Krüppel-like factor 6 involvement in the endoplasmic reticulum homeostasis of extravillous trophoblasts.

INTRODUCTION: Trophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis. MATERIALS AND METHODS: Protein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized. RESULTS: KLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased. DISCUSSION: Results provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.

Unfolded protein response markers Grp78 and eIF2alpha are upregulated with increasing alpha-synuclein levels in Lewy body disease.

AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

The role of interleukin-10 in mitigating endoplasmic reticulum stress in aged mice through exercise.

Although unfolded protein response (UPR) is essential for cellular protection, its prolonged activation may induce apoptosis, compromising cellular longevity. The aging process increases the endoplasmic reticulum (ER) stress in skeletal muscle. However, whether combined exercise can prevent age-induced ER stress in skeletal muscle remains unknown. Evidence suggests that ER stress may increase inflammation by counteracting the positive effects of interleukin-10 (IL-10), whereas its administration in cells inhibits ER stress and apoptosis. This study verified the effects of aging and combined exercise on physical performance, ER stress markers, and inflammation in the quadriceps of mice. Moreover, we verified the effects of IL-10 on ER stress markers. C57BL/6 mice were distributed into young (Y, 6 mo old), old sedentary (OS, sedentary, 24 mo old), and old trained group (OT, submitted to short-term combined exercise, 24 mo old). To clarify the role of IL-10 in UPR pathways, knockout mice lacking IL-10 were used. The OS mice presented worse physical performance and higher ER stress-related proteins, such as C/EBP homologous protein (CHOP) and phospho-eukaryotic translation initiation factor 2 alpha (p-eIF2α/eIF2α). The exercise protocol increased muscle strength and IL-10 protein levels in OT while inducing the downregulation of CHOP protein levels compared with OS. Furthermore, mice lacking IL-10 increased BiP, CHOP, and p-eIF2α/eIF2α protein levels, indicating this cytokine can regulate the ER stress response in skeletal muscle. Bioinformatics analysis showed that endurance and resistance training downregulated DNA damage inducible transcript 3 (DDIT3) and XBP1 gene expression in the vastus lateralis of older people, reinforcing our findings. Thus, combined exercise is a potential therapeutic intervention for promoting adjustments in ER stress markers in aged skeletal muscle.NEW & NOTEWORTHY Aging elevates endoplasmic reticulum (ER) stress in skeletal muscle, potentially heightening inflammation by opposing interleukin-10 (IL-10) effects. This study found that short-term combined exercise boosted strength and IL-10 protein levels while reducing CHOP protein levels in older mice. In addition, IL-10-deficient mice exhibited increased ER stress markers, highlighting IL-10's role in regulating ER stress in skeletal muscle. Consequently, combined exercise emerges as a therapeutic intervention to elevate IL-10 and adjust ER stress markers in aging.

Endoplasmic reticulum stress in pancreatic ß-cell dysfunctionality and diabetes mellitus: a promising target for generation of functional hPSC-derived ß-cells in vitro.

Diabetes mellitus (DM), is a chronic disorder characterized by impaired glucose homeostasis that results from the loss or dysfunction of pancreatic ß-cells leading to type 1 diabetes (T1DM) and type 2 diabetes (T2DM), respectively. Pancreatic ß-cells rely to a great degree on their endoplasmic reticulum (ER) to overcome the increased secretary need for insulin biosynthesis and secretion in response to nutrient demand to maintain glucose homeostasis in the body. As a result, ß-cells are potentially under ER stress following nutrient levels rise in the circulation for a proper pro-insulin folding mediated by the unfolded protein response (UPR), underscoring the importance of this process to maintain ER homeostasis for normal ß-cell function. However, excessive or prolonged increased influx of nascent proinsulin into the ER lumen can exceed the ER capacity leading to pancreatic ß-cells ER stress and subsequently to ß-cell dysfunction. In mammalian cells, such as ß-cells, the ER stress response is primarily regulated by three canonical ER-resident transmembrane proteins: ATF6, IRE1, and PERK/PEK. Each of these proteins generates a transcription factor (ATF4, XBP1s, and ATF6, respectively), which in turn activates the transcription of ER stress-inducible genes. An increasing number of evidence suggests that unresolved or dysregulated ER stress signaling pathways play a pivotal role in ß-cell failure leading to insulin secretion defect and diabetes. In this article we first highlight and summarize recent insights on the role of ER stress and its associated signaling mechanisms on ß-cell function and diabetes and second how the ER stress pathways could be targeted in vitro during direct differentiation protocols for generation of hPSC-derived pancreatic ß-cells to faithfully phenocopy all features of bona fide human ß-cells for diabetes therapy or drug screening.

