A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride-induced neuronal death.

Autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis. Dysfunction of autophagy is associated with the progression of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although one of the typical features of brain aging is an accumulation of redox-active metals that eventually lead to neurodegeneration, a plausible link between trace metal-induced neurodegeneration and dysregulated autophagy has not been clearly determined. Here, we used a cupric chloride-induced neurodegeneration model in MN9D dopaminergic neuronal cells along with ultrastructural and biochemical analyses to demonstrate impaired autophagic flux with accompanying lysosomal dysfunction. We found that a surge of cytosolic calcium was involved in cupric chloride-induced dysregulated autophagy. Consequently, buffering of cytosolic calcium by calbindin-D28K overexpression or co-treatment with the calcium chelator BAPTA attenuated the cupric chloride-induced impairment in autophagic flux by ameliorating dysregulation of lysosomal function. Thus, these events allowed the rescue of cells from cupric chloride-induced neuronal death. These phenomena were largely confirmed in cupric chloride-treated primary cultures of cortical neurons. Taken together, these results suggest that abnormal accumulation of trace metal elements and a resultant surge of cytosolic calcium leads to neuronal death by impairing autophagic flux at the lysosomal level.

Nephrin expression in human epidermal keratinocytes and its implication in poor wound closure.

Nephrin is a type-1 transmembrane protein and a component of the slit diaphragm renal-filtration barrier. It has several functions in actin remodeling and cell-cell adhesion. Nephrin is principally located in the kidney glomerulus, but several studies have reported that nephrin is found in the pancreas, brain, and placenta. However, nephrin expression and its role in human skin have not yet been reported. First, using single-cell RNA sequencing, immunohistochemistry, and immuno-electron microscopy, nephrin expression was confirmed in human-skin epidermal keratinocytes. Nephrin expression colocalized with the expression of zonula occludens-1 in keratinocytes and was closely related to keratinocyte cell density, proliferation, and migration. High glucose treatment decreased nephrin expression and compromised keratinocyte cell migration without yes-associated protein nuclear entry. This reduced cell migration under high glucose conditions was improved in nephrin-overexpressing keratinocytes. Nephrin was highly expressed on the margins of re-epithelized epidermis based on in vivo mice and ex vivo human skin wound models. The results demonstrate that nephrin is expressed in human-skin keratinocytes and functions in cell adhesion, proliferation, and migration. In conclusion, this study suggests that nephrin may have a variety of physiological roles in human skin.

A Novel Knitted Polytetrafluoroethylene Patch for Cardiovascular Surgery.

Polytetrafluoroethylene (PTFE) is widely used in cardiovascular surgeries; however, postoperative complications such as thrombosis, calcification, and neointimal hyperplasia are yet to be resolved in patients. We developed two types of novel knitted PTFE patches and evaluated them using a swine model. Both patches were composed of knitted PTFE impregnated with micro-PTFE particles, and one of them was pressed after PTFE impregnation. Twenty micromini pigs were used in this study. After left lateral thoracotomy, the new patches (n = 8 for each type of patch) were implanted into the descending aorta and left atrium for the high- and low-pressure models, respectively. Clinically used expanded PTFE (ePTFE) patches were used as the control material (n = 4). The patches were explanted and histopathologically examined at 4, 12, and 24 weeks after implantation. A tensile test was also applied to the high-pressure model at 12 and 24 weeks. As a result, there was no significant difference noted in the tensile test, intimal hyperplasia thickness, or endothelialization among the three patches. In contrast, the degree of macrophage infiltration into the patches and the degree of macrophage, lymphocyte, and granulocyte infiltration outside the patches were lower in the new patches than in the control ePTFE. The degree of cellular infiltration outside new patches decreased over time. There were no significant differences between the two new patch types in these results. In conclusion, our novel knitted PTFE patch showed noninferiority in durability and intensity and less inflammatory responses than a clinically used ePTFE patch.

Impact of Complete Revascularization on Long-Term Outcomes After Coronary Artery Bypass Grafting in Patients With Left Ventricular Dysfunction.

