Small Extracellular Vesicles and Oxidative Pathophysiological Mechanisms in Retinal Degenerative Diseases.

This review focuses on the role of small extracellular vesicles in the pathophysiological mechanisms of retinal degenerative diseases. Many of these mechanisms are related to or modulated by the oxidative burden of retinal cells. It has been recently demonstrated that cellular communication in the retina involves extracellular vesicles and that their rate of release and cargo features might be affected by the cellular environment, and in some instances, they might also be mediated by autophagy. The fate of these vesicles is diverse: they could end up in circulation being used as markers, or target neighbor cells modulating gene and protein expression, or eventually, in angiogenesis. Neovascularization in the retina promotes vision loss in diseases such as diabetic retinopathy and age-related macular degeneration. The importance of micro RNAs, either as small extracellular vesicles' cargo or free circulating, in the regulation of retinal angiogenesis is also discussed.

Role of Exosomal miR-205-5p Cargo in Angiogenesis and Cell Migration.

Exosomes or small extracellular vesicles (sEVs) represent a pivotal component in intercellular communication, carrying a diverse array of biomolecules. Several factors can affect sEVs release dynamics, as occurs in hyperglycemia or inflammation. In fact, sEVs release has been associated with the promotion of physio-pathological processes. Among the sEVs cargo, microRNAs play an essential role in cell-to-cell regulation. More concretely, miR-205-5p is related to angiogenesis and cell proliferation. The aim of this study is to understand the specific role of sEVs containing miR-205-5p under high glucose conditions. ARPE-19 cells were cultured with high glucose (HG) for 5 days. sEVs were isolated and characterized. sEVs from ARPE-19 were used for angiogenesis and cell proliferation. HG increased sEVs release but downregulated miR-205-5p cargo expression compared to the control. sEVs from HG-treated ARPE-19 cells promoted tube formation and migration processes. In contrast, miR-205-5p overexpression (by mimic transfection) decreased angiogenesis and cell migration. Our results demonstrate how ARPE-19 cells respond to HG challenge by increasing sEVs with weak miR-205-5p cargo. The absence of this miRNA in sEVs is enough to promote angiogenesis. In contrast, restoring sEVs-miR-205-5p levels decreased it. These findings open new possibilities in sEVs-based therapies containing miR-205-5p against angiogenesis.

The Added Value of Somatosensory Potential N70 in Neurological Prognosis After Coma by Acute Brain Structural Injury: A Retrospective Study.

Background: Coma after acute brain structural injury (ABI) are associated with high mortality and disability. Somatosensory evoked potentials (SSEP) N20 and N70 are used to predict prognosis. Purpose: We assessed the utility of SSEP (N20-N70) as an early indicator of long-term functional prognosis in these patients. Methods: We conducted a retrospective cohort study of patients admitted to the intensive care unit (ICU) with a diagnosis of coma after ABI (n=60). An SSEP study including N20 and N70 was performed 24-72 hours after coma onset. Functional recovery was evaluated 6 to 12 months later using the Modified Glasgow Scale (mGS). The study was approved by our local research ethics committee. Results: The absence of N20 (41% specificity=100%) or N70 (78%) was a strong indicator of a poor outcome. In contrast, the presence of N70 was an indicator of a good outcome (specificity=64.2% sensitivity=91.3%). Conclusion: SSEP N20 and N70 are useful early prognostic markers with high specificity (N20) and sensitivity (N70). N70 has potential additional value for improving the prediction of good functional outcomes in the long term.

Functional and Prognostic Assessment in Comatose Patients: A Study Using Somatosensory Evoked Potentials.

Aim: The functional prognosis of patients after coma following either cardiac arrest (CA) or acute structural brain injury (ABI) is often uncertain. These patients are associated with high mortality and disability. N20 and N70 somatosensory evoked potentials (SSEP) are used to predict prognosis. We evaluated the utility of SSEP (N20-N70) as an early indicator of long-term prognosis in these patients. Methods: This was a retrospective cohort study of patients (n = 120) admitted to the intensive care unit (ICU) with a diagnosis of coma after CA (n = 60) or ABI (n = 60). An SSEP study was performed, including N20 and N70 at 24-72 h, after coma onset. Functional recovery was assessed 6-12 months later using the modified Glasgow scale (mGS). The study was approved by our local research ethics committee. Results: In the CA and ABI groups, the absence of N20 (36% of CA patients and 41% of ABI patients; specificity = 100%) or N70 (68% of CA patients and 78% of ABI patients) was a strong indicator of poor outcome. Conversely, the presence of N70 was an indicator of a good outcome (AC: specificity = 84.2%, sensitivity = 92.7%; ABI: specificity = 64.2% sensitivity = 91.3%). Conclusion: Somatosensory evoked potentials are useful early prognostic markers with high specificity (N20) and sensitivity (N70). Moreover, N70 has additional potential value for improving the prediction of good long-term functional outcomes. Clinical Trial Registration: [https://clinicaltrials.gov/], identifier [2018/01/001].