The unfolded protein response machinery in glioblastoma genesis, chemoresistance and as a druggable target.

BACKGROUND: The role of the unfolded protein response (UPR) has been progressively unveiled over the last decade and several studies have investigated its implication in glioblastoma (GB) development. The UPR restores cellular homeostasis by triggering the folding and clearance of accumulated misfolded proteins in the ER consecutive to endoplasmic reticulum stress. In case it is overwhelmed, it induces apoptotic cell death. Thus, holding a critical role in cell fate decisions. METHODS: This article, reviews how the UPR is implicated in cell homeostasis maintenance, then surveils the evidence supporting the UPR involvement in GB genesis, progression, angiogenesis, GB stem cell biology, tumor microenvironment modulation, extracellular matrix remodeling, cell fate decision, invasiveness, and grading. Next, it concurs the evidence showing how the UPR mediates GB chemoresistance-related mechanisms. RESULTS: The UPR stress sensors IRE1, PERK, and ATF6 with their regulator GRP78 are upregulated in GB compared to lower grade gliomas and normal brain tissue. They are activated in response to oncogenes and are implicated at different stages of GB progression, from its genesis to chemoresistance and relapse. The UPR arms can be effectors of apoptosis as mediators or targets. CONCLUSION: Recent research has established the role of the UPR in GB pathophysiology and chemoresistance. Targeting its different sensors have shown promising in overcoming GB chomo- and radioresistance and inducing apoptosis.

Mitochondrial unfolded protein response mechanism and its cardiovascular protective effects.

The mitochondrial unfolded protein response (UPRmt) is a cytoprotective response in response to cellular stress that is activated in response to mitochondrial stress to maintain intra-protein homeostasis, thereby protecting the cell from a variety of stimuli. The activation of this response has been linked to cardiovascular diseases. Here, we reviewed the current understanding of UPRmt and discussed its specific molecular mechanism, mainly in mammals, as well as addressing its protective role against cardiovascular diseases, so as to provide direction for further research on UPRmt and therapies targeting cardiovascular diseases in the future.

Endoplasmic reticulum stress mechanisms and exercise intervention in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a metabolic disease primarily characterized by insulin resistance (IR) and insufficient insulin secretion. The unfolded protein response (UPR) overactivation induced by endoplasmic reticulum stress (ERS) appears to play a key role in this process, although the exact pathogenesis of T2DM is not fully understood. Studies have demonstrated that appropriate exercise can regulate ERS in the heart, liver, pancreas, skeletal muscle, and other body tissues leading to an improvement in diabetes and its complications. However, the exact mechanism remains unclear. By analyzing the relationship between ERS, T2DM pathology, and exercise intervention, this review concludes that exercise can increase insulin sensitivity, inhibit IR, promote insulin secretion and alleviate T2DM by regulating ERS. This paper specifically reviews the signaling pathways by which ERS induces diabetes, the mechanisms of exercise regulation of ERS in diabetes, and the varying effects of different types of exercise on diabetes improvement through ERS mechanisms. Physical exercise is an effective non-pharmacological intervention for T2DM. Thus, further exploration of how exercise regulates ERS in diabetes could refine "precision exercise medicine" for diabetes and identify new drug targets.

The role of taurine through endoplasmic reticulum in physiology and pathology.

Taurine is a sulfur-containing amino acid found in many cell organelles that plays a wide range of biological roles, including bile salt production, osmoregulation, oxidative stress reduction, and neuromodulation. Taurine treatments have also been shown to ameliorate the onset and development of many diseases, including hypertension, fatty liver, neurodegenerative diseases and ischemia-reperfusion injury, by exerting antioxidant, anti-inflammatory, and antiapoptotic effects. The endoplasmic reticulum (ER) is a dynamic organelle involved in a wide range of cellular functions, including lipid metabolism, calcium storage and protein stabilization. Under stress, the disruption of the ER environment leads to the accumulation of misfolded proteins and a characteristic stress response called the unfolded protein response (UPR). The UPR protects cells from stress and helps to restore cellular homeostasis, but its activation promotes cell death under prolonged ER stress. Recent studies have shown that ER stress is closely related to the onset and development of many diseases. This article reviews the beneficial effects and related mechanisms of taurine by regulating the ER in different physiological and pathological states, with the aim of providing a reference for further research and clinical applications.

