Dengue virus exploits autophagy vesicles and secretory pathways to promote transmission by human dendritic cells.

Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.

Secretory autophagy - a new paradigm regulating synaptic plasticity.

When exposed to new experiences or changes in the environment, neurons rapidly remodel their synaptic structure and function in a process called activity-induced synaptic remodeling. This process is necessary for transforming transient experiences into stable, lasting memories. The molecular mechanisms underlying acute, activity-dependent synaptic changes are not well understood, partly because processes regulating synaptic plasticity and neurodevelopment are intricately linked. By using an RNAi screen in Drosophila targeting genes associated with human nervous system function, we found that while macroautophagy (referred to as autophagy) is fundamental for both synapse development and synaptic plasticity, activity-induced synaptic remodeling does not rely on genes associated with lysosomal degradation. These findings suggest a requirement for the unconventional secretory autophagy pathway in regulating synaptic plasticity, wherein autophagosomes, instead of fusing with lysosomes for degradation, fuse with the plasma membrane to release their contents extracellularly. To test this hypothesis, we knocked down Sec22, Snap29, and Rab8, molecular components required for secretory autophagy, all of which disrupted structural and functional plasticity. Additionally, by monitoring autophagy, we demonstrated that neuronal activity suppresses degradative autophagy to shift the pathway toward secretory autophagy release. Our work unveils secretory autophagy as a novel trans-synaptic signaling mechanism crucial for activity-induced synaptic remodeling.

SKArred 2 death: neuroinflammatory breakdown of the hippocampus.

A multitude of cellular responses to intrinsic and extrinsic signals converge on macroautophagy/autophagy, a conserved catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, particularly during starvation or stress. In addition to protein degradation, autophagy is deeply interconnected with unconventional protein secretion and polarized sorting at multiple levels within eukaryotic cells. Secretory autophagy (SA) has been recognized as a novel mechanism in which autophagosomes fuse with the plasma membrane and actively participate in the secretion of a series of cytosolic proteins, ranging from tissue remodeling factors to inflammatory molecules of the IL1 family. SA is partially controlled by the glucocorticoid-responsive, HSP90 co-chaperone FKBP5 and members of the SNARE proteins, SEC22B, SNAP23, SNAP29, STX3 and STX4. SA deregulation is implicated in several inflammatory pathologies, including cancer, cell death and degeneration. However, the key molecular mechanisms governing SA and its regulation remain elusive, as does its role in neuroinflammation and neurodegeneration. To further characterize SA and pinpoint its involvement in neuroinflammatory processes, we studied SA-relevant protein interaction networks in mouse brain, microglia and human postmortem brain tissue from control subjects and Alzheimer disease cases. We demonstrate that SA regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling.

Revealing potential Rab proteins participate in regulation of secretory autophagy machinery.

Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy-mediated exocytosis of IL-1ß, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic ß-cell "Min-6" and human lung cancer cell "CL1-5-Q89L" with high secretory autophagy tendency by LC-MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy-related vesicle trafficking. We used CL1-5-Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy-related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.

Molecular mechanisms of secretory autophagy and its potential role in diseases.

Autophagy is a cellular degradation system that recycles or degrades damaged organelles, viral particles, and aggregated proteins through the lysosomal pathway. Autophagy plays an indispensable role in cellular homeostasis and communication processes. An interesting aspect is that autophagy also mediates the secretion of cellular contents, a process known as secretory autophagy. Secretory autophagy differs from macroautophagy, which sequesters recruited proteins, organelles, or viral particles into autophagosomes and degrades these sequesters in lysosomes, while the secretory autophagy pathway participates in the extracellular export of cellular contents sequestered by autophagosomes through autophagy and endosomal modulators. Recent evidence reveals that secretory autophagy is pivotal in the occurrence and progression of diseases. In this review, we summarize the molecular mechanisms of secretory autophagy. Furthermore, we review the impact of secretory autophagy on diseases, including cancer, viral infectious diseases, neurodegenerative diseases, and cardiovascular diseases. Considering the pleiotropic actions of secretory autophagy on diseases, studying the mechanism of secretory autophagy may help to understand the relevant pathophysiological processes.

