Xiucui Ma

Impaired Autophagosome Clearance Contributes to Cardiomyocyte Death in Ischemia-Reperfusion Injury

Background— In myocardial ischemia, induction of autophagy via the AMP-induced protein kinase pathway is protective, whereas reperfusion stimulates autophagy with BECLIN-1 upregulation and is implicated in causing cell death. We examined flux through the macroautophagy 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 green fluorescent protein–tagged microtubule–associated protein light chain-3 to induce ischemia (120 minutes) or ischemia/reperfusion (30–90 minutes) with saline or chloroquine pretreatment (n=4 per group). Autophagosome clearance, assessed as the ratio of punctate light chain-3 abundance in saline to chloroquine-treated samples, was markedly impaired with ischemia/reperfusion compared with sham controls. Reoxygenation increased cell death in neonatal rat cardiomyocytes compared with hypoxia alone, markedly increased autophagosomes but not autolysosomes (assessed as punctate dual fluorescent mCherry-green fluorescent protein tandem-tagged light chain-3 expression), and impaired clearance of polyglutamine aggregates, indicating impaired autophagic flux. The resultant autophagosome accumulation was associated with increased reactive oxygen species and mitochondrial permeabilization, leading to cell death, which was attenuated by cyclosporine A pretreatment. Hypoxia-reoxygenation injury was accompanied by reactive oxygen species–mediated BECLIN-1 upregulation and a reduction in lysosome-associated membrane protein-2, a critical determinant of autophagosome-lysosome fusion. Restoration of lysosome-associated membrane protein-2 levels synergizes with partial BECLIN-1 knockdown to restore autophagosome processing and to attenuate cell death after hypoxia-reoxygenation. Conclusion— Ischemia/reperfusion injury impairs autophagosome clearance mediated in part by reactive oxygen species–induced decline in lysosome-associated membrane protein-2 and upregulation of BECLIN-1, contributing to increased cardiomyocyte death.

Enhancing Astrocytic Lysosome Biogenesis Facilitates Aβ Clearance and Attenuates Amyloid Plaque Pathogenesis

In sporadic Alzheimers disease (AD), impaired Aβ removal contributes to elevated extracellular Aβ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood–brain barrier, and cellular uptake facilitate physiologic Aβ clearance. Astrocytes can take up and degrade Aβ, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aβ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aβ 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 Aβ 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 Aβ42, 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 Aβ. 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 Aβ 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 Aβ removal and counter amyloid plaque pathogenesis in AD.

Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death

Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

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 in-vivo 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 TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice

Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to alpha-L-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via mechanisms that are unclear, but may result from the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Elastin fragmentation causes aortic dilatation and valvular insufficiency, which can result in cardiovascular disease. The pathophysiology of aortic disease was evaluated in MPS I mice. MPS I mice have normal elastic fiber structure and aortic compliance at early ages, which suggests that elastin assembly is normal. Elastin fragmentation and aortic dilatation are severe at 6 months, which is temporally associated with marked increases in mRNA and enzyme activity for two elastin-degrading proteins, matrix metalloproteinase-12 (MMP-12) and cathepsin S. Upregulation of these genes likely involves activation of STAT proteins, which may be induced by structural stress to smooth muscle cells from accumulation of glycosaminoglycans in lysosomes. Neonatal intravenous injection of a retroviral vector normalized MMP-12 and cathepsin S mRNA levels and prevented aortic disease. We conclude that aortic dilatation in MPS I mice is likely due to degradation of elastin by MMP-12 and/or cathepsin S. This aspect of disease might be ameliorated by inhibition of the signal transduction pathways that upregulate expression of elastase proteins, or by inhibition of elastase activity. This could result in a treatment for patients with MPS I, and might reduce aortic aneurism formation in other disorders.

Autophagy is impaired in cardiac ischemia-reperfusion injury

Accumulating 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.