Beyond insulin: Unraveling the complex interplay of ER stress, oxidative damage, and CFTR modulation in CFRD.

CF-related diabetes (CFRD) is a prevalent comorbidity in people with Cystic Fibrosis (CF), significantly impacting morbidity and mortality rates. This review article critically evaluates the current understanding of CFRD molecular mechanisms, including the role of CFTR protein, oxidative stress, unfolded protein response (UPR) and intracellular communication. CFRD manifests from a complex interplay between exocrine pancreatic damage and intrinsic endocrine dysfunction, further complicated by the deleterious effects of misfolded CFTR protein on insulin secretion and action. Studies indicate that ER stress and subsequent UPR activation play critical roles in both exocrine and endocrine pancreatic cell dysfunction, contributing to ß-cell loss and insulin insufficiency. Additionally, oxidative stress and altered calcium flux, exacerbated by CFTR dysfunction, impair ß-cell survival and function, highlighting the significance of antioxidant pathways in CFRD pathogenesis. Emerging evidence underscores the importance of exosomal microRNAs (miRNAs) in mediating inflammatory and stress responses, offering novel insights into CFRD's molecular landscape. Despite insulin therapy remaining the cornerstone of CFRD management, the variability in response to CFTR modulators underscores the need for personalized treatment approaches. The review advocates for further research into non-CFTR therapeutic targets, emphasizing the need to address the multifaceted pathophysiology of CFRD. Understanding the intricate mechanisms underlying CFRD will pave the way for innovative treatments, moving beyond insulin therapy to target the disease's root causes and improve the quality of life for individuals with CF.

Oscillations in Neuronal Activity: A Neuron-Centered Spatiotemporal Model of the Unfolded Protein Response in Prion Diseases.

Many neurodegenerative diseases (NDs) are characterized by the slow spatial spread of toxic protein species in the brain. The toxic proteins can induce neuronal stress, triggering the Unfolded Protein Response (UPR), which slows or stops protein translation and can indirectly reduce the toxic load. However, the UPR may also trigger processes leading to apoptotic cell death and the UPR is implicated in the progression of several NDs. In this paper, we develop a novel mathematical model to describe the spatiotemporal dynamics of the UPR mechanism for prion diseases. Our model is centered around a single neuron, with representative proteins P (healthy) and S (toxic) interacting with heterodimer dynamics (S interacts with P to form two S's). The model takes the form of a coupled system of nonlinear reaction-diffusion equations with a delayed, nonlinear flux for P (delay from the UPR). Through the delay, we find parameter regimes that exhibit oscillations in the P- and S-protein levels. We find that oscillations are more pronounced when the S-clearance rate and S-diffusivity are small in comparison to the P-clearance rate and P-diffusivity, respectively. The oscillations become more pronounced as delays in initiating the UPR increase. We also consider quasi-realistic clinical parameters to understand how possible drug therapies can alter the course of a prion disease. We find that decreasing the production of P, decreasing the recruitment rate, increasing the diffusivity of S, increasing the UPR S-threshold, and increasing the S clearance rate appear to be the most powerful modifications to reduce the mean UPR intensity and potentially moderate the disease progression.

Tissue-specific roles of mitochondrial unfolded protein response during obesity.

Obesity is a worldwide multifactorial disease caused by an imbalance in energy metabolism, increasing adiposity, weight gain, and promoting related diseases such as diabetes, cardiovascular diseases, neurodegeneration, and cancer. Recent findings have reported that metabolic stress related to obesity induces a mitochondrial stress response called mitochondrial unfolded protein response (UPRmt), a quality control pathway that occurs in a nuclear DNA-mitochondria crosstalk, causing transduction of chaperones and proteases under stress conditions. The duality of UPRmt signaling, with both beneficial and detrimental effects, acts in different contexts depending on the tissue, cell type, and physiological states, affecting the mitochondrial function and efficiency and the metabolism homeostasis during obesity, which remains not fully clarified. Therefore, this review discusses the most recent findings regarding UPRmt signaling during obesity, bringing an overview of UPRmt across different metabolic tissues.