Intercellular mitochondrial component transfer triggers ischemic cardiac fibrosis.

Myocardial fibrosis is the villain of sudden cardiac death. Myocardial ischemia/reperfusion (MI/R) injury induces cardiomyocyte damage or even death, which in turn stimulates fibroblast activation and fibrosis, but the intercellular communication mechanism remains unknown. Recent studies have shown that small extracellular vesicles (sEVs) significantly contribute to intercellular communication. Whether and how sEV might mediate post-MI/R cardiomyocyte/fibroblasts communication remain unknown. Here, in vivo and in vitro MI/R models were established. We demonstrate that sEVs derived from cardiomyocyte (Myo-sEVs) carry mitochondrial components, which enter fibroblasts to initiate myocardial fibrosis. Based on bioinformatics screening and experimental verification, the activating molecule in Beclin1-regulated autophagy protein 1 (autophagy/beclin-1 regulator 1, Ambra1) was found to be a critical component of these sEV and might be a new marker for Myo-sEVs. Interestingly, release of Ambra1+-Myo-sEVs was caused by secretory rather than canonical autophagy after MI/R injury and thereby escaped degradation. In ischemic and peripheral areas, Ambra1+-Myo-sEVs were internalized by fibroblasts, and the delivered mtDNA components to activate the fibroblast cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to promote fibroblast activation and proliferation. In addition, our data show that Ambra1 is expressed on the EV surface and cardiac-specific Ambra1 down regulation inhibits the Ambra1+-Myo-sEVs release and fibroblast uptake, effectively inhibiting ischemic myocardial fibrosis. This finding newly provides the evidence that myocardial secretory autophagy plays a role in intercellular communication during cardiac fibrosis. Ambra1 is a newly characterized molecule with bioactivity and might be a marker for Myo-sEVs, providing new therapeutic targets for cardiac remodeling.

Repurposing thioridazine for inducing immunogenic cell death in colorectal cancer via eIF2α/ATF4/CHOP and secretory autophagy pathways.

BACKGROUND: Colorectal cancer (CRC) is a highly prevalent cancer type with limited targeted therapies available and 5-year survival rate, particularly for late-stage patients. There have been numerous attempts to repurpose drugs to tackle this problem. It has been reported that autophagy inducers could augment the effect of certain chemotherapeutic agents by enhancing immunogenic cell death (ICD). METHODS: In this study, we employed bioinformatics tools to identify thioridazine (THD), an antipsychotic drug, and found that it could induce autophagy and ICD in CRC. Then in vitro and in vivo experiments were performed to further elucidate the molecular mechanism of THD in CRC. RESULTS: THD was found to induce endoplasmic reticulum (ER) stress in CRC cells by activating the eIF2α/ATF4/CHOP axis and facilitating the accumulation of secretory autophagosomes, leading to ICD. In addition, THD showed a remarkable ICD-activating effect when combined with oxaliplatin (OXA) to prevent tumor progression in the mouse model. CONCLUSIONS: Together, our findings suggest that the repurposed function of THD in inhibiting CRC involves the upregulation of autophagosomes and ER stress signals, promoting the release of ICD markers, and providing a potential candidate to enhance the clinical outcome for CRC treatment. Video Abstract.

Autophagy orchestrates the crosstalk between cells and organs.

Over the recent years, it has become apparent that a deeper understanding of cell-to-cell and organ-to-organ communication is necessary to fully comprehend both homeostatic and pathological states. Autophagy is indispensable for cellular development, function, and homeostasis. A crucial aspect is that autophagy can also mediate these processes through its secretory role. The autophagy-derived secretome relays its extracellular signals in the form of nutrients, proteins, mitochondria, and extracellular vesicles. These crosstalk mediators functionally shape cell fate decisions, tissue microenvironment and systemic physiology. The diversity of the secreted cargo elicits an equally diverse type of responses, which span over metabolic, inflammatory, and structural adaptations in disease and homeostasis. We review here the emerging role of the autophagy-derived secretome in the communication between different cell types and organs and discuss the mechanisms involved.

Immune modulation through secretory autophagy.

