Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.

Neuronal synapse dysfunction is a key characteristic of several neurodegenerative disorders, such as Alzheimer's disease, spinocerebellar ataxias, and Huntington's disease. Modeling these disorders to study synaptic dysfunction requires a robust and reproducible method for assaying the subtle changes associated with synaptopathies in terms of structure and function of the synapses. Drosophila melanogaster neuromuscular junctions (NMJs) serve as good models to study such alterations. Further, modifications in the microenvironment of synapses can sometimes reflect in the behavior of the animal, which can also be assayed in a high-throughput manner. The methods outlined in this chapter highlight assays to study the behavioral changes associated with synaptic dysfunction in a spinocerebellar ataxia type 3 (SCA3) model. Further, molecular assessment of alterations in NMJ structure and function is also summarized, followed by effects of autophagy pathway upregulation in providing neuroprotection. These methods can be further extended and modified to study the therapeutic effects of drugs or small molecules in providing neuroprotection for any synaptopathy models.

Decoding the mitochondria without a code: mechanistic insights into mitochondrial DNA depletion syndromes.

Mitochondrial DNA depletion syndromes (MDS) encompass a wide spectrum of rare genetic disorders caused by severe reduction in mitochondrial DNA (mtDNA), and exhibit heterogenous phenotypes classified as myopathic, encephalomyopathic, hepatocerebral, and neurogastrointestinal. Prognosis for such a spectrum of diseases is poor and is majorly dependent on symptomatic treatment and nutritional supplementation. Understanding the mechanistic aspect of mtDNA depletion can help bring forth a new era of medicine, moving beyond symptomatic treatment and focusing more on organelle-targeted therapies. In this review, we highlight some of the proposed mechanistic bases of mtDNA depletion and the latest therapeutic measures used to treat MDS.

Driving autophagy - the role of molecular motors.

Most of the vesicular transport pathways inside the cell are facilitated by molecular motors that move along cytoskeletal networks. Autophagy is a well-explored catabolic pathway that is initiated by the formation of an isolation membrane known as the phagophore, which expands to form a double-membraned structure that captures its cargo and eventually moves towards the lysosomes for fusion. Molecular motors and cytoskeletal elements have been suggested to participate at different stages of the process as the autophagic vesicles move along cytoskeletal tracks. Dynein and kinesins govern autophagosome trafficking on microtubules through the sequential recruitment of their effector proteins, post-translational modifications and interactions with LC3-interacting regions (LIRs). In contrast, myosins are actin-based motors that participate in various stages of the autophagic flux, as well as in selective autophagy pathways. However, several outstanding questions remain with regard to how the dominance of a particular motor protein over another is controlled, and to the molecular mechanisms that underlie specific disease variants in motor proteins. In this Review, we aim to provide an overview of the role of molecular motors in autophagic flux, as well as highlight their dysregulation in diseases, such as neurodegenerative disorders and pathogenic infections, and ageing.

Leaderless secretory proteins of the neurodegenerative diseases via TNTs: a structure-function perspective.

Neurodegenerative disease-causing proteins such as alpha-synuclein, tau, and huntingtin are known to traverse across cells via exosomes, extracellular vesicles and tunneling nanotubes (TNTs). There seems to be good synergy between exosomes and TNTs in intercellular communication. Interestingly, many of the known major neurodegenerative proteins/proteolytic products are leaderless and are also reported to be secreted out of the cell via unconventional protein secretion. Such classes contain intrinsically disordered proteins and regions (IDRs) within them. The dynamic behavior of these proteins is due to their heterogenic conformations that is exhibited owing to various factors that occur inside the cells. The amino acid sequence along with the chemical modifications has implications on the functional roles of IDRs inside the cells. Proteins that form aggregates resulting in neurodegeneration become resistant to degradation by the processes of autophagy and proteasome system thus leading to Tunneling nanotubes, TNT formation. The proteins that traverse across TNTs may or may not be dependent on the autophagy machinery. It is not yet clear whether the conformation of the protein plays a crucial role in its transport from one cell to another without getting degraded. Although there is some experimental data, there are many grey areas which need to be revisited. This review provides a different perspective on the structural and functional aspects of these leaderless proteins that get secreted outside the cell. In this review, attention has been focused on the characteristic features that lead to aggregation of leaderless secretory proteins (from structural-functional aspect) with special emphasis on TNTs.

Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease.

Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, we expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, we examined the contribution of autophagy in mitigating toxicity caused by mutant HTT protein. We found that targeted overexpression of an autophagy gene, Atg8a in male flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, we demonstrate that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. We show that the rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, our study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.SIGNIFICANCE STATEMENT Defects in sleep and circadian rhythms are well documented in Huntington's disease. Recent literature suggests that circadian and sleep disturbances can exacerbate neurodegenerative phenotypes. Hence, identifying potential modifiers that can improve the functioning of these circuits could greatly improve disease management. We used a genetic approach to enhance cellular proteostasis and found that overexpression of a crucial autophagy gene, Atg8a, induces the autophagy pathway in the Drosophila circadian and sleep neurons and rescues sleep and activity rhythm. We demonstrate that the Atg8a improves synaptic function of these circuits by possibly enhancing the aggregation of the mutant protein in neurons. Further, our results suggest that differences in basal levels of protein homeostatic pathways is a factor that determines selective susceptibility of neurons.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Glutamatergic Synapse Dysfunction in Drosophila Neuromuscular Junctions Can Be Rescued by Proteostasis Modulation.

Glutamate is the major excitatory neurotransmitter in the nervous system, and the Drosophila glutamatergic neuromuscular junctions (NMJs) offer a tractable platform to understand excitatory synapse biology both in health and disease. Synaptopathies are neurodegenerative diseases that are associated with synaptic dysfunction and often display compromised proteostasis. One such rare, progressive neurodegenerative condition, Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph Disease (MJD), is characterized by cerebellar ataxia, Parkinsonism, and degeneration of motor neuron synapses. While the polyQ repeat mutant protein ataxin-3 is implicated in MJD, it is unclear how it leads to impaired synaptic function. In this study, we indicated that a Drosophila model of MJD recapitulates characteristics of neurodegenerative disorders marked by motor neuron dysfunction. Expression of 78 polyQ repeats of mutant ataxin-3 protein in Drosophila motor neurons resulted in behavioral defects, such as impaired locomotion in both larval and adult stages. Furthermore, defects in eclosion and lifespan were observed in adult flies. Detailed characterization of larval glutamatergic neuromuscular junctions (NMJs) revealed defects in morphological features along with compromised NMJ functioning. Autophagy, one of the key proteostasis pathways, is known to be impaired in the case of several synaptopathies. Our study reveals that overexpression of the autophagy-related protein Atg8a rescued behavioral defects. Thus, we present a model for glutamatergic synapse dysfunction that recapitulates synaptic and behavioral deficits and show that it is an amenable system for carrying out genetic and chemical biology screens to identify potential therapeutic targets for synaptopathies.

Early Bioinformatic Implication of Triacidic Amino Acid Motifs in Autophagy-Dependent Unconventional Secretion of Mammalian Proteins.

Several proteins are secreted outside the cell, and in many cases, they may be identified by a characteristic signal peptide. However, more and more studies point to the evidence for an "unconventional" secretion, where proteins without a hitherto unknown signal are secreted, possibly in conditions of starvation. In this work, we analyse a set of 202 RNA binding mammalian proteins, whose unconventional secretion has recently been established. Analysis of these proteins secreted by LC3 mediation, the largest unconventionally secreted dataset to our knowledge, identifies the role of KKX motif as well as triacidic amino acid motif in unconventional secretion, the latter being an extension of the recent implicated diacidic amino acid motif. Further data analysis evolves a hypothesis on the sequence or structural proximity of the triacidic or KKX motifs to the LC3 interacting region, and a phosphorylatable amino acid such as serine as a statistically significant feature among these unconventionally secreted proteins. This hypothesis, although needs to be validated in experiments that challenge the specific details of each of these aspects, appears to be one of the early steps in defining what may be a plausible signal for unconventional protein secretion.

Diacylglycerol kinase alleviates autophagic degradation of the endoplasmic reticulum in SPT10-deficient yeast to enhance triterpene biosynthesis.

A recent study showed that deletion of the gene encoding the transcription regulator SuPpressor of Ty10 (SPT10) increases total phospholipids, and our previous study established a critical link between phospholipids and the mevalonate/ergosterol (MEV/ERG) pathway, which synthesises triterpenes. This study aims to use spt10Δ yeast to improve triterpene production. Though MEV/ERG pathway was highly expressed in spt10Δ yeast, results showed insufficient accumulation of key metabolites and also revealed massive endoplasmic reticulum (ER) degradation. We found a stable, massive ER structure when we overexpressed diacylglycerol kinase1 (DGK1OE ) in spt10Δ yeast. Analyses of ER-stress and autophagy suggest that DGK1OE in the spt10Δ strain decreased autophagy, resulting in increased MEV/ERG pathway activity. Heterologous expression of ß-amyrin synthase showed significant production of the triterpene ß-amyrin in DGK1OE spt10Δ yeast. Overall, our study provides a strategic approach to improve triterpene production by increasing ER biogenesis while limiting ER degradation.