Newsights of endoplasmic reticulum in hypoxia.

The endoplasmic reticulum (ER) is important to cells because of its essential functions, including synthesizing three major nutrients and ion transport. When cellular homeostasis is disrupted, ER quality control (ERQC) system is activated effectively to remove misfolded and unfolded proteins through ER-phagy, ER-related degradation (ERAD), and molecular chaperones. When unfolded protein response (UPR) and ER stress are activated, the cell may be suffering a huge blow, and the most probable consequence is apoptosis. The membrane contact points between the ER and sub-organelles contribute to communication between the organelles. The decrease in oxygen concentration affects the morphology and structure of the ER, thereby affecting its function and further disrupting the stable state of cells, leading to the occurrence of disease. In this study, we describe the functions of ER-, ERQC-, and ER-related membrane contact points and their changes under hypoxia, which will help us further understand ER and treat ER-related diseases.

The Role of Endoplasmic Reticulum Stress in Metabolic Diseases.

The endoplasmic reticulum (ER), the center of protein folding, also controls the cell's life-and-death signaling mechanisms. ER stress caused by unfolded or misfolded proteins leads to the activation of the unfolded protein response (UPR) in the cell. The UPR utilizes three main signaling pathways to restore disrupted ER homeostasis. These signaling pathways are protein kinase R-like endoplasmic reticulum kinase, inositol-requiring enzyme 1, and activating transcription factor 6. Studies have reported that ER stress (ERS) plays a role in the pathogenesis of metabolic disorders such as diabetes, obesity, atherosclerosis, and nonalcoholic liver disease. This review will briefly discuss the ERS response in these metabolic diseases.

Gene-expression profiling of individuals resilient to Alzheimer's disease reveals higher expression of genes related to metallothionein and mitochondrial processes and no changes in the unfolded protein response.

Some individuals show a discrepancy between cognition and the amount of neuropathological changes characteristic for Alzheimer's disease (AD). This phenomenon has been referred to as 'resilience'. The molecular and cellular underpinnings of resilience remain poorly understood. To obtain an unbiased understanding of the molecular changes underlying resilience, we investigated global changes in gene expression in the superior frontal gyrus of a cohort of cognitively and pathologically well-defined AD patients, resilient individuals and age-matched controls (n = 11-12 per group). 897 genes were significantly altered between AD and control, 1121 between resilient and control and 6 between resilient and AD. Gene set enrichment analysis (GSEA) revealed that the expression of metallothionein (MT) and of genes related to mitochondrial processes was higher in the resilient donors. Weighted gene co-expression network analysis (WGCNA) identified gene modules related to the unfolded protein response, mitochondrial processes and synaptic signaling to be differentially associated with resilience or dementia. As changes in MT, mitochondria, heat shock proteins and the unfolded protein response (UPR) were the most pronounced changes in the GSEA and/or WGCNA, immunohistochemistry was used to further validate these processes. MT was significantly increased in astrocytes in resilient individuals. A higher proportion of the mitochondrial gene MT-CO1 was detected outside the cell body versus inside the cell body in the resilient compared to the control group and there were higher levels of heat shock protein 70 (HSP70) and X-box-binding protein 1 spliced (XBP1s), two proteins related to heat shock proteins and the UPR, in the AD donors. Finally, we show evidence for putative sex-specific alterations in resilience, including gene expression differences related to autophagy in females compared to males. Taken together, these results show possible mechanisms involving MTs, mitochondrial processes and the UPR by which individuals might maintain cognition despite the presence of AD pathology.

Acute Exercise Increases GDF15 and Unfolded Protein Response/Integrated Stress Response in Muscle in Type 2 Diabetes.