BACKGROUND: The long-term outcomes of complete revascularization (CR) in patients with left ventricular (LV) dysfunction undergoing coronary artery bypass grafting (CABG) remain unclear. Methods and Results: We evaluated a consecutive series of 111 patients with LV ejection fraction ≤35% who underwent isolated first-time CABG: 63 underwent CR and 48 underwent incomplete revascularization (IR). At a median follow-up of 10.1 years, the rates of death from any cause, cardiac death, and major adverse cardiac and cerebrovascular events (MACCE) were significantly greater in the IR group. After adjusting for propensity score, no significant difference was found between the CR and IR groups regarding death from any cause (hazard ratio [HR], 1.45; 95% CI: 0.75-2.81; P=0.271) and cardiac death (HR, 1.45; 95% CI: 0.68-3.10; P=0.337). In contrast, IR increased the risk of MACCE (HR, 1.92; 95% CI: 1.08-3.41; P=0.027), which was principally attributed to an increased risk of repeat revascularization (HR, 3.92; 95% CI: 1.34-11.44; P=0.013). CONCLUSIONS: Although IR was not significantly associated with an increased risk of long-term mortality in patients with LV dysfunction who underwent CABG, CR might reduce the risks of repeat revascularization and subsequent MACCE.

Surgical Treatment of Coronary to Pulmonary Artery Fistulas in Adults.

Coronary to pulmonary artery fistulas (CPAFs) are abnormal communications between the coronary and pulmonary arteries. They are an uncommon congenital heart disease and usually remain asymptomatic until later in life. However, there is no consensus on their management. We present four adult patients who required surgery for coronary to pulmonary artery fistulas to illuminate this issue. The clinical presentations were variable depending on the anatomical features of coronary to pulmonary artery fistulas and the presence or absence of other cardiac diseases. We successfully performed surgical closure of the coronary to pulmonary artery fistulas in each of the cases. In this report, we describe our experience with these cases and outline the available therapeutic strategies and treatment options for coronary to pulmonary artery fistulas.

[Coronary-pulmonary Artery Fistula in a Hemodialysis Patient].

We report a case of coronary-pulmonary artery fistula in a hemodialysis patient treated surgically. A 47-year-old man was admitted with chest pain and exertional dyspnea that progressively worsened over the previous 2 months. He had a history of end-stage renal disease and has been on hemodialysis for 5 years. Coronary angiography and computed tomography revealed coronary-pulmonary artery fistula that originated from both the left anterior descending coronary artery and the right coronary artery. Under cardiopulmonary bypass, we opened the pulmonary artery and closed the fistula. The symptoms of the patient improved after the surgery, and he was discharged without major complications. Coronary-pulmonary artery fistulas in hemodialysis patients are rare. The presence of additional arteriovenous fistula used for dialysis in these patients increases the left-to-right shunt flow and might cause progression of heart failure. Even if the left-to-right shunt volume is small, surgical treatment is highly recommended at an early stage of the coronary-pulmonary artery fistula in hemodialysis patients.

CXXC5 plays a role as a transcription activator for myelin genes on oligodendrocyte differentiation.

Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC-type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/ß-catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA-binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5(-/-) mice, the expressions of myelin genes were simultaneously reduced. The CXXC5(-/-) mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5(-/-) mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum.

O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster.

O-GlcNAcylation is a dynamic post-translational modification that takes place on ser/thr residues of nucleocytoplasmic proteins. O-GlcNAcylation regulates almost all cellular events as a nutrient sensor, a transcriptional and translational regulator, and a disease-related factor. Although the role of O-GlcNAcylation in insulin signaling and metabolism are well established, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we manipulated O-GlcNAcylation in Drosophila and found that it regulates autophagy through Akt/dFOXO signaling. We demonstrate that O-GlcNAcylation and the levels of O-GlcNAc transferase (OGT) are increased during starvation. Furthermore, Atg proteins and autolysosomes are increased in OGT-reduced flies without fasting. Atg proteins and autophagosomes are reduced in OGT-overexpressing flies. Our results suggest that not only autophagy gene expression but also autophagic structures are regulated by OGT through Akt and dFOXO. These data imply that O-GlcNAcylation is important in modulating autophagy as well as insulin signaling in Drosophila.