Mitophagy and Neurodegeneration: Between the Knowns and the Unknowns.

Macroautophagy (henceforth autophagy) an evolutionary conserved intracellular pathway, involves lysosomal degradation of damaged and superfluous cytosolic contents to maintain cellular homeostasis. While autophagy was initially perceived as a bulk degradation process, a surfeit of studies in the last 2 decades has revealed that it can also be selective in choosing intracellular constituents for degradation. In addition to the core autophagy machinery, these selective autophagy pathways comprise of distinct molecular players that are involved in the capture of specific cargoes. The diverse organelles that are degraded by selective autophagy pathways are endoplasmic reticulum (ERphagy), lysosomes (lysophagy), mitochondria (mitophagy), Golgi apparatus (Golgiphagy), peroxisomes (pexophagy) and nucleus (nucleophagy). Among these, the main focus of this review is on the selective autophagic pathway involved in mitochondrial turnover called mitophagy. The mitophagy pathway encompasses diverse mechanisms involving a complex interplay of a multitude of proteins that confers the selective recognition of damaged mitochondria and their targeting to degradation via autophagy. Mitophagy is triggered by cues that signal the mitochondrial damage such as disturbances in mitochondrial fission-fusion dynamics, mitochondrial membrane depolarisation, enhanced ROS production, mtDNA damage as well as developmental cues such as erythrocyte maturation, removal of paternal mitochondria, cardiomyocyte maturation and somatic cell reprogramming. As research on the mechanistic aspects of this complex pathway is progressing, emerging roles of new players such as the NIPSNAP proteins, Miro proteins and ER-Mitochondria contact sites (ERMES) are being explored. Although diverse aspects of this pathway are being investigated in depth, several outstanding questions such as distinct molecular players of basal mitophagy, selective dominance of a particular mitophagy adapter protein over the other in a given physiological condition, molecular mechanism of how specific disease mutations affect this pathway remain to be addressed. In this review, we aim to give an overview with special emphasis on molecular and signalling pathways of mitophagy and its dysregulation in neurodegenerative disorders.

Interactions of Autophagy and the Immune System in Health and Diseases.

Autophagy is a highly conserved process that utilizes lysosomes to selectively degrade a variety of intracellular cargo, thus providing quality control over cellular components and maintaining cellular regulatory functions. Autophagy is triggered by multiple stimuli ranging from nutrient starvation to microbial infection. Autophagy extensively shapes and modulates the inflammatory response, the concerted action of immune cells, and secreted mediators aimed to eradicate a microbial infection or to heal sterile tissue damage. Here, we first review how autophagy affects innate immune signaling, cell-autonomous immune defense, and adaptive immunity. Then, we discuss the role of non-canonical autophagy in microbial infections and inflammation. Finally, we review how crosstalk between autophagy and inflammation influences infectious, metabolic, and autoimmune disorders.

Pharmacological intervention in young adolescents rescues synaptic physiology and behavioural deficits in Syngap1+/- mice.

Haploinsufficiency in SYNGAP1 is implicated in intellectual disability (ID) and autism spectrum disorder (ASD) and affects the maturation of dendritic spines. The abnormal spine development has been suggested to cause a disbalance of excitatory and inhibitory (E/I) neurotransmission at distinct developmental periods. In addition, E/I imbalances in Syngap1+/- mice might be due to abnormalities in K+-Cl- co-transporter function (NKCC1, KCC2), in a maner similar to the murine models of Fragile-X and Rett syndromes. To study whether an altered intracellular chloride ion concentration represents an underlying mechanism of modified function of GABAergic synapses in Dentate Gyrus Granule Cells of Syngap1+/- recordings were performed at different developmental stages of the mice. We observed depolarised neurons at P14-15 as illustrated by decreased Cl- reversal potential in Syngap1+/- mice. The KCC2 expression was decreased compared to Wild-type (WT) mice at P14-15. The GSK-3ß inhibitor, 6-bromoindirubin-3'-oxime (6BIO) that crosses the blood-brain barrier, was tested to restore the function of GABAergic synapses. We discovered that the intraperitoneal administration of 6BIO during the critical period or young adolescents [P30 to P80 (4-week to 10-week)] normalised an altered E/I balance, the deficits of synaptic plasticity, and behavioural performance like social novelty, anxiety, and memory of the Syngap1+/- mice. In summary, altered GABAergic function in Syngap1+/- mice is due to reduced KCC2 expression leading to an increase in the intracellular chloride concentration that can be counteracted by the 6BIO, which restored cognitive, emotional, and social symptoms by pharmacological intervention, particularly in adulthood.

