is the best known trigger for autophagy stimulation. In addition, intracellular Ca²⁺ regulates autophagy, but its exact role remains ambiguous. Here, we report that the mTOR inhibitor rapamycin, while enhancing autophagy, also remodeled the intracellular Ca²⁺-signaling machinery. These alterations include a) an increase in the endoplasmic-reticulum (ER) Ca²⁺-store content, b) a decrease in the ER Ca²⁺-leak rate, and c) an increased Ca²⁺ release through the inositol 1,4,5-trisphosphate receptors (IP₃Rs), the main ER-resident Ca²⁺-release channels. Importantly, buffering cytosolic Ca²⁺ with BAPTA impeded rapamycin-induced autophagy. These results reveal intracellular Ca²⁺ signaling as a crucial component in the canonical mTOR-dependent autophagy pathway.     

Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca²⁺ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca²⁺ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca²⁺, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca²⁺-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca²⁺. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca²⁺ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.     

Ca2+ transfer via the ER-mitochondria tethering complex in neuronal cells contribute to cadmium-induced autophagy

Mitochondrial-associated endoplasmic reticulum (ER) membranes (MAMs) play a key role in several physiological functions, including calcium ion (Ca²⁺) transfer and autophagy; however, the molecular mechanism controlling this interaction in cadmium (Cd)-induced neurotoxicity is unknown. This study shows that Cd induces alterations in MAMs and mitochondrial Ca²⁺ levels in PC12 cells and primary neurons. Ablation or silencing of mitofusin 2 (Mfn2) in PC12 cells or primary neurons blocks the colocalization of ER and mitochondria while reducing the efficiency of mitochondrial Ca²⁺ uptake. Moreover, Mfn2 defects reduce interactions or colocalization between GRP75 and VDAC1. Interestingly, the enhancement of autophagic protein levels, colocalization of LC3 and Lamp2, and GFP-LC3 puncta induced by Cd decreased in Mfn2^?/? or Grp75^?/? PC12 cells and Mfn2- or Grp75-silenced primary neurons. Notably, the specific Ca²⁺ uniporter inhibitor RuR blocked both mitochondrial Ca²⁺ uptake and autophagy induced by Cd. Finally, this study proves that the mechanism by which IP3R-Grp75-VDAC1 tethers in MAMs is associated with the regulation of autophagy by Mfn2 and involves their role in mediating mitochondrial Ca²⁺ uptake from ER stores. These results give new evidence into the organelle metabolic process by demonstrating that Ca²⁺ transport between ER-mitochondria is important in autophagosome formation in Cd-induced neurodegeneration.

Graphical abstract

     

Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling

Macroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq^?/?) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq^?/? largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq^?/? mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.     

CCCP induces autophagy in an AMPK-independent manner

AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.     

Human WIPI-1 puncta-formation: a novel assay to assess mammalian autophagy

Autophagy depends on the activity of phosphoinositide-3 kinase class III to generate PI(3)P. We identified the human WIPI protein family of PI(3)P-binding factors and showed that WIPI-1 (Atg18) is linked to autophagy in human cells. Induction of autophagy by rapamycin, gleevec, thapsigargin and amino acid deprivation led to an accumulation of WIPI-1 at LC3-positive membrane structures (WIPI-1 puncta-formation), suggested to represent autophagosomal isolation membranes. WIPI-1 puncta-formation is inhibited by wortmannin and LY294002, and PI(3)P-binding-deficient WIPI-1 is puncta-formation-incompetent. Quantification of WIPI-1 puncta should be suitable to assay mammalian autophagy.     

ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

BAFF inhibits autophagy promoting cell proliferation and survival by activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells

B cell activating factor from the TNF family (BAFF) is implicated in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Autophagy plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how excessive BAFF mediates autophagy contributing to B-cell proliferation and survival. Here, we show that excessive human soluble BAFF (hsBAFF) inhibited autophagy with a concomitant reduction of LC3-II in normal and B-lymphoid (Raji) cells. Knockdown of LC3 not only potentiated hsBAFF inhibition of autophagy, but also attenuated hsBAFF activation of Akt/mTOR pathway, thereby diminishing hsBAFF-induced B-cell proliferation/viability. Further, we found that hsBAFF inhibition of autophagy was Akt/mTOR-dependent. This is supported by the findings that hsBAFF increased mTORC1-mediated phosphorylation of ULK1 (Ser757); Akt inhibitor X, mTORC1 inhibitor rapamycin, mTORC1/2 inhibitor PP242, expression of dominant negative Akt, or knockdown of mTOR attenuated hsBAFF-induced phosphorylation of ULK1, decrease of LC3-II level, and increase of cell proliferation/viability. Chelating intracellular free Ca²⁺ ([Ca²⁺]_i) with BAPTA/AM or preventing [Ca²⁺]_i elevation using EGTA or 2-APB profoundly blocked hsBAFF-induced activation of Akt/mTOR, phosphorylation of ULK1 and decrease of LC3-II, as well as increase of cell proliferation/viability. Similar effects were observed in the cells where CaMKII was inhibited by KN93 or knocked down by CaMKII shRNA. Collectively, these results indicate that hsBAFF inhibits autophagy promoting cell proliferation and survival through activating Ca²⁺-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Our findings suggest that manipulation of intracellular Ca²⁺ level or CaMKII, Akt, or mTOR activity to promote autophagy may be exploited for prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.     

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis,Autophagy, and CLN3 Protein Function

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) Ca²⁺ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca²⁺-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca²⁺ chelation. Interrogation of intracellular Ca²⁺ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca²⁺ pools and in store-operated Ca²⁺ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca²⁺ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.     

Activation of autophagy via Ca2+-dependent AMPK/mTOR pathway in rat notochordal cells is a cellular adaptation under hyperosmotic stress

Nucleus pulposus (NP) cells experience hyperosmotic stress in spinal discs; however, how these cells can survive in the hostile microenvironment remains unclear. Autophagy has been suggested to maintain cellular homeostasis under different stresses by degrading the cytoplasmic proteins and organelles. Here, we explored whether autophagy is a cellular adaptation in rat notochordal cells under hyperosmotic stress. Hyperosmotic stress was found to activate autophagy in a dose- and time-dependent manner. SQSTM1/P62 expression was decreased as the autophagy level increased. Transient Ca²⁺ influx from intracellular stores and extracellular space was stimulated by hyperosmotic stress. Activation of AMPK and inhibition of p70S6K were observed under hyperosmotic conditions. However, intercellular Ca²⁺ chelation inhibited the increase of LC3-II and partly reversed the decrease of p70S6K. Hyperosmotic stress decreased cell viability and promoted apoptosis. Inhibition of autophagy led to SQSTM1/P62 accumulation, reduced cell viability, and accelerated apoptosis in notochordal cells under this condition. These evidences suggest that autophagy induction via the Ca²⁺-dependent AMPK/mTOR pathway might occur as an adaptation mechanism for notochordal cells under hyperosmotic stress. Thus, activating autophagy might be a promising approach to improve viability of notochordal cells in intervertebral discs.     

Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins

Autophagy is an intracellular degradation process, through which cytosolic materials are delivered to the lysosome. Despite recent identification of many autophagy-related genes, how autophagosomes are generated remains unclear. Here, we examined the hierarchical relationships among mammalian Atg proteins. Under starvation conditions, ULK1, Atg14, WIPI-1, LC3 and Atg16L1 target to the same compartment, whereas DFCP1 localizes adjacently to these Atg proteins. In terms of puncta formation, the protein complex including ULK1 and FIP200 is the most upstream unit and is required for puncta formation of the Atg14-containing PI3-kinase complex. Puncta formation of both DFCP1 and WIPI-1 requires FIP200 and Atg14. The Atg12-Atg5-Atg16L1 complex and LC3 are downstream units among these factors. The punctate structures containing upstream Atg proteins such as ULK1 and Atg14 tightly associate with the ER, where the ER protein Vacuole membrane protein 1 (VMP1) also transiently localizes. These structures are formed even when cells are treated with wortmannin to suppress autophagosome formation. These hierarchical analyses suggest that ULK1, Atg14 and VMP1 localize to the ER-associated autophagosome formation sites in a PI3-kinase activity-independent manner.     

Capsaicin mimics mechanical load-induced intracellular signaling events

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca²⁺]_i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca²⁺]_i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.