CCCTC-binding factor controls the homeostatic maintenance and migration of Langerhans cells.

BACKGROUND: Langerhans cells (LCs) are skin-resident dendritic cells (DCs) that orchestrate skin immunity. CCCTC-binding factor (CTCF) is a highly conserved DNA-binding protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. OBJECTIVE: We sought to clarify a possible role for CTCF in LC homeostasis and function in vivo. METHODS: We used a conditional gene deletion mouse system to generate DC- and LC-specific CTCF-ablated mice. Short hairpin RNA-mediated RNA interference was used to silence CTCF expression in human monocyte-derived Langerhans cells. DC populations were assessed by using flow cytometry and immunofluorescence. Gene expression arrays were performed to identify genes regulated by CTCF in LCs. Contact hypersensitivity and epicutaneous sensitization responses were measured to examine the functional significance of CTCF ablation. RESULTS: DC-specific CTCF deletion led to a reduced pool of systemic DCs, with LCs most severely affected. Decreases in epidermal LC numbers were specifically associated with self-turnover defects. Interestingly, CTCF-deficient LCs demonstrated impaired migration out of the epidermis. Whole-transcriptome analyses revealed that genes that promoted cell adhesion were highly expressed, but CCR7 was downregulated in CTCF-depleted LCs. Hapten-induced contact hypersensitivity responses were more sustained in LC-specific CTCF-deficient mice, whereas epicutaneous sensitization to protein antigen was attenuated, indicating that CTCF-dependent LC homeostasis is required for optimal immune function of LCs in a context-dependent manner. CONCLUSION: Our results show that CTCF positively regulates the homeostatic pool and the efficient emigration of LCs, which are required for modulating the functional immune network of the skin.

[Unroofed Coronary Sinus in an Adult; Report of a Case].

Unroofed coronary sinus( UCS) is a rare cardiac anomaly and the most uncommon type of atrial septal defect. It is rarely diagnosed by only echocardiography prior to surgery. We herein report a case of UCS (partially unroofed terminal portion) without persistent left superior vena cava that was precisely diagnosed by cardiac multi-detector low computed tomography (MDCT). A 50-year-old asymptomatic man was admitted for cardiac evaluation. A transesophageal echocardiography showed a left to right shunt at the atrial level. MDCT clearly showed a defect in the coronary sinus and lower part of the interatrial septum. At the time of surgery, the defect was closed using a Gore-Tex patch, such that the coronary sinus drained entirely into the left atrium. The postoperative course was uneventful. Cardiac MDCT provides accurate anatomic details about defects as well as the associated anomalies of the heart and pulmonary vasculature.

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase.

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407-4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434-1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy.

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.

Coronary artery spasm following off-pump coronary artery bypass surgery.

Coronary artery spasm after coronary artery bypass surgery may result in life-threatening arrhythmias, circulatory collapse, or death. We report two cases of coronary artery spasm after coronary artery bypass surgery, one of which developed ventricular fibrillation requiring extracorporeal membrane oxygenation support. Both patients were discharged in good condition and are currently followed as outpatients. Unexpected sudden hemodynamic compromise could be due to coronary vasospasm, and this should be considered as one of the possible differential diagnoses. We were able to prevent the lethal consequences seen with coronary artery spasm by early diagnosis and management.

Intraoperative assessment for mitral valve competency in a beating heart under retrograde coronary perfusion.

Intraoperative assessment of a repaired mitral valve is of paramount importance for reparative mitral surgery. From September 2010 through November 2012, 20 consecutive patients underwent mitral valve plasty for mitral regurgitation. The patients who underwent surgery after June 2012 received assessment of the repair with the heart beating (HB group, n = 10), and the patients who underwent the operation before May 2012 were assessed for the repair only under cardioplegic heart arrest (non-HB group, n = 10). Intermittent cold retrograde blood cardioplegia was used in all patients. In the HB-group, after completion of the procedures, pump blood without a crystalloid additive was delivered into the coronary sinus. The function of the mitral valve was assessed under beating conditions. There were no differences between the two groups in aortic cross clamp time and operation time, although operative and concomitant procedures were slightly more complicated in the HB group than in the non-HB group. Postoperative echocardiography revealed none or mild mitral regurgitation in all the patients in both groups. Reopening of the closed left atrium for additional repair was necessary only in one patient in the HB group and 3 patients in the non-HB group. In conclusion, the method of perfusing the myocardium retrogradely via the coronary sinus with warm blood is safe and effective for assessing the competency of the mitral valve in a beating heart.

