ACBP/DBI protein neutralization confers autophagy-dependent organ protection through inhibition of cell loss, inflammation, and fibrosis.

Acyl-coenzyme A (CoA)-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2F77I mutation that abolishes ACBP/DBI binding to the GABAA receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b. Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

The Role of Alternative Mitophagy in Heart Disease.

Autophagy is essential for maintaining cellular homeostasis through bulk degradation of subcellular constituents, including misfolded proteins and dysfunctional organelles. It is generally governed by the proteins Atg5 and Atg7, which are critical regulators of the conventional autophagy pathway. However, recent studies have identified an alternative Atg5/Atg7-independent pathway, i.e., Ulk1- and Rab9-mediated alternative autophagy. More intensive studies have identified its essential role in stress-induced mitochondrial autophagy, also known as mitophagy. Alternative mitophagy plays pathophysiological roles in heart diseases such as myocardial ischemia and pressure overload. Here, this review discusses the established and emerging mechanisms of alternative autophagy/mitophagy that can be applied in therapeutic interventions for heart disorders.

The role of autophagic cell death in cardiac disease.

Cardiomyocytes undergo various forms of cell death during heart disease such as myocardial infarction and heart failure. Understanding the mechanisms of cell death in cardiomyocytes is one of the most fundamental issues in the treatment of heart failure. Among the several kinds of cell death mechanisms, this review will focus on autophagy-related cardiomyocyte cell death. Although autophagy plays an essential role in mediating cellular quality control mechanisms for cell survival, dysregulation of autophagy can cause cell death, referred to as autophagy-dependent cell death or type II programmed cell death. The recent discovery of autosis as a modality of autophagy-dependent cell death with unique morphological and biochemical features has allowed us to broaden our understanding of the mechanistic role of autophagy in cell death. Here, we discuss autophagy-dependent cardiomyocyte cell death, including autosis, in pathophysiological conditions of the heart.

Differential dynamics of cullin deneddylation via COP9 signalosome subunit 5 interaction.

Cullin-RING E3 ubiquitin ligases (CRLs) spatiotemporally regulate the proteasomal degradation of numerous cellular proteins involved in cell cycle control, DNA replication, and maintenance of genome stability. Activation of CRLs is controlled via neddylation by NEDD8-activating, -conjugating, and -attaching enzymes to the C-terminus of scaffold cullins (CULs), whereas the COP9 signalosome (CSN) inactivates CRLs via deneddylation. Here, we show that the deneddylation rate of each CUL is differentially modulated. Dose- or time-dependent treatment with pevonedistat, a small molecule inhibitor of NEDD8-activating enzyme (NAE), rapidly inhibits neddylation in most CULs, including CUL1, CUL3, CUL4A/B, and CUL5, whereas the deneddylation of CUL2 is slowly increased. We revealed that the different deneddylation speeds of each CUL depend on its binding strength with CSN5, the catalytic core of the CSN complex. Immunoprecipitation analysis revealed that CUL2 has a lower binding affinity for CSN5 than other CULs. Consistently, released cells treated with CSN5 inhibitor showed that CUL2 was slowly converted to the deneddylated form compared to the rapid deneddylation of other CULs. These findings provide mechanistic insights into the different dynamics of CULs in neddylation-deneddylation conversion.

Pimozide reduces toxic forms of tau in TauC3 mice via 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease-associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non-mTOR pathways is a new option for autophagy-based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK-Unc-51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase-cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40-insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR-independent AMPK-ULK1 axis.

Dual-specificity phosphatase 26 (DUSP26) stimulates Aß42 generation by promoting amyloid precursor protein axonal transport during hypoxia.

Amyloid beta peptide (Aß) is a pathological hallmark of Alzheimer's disease (AD) and is generated through the sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. Hypoxia is a known risk factor for AD and stimulates Aß generation by γ-secretase; however, the underlying mechanisms remain unclear. In this study, we showed that dual-specificity phosphatase 26 (DUSP26) regulates Aß generation through changes in subcellular localization of the γ-secretase complex and its substrate C99 under hypoxic conditions. DUSP26 was identified as a novel γ-secretase regulator from a genome-wide functional screen using a cDNA expression library. The phosphatase activity of DUSP26 was required for the increase in Aß42 generation through γ-secretase, but this regulation did not affect the amount of the γ-secretase complex. Interestingly, DUSP26 induced the accumulation of C99 in the axons by stimulating anterograde transport of C99-positive vesicles. Additionally, DUSP26 induced c-Jun N-terminal kinase (JNK) activation for APP processing and axonal transport of C99. Under hypoxic conditions, DUSP26 expression levels were elevated together with JNK activation, and treatment with JNK inhibitor SP600125, or the DUSP26 inhibitor NSC-87877, reduced hypoxia-induced Aß generation by diminishing vesicle trafficking of C99 to the axons. Finally, we observed enhanced DUSP26 expression and JNK activation in the hippocampus of AD patients. Our results suggest that DUSP26 mediates hypoxia-induced Aß generation through JNK activation, revealing a new regulator of γ-secretase-mediated APP processing under hypoxic conditions. We propose the role of phosphatase dual-specificity phosphatase 26 (DUSP26) in the selective regulation of Aß42 production in neuronal cells under hypoxic stress. Induction of DUSP26 causes JNK-dependent shift in the subcellular localization of γ-secretase and C99 from the cell body to axons for Aß42 generation. These findings provide a new strategy for developing new therapeutic targets to arrest AD progression.