Cross-talk between autophagy and sporulation in Saccharomyces cerevisiae.

Unicellular organisms, like yeast, have developed mechanisms to overcome environmental stress conditions like nutrient starvation. Autophagy and sporulation are two such mechanisms employed by yeast cells. Autophagy is a well-conserved, catabolic process that degrades excess and unwanted cytoplasmic materials and provides building blocks during starvation conditions. Thus, autophagy maintains cellular homeostasis at basal conditions and acts as a survival mechanism during stress conditions. Sporulation is an essential process that, like autophagy, is triggered due to stress conditions in yeast. It involves the formation of ascospores that protect the yeast cells during extreme conditions and germinate when the conditions are favorable. Studies show that autophagy is required for the sporulation process in yeast. However, the exact mechanism of action is not clear. Furthermore, several of the core autophagy gene knockouts do not sporulate and at what stage of sporulation they are involved is not clear. Besides, many overlapping proteins function in both sporulation and autophagy and it is unclear how the pathway-specific roles of these proteins are determined. All these observations suggest that the two processes cross-talk. Individually, some key features from both the processes remain to be studied with respect to the source of membrane for autophagosomes, prospore membrane (PSM) formation, and closure of the membranes. Therefore, it becomes crucial to study the cross-talk between autophagy and sporulation. In this review, the cross-talk between the two pathways, the common protein machineries have been discussed.

Spatiotemporal analysis of soluble aggregates and autophagy markers in the R6/2 mouse model.

Maintenance of cellular proteostasis is vital for post-mitotic cells like neurons to sustain normal physiological function and homeostasis, defects in which are established hallmarks of several age-related conditions like AD, PD, HD, and ALS. The Spatio-temporal accumulation of aggregated proteins in the form of inclusion bodies/plaques is one of the major characteristics of many neurodegenerative diseases, including Huntington's disease (HD). Toxic accumulation of HUNTINGTIN (HTT) aggregates in neurons bring about the aberrant phenotypes of HD, including severe motor dysfunction, dementia, and cognitive impairment at the organismal level, in an age-dependent manner. In several cellular and animal models, aggrephagy induction has been shown to clear aggregate-prone proteins like HTT and ameliorate disease pathology by conferring neuroprotection. In this study, we used the mouse model of HD, R6/2, to understand the pathogenicity of mHTT aggregates, primarily focusing on autophagy dysfunction. We report that basal autophagy is not altered in R6/2 mice, whilst being functional at a steady-state level in neurons. Moreover, we tested the efficacy of a known autophagy modulator, Nilotinib (Tasigna™), presently in clinical trials for PD, and HD, in curbing mHTT aggregate growth and their potential clearance, which was ineffective in both inducing autophagy and rescuing the pathological phenotypes in R6/2 mice.

XCT 790 is a pharmacological aggrephagy inducer in a yeast model of proteotoxicity.

Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfolded protein aggregates such as α-synuclein. Here, we identify and demonstrate that the small molecule, XCT 790 alleviates α-synuclein-mediated adverse effects in a yeast model of proteotoxicity. XCT 790 induced general autophagy and also enhanced starvation-induced autophagy. Mechanistically, we showed that XCT 790 clears toxic α-synuclein aggregates in an autophagy-dependent manner. Interestingly, XCT 790 did not demonstrate a synergistic effect on autophagy induction in the presence of another autophagy inducer such as 6-Bio.

TFEB - at the crossroads of host-pathogen interactions.

The existence of constantly evolving dynamic interactions between the host and the pathogen determines their fate in this continuous arms race. Hence, identifying the molecular basis of processes that reinforce host defensive strategies to eliminate intracellular pathogens is of utmost significance. Pathogenic intrusion activates autophagy and phagocytic pathways that culminate in the lysosome, a vital organelle responsible for pathogen clearance. The transcription factor TFEB plays a pivotal role in autophagy-lysosomal function. Although TFEB is an emerging transcription factor in the field of immune signaling pathways, its role in infectious diseases remains contentious. Recent evidence suggests that infection with certain bacterial and viral pathogens causes TFEB, which is normally located in the cytoplasm, to translocate to the nucleus. There, it activates the transcription of genes that trigger the autophagy-lysosomal and inflammatory pathways to target intracellular pathogens. It is known that some pathogens modulate TFEB to establish themselves inside the host; in some cases, pathogens restrict TFEB to the cytoplasm, whereas in others, functional TFEB fuels pathogen survival and replication. However, the key regulators and molecular mechanisms that decide the outcome of TFEB function during intracellular infection are not clear. In this Review, we attempt to dissect the complex functions of TFEB in host-pathogen interactions and explore the suitability of TFEB as a therapeutic target of clinical relevance.