Outcomes of surgery for acute type A dissection in octogenarians versus non-octogenarians: a systematic review and meta analysis.

INTRODUCTION: Acute Type A Aortic Dissection (ATAAD) is a cardiothoracic emergency that requires urgent intervention. Elderly status, particularly age over 80, is an independent risk factor for mortality and morbidity. The mid-term outcomes of this age group are also unknown. This systematic review and meta-analysis of observational studies was therefore performed to analyse short- and mid-term mortality and morbidity in octogenarians following surgery for ATAAD. METHODS: A systematic review was conducted for studies published since January 2000. The primary endpoint was short-term mortality, either reported as 30-day mortality or in-hospital mortality and medium-term (five year) survival. Secondary endpoints were rates of postoperative complications, namely stroke, acute renal failure (ARF), re-exploration and intensive care unit (ICU) length of stay (LOS). RESULTS: A total of 16 retrospective studies, with a total of 16, 641 patients were included in the systematic review and meta-analysis. Pooled analysis demonstrated that octogenarian cohorts are at significantly higher risk of short-term mortality than non-octogenarians (OR 1.93; 95% CI 1.33-2.81; P < 0.001). Actuarial survival was significantly lower in the octogenarian cohort, with a five-year survival in the octogenarian cohort of 54% compared to 76% in the non-octogenarian cohort (P < 0.001). There were no significant differences between the cohorts in terms of secondary outcomes: stroke, ARF, re-exploration or ICU LOS. CONCLUSION: Octogenarians are twice as likely to die in the short-term following surgery for ATAAD and demonstrate a significantly lower five-year actuarial survival. Patients and family members should be well informed of the risks of surgery and suitable octogenarians selected for surgery.

Buffering of cytosolic calcium plays a neuroprotective role by preserving the autophagy-lysosome pathway during MPP+-induced neuronal death.

Parkinson's disease (PD) is a chronic neurodegenerative disease with no cure. Calbindin, a Ca2+-buffering protein, has been suggested to have a neuroprotective effect in the brain tissues of PD patients and in experimental models of PD. However, the underlying mechanisms remain elusive. Here, we report that in 1-methyl-4-phenylpyridinium (MPP+)-induced culture models of PD, the buffering of cytosolic Ca2+ by calbindin-D28 overexpression or treatment with a chemical Ca2+ chelator reversed impaired autophagic flux, protecting cells against MPP+-mediated neurotoxicity. When cytosolic Ca2+ overload caused by MPP+ was ameliorated, the MPP+-induced accumulation of autophagosomes decreased and the autophagic flux significantly increased. In addition, the accumulation of damaged mitochondria and p62-positive ubiquitinated protein aggregates, following MPP+ intoxication, was alleviated by cytosolic Ca2+ buffering. We showed that MPP+ treatment suppressed autophagic degradation via raising the lysosomal pH and therefore reducing cytosolic Ca2+ elevation restored the lysosomal pH acidity and normal autophagic flux. These results support the notion that functional lysosomes are required for Ca2+-mediated cell protection against MPP+-mediated neurotoxicity. Thus, our data suggest a novel process in which the modulation of Ca2+ confers neuroprotection via the autophagy-lysosome pathway. This may have implications for the pathogenesis and future therapeutic targets of PD.

Induction of IL-8 in periodontal ligament cells by H(2)O (2).