Caspase-cleaved tau exhibits rapid memory impairment associated with tau oligomers in a transgenic mouse model.

In neurodegenerative diseases like AD, tau forms neurofibrillary tangles, composed of tau protein. In the AD brain, activated caspases cleave tau at the 421th Asp, generating a caspase-cleaved form of tau, TauC3. Although TauC3 is known to assemble rapidly into filaments in vitro, a role of TauC3 in vivo remains unclear. Here, we generated a transgenic mouse expressing human TauC3 using a neuron-specific promoter. In this mouse, we found that human TauC3 was expressed in the hippocampus and cortex. Interestingly, TauC3 mice showed drastic learning and spatial memory deficits and reduced synaptic density at a young age (2-3months). Notably, tau oligomers as well as tau aggregates were found in TauC3 mice showing memory deficits. Further, i.p. or i.c.v. injection with methylene blue or Congo red, inhibitors of tau aggregation in vitro, and i.p. injection with rapamycin significantly reduced the amounts of tau oligomers in the hippocampus, rescued spine density, and attenuated memory impairment in TauC3 mice. Together, these results suggest that TauC3 facilitates early memory impairment in transgenic mice accompanied with tau oligomer formation, providing insight into the role of TauC3 in the AD pathogenesis associated with tau oligomers and a useful AD model to test drug candidates.

Low levels of methyl ß-cyclodextrin disrupt GluA1-dependent synaptic potentiation but not synaptic depression.

Methyl-ß-cyclodextrin (MßCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol-enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MßCD. However, how MßCD changes synaptic function, such as N-methyl-d-aspartate receptor (NMDA-R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MßCD. At low concentrations (0.5 mg/mL), MßCD decreased basal synaptic transmission and miniature excitatory post-synaptic current without changing NMDA-R-mediated synaptic transmission and the paired-pulse facilitation ratio. Interestingly, low doses of MßCD failed to deplete cholesterol or affect α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) and NMDA-R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MßCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2-lacking AMPA-R. MßCD successfully decreased NMDA-R-mediated long-term potentiation but did not affect the formation of either NMDA-R-mediated or group I metabotropic glutamate receptor-dependent long-term depression. MßCD inhibited de-depression without affecting de-potentiation. These results suggest that MßCD regulates GluA1-dependent synaptic potentiation but not synaptic depression in a cholesterol-independent manner.

Essential role of POLDIP2 in Tau aggregation and neurotoxicity via autophagy/proteasome inhibition.

In Alzheimer's disease and other tauopathy, abnormal Tau proteins form intracellular aggregates and Tau filaments. However, the mechanisms that regulate Tau aggregation are not fully understood. In this paper, we show that POLDIP2 is a novel regulator of Tau aggregation. From a cell-based screening using cDNA expression library, we isolated POLDIP2 which increased Tau aggregation. Expression of POLDIP2 was increased in neuronal cells by the multiple stresses, including Aß, TNF-α and H2O2. Accordingly, ectopic expression of POLDIP2 enhanced the formation of Tau aggregates without affecting Tau phosphorylation, while down-regulation of POLDIP2 alleviated ROS-induced Tau aggregation. Interestingly, we found that POLDIP2 overexpression induced impairments of autophagy activity and partially proteasome activity and this activities were retained in DUF525 domain of POLDIP2. In a drosophila model of human tauopathy, knockdown of the drosophila POLDIP2 homolog, CG12162, attenuated rough eye phenotype induced by Tau overexpression. Further, the lifespan of neural-Tau(R406W) transgenic files were recovered by CG12162 knockdown. Together, these observations indicate that POLDIP2 plays a crucial role in Tau aggregation via the impairment of autophagy activity, providing insight into Tau aggregation in Tau pathology.