Oxidative-Induced Angiogenesis Is Modulated by Small Extracellular Vesicle miR-302a-3p Cargo in Retinal Pigment Epithelium Cells.

Extracellular vesicles are released from cells under diverse conditions. Widely studied in cancer, they are associated with different diseases playing major roles. Recent reports indicate that oxidative damage promotes the release of small extracellular vesicle (sEVs) from the retinal pigment epithelium (RPE), with an angiogenic outcome and changes in micro-RNA (miRNA) levels. The aim of this study was to determine the role of the miRNA miR-302a-3p, included within RPE-released sEVs, as an angiogenic regulator in cultures of endothelial cells (HUVEC). ARPE-19 cell cultures, treated with H2O2 to cause an oxidative insult, were transfected with a miR-302a-3p mimic. Later, sEVs from the medium were isolated and added into HUVEC or ARPE-19 cultures. sEVs from ARPE-19 cells under oxidative damage presented a decrease of miR-302a-3p levels and exhibited proangiogenic properties. In contrast, sEVs from miR-302a-3p-mimic transfected cells resulted in control angiogenic levels. The results herein indicate that miR-302a-3p contained in sEVs can modify VEGFA mRNA expression levels as part of its antiangiogenic features.

Usability and value of a digital learning resource in nursing education across European countries: a cross-sectional exploration.

BACKGROUND: Higher education is responsible for providing education that meets international benchmarks relevant to the needs of the international community. Due to the increase of digital tools in higher education, the possibility of sharing learning resources across nations has expanded. In the current project, a Norwegian university invited universities in Spain and the United Kingdom to adapt and translate e-learning resources originally developed for Norwegian nursing students for use within their respective Bachelor in Nursing programmes. AIM: The aim of the current study was to gain insights into the usability and value for learning of e-compendiums shared and implemented across three European universities. METHODS: The study adopted a descriptive cross-sectional design and included nursing students from the University of Nottingham, Valencia Catholic University, and the University of Stavanger. Data were collected in Autumn 2017 through a questionnaire adapted from the validated "Centre for Excellence in Teaching and Learning Reusable Learning Object evaluation questionnaire" The questionnaire consisted of 19 items that included two aspects: e-compendiums' value for learning and e-compendiums' usability. The different study sites were compared using a binary logistic regression analysis. Subgroups of students were compared based on their gender and age. RESULTS: A total of 480 nursing students participated in the study. The e -compendiums were overall positively rated, especially for reinforcing and retaining knowledge. Compared to the students from the University of Stavanger, students from Valencia Catholic University rated the e-compendiums more positively in most aspects of learning. Students from University of Nottingham found the e-compendiums to be more important for learning engagement compared to students at the Norwegian study site, and no differences were found in any other aspects of learning. Younger students rated the interactivity and visual components as more important compared to older students. CONCLUSIONS: Students from the University of Nottingham and Valencia Catholic University seem to accept the e-compendiums despite the fact that they were originally developed for use in another country. We argue that, when sharing e-learning resources across countries, an adaptation and translation process that includes a multicultural and multidisciplinary perspective should be carried out.

Ethanol-Induced Oxidative Stress Modifies Inflammation and Angiogenesis Biomarkers in Retinal Pigment Epithelial Cells (ARPE-19): Role of CYP2E1 and its Inhibition by Antioxidants.

The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.

Oxidative stress-induced angiogenesis is mediated by miR-205-5p.

miR-205-5p is known to be involved in VEGF-related angiogenesis and seems to regulate associated cell signalling pathways, such as cell migration, proliferation and apoptosis. Therefore, several studies have focused on the potential role of miR-205-5p as an anti-angiogenic factor. Vascular proliferation is observed in diabetic retinopathy and the 'wet' form of age-related macular degeneration. Today, the most common treatments against these eye-related diseases are anti-VEGF therapies. In addition, both AMD and DR are typically associated with oxidative stress; hence, the use of antioxidant agents is accepted as a co-adjuvant therapy for these patients. According to previous data, ARPE-19 cells release pro-angiogenic factors when exposed to oxidative insult, leading to angiogenesis. Matching these data, results reported here, indicate that miR-205-5p is modulated by oxidative stress and regulates VEGFA-angiogenesis. Hence, miR-205-5p is proposed as a candidate against eye-related proliferative diseases.

miR302a and 122 are deregulated in small extracellular vesicles from ARPE-19 cells cultured with H2O2.