Periodontitis is an inflammatory disease caused by bacteria. In periodontitis, reactive oxygen species (ROS) are released from inflammatory cells in response to bacteria. Interleukin (IL)-8 is one of pro-inflammatory cytokines. To investigate the role of ROS in pathogenesis of periodontitis, we estimated the effect of H(2)O(2), one of ROS, on the expression of IL-8 in human periodontal ligament (PDL) cells. PDL cells were treated with H(2)O(2). IL-8 expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38) and c-jun NH(2)-terminal kinase (JNK) was estimated by Western blotting. Treatment with H(2)O(2) at concentration of up to 250 microM increased IL-8 mRNA expression and production in a concentration-dependent manner. However, treatment with 500 microM H(2)O(2) did not increase IL-8 production. Catalase, an inhibitor of H(2)O(2), down-regulated the production of IL-8 induced by H(2)O(2). H(2)O(2) increased the phosphorylation of ERK, p38, and JNK. Pretreatment with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor) decreased the IL-8 production induced by H(2)O(2). These results indicate that H(2)O(2) acts as an inducer of IL-8 secretion via activation of ERK, p38, and JNK in PDL cells. H(2)O(2) deposited in periodontal tissue during inflammation against bacteria may accelerate tissue destruction via induction of IL-8 in PDL cells.

Effectiveness and long-term outcomes of surgical intervention for constrictive epicardium in constrictive pericarditis.

OBJECTIVE: Epicardial resection is considered effective in patients with constrictive pericarditis accompanied by epicardial constriction, but few publications have evaluated its long-term outcomes. The study objective was to evaluate early and late results of epicardial resection. METHODS: A total of 22 patients with pericardiectomy for constrictive pericarditis were treated at our institution between 1989 and 2016. They were stratified by the presence or absence of constrictive epicardium and the surgical intervention performed. Group A (n = 7) included those with constrictive epicardium and epicardial resection in addition to pericardiectomy. Group B (n = 5) included those with constrictive epicardium and single pericardiectomy. Group C (n = 10) included those without constrictive epicardium and with a single pericardiectomy. Postsurgical changes were evaluated by echocardiography. RESULTS: No significant differences were found in preoperative baseline characteristics, operative details, and operative mortality among the groups. The median postoperative follow-up was 80 months. Group A patients did not experience cardiac events, and left ventricular end-diastolic volume and stroke volume were significantly improved at late follow-up compared with before and soon after surgery. The early postsurgical parameters were significantly improved in Group C patients, but the improvements were attenuated on late follow-up. Group B patients had the highest occurrence of cardiac events and no improvement in echocardiographic parameters. CONCLUSION: Surgical intervention in constrictive epicardium improved echocardiographic parameters and was free of cardiac events during long-term follow-up.

Dysregulated autophagy contributes to caspase-dependent neuronal apoptosis.

Autophagy is a regulated, intracellular degradation process that delivers unnecessary or dysfunctional cargo to the lysosome. Autophagy has been viewed as an adaptive survival response to various stresses, whereas in other cases, it promotes cell death. Therefore, both deficient and excessive autophagy may lead to cell death. In this study, we specifically attempted to explore whether and how dysregulated autophagy contributes to caspase-dependent neuronal cell death induced by the neurotoxin 6-hydroxydopamine (6-OHDA). Ultrastructural and biochemical analyses indicated that MN9D neuronal cells and primary cultures of cortical neurons challenged with 6-OHDA displayed typical features of autophagy. Cotreatment with chloroquine and monitoring autophagic flux by a tandem mRFP-EGFP-tagged LC3 probe indicated that the autophagic phenomena were primarily caused by dysregulated autophagic flux. Consequently, cotreatment with an antioxidant but not with a pan-caspase inhibitor significantly blocked 6-OHDA-stimulated dysregulated autophagy. These results indicated that 6-OHDA-induced generation of reactive oxygen species (ROS) played a critical role in triggering neuronal death by causing dysregulated autophagy and subsequent caspase-dependent apoptosis. The results of the MTT reduction, caspase-3 activation, and TUNEL assays indicated that pharmacological inhibition of autophagy using 3-methyladenine or deletion of the autophagy-related gene Atg5 significantly inhibited 6-OHDA-induced cell death. Taken together, our results suggest that abnormal induction of autophagic flux promotes apoptotic neuronal cell death, and that the treatments limiting dysregulated autophagy may have a strong neuroprotective potential.

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR.

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl- channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.