Facets of Autophagy Based Unconventional Protein Secretion-The Road Less Traveled.

Unconventional protein secretion (UCPS) of leaderless proteins bypasses the conventional endoplasmic reticulum (ER)-Golgi route. The proportion of UCPS in the secretome varies tremendously across eukaryotes. Interestingly, macroautophagy, an intracellular recycling process that is generally involved in cargo degradation, also participates in UCPS. This emerging field of secretory mode of autophagy is underexplored and has several unanswered questions regarding the composition of players, cargo, and the mechanisms that drive it. As secretomes vary considerably across cell types and physiological conditions, the contribution of secretory autophagy in healthy and pathophysiological states remain to be elucidated. Recent studies have begun to shed light on this enigmatic process.

Soluble 4R0N Tau Abrogates Endocytic Vesicular Dynamics.

Aggregated tau is a hallmark neuropathological feature in numerous neurodegenerative disorders. Previous studies aiming to validate aggregated tau pathology as a pathogenic driver of neurodegeneration in correlation to characteristic behavioral phenotypes have had shortcomings. Although studies on soluble tau pathology have effectively addressed these shortcomings, the role of soluble tau in the molecular pathogenesis of neurodegeneration is not yet unequivocally established. In sporadic Alzheimer's disease (AD), the relevance of soluble tau pathology in endolysosomal dysfunction and autophagic stress, some of the earliest disease manifestations, is unclear. In this study, we report that soluble 4R0N tau overexpression affects the expression levels of certain markers associated with the endolysosomal system and autophagy. Moreover, through live-cell imaging, we found that the vesicular dynamics of early endosomes were affected with respect to spatiotemporal parameters and vesicle maturation. Additionally, we observed the localization of amyloid precursor protein (APP) along the endocytic pathway and found that upon overexpression of soluble 4R0N tau, APP was preferentially localized to the endocytic compartments implicated in the amyloidogenic pathway. Overall, our observations indicate that soluble 4R0N tau abrogates the dynamics of the endolysosomal system, autophagy, and affects the trafficking of APP. Since the amyloidogenic processing of APP occurs during its progression through the endocytic pathway, our results suggest that the generation of amyloid-ß (Aß) might also be modulated.

Surface-Enhanced Raman Spectroscopy as a Tool for Distinguishing Extracellular Vesicles under Autophagic Conditions: A Marker for Disease Diagnostics.

Extracellular vesicles (EVs) laden with lipids, proteins, DNA, and micro-RNAs play important biological functions in intercellular communication and have pivotal roles in pathophysiological conditions. Characterization of the EVs has always been a multistep process involving large volumes, and they are heterogeneous in size and properties. A multitude of approaches is used to distinguish the EVs. Here, we report simple citrate reduced silver nanoparticles assisted surface-enhanced Raman spectroscopy (SERS) as a tool to distinguish EVs extracted from several cell lines isolated under autophagic conditions (nitrogen starvation). This study is the first report of its kind in characterizing EVs from cells under autophagic conditions using SERS. We used two cancerous cell lines, HeLa, its corresponding autophagy-deficient cell line (Atg5-/-), and a noncancerous cell line, HEK293, to isolate EVs. Our study helps in the facile detection and differentiation of EVs isolated between two closely related human cell lines that differ by their autophagic ability. The principal component analysis (PCA) of the SERS spectra of these EVs consistently showed the presence of distinct chemical compositions of the EVs. SERS of EVs can help in probing more into the molecular level information from EVs and could become a powerful tool once coupled with improved microscopy techniques for diagnosis and therapy.

Foot-and-mouth disease virus induces PERK-mediated autophagy to suppress the antiviral interferon response.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication-associated viral protein expression induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP (also known as HSPA5 and GRP78), a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through the PKR-like ER kinase (PERK; also known as EIF2AK3)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II [the lipidated form of LC3B (also known as MAP1LC3B)]. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK-mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation of functional autophagy. Modulation of the UPR and autophagy by pharmacological and genetic approaches resulted in reduced numbers of viral progeny, by enhancing the antiviral interferon response. Taken together, this study underscores the prospect of exploring PERK-mediated autophagy as an antiviral target.