Regulation of the Transcription Factor EB-PGC1α Axis by Beclin-1 Controls Mitochondrial Quality and Cardiomyocyte Death under Stress

ABSTRACT In cardiac ischemia-reperfusion injury, reactive oxygen species (ROS) generation and upregulation of the hypoxia-inducible protein BNIP3 result in mitochondrial permeabilization, but impairment in autophagic removal of damaged mitochondria provokes programmed cardiomyocyte death. BNIP3 expression and ROS generation result in upregulation of beclin-1, a protein associated with transcriptional suppression of autophagy-lysosome proteins and reduced activation of transcription factor EB (TFEB), a master regulator of the autophagy-lysosome machinery. Partial beclin-1 knockdown transcriptionally stimulates lysosome biogenesis and autophagy via mTOR inhibition and activation of TFEB, enhancing removal of depolarized mitochondria. TFEB activation concomitantly stimulates mitochondrial biogenesis via PGC1α induction to restore normally polarized mitochondria and attenuate BNIP3- and hypoxia-reoxygenation-induced cell death. Conversely, overexpression of beclin-1 activates mTOR to inhibit TFEB, resulting in declines in lysosome numbers and suppression of PGC1α transcription. Importantly, knockdown of endogenous TFEB or PGC1α results in a complete or partial loss, respectively, of the cytoprotective effects of partial beclin-1 knockdown, indicating a critical role for both mitochondrial autophagy and biogenesis in ensuring cellular viability. These studies uncover a transcriptional feedback loop for beclin-1-mediated regulation of TFEB activation and implicate a central role for TFEB in coordinating mitochondrial autophagy with biogenesis to restore normally polarized mitochondria and prevent ischemia-reperfusion-induced cardiomyocyte death.

Improved Retroviral Vector Design Results in Sustained Expression after Adult Gene Therapy in Mucopolysaccharidosis I Mice

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to α‐L‐iduronidase (IDUA) deficiency that results in the accumulation of glycosaminoglycans (GAG). Gene therapy can reduce most clinical manifestations, but mice that receive transfer as adults lose expression unless they receive immunosuppression. Increasing liver specificity of transgene expression has reduced immune responses to other genes.

A Self-inactivating γ-Retroviral Vector Reduces Manifestations of Mucopolysaccharidosis I in Mice

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in α-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a γ-retroviral vector (γ-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) γ-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective. This report demonstrates that intravenous (i.v.) injection of a SIN γ-RV expressing canine IDUA from the liver-specific human α1-antitrypsin promoter into adult or newborn MPS I mice completely prevents biochemical abnormalities in several organs, and improved bone disease, vision, hearing, and aorta to a similar extent as was seen with administration of the LTR-intact vector to adults. Improvements were less profound than when using an LTR-intact γ-RV in newborns, which likely reflects a lower level of transduction and expression for the SIN vector-transduced mice, and might be overcome by using a higher dose of SIN vector. A SIN γ-RV vector ameliorates clinical manifestations of MPS I in mice and should be safer than an LTR-intact γ-RV.Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in alpha-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a gamma-retroviral vector (gamma-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) gamma-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective. This report demonstrates that intravenous (i.v.) injection of a SIN gamma-RV expressing canine IDUA from the liver-specific human alpha(1)-antitrypsin promoter into adult or newborn MPS I mice completely prevents biochemical abnormalities in several organs, and improved bone disease, vision, hearing, and aorta to a similar extent as was seen with administration of the LTR-intact vector to adults. Improvements were less profound than when using an LTR-intact gamma-RV in newborns, which likely reflects a lower level of transduction and expression for the SIN vector-transduced mice, and might be overcome by using a higher dose of SIN vector. A SIN gamma-RV vector ameliorates clinical manifestations of MPS I in mice and should be safer than an LTR-intact gamma-RV.