CONTEXT: Regular exercise is a key prevention strategy for obesity and type 2 diabetes (T2D). Exerkines secreted in response to exercise or recovery may contribute to improved systemic metabolism. Conversely, an impaired exerkine response to exercise and recovery may contribute to cardiometabolic diseases. OBJECTIVE: We investigated if the exercise-induced regulation of the exerkine, growth differentiation factor 15 (GDF15) and its putative upstream regulators of the unfolded protein response (UPR)/integrated stress response (ISR) is impaired in skeletal muscle in patients with T2D compared with weight-matched glucose-tolerant men. METHODS: Thirteen male patients with T2D and 14 age- and weight-matched overweight/obese glucose-tolerant men exercised at 70% of VO2max for 1 hour. Blood and skeletal muscle biopsies were sampled before, immediately after, and 3 hours into recovery. Serum and muscle transcript levels of GDF15 and key markers of UPR/ISR were determined. Additionally, protein/phosphorylation levels of key regulators in UPR/ISR were investigated. RESULTS: Acute exercise increased muscle gene expression and serum GDF15 levels in both groups. In recovery, muscle expression of GDF15 decreased toward baseline, whereas serum GDF15 remained elevated. In both groups, acute exercise increased the expression of UPR/ISR markers, including ATF4, CHOP, EIF2K3 (encoding PERK), and PPP1R15A (encoding GADD34), of which only CHOP remained elevated 3 hours into recovery. Downstream molecules of the UPR/ISR including XBP1-U, XBP1-S, and EDEM1 were increased with exercise and 3 hours into recovery in both groups. The phosphorylation levels of eIF2α-Ser51, a common marker of unfolded protein response (UPR) and ISR, increased immediately after exercise in controls, but decreased 3 hours into recovery in both groups. CONCLUSION: In conclusion, exercise-induced regulation of GDF15 and key markers of UPR/ISR are not compromised in patients with T2D compared with weight-matched controls.

Potential Role of Endoplasmic Reticulum Stress in Modulating Protein Homeostasis in Oligodendrocytes to Improve White Matter Injury in Preterm Infants.

Preterm white matter injury (WMI) is a demyelinating disease with high incidence and mortality in premature infants. Oligodendrocyte cells (OLs) are a specialized glial cell that produces myelin proteins and adheres to the axons providing energy and metabolic support which susceptible to endoplasmic reticulum protein quality control. Disruption of cellular protein homeostasis led to OLs dysfunction and cell death, immediately, the unfolded protein response (UPR) activated to attempt to restore the protein homeostasis via IRE1/XBP1s, PERK/eIF2α and ATF6 pathway that reduced protein translation, strengthen protein-folding capacity, and degraded unfolding/misfolded protein. Moreover, recent works have revealed the conspicuousness function of ER signaling pathways in regulating influenced factors such as calcium homeostasis, mitochondrial reactive oxygen generation, and autophagy activation to regulate protein hemostasis and improve the myelination function of OLs. Each of the regulation modes and their corresponding molecular mechanisms provides unique opportunities and distinct perspectives to obtain a deep understanding of different actions of ER stress in maintaining OLs' health and function. Therefore, our review focuses on summarizing the current understanding of ER stress on OLs' protein homeostasis micro-environment in myelination during white matter development, as well as the pathophysiology of WMI, and discussing the further potential experimental therapeutics targeting these factors that restore the function of the UPR in OLs myelination function.

A cytosolic surveillance mechanism activates the mitochondrial UPR.

The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. METHODS: We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. RESULTS: ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. CONCLUSIONS: ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

C9orf72 regulates the unfolded protein response and stress granule formation by interacting with eIF2α.

Rationale: A C9orf72 hexanucleotide repeat expansion (GGGGCC) is the most common genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Haploinsufficiency of C9orf72 has been proposed as a possible disease mechanism (loss-of-function mechanism). Additionally, the aberrantly activated unfolded protein response (UPR) and stress granule (SG) formation are associated with the etiopathology of both ALS and FTD. However, the molecular determinants in this pathogenesis are not well characterized. Methods: We performed an immunoprecipitation-mass spectrometry (IP-MS) assay to identify potential proteins interacting with the human C9orf72 protein. We used C9orf72 knockout cell and rat models to determine the roles of C9orf72 in translation initiation and the stress response. Results: Here, we show that C9orf72, which is genetically and pathologically related to ALS and FTD, interacts with eukaryotic initiation factor 2 subunit alpha (eIF2α) and regulates its function in translation initiation. C9orf72 knockout weakens the interaction between eIF2α and eIF2B5, leading to global translation inhibition. Moreover, the loss of C9orf72 results in primary ER stress with activated UPR in rat spleens, which is one of the causes of splenomegaly with inflammation in C9orf72 -/- rats. Finally, C9orf72 delays SG formation by interacting with eIF2α in stressed cells. Conclusions: In summary, these data reveal that C9orf72 modulates translation initiation, the UPR and SG formation, which have implications for understanding ALS/FTD pathogenesis.