Autophagy is a central mechanism of cellular homeostasis through the degradation of a wide range of cellular constituents. However, recent evidence suggests that autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy couples the autophagy machinery to the secretion of cellular content via extracellular vesicles (EVs). EVs carry a variety of cargo, that reflect the pathophysiological state of the originating cells and have the potential to change the functional profile of recipient cells, to modulate cell biology. The immune system has evolved to maintain local and systemic homeostasis. It is able to sense a wide array of molecules signaling disturbed homeostasis, including EVs and their content. In this review, we explore the emerging concept of secretory autophagy as a means to communicate cellular, and in total tissue pathophysiological states to the immune system to initiate the restoration of tissue homeostasis. Understanding how autophagy mediates the secretion of immunogenic factors may hold great potential for therapeutic intervention.

Secretory autophagy-promoted cargo exocytosis requires active RAB37.

RAB37 GTPase regulates cargo exocytosis by cycling between an inactive GDP-bound form and an active GTP-bound form. We reveal that RAB37 simultaneously regulates autophagy activation and tissue inhibitor of metalloproteinase 1 (TIMP1) secretion in lung cancer cells under starvation conditions. TIMP1, an inflammatory cytokine, is a known inhibitory molecule of matrix metalloproteinases matrix metalloproteinase 9 and suppresses the mobility of lung cancer cells both in vitro and in vivo through conventional exocytosis under serum-free conditions. Notably, we disclosed that secretory autophagy participates in TIMP1 secretion in a RAB37- and Sec22b-dependent manner. Sec22b, a SNARE family protein, participates in vesicle and membrane fusion of secretory autophagy. Knockdown of Sec22b decreased TIMP1 secretion and cell motility but did not affect cell proliferation under starvation conditions. We confirmed that starvation-activated RAB37 accompanied by Sec22b is essential for secretory autophagy to further enhance TIMP1 exocytosis. We further use an off-label drug amiodarone to demonstrate that autophagy induction facilitates TIMP1 secretion and suppresses the motility and metastasis of lung cancer cells in a RAB37-dependent manner in the lung-to-lung mouse model. In conclusion, we demonstrated that the RAB37 activation plays a pivotal regulatory role in secretory autophagy for TIMP1 secretion in lung cancer.Abbreviations: ATG: autophagy-related gene; GDP: guanosine diphosphate; GTP: guanosine triphosphate; LC3: microtubule-associated protein 1A/1B-light chain 3; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TIMP1: tissue inhibitor matrix metalloproteinase 1.

Non-canonical autophagy in aging and age-related diseases.

Autophagy, one of the arms of proteostasis, influences aging and age-related diseases. Recently, the discovery of additional roles of autophagy-related proteins in non-canonical degradation and secretion has revealed alternative fates of autophagic cargo. Some of these non-canonical pathways have been linked to neurodegenerative diseases and improving the understanding of this link is crucial for their potential targetability in aging and age-related diseases. This review discusses recent investigations of the involvement of non-canonical autophagy players and pathways in age-related diseases that are now beginning to be discovered. Unraveling these pathways and their relation to classical autophagy could unearth a fascinating new layer of proteostasis regulation during normal aging and in longevity.

RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) serve as critical mediators of cell-to-cell communication. Manzamine A (MA), a unique marine-derived alkaloid with various bioactivities, exerts anticancer effects against several kinds of tumors, but it remains unclear whether it has the same activity against breast cancer. Here, we proved that MA inhibits MDA-MB-231 and MCF-7 cell proliferation, migration, and invasion in a time- and dose-dependent manner. In addition, MA promotes autophagosome formation but suppresses autophagosome degradation in breast cancer cells. Importantly, we also found that MA stimulates sEVs secretion and increases autophagy-related protein accumulation in secreted sEVs, further potentiated by autophagy inhibitor chloroquine (CQ). Mechanistically, MA decreases the expression level of RIP1, the key upstream regulator of the autophagic pathway, and reduces the acidity of lysosome. Overexpression of RIP1 activated AKT/mTOR signaling, thus attenuating MA-induced autophagy and the corresponding secretion of autophagy-associated sEVs. Collectively, these data suggested that MA is a potential inhibitor of autophagy by preventing autophagosome turnover, and RIP1 mediates MA-induced secretory autophagy, which may be efficacious for breast cancer treatment.

LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles.

LC3-dependent EV loading and secretion (LDELS) is a secretory autophagy pathway in which the macroautophagy/autophagy machinery facilitates the packaging of cytosolic cargos, such as RNA-binding proteins, into extracellular vesicles (EVs) for secretion outside of the cell. Here, we identify TFRC (transferrin receptor), one of the first proteins found to be secreted via EVs, as a transmembrane cargo of the LDELS pathway. Similar to other LDELS targets, TFRC secretion via EVs genetically requires components of the MAP1LC3/LC3-conjugation machinery but is independent of other ATGs involved in classical autophagosome formation. Furthermore, the packaging and secretion of this transmembrane protein into EVs depends on multiple ESCRT pathway components and the small GTPase RAB27A. Based on these results, we propose that the LDELS pathway promotes TFRC incorporation into EVs and its secretion outside the cell.Abbreviations: ATG: autophagy related; ESCRT: endosomal sorting complexes required for transport; EV: extracellular vesicle; EVP: extracellular vesicle and particle; ILV: intralumenal vesicle; LDELS: LC3-dependent EV loading and secretion; LIR: LC3-interacting region; MVE: multivesicular endosome; RBP: RNA-binding protein; TMT: tandem mass tag; TFRC: transferrin receptor.

Secretory autophagy promotes RAB37-mediated insulin secretion under glucose stimulation both in vitro and in vivo.

High blood glucose is one of the risk factors for metabolic disease and INS (insulin) is the key regulatory hormone for glucose homeostasis. Hypoinsulinemia accompanied with hyperglycemia was diagnosed in mice with pancreatic ß-cells exhibiting autophagy deficiency; however, the underlying mechanism remains elusive. The role of secretory autophagy in the regulation of metabolic syndrome is gaining more attention. Our data demonstrated that increased macroautophagic/autophagic activity leads to induction of insulin secretion in ß-cells both in vivo and in vitro under high-glucose conditions. Moreover, proteomic analysis of purified autophagosomes from ß-cells identified a group of vesicular transport proteins participating in insulin secretion, implying that secretory autophagy regulates insulin exocytosis. RAB37, a small GTPase, regulates vesicle biogenesis, trafficking, and cargo release. We demonstrated that the active form of RAB37 increased MAP1LC3/LC3 lipidation (LC3-II) and is essential for the promotion of insulin secretion by autophagy, but these phenomena were not observed in rab37 knockout (rab37-/-) cells and mice. Unbalanced insulin and glucose concentration in the blood was improved by manipulating autophagic activity using a novel autophagy inducer niclosamide (an antihelminthic drug) in a high-fat diet (HFD)-obesity mouse model. In summary, we reveal that secretory autophagy promotes RAB37-mediated insulin secretion to maintain the homeostasis of insulin and glucose both in vitro and in vivo.

Regulation of hepatitis B virus replication by autophagic membranes.

Autophagy (i.e. macroautophagy) plays a significant role in the replication of hepatitis B virus (HBV). In our recent study, we examined the underlying mechanism and discovered that autophagic membranes participated in different steps of the HBV life cycle. We found that phagophores are involved in the assembly of HBV nucleocapsids, autophagosomes participate in the trafficking of HBV nucleocapsids, amphisomes likely participate in the maturation and egress of mature HBV particles, and autolysosomes negatively regulate HBV replication. Our work provides important insights for understanding the relationship between autophagic membranes and HBV replication and raises the possibility of targeting the autophagic pathway for the development of novel drugs against HBV.

Autophagic secretion of mitochondria (ASM): an alternative way for getting rid of damaged mitochondria.