OCIAD2 activates γ-secretase to enhance amyloid ß production by interacting with nicastrin.

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aß), a key molecule that causes major neurotoxicity during the early stage of Alzheimer's disease (AD). However, despite its important role in Aß production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aß production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aß production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aß generation.

Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models.

Rationale: Promotion of mitophagy is considered a promising strategy for the treatment of neurodegenerative diseases including Alzheimer's disease (AD). The development of mitophagy-specific inducers with low toxicity and defined molecular mechanisms is essential for the clinical application of mitophagy-based therapy. The aim of this study was to investigate the potential of a novel small-molecule mitophagy inducer, ALT001, as a treatment for AD. Methods: ALT001 was developed through chemical optimization of an isoquinolium scaffold, which was identified from a chemical library screening using a mitophagy reporter system. In vitro and in vivo experiments were conducted to evaluate the potential of ALT001 as a mitophagy-targeting therapeutic agent and to investigate the molecular mechanisms underlying ALT001-induced mitophagy. The therapeutic effect of ALT001 was assessed in SH-SY5Y cells expressing mutant APP and mouse models of AD (5×FAD and PS2APP) by analyzing mitochondrial dysfunction and cognitive defects. Results: ALT001 specifically induces mitophagy both in vitro and in vivo but is nontoxic to mitochondria. Interestingly, we found that ALT001 induces mitophagy through the ULK1-Rab9-dependent alternative mitophagy pathway independent of canonical mitophagy pathway regulators such as ATG7 and PINK1. Importantly, ALT001 reverses mitochondrial dysfunction in SH-SY5Y cells expressing mutant APP in a mitophagy-dependent manner. ALT001 induces alternative mitophagy in mice and restores the decreased mitophagy level in a 5×FAD AD model mouse. In addition, ALT001 reverses mitochondrial dysfunction and cognitive defects in the PS2APP and 5×FAD AD mouse models. AAV-mediated silencing of Rab9 in the hippocampus further confirmed that ALT001 exerts its therapeutic effect through alternative mitophagy. Conclusion: Our results highlight the therapeutic potential of ALT001 for AD via alleviation of mitochondrial dysfunction and indicate the usefulness of the ULK1-Rab9 alternative mitophagy pathway as a therapeutic target.

The role of the Hippo pathway in autophagy in the heart.

The Hippo pathway, an evolutionarily conserved signalling mechanism, controls organ size and tumourigenesis. Increasing lines of evidence suggest that autophagy, an important mechanism of lysosome-mediated cellular degradation, is regulated by the Hippo pathway, which thereby profoundly affects cell growth and death responses in various cell types. In the heart, Mst1, an upstream component of the Hippo pathway, not only induces apoptosis but also inhibits autophagy through phosphorylation of Beclin 1. YAP/TAZ, transcription factor co-factors and the terminal effectors of the Hippo pathway, affect autophagy through transcriptional activation of TFEB, a master regulator of autophagy and lysosomal biogenesis. The cellular abundance of YAP is negatively regulated by autophagy and suppression of autophagy induces accumulation of YAP, which, in turn, acts as a feedback mechanism to induce autophagosome formation. Thus, the Hippo pathway and autophagy regulate each other, thereby profoundly affecting cardiomyocyte survival and death. This review discusses the interaction between the Hippo pathway and autophagy and its functional significance during stress conditions in the heart and the cardiomyocytes therein.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Tfeb-Mediated Transcriptional Regulation of Autophagy Induces Autosis during Ischemia/Reperfusion in the Heart.

Autosis is a unique form of cell death with characteristic morphological and biochemical features caused by dysregulated autophagy. Autosis is observed in the heart during the late phase of ischemia/reperfusion (I/R), when marked accumulation of autophagosomes is induced. We previously showed that the excessive accumulation of autophagosomes promotes autosis in cardiomyocytes. Although the inhibition of autophagic flux via the upregulation of Rubicon induces the accumulation of autophagosomes during I/R, it appears that additional mechanisms exacerbating autophagosome accumulation are required for the induction of autosis. Here, we show that Tfeb contributes to the induction of autosis during the late phase of I/R in the heart. During myocardial reperfusion, Tfeb is activated and translocated into the nucleus, which in turn upregulates genes involved in autophagy and lysosomal function. The overexpression of Tfeb enhanced cardiomyocyte death induced by a high dose of TAT-Beclin 1, an effect that was inhibited by the downregulation of Atg7. Conversely, the knockdown of Tfeb attenuated high-dose TAT-Beclin1-induced death in cardiomyocytes. Although the downregulation of Tfeb in the heart significantly decreased the number of autophagic vacuoles and inhibited autosis during I/R, the activation of Tfeb activity via 3,4-dimethoxychalcone, an activator of Tfeb, aggravated myocardial injury during I/R. These findings suggest that Tfeb promotes cardiomyocyte autosis during the late phase of reperfusion in the heart.

