Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models. However, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular survival under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate whether the cell lives or dies. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.

Adaptation to the shortage in free amino acids (AA) is mediated by 2 pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the 2 pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.

Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.

Vesicle-associated membrane protein-associated protein-B (VAPB) is an ER membrane bound protein. VAPB P56S causes a dominant, familial form of amyotrophic lateral sclerosis (ALS), however, the mechanism through which this mutation causes motor neuron (MN) disease remains unknown. Using inducible wild type (WT) and VAPB P56S expressing iPSC-derived MNs we show that VAPB P56S, but not WT, protein decreased neuronal firing and mitochondrial-ER contact (MERC) with an associated age-dependent decrease in mitochondrial membrane potential (MMP); all typical characteristics of MN-disease. We further show that VAPB P56S expressing iPSC-derived MNs have enhanced age-dependent sensitivity to ER stress. We identified elevated expression of the master regulator of the Integrated Stress Response (ISR) marker ATF4 and decreased protein synthesis in the VAPB P56S iPSC-derived MNs. Chemical inhibition of ISR with the compound, ISRIB, rescued all MN disease phenotype in VAPB P56S MNs. Thus, our results not only support ISR inhibition as a potential therapeutic target for ALS patients, but also provides evidence to pathogenesis.

Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models; however, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular fate under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate cell fate. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby successfully avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Mitochondrial ncRNA LDL-805 declines in alveolar epithelial type 2 cells of chronic obstructive pulmonary disease patients.

Rationale: We showed that levels of a murine mitochondrial noncoding RNA, mito-ncR-LDL805 , increase in alveolar epithelial type 2 cells exposed to extracts from cigarette smoke. The transcripts translocate to the nucleus, upregulating nucleus-encoded mitochondrial genes and mitochondrial bioenergetics. This response is lost after chronic exposure to smoke in a mouse model of chronic obstructive pulmonary disease. Objectives: To determine if mito-ncR-LDL805 plays a role in human disease, this study aimed to (i) identify the human homologue, (ii) test if the smoke-induced response occurs in human cells, (ii) determine causality between the subcellular localization of the transcript and increased mitochondrial bioenergetics, and (iii) analyze mito-ncR-LDL805 transcript levels in samples from patients with chronic obstructive pulmonary disease. Methods: Levels and subcellular localization of the human homologue identified from an RNA transcript library were assessed in human alveolar epithelial type 2 cells exposed to smoke extract. Lipid nanoparticles were used for nucleus-targeted delivery of mito-ncR-LDL805 transcripts. Analyses included in situ hybridization, quantitative PCR, cell growth, and Seahorse mitochondrial bioenergetics assays. Measurements and Main Results: The levels of human homologue transiently increased and the transcripts translocated to the nuclei in human cells exposed to smoke extract. Targeted nuclear delivery of transcripts increased mitochondrial bioenergetics. Alveolar cells from humans with chronic obstructive pulmonary disease had reduced levels of the mito-ncR-LDL805 . Conclusions: mito-ncR-LDL805 mediates mitochondrial bioenergetics in murine and human alveolar epithelial type 2 cells in response to cigarette smoke exposure, but this response is likely lost in diseases associated with chronic smoking, such as chronic obstructive pulmonary disease, due to its diminished levels. Impact: This study describes a novel mechanism by which epithelial cells in the lungs adapt to the mitochondrial stress triggered by exposure to cigarette smoke. We show that a noncoding RNA in mitochondria is upregulated and translocated to the nuclei of alveolar epithelial type 2 cells to trigger expression of genes that restore mitochondrial bioenergetics. Mitochondria function and levels of the noncoding RNA decrease under conditions that lead to chronic obstructive pulmonary disease, suggesting that the mitochondrial noncoding RNA can serve as potential therapeutic target to restore function to halt disease progression.

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.

Stress-induced ß cell early senescence confers protection against type 1 diabetes.

During the progression of type 1 diabetes (T1D), ß cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve ß cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in ß cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the ß cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced ß cells, the reduction of ß cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual ß cells of T1D patients. Our findings reveal a previously unrecognized link between ß cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8 h hypoxia followed by 16 h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72 h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

Adaptation to chronic ER stress enforces pancreatic ß-cell plasticity.

Pancreatic ß-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise ß-cell identity is unknown. We show here under reversible, chronic stress conditions ß-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of ß-cell function and identity. Upon recovery from stress, ß-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while ß-cells show resilience to episodic ER stress, when episodes exceed a threshold, ß-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest ß-cell adaptive exhaustion contributes to diabetes pathogenesis.

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics.

Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the "functional bit". Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.

eIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

The integrated stress response is tumorigenic and constitutes a therapeutic liability in KRAS-driven lung cancer.

The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. Here we demonstrate that ISR is pivotal in lung adenocarcinoma (LUAD) development, the most common histological type of lung cancer and a leading cause of cancer death worldwide. Increased phosphorylation of the translation initiation factor eIF2 (p-eIF2α), the focal point of ISR, is related to invasiveness, increased growth, and poor outcome in 928 LUAD patients. Dissection of ISR mechanisms in KRAS-driven lung tumorigenesis in mice demonstrated that p-eIF2α causes the translational repression of dual specificity phosphatase 6 (DUSP6), resulting in increased phosphorylation of the extracellular signal-regulated kinase (p-ERK). Treatments with ISR inhibitors, including a memory-enhancing drug with limited toxicity, provides a suitable therapeutic option for KRAS-driven lung cancer insofar as they substantially reduce tumor growth and prolong mouse survival. Our data provide a rationale for the implementation of ISR-based regimens in LUAD treatment.

Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR.

In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPß, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPß is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.

Autophagy impairment as a key feature for acetaminophen-induced ototoxicity.

Macroautophagy/autophagy is a highly conserved self-digestion pathway that plays an important role in cytoprotection under stress conditions. Autophagy is involved in hepatotoxicity induced by acetaminophen (APAP) in experimental animals and in humans. APAP also causes ototoxicity. However, the role of autophagy in APAP-induced auditory hair cell damage is unclear. In the present study, we investigated autophagy mechanisms during APAP-induced cell death in a mouse auditory cell line (HEI-OC1) and mouse cochlear explant culture. We found that the expression of LC3-II protein and autophagic structures was increased in APAP-treated HEI-OC1 cells; however, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence, and the activity of lysosomal enzymes decreased in APAP-treated HEI-OC1 cells. The degradation of p62 protein and the expression of lysosomal enzymes also decreased in APAP-treated mouse cochlear explants. These data indicate that APAP treatment compromises autophagic degradation and causes lysosomal dysfunction. We suggest that lysosomal dysfunction may be directly responsible for APAP-induced autophagy impairment. Treatment with antioxidant N-acetylcysteine (NAC) partially alleviated APAP-induced autophagy impairment and apoptotic cell death, suggesting the involvement of oxidative stress in APAP-induced autophagy impairment. Inhibition of autophagy by knocking down of Atg5 and Atg7 aggravated APAP-induced ER and oxidative stress and increased apoptotic cell death. This study provides a better understanding of the mechanism responsible for APAP ototoxicity, which is important for future exploration of treatment strategies for the prevention of hearing loss caused by ototoxic medications.