Background In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase (AMPK) pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation, and is implicated in causing cell death. We examined flux through the macro-autophagy pathway as a determinant of the discrepant outcomes in cardiomyocyte cell death in this setting Methods and Results Reversible left anterior descending coronary artery ligation was performed in mice with cardiomyocyte-restricted expression of GFP-tagged microtubule associated protein light chain-3 (LC3) to induce ischemia (120 minutes) or ischemia-reperfusion (IR, 30–90 minutes) with saline or chloroquine (CQ) pretreatment (n=4/group). Autophagosome clearance, assessed as the ratio of punctate LC3 abundance in saline to CQ treated samples was markedly impaired with IR as compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes (NRCMs) as compared with hypoxia alone; markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-GFP tandem tagged LC3 expression); and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species (ROS) and mitochondrial permeabilization leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by ROS-mediated BECLIN-1 upregulation and reduction in Lysosome Associated Membrane Protein-2 (LAMP2), a critical determinant of autophagosome-lysosome fusion. Restoration of LAMP2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and attenuate cell death following hypoxia-reoxygenation. Conclusions Ischemia-reperfusion injury impairs autophagosome clearance mediated in part by ROS-induced decline in LAMP2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.

In sporadic Alzheimer's disease (AD), impaired A␤ removal contributes to elevated extracellular A␤ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood-brain barrier, and cellular uptake facilitate physiologic A␤ clearance. Astrocytes can take up and degrade A␤, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate A␤ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein A␤ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote A␤ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied A␤42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of A␤. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced A␤ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate A␤ removal and counter amyloid plaque pathogenesis in AD.

In AD, an imbalance between A␤ production and removal drives elevated brain A␤ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via ␣-cleavage at the plasma membrane, amyloidogenic ␤-and ␥-cleavage within endosomes to generate A␤, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce A␤ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adenoassociated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the ␣ and ␤ C-terminal APP fragments (CTFs), and in the steady-state A␤ levels in the brain interstitial fluid. In aged mice, total A␤ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and ␣-and ␤-CTFs, and attenuated A␤ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce A␤ generation. Activation of TFEB in neurons is an effective strategy to attenuate A␤ generation and attenuate amyloid plaque deposition in AD.

(2015) Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemiareperfusion injury, Autophagy, 11:9, 1537-1560, DOI: 10.1080/15548627.2015 Keywords: autophagy, fasting, ischemia-reperfusion, lysosome, myocardial infarctionAbbreviations: CMA, chaperone-mediated autophagy; CQ, chloroquine; GFP, green fluorescent protein; LAD, left anterior descending; LAMP2, lysosomal-associated membrane protein 2; MOI, multiplicity of infection; NRCMs, neonatal rat ventricular cardiac myocytes; q4D, quaque qutra die/every fourth day; qOD, quaque otra die/every other day; TFEB, transcription factor EB; MAP1LC3B (also abbreviated as LC3), microtubule-associated protein 1 light chain 3, isoform B; TTC, triphenyl tetrazolium chloride; LV, left ventricle; AAR, area at risk; WT, wild type.Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from invivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEBmediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fastinginduced autophagy in myocardial ischemia-reperfusion injury.

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Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