Ulk1-dependent alternative mitophagy plays a protective role during pressure overload in the heart.

AIMS: Well-controlled mitochondrial homeostasis, including a mitochondria-specific form of autophagy (hereafter referred to as mitophagy), is essential for maintaining cardiac function. The molecular mechanism mediating mitophagy during pressure overload (PO) is poorly understood. We have shown previously that mitophagy in the heart is mediated primarily by Atg5/Atg7-independent mechanisms, including Unc-51-like kinase 1 (Ulk1)-dependent alternative mitophagy, during myocardial ischaemia. Here, we investigated the role of alternative mitophagy in the heart during PO-induced hypertrophy. METHODS AND RESULTS: Mitophagy was observed in the heart in response to transverse aortic constriction (TAC), peaking at 3-5 days. Whereas mitophagy is transiently up-regulated by TAC through an Atg7-dependent mechanism in the heart, peaking at 1 day, it is also activated more strongly and with a delayed time course through an Ulk1-dependent mechanism. TAC induced more severe cardiac dysfunction, hypertrophy, and fibrosis in ulk1 cardiac-specific knock-out (cKO) mice than in wild-type mice. Delayed activation of mitophagy was characterized by the co-localization of Rab9 dots and mitochondria and phosphorylation of Rab9 at Ser179, major features of alternative mitophagy. Furthermore, TAC-induced decreases in the mitochondrial aspect ratio were abolished and the irregularity of mitochondrial cristae was exacerbated, suggesting that mitochondrial quality control mechanisms are impaired in ulk1 cKO mice in response to TAC. TAT-Beclin 1 activates mitophagy even in Ulk1-deficient conditions. TAT-Beclin 1 treatment rescued mitochondrial dysfunction and cardiac dysfunction in ulk1 cKO mice during PO. CONCLUSION: Ulk1-mediated alternative mitophagy is a major mechanism mediating mitophagy in response to PO and plays an important role in mediating mitochondrial quality control mechanisms and protecting the heart against cardiac dysfunction.

The roles of the inhibitory autophagy regulator Rubicon in the heart: A new therapeutic target to prevent cardiac cell death.

Autophagy contributes to the maintenance of cardiac homeostasis. The level of autophagy is dynamically altered in heart disease. Although autophagy is a promising therapeutic target, only a few selective autophagy activator candidates have been reported thus far. Rubicon is one of the few endogenous negative regulators of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon was initially identified as a component of the Class III PI3K complex, and it has multiple functions, not only in canonical autophagy but also in endosomal trafficking and inflammatory responses. This review summarizes the molecular action of Rubicon in canonical and noncanonical autophagy. We discuss the roles of Rubicon in cardiac stress and the therapeutic potential of Rubicon in cardiac diseases through its modulation of autophagy.

YAP plays a crucial role in the development of cardiomyopathy in lysosomal storage diseases.

Lysosomal dysfunction caused by mutations in lysosomal genes results in lysosomal storage disorder (LSD), characterized by accumulation of damaged proteins and organelles in cells and functional abnormalities in major organs, including the heart, skeletal muscle, and liver. In LSD, autophagy is inhibited at the lysosomal degradation step and accumulation of autophagosomes is observed. Enlargement of the left ventricle (LV) and contractile dysfunction were observed in RagA/B cardiac-specific KO (cKO) mice, a mouse model of LSD in which lysosomal acidification is impaired irreversibly. YAP, a downstream effector of the Hippo pathway, was accumulated in RagA/B cKO mouse hearts. Inhibition of YAP ameliorated cardiac hypertrophy and contractile dysfunction and attenuated accumulation of autophagosomes without affecting lysosomal function, suggesting that YAP plays an important role in mediating cardiomyopathy in RagA/B cKO mice. Cardiomyopathy was also alleviated by downregulation of Atg7, an intervention to inhibit autophagy, whereas it was exacerbated by stimulation of autophagy. YAP physically interacted with transcription factor EB (TFEB), a master transcription factor that controls autophagic and lysosomal gene expression, thereby facilitating accumulation of autophagosomes without degradation. These results indicate that accumulation of YAP in the presence of LSD promotes cardiomyopathy by stimulating accumulation of autophagosomes through activation of TFEB.