Age related macular degeneration (AMD) is a common retina-related disease leading to blindness. Little is known on the origin of the disease, but it is well documented that oxidative stress generated in the retinal pigment epithelium and choroid neovascularization are closely involved. The study of circulating miRNAs is opening new possibilities in terms of diagnosis and therapeutics. miRNAs can travel associated to lipoproteins or inside small Extracellular Vesicles (sEVs). A number of reports indicate a significant deregulation of circulating miRNAs in AMD and experimental approaches, but it is unclear whether sEVs present a significant miRNA cargo. The present work studies miRNA expression changes in sEVs released from ARPE-19 cells under oxidative conditions (i.e. hydrogen peroxide, H2O2). H2O2 increased sEVs release from ARPE-19 cells. Moreover, 218 miRNAs could be detected in control and H2O2 induced-sEVs. Interestingly, only two of them (hsa-miR-302a and hsa-miR-122) were significantly under-expressed in H2O2-induced sEVs. Results herein suggest that the down regulation of miRNAs 302a and 122 might be related with previous studies showing sEVs-induced neovascularization after oxidative challenge in ARPE-19 cells.

Release of Retinal Extracellular Vesicles in a Model of Retinitis Pigmentosa.

Extracellular vesicles (EVs) are membranous structures released by cells, including those of the retinal pigment epithelium (RPE) and photoreceptors. The cargo of EVs includes genetic material and proteins, making these vesicles essential in cell communication. Among the genetic materials, we find a large number of microRNAs (miRNAs), small chains of noncoding RNA. In the case of EVs from the retina, changes have also been observed in the number and cargo of EVs.Our group confirmed that damaged RPE cells in vitro release a greater number of EVs with a higher pro-angiogenic factor (VEGFR-1 and VEGFR-2) than control non-damaged cells, thus increasing neovascularization in endothelial cell cultures. This indicates that something similar could happen in patients suffering from some types of retinal degeneration that occur with angiogenesis, such as wet AMD or RD.Here, we investigated the role of EVs in photoreceptor degeneration, and we report for the first time on CD9 and CD81, closely related tetraspanins, in wild-type and rd1 retinae. Our study demonstrates the involvement of EVs in the process of inherited photoreceptor degeneration in a PDE6 mutation.

Animal-Assisted Therapy in the Residential Treatment of Dual Pathology.

Animal-assisted therapy (AAT) is a complementary intervention of therapy that has shown positive results in the treatment of various pathologies. This study assesses the viability of the implementation and the effectiveness of an AAT program in patients diagnosed with substance abuse disorder and associated mental disorders (dual pathology). For the study, a dynamic prospective cohort was used, consisting of 43 patients in residential treatment. The program consisted of 10 sessions with a duration of about 60 min, where data was collected in the 3rd, 6th and 10th sessions. The Life Skills Profile questionnaire (LSP) and the Barratt Impulsiveness Scale (BIS-11) were used for subsequent evaluation. Patients who participated in the program showed an improvement in daily skills, which favoured a better quality of life and decreased impulsiveness, enabling them to regain self-control. These results suggest that the dog can be a multi-sensory stimulus that captures attention, and improves motivation, cooperation and patient involvement in therapy. It was concluded that AAT can serve as an adjunctive therapy in the rehabilitation processes of people diagnosed with dual pathology.

Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a RhoD190N mutation.

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

Poly ADP ribosylation and extracellular vesicle activity in rod photoreceptor degeneration.

Retinitis Pigmentosa is a group of inherited neurodegenerative diseases that result in selective cell death of photoreceptors. In the developed world, RP is regarded as the main cause of blindness among the working age population. The precise mechanisms eventually leading to cell death remain unknown and to date no adequate treatment for RP is available. Poly ADP ribose polymerase (PARP) over activity is involved in photoreceptor degeneration and pharmacological inhibition or genetic knock-down PARP1 activity protect photoreceptors in mice models, the mechanism of neuroprotection is not clear yet. Our result indicated that olaparib, a PARP1 inhibitor, significantly rescued photoreceptor cells in rd10 retina. Extracellular vesicles (EVs) were previously recognized as a mechanism for discharging useless cellular components. Growing evidence has elucidated their roles in cell-cell communication by carrying nucleic acids, proteins and lipids that can, in turn, regulate behavior of the target cells. Recent research suggested that EVs extensively participate in progression of diverse blinding diseases, such as age-related macular (AMD) degeneration. Our study demonstrates the involvement of EVs activity in the process of photoreceptor degeneration in a PDE6 mutation. PARP inhibition protects photoreceptors via regulation of the EVs activity in rod photoreceptor degeneration in a PDE6b mutation.

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5.

