Nathan R. Brady

Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3ΔTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3ΔTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

Modulation of Serines 17 and 24 in the LC3-interacting Region of Bnip3 Determines Pro-survival Mitophagy versus Apoptosis

Background: Bnip3 is both a pro-apoptotic BH3-only protein and a mitochondrial autophagy receptor. Results: Serine phosphorylation of the Bnip3 LC3-interacting region (LIR) increased binding to Atg8 members and consequently mitophagy, in a manner positively regulated by Bcl-xL. Conclusion: The Bnip3 LIR activity state determines either pro-survival mitophagy or mitochondrial apoptosis. Significance: Bnip3-induced mitophagy is serine kinase-regulated and thus a targetable pathway. BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-xL positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production

The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment.

Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-α and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy.

Mitochondria are an essential source of ATP for cellular function, but when damaged, mitochondria generate a plethora of stress signals, which lead to cellular dysfunction and eventually programmed cell death. Thus, a major component of maintaining cellular homeostasis is the recognition and removal of dysfunctional mitochondria through autophagy-mediated degradation, i.e., mitophagy. Mitophagy further constitutes a developmental program, and undergoes a high degree of crosstalk with apoptosis. Reduced mitochondrial quality control is linked to disease pathogenesis, suggesting the importance of process elucidation as a clinical target. Recent work has revealed multiple mitophagy programs that operate independently or undergo crosstalk, and require modulated autophagy receptor activities at outer membranes of mitochondria. Here, we review these mitophagy programs, focusing on pathway mechanisms which recognize and target mitochondria for sequestration by autophagosomes, as well as mechanisms controlling pathway activities. Furthermore, we provide an introduction to the currently available methods for detecting mitophagy.

Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.

BackgroundAutophagy mediates lysosomal degradation of cytosolic components. Recent work has associated autophagic dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity.ResultsWe developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Our assay allowed for time-resolved individual measurements of autolysosome formation and degradation, and endolysosomal activities under both basal and activated autophagy conditions. As proof of concept, we analyzed conventional autophagy regulators, including cardioprotective compounds aminoimidazole carboxamide ribonucleotide (AICAR), rapamycin, and resveratrol, and revealed striking conditional dependencies of rapamycin and autophagy inhibitor 3-methyladenine (3-MA). To identify novel autophagy modulators with translational potential, we screened the Prestwick Chemical Library of 1,120 US Food and Drug Administration (FDA)-approved compounds for impact on autolysosome formation. In all, 38 compounds were identified as potential activators, and 36 as potential inhibitors of autophagy. Notably, amongst the autophagy enhancers were cardiac glycosides, from which we selected digoxin, strophanthidin, and digoxigenin for validation by standard biochemical and imaging techniques. We report the induction of autophagic flux by these cardiac glycosides, and the concentrations allowing for specific enhancement of autophagic activities without impact on endolysosomal activities.ConclusionsOur systematic analysis of autophagic and endolysosomal activities outperformed conventional autophagy assays and highlights the complexity of drug influence on autophagy. We demonstrate conditional dependencies of established regulators. Moreover, we identified new autophagy regulators and characterized cardiac glycosides as novel potent inducers of autophagic flux.

Autophagy as a Protective Response to Bnip3-Mediated Apoptotic Signaling in the Heart

Bnip3 is a member of the ‘BH3-only’ Bcl-2 subfamily which has been implicated in apoptotic, necrotic, and autophagic cell death. We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R). Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.

Histone deacetylase 10 promotes autophagy-mediated cell survival

Significance Resistance to chemotherapy is one of the major challenges in oncology. Neuroblastoma is the most common extracranial solid tumor in childhood, and the successful response of high-risk patients to chemotherapy remains poor. Our work showed that the so far poorly studied histone deacetylase (HDAC)10 promotes autophagy-mediated cell survival and signals poor outcome in independent high-risk patient cohorts. Inhibition of HDAC10 sensitized tumor cells for cytotoxic drug treatment. These results offer HDAC10 as a potential biomarker for treatment response of high-risk tumors and open new avenues for developing selective treatment strategies to bypass drug resistance of these tumors. Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

Divergent Roles of Autophagy in Virus Infection

Viruses have played an important role in human evolution and have evolved diverse strategies to co-exist with their hosts. As obligate intracellular pathogens, viruses exploit and manipulate different host cell processes, including cellular trafficking, metabolism and immunity-related functions, for their own survival. In this article, we review evidence for how autophagy, a highly conserved cellular degradative pathway, serves either as an antiviral defense mechanism or, alternatively, as a pro-viral process during virus infection. Furthermore, we highlight recent reports concerning the role of selective autophagy in virus infection and how viruses manipulate autophagy to evade lysosomal capture and degradation.