A ccumulating evidence attests to a prosurvival role for autophagy under stress, by facilitating removal of damaged proteins and organelles and recycling basic building blocks, which can be utilized for energy generation and targeted macromolecular synthesis to shore up cellular defenses. These observations are difficult to reconcile with the dichotomous prosurvival and death-inducing roles ascribed to macroautophagy in cardiac ischemia and reperfusion injury, respectively. A careful reexamination of 'flux' through the macroautophagy pathway reveals that autophagosome clearance is markedly impaired with reperfusion (reoxygenation) in cardiomyocytes following an ischemic (hypoxic) insult, resulting from reactive oxygen species (ROS)-mediated decline in LAMP2 and increase in BECN1 abundance. This results in impaired autophagy that is 'ineffective' in protecting against cell death with ischemia-reperfusion injury. Restoration of autophagosome clearance and by inference, 'adequate' autophagy, attenuates reoxygenation-induced cell death.Myocardial infarction most commonly results from thrombotic occlusion of a coronary artery resulting in ischemia; and spontaneous or therapeutic reperfusion offers the best hope for myocardial salvage. However, reperfusion is accompanied by a burst of ROS generation and further injury, causing cardiomyocyte death and myocardial dysfunction. Despite significant research efforts, therapeutic options for reperfusion injury are limited to preventive pre-administration of pharmacological agents, which is impractical in the clinical setting. Understanding the Autophagy is impaired in cardiac ischemia-reperfusion injuryXiucui Ma, Haiyan Liu, Sarah R. Foyil, Rebecca J. Godar, Carla J. Weinheimer and Abhinav Diwan* Center for Cardiovascular Research; Division of Cardiology; Department of Internal Medicine; Washington University School of Medicine, St. Louis, MO USA role of autophagy in cardiomyocyte death in myocardial infarction, therefore, holds tremendous promise in development of strategies to promote myocardial salvage. Contemporaneous studies have employed autophagosome abundance as a readout for autophagy, and conclude that induction of autophagy via activation of adenosine monophosphate-activated kinase (AMPK) is beneficial during the ischemic phase, but further activation of autophagy, i.e. an increase in autophagosome prevalence, by BECN1 upregulation causes cell death during reperfusion, thus presenting a conundrum in the development of a therapeutic approach targeting autophagy in ischemia-reperfusion injury.Since 'autophagy' is a process, and assessment of its efficiency should ideally require assessment of the rate of degradation of its substrates (such as damaged proteins and organelles) and/or the rate of generation of the end products (amino acids, sugars and lipids recycled back into the cytosol from the lysosomes), reliance on the levels of a single intermediate, such as autophagosomes, may not yield accurate conclusions. To overcome the limitation of the lack of a suitable ...

Background Tumor Necrosis Factor (TNF) signaling protects against ischemia-reperfusion-induced cardiomyocyte death, in-vitro, ex-vivo and in-vivo. TNF-Receptor Associated Factor-2 (TRAF2), an E3 ubiquitin ligase, coordinates cytoprotective signaling downstream of both TNF receptors, via unclear mechanisms. Noting that TRAF2 is recruited to mitochondria, and that autophagic removal of ubiquitin-tagged damaged mitochondria is cytoprotective, we tested the hypothesis that TRAF2 mediates mitochondrial autophagy. Methods and Results TRAF2 localizes to the mitochondria in neonatal rat cardiac myocytes (NRCMs) and TNF treatment transcriptionally upregulates TRAF2 abundance in the mitochondrial sub-fraction. TRAF2 co-localizes with ubiquitin, p62 adaptor protein, and mitochondria within LC3-bound autophagosomes; and exogenous TRAF2 enhances autophagic removal of mitochondria. TRAF2 knockdown with adenoviral shRNA transduction induces accumulation of depolarized mitochondria in resting NRCMs, as well as in those treated with TNF or uncoupling agent CCCP, suggesting an essential role for TRAF2 in homeostatic and stress-induced mitochondrial autophagy. TRAF2 also co-localizes with and interacts with PARKIN, a previously described E3 ubiquitin ligase and mitophagy effector, on depolarized mitochondria in NRCMs. Exogenous expression of TRAF2, but not its E3 ligase-deficient mutants, is sufficient to partially restore mitophagy in the setting of PARKIN knockdown, suggesting redundancy in their ubiquitin ligase roles. TRAF2 abundance increases in the mitochondrial sub-fraction of ischemia-reperfusion-modeled hearts; and exogenous TRAF2, but not its E3 ligase-deficient mutants, reduces depolarized mitochondria and rescues cell death in NRCMs subjected to hypoxia-reoxygenation. Conclusions Taken together, these data indicate an essential role for TRAF2 in concert with PARKIN as a mitophagy effector, which contributes to TRAF2-induced cytoprotective signaling.