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR5) PG-901 (10-10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR1 agonist BMS-470539 (10-5 M) or with the mixed MCR3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10-10M) but not by the MCR3/4 agonist MTII (0.30 nmol) or the MCR1 agonist BMS-470539 (10-5 M). The MCR5-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR5 agonist in the ARPE-19 cells. Altogether, these data suggest that MCR5 is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.

Role of retinal pigment epithelium-derived exosomes and autophagy in new blood vessel formation.

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age-related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE-19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE-19 cells were treated with a siRNA-targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF-which is critical for the development of new blood vessels-was higher in exosome populations released from stressed ARPE-19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE-19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF-independent pathway. We propose that abnormal vessel growth correlates with VEGFR2-expressing exosomes release from stressed ARPE-19 cells, and is directly linked to autophagy.

Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells.

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell-to-cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT-PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.

Rotenone Induces the Formation of 4-Hydroxynonenal Aggresomes. Role of ROS-Mediated Tubulin Hyperacetylation and Autophagic Flux Disruption.

Oxidative stress causes cellular damage by (i) altering protein stability, (ii) impairing organelle function, or (iii) triggering the formation of 4-HNE protein aggregates. The catabolic process known as autophagy is an antioxidant cellular response aimed to counteract these stressful conditions. Therefore, autophagy might act as a cytoprotective response by removing impaired organelles and aggregated proteins. In the present study, we sought to understand the role of autophagy in the clearance of 4-HNE protein aggregates in ARPE-19 cells under rotenone exposure. Rotenone induced an overproduction of reactive oxygen species (ROS), which led to an accumulation of 4-HNE inclusions, and an increase in the number of autophagosomes. The latter resulted from a disturbed autophagic flux rather than an activation of the autophagic synthesis pathway. In compliance with this, rotenone treatment induced an increase in LC3-II while upstream autophagy markers such as Beclin- 1, Vsp34 or Atg5-Atg12, were decreased. Rotenone reduced the autophagosome-to-lysosome fusion step by increasing tubulin acetylation levels through a ROS-mediated pathway. Proof of this is the finding that the free radical scavenger, N-acetylcysteine, restored autophagy flux and reduced rotenone-induced tubulin hyperacetylation. Indeed, this dysfunctional autophagic response exacerbates cell death triggered by rotenone, since 3-methyladenine, an autophagy inhibitor, reduced cell mortality, while rapamycin, an inductor of autophagy, caused opposite effects. In summary, we shed new light on the mechanisms involved in the autophagic responses disrupted by oxidative stress, which take place in neurodegenerative diseases such as Huntington or Parkinson diseases, and age-related macular degeneration.

CYP2E1 in the Human Retinal Pigment Epithelium: Expression, Activity, and Induction by Ethanol.

PURPOSE: Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme with particular affinity for ethanol (EtOH) expressed in several tissues. Although CYP2E1 has been identified in human RPE, nothing is known about its metabolic activity. Expression of CYP2E1 and activity after EtOH exposure have been studied in human RPE and ARPE-19 cells. METHODS: Ethanol-induced CYP2E1 mRNA expression was analyzed by RT-PCR and quantitative PCR (qPCR) from human donor RPE as well as from ARPE-19 cells. Expression of CYP2E1 protein was determined by Western blot. Cytoplasmic CYP2E1 location also was demonstrated by immunocytochemistry. Cell viability was studied by the colorimetric assay XTT after EtOH treatment. Diallyl sulfide (DAS) was used to inhibit CYP2E1 activity. The microsomal CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation through HPLC. RESULTS: Relevant CYP2E1 mRNA levels are present in human RPE. Ethanol augmented the formation of reactive oxygen species (ROS) in ARPE-19 cells. Expression of CYP2E1 mRNA, CYP2E1 protein activity, and ROS production were induced by ethanol in a concentration-dependent manner. Interestingly, the treatment with DAS reduced all the aforementioned increased values. The presence of CYP2E1 in both hRPE and ARPE-19 cells reinforces the protective role of the RPE and strongly suggests additional roles for CYP2E1 related to vision.

Matching Diabetes and Alcoholism: Oxidative Stress, Inflammation, and Neurogenesis Are Commonly Involved.

Diabetes and alcohol misuse are two of the major challenges in health systems worldwide. These two diseases finally affect several organs and systems including the central nervous system. Hippocampus is one of the most relevant structures due to neurogenesis and memory-related processing among other functions. The present review focuses on the common profile of diabetes and ethanol exposure in terms of oxidative stress and proinflammatory and prosurvival recruiting transcription factors affecting hippocampal neurogenesis. Some aspects around antioxidant strategies are also included. As a global conclusion, the present review points out some common hits on both diseases giving support to the relations between alcohol intake and diabetes.

On the mechanism underlying ethanol-induced mitochondrial dynamic disruption and autophagy response.

We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600 mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.