Mitochondrial glutathione: Features, regulation and role in disease

El pdf del artículo es la versión post-print.[Background]: Mitochondria are the powerhouse of mammalian cells and the main source of reactive oxygen species (ROS) associated with oxygen consumption. In addition, they also play a strategic role in controlling the fate of cells through regulation of death pathways. Mitochondrial ROS production fulfills a signaling role through regulation of redox pathways, but also contributes to mitochondrial damage in a number of pathological states. [Scope of review]: Mitochondria are exposed to the constant generation of oxidant species, and yet the organelle remains functional due to the existence of an armamentarium of antioxidant defense systems aimed to repair oxidative damage, of which mitochondrial glutathione (mGSH) is of particular relevance. Thus, the aim of the review is to cover the regulation of mGSH and its role in disease. [Major conclusions]: Cumulating evidence over recent years has demonstrated the essential role for mGSH in mitochondrial physiology and disease. Despite its high concentration in the mitochondrial matrix, mitochondria lack the enzymes to synthesize GSH de novo, so that mGSH originates from cytosolic GSH via transport through specific mitochondrial carriers, which exhibit sensitivity to membrane dynamics. Depletion of mGSH sensitizes cells to stimuli leading to oxidative stress such as TNF, hypoxia or amyloid β-peptide, thereby contributing to disease pathogenesis. [General significance]: Understanding the regulation of mGSH may provide novel insights to disease pathogenesis and toxicity and the opportunity to design therapeutic targets of intervention in cell death susceptibility and disease. This article is part of a Special Issue entitled Cellular functions of glutathione. © 2012 Elsevier B.V.The work was supported by grants: SAF2009-11417, SAF2010-15760, and SAF2011-23031 (Plan Nacional de I + D), Proyectos de Investigación en Salud PI10/02114 and PS09/00056 (Instituto de Salud Carlos III), P50-AA-11999 (Research Center for Liver and Pancreatic Diseases, US National Institute on Alcohol Abuse and