PINK1-PRKN/Parkin-mediated mitophagy represents an important mitochondrial quality control (MQC) pathway that clears damaged/dysfunctional mitochondria. Although the conjugation of mammalian Atg8-family proteins (mATG8s) to phosphatidylethanolamine (PE) is a defining step in autophagy, its role in mitophagy remains unclear. In our recent study, we found that the mATG8 conjugation system is not required for PINK1-PRKN-mediated mitochondria clearance. Instead, mATG8 conjugation system-independent mitochondria clearance relies on secretory autophagy, a process we term as the autophagic secretion of mitochondria (ASM). As ASM results in the spurious activation of the CGAS-STING1 pathway, we propose that defects in mATG8 lipidation may promote inflammation through ASM.

Panobinostat inhibits breast cancer progression via Vps34-mediated exosomal pathway.

Exosomes play crucial roles in intercellular communication, including tumor metastasis. Panobinostat (LBH589), a histone deacetylases (HDAC) inhibitor, is an emerging anti-tumor drug with promising efficacy in cancer therapy. This study was set out from recent evidence that exosome was a mechanism of intercellular drug transfer with significant pharmacological consequences. It enlightened us LBH589 might regulate tumor growth through exosomal secretion. Here we demonstrated LBH589 induced autophagy and facilitated secretory autophagy. Furthermore, LBH589 dose- and time-dependently stimulated exosomal release mediated by Vps34/Rab5C pathway, documented by the ablation of Vps34 and/or Rab5C in breast cancer cells. Additionally, the findings also presented LBH589 inhibited breast cancer progression via exosomes. Altogether, we revealed a novel mechanism of LBH589 in exosome-mediated anti-tumor effects in breast cancer. The schematic diagram of signaling pathways involved in the suppression of breast cancer progression by LBH589 via exosomes.

Secretory autophagy: a turn key for understanding AMD pathology and developing new therapeutic targets?

INTRODUCTION: Age-related macular degeneration (AMD) is an eye disease leading to vision loss with poorly known pathogenesis and limited therapeutic options. Degradative autophagy (DA) is impaired in AMD, but emerging evidence points to secretary autophagy (SA) as a key element in AMD pathogenesis. AREAS COVERED: SA may cause the release of proteins and protein aggregates, lipofuscin, beta amyloid, faulty mitochondria, pro-inflammatory and pro-angiogenic factors from the retinal pigment epithelium (RPE) that may contribute to drusen formation and choroidal neovascularization. SA may replace DA, when formation of autolysosome is impaired, and then a harmful cargo, instead of being degraded, is extruded from the RPE contributing to drusen and/or angiogenic environment. Therefore, the interplay between DA and SA may be critical for drusen formation and choroidal neovascularization, so it can be a turn key to understand AMD pathogenesis. EXPERT OPINION: Although SA fulfills some beneficial functions, it is detrimental for the retina in many cases. Therefore, inhibiting SA may be a therapeutic strategy in AMD, but it is challenged by the development of selective SA inhibitors that would not affect DA. The TRIM16, SEC22B and RAB8A proteins, specific for secretory autophagosome, may be primary candidates as therapeutic targets, but their action is not limited to autophagy and therefore requires further studies.

Recent advances in conventional and unconventional vesicular secretion pathways in the tumor microenvironment.

All cells in the changing tumor microenvironment (TME) need a class of checkpoints to regulate the balance among exocytosis, endocytosis, recycling and degradation. The vesicular trafficking and secretion pathways regulated by the small Rab GTPases and their effectors convey cell growth and migration signals and function as meditators of intercellular communication and molecular transfer. Recent advances suggest that Rab proteins govern conventional and unconventional vesicular secretion pathways by trafficking widely diverse cargoes and substrates in remodeling TME. The mechanisms underlying the regulation of conventional and unconventional vesicular secretion pathways, their action modes and impacts on the cancer and stromal cells have been the focus of much attention for the past two decades. In this review, we discuss the current understanding of vesicular secretion pathways in TME. We begin with an overview of the structure, regulation, substrate recognition and subcellular localization of vesicular secretion pathways. We then systematically discuss how the three fundamental vesicular secretion processes respond to extracellular cues in TME. These processes are the conventional protein secretion via the endoplasmic reticulum-Golgi apparatus route and two types of unconventional protein secretion via extracellular vesicles and secretory autophagy. The latest advances and future directions in vesicular secretion-involved interplays between tumor cells, stromal cell and host immunity are also described.