Tumor Necrosis Factor Receptor–Associated Factor 2 Mediates Mitochondrial Autophagy

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, and TNF treatment transcriptionally upregulates TRAF2 abundance in the mitochondrial subfraction. TRAF2 colocalizes 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 neonatal rat cardiac myocytes, as well as in those treated with TNF or uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone, suggesting an essential role for TRAF2 in homeostatic and stress-induced mitochondrial autophagy. TRAF2 also colocalizes and interacts with PARKIN, a previously described E3 ubiquitin ligase and mitophagy effector, on depolarized mitochondria in neonatal rat cardiac myocytes. 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 subfraction of ischemia/reperfusion–modeled hearts; and exogenous TRAF2, but not its E3 ligase-deficient mutants, reduces depolarized mitochondria and rescues cell death in neonatal rat cardiac myocytes 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.Emerging evidence indicates that activation of innate immunity signaling is critical for myocardial adaptation to stress (reviewed in 1). One such highly evolutionary conserved pathway is activated by Tumor Necrosis Factor (TNF), the prototypical member of the TNF superfamily of ligands.2 Indeed, TNF pretreatment, or activation of either TNFR1 or TNFR2 receptor, prevents hypoxia-reperfusion-induced cell death in mammalian cardiomyocytes, in-vitro;3 and transgenic expression or exogenous administration of low doses of TNF attenuates ex-vivo cardiac ischemia-reperfusion (IR) injury.4, 5 Also, absence of both TNFR1 and TNFR2 receptors increases IR-induced cardiomyocyte death, ex-vivo4 and results in marked increase in infarct size with in-vivo coronary ligation as compared with controls;6 pointing to a redundancy in cytoprotective signaling triggered by TNF via its cognate receptors. In this context, it is notable that TNF-receptor associated factor-2 (TRAF2), a scaffolding protein, is recruited to both TNF receptors upon their activation,7 and its transgenic expression (at low levels) attenuates cardiomyocyte death with experimental ex-vivo ischemia-reperfusion injury.4 While these data suggest that TRAF2 may facilitate cytoprotective signaling downstream of both TNF receptors, the underlying mechanisms remain largely unknown, despite extensive investigation.3-5 Ischemia-reperfusion injury results in generation of reactive oxygen species, which provoke mitochondrial permeabilization leading to programmed cardiomyocyte death.8 Autophagy is an evolutionarily conserved pro-survival pathway that sequesters damaged mitochondria within autophagosomes resulting in their intralysosomal degradation (by mitophagy), which is essential to protect against cardiomyocyte death in myocardial infarction.9, 10 Activation of mitophagy also plays a central role in ischemia preconditioning.11 TNF signaling is implicated in induction of cardiomyocyte autophagy, which is cytoprotective against LPS-induced cell death.12 Whether TNF induces mitophagy or signals via TRAF2 to promote mitochondrial autophagy, is not known. Ubiquitination of mitochondrial proteins in response to mitochondrial damage is essential for their sequestration and degradation within the lysosomes.13 PARKIN, an E3 ubiquitin ligase is recruited to damaged mitochondria via activation by PINK1 (PTEN-induced putative kinase 1, a serine-heroine kinase), and ubiquitinates mitochondrial proteins.14, 15 However, while targeted ablation of PINK1 in cardiac myocytes results in mitochondrial abnormalities and cardiomyopathy,16 loss of PARKIN is well tolerated in the unstressed state,17 suggesting that other E3 ubiquitin ligases may be involved in removal of damaged mitochondria. Relevant to this discussion is the observation that TRAF2, an E3 ubiquitin ligase,18 is recruited to the mitochondria by MAVS (Mitochondrial Anti-Viral-Signalosome), a mitochondrially localized protein with multiple scaffolding domains,19-21 following activation of innate immune signaling pathways. Therefore, we tested the hypothesis that TRAF2 mediates mitochondrial autophagy. Here, we show for the first time that TRAF2 is present on the mitochondria in resting cardiac myocytes, and functions in concert with PARKIN as an E3 ubiquitin ligase to facilitate autophagic removal of damaged mitochondria, raising the intriguing possibility that TRAF2-mediated mitophagy may be an important determinant of cytoprotective TNF signaling in ischemia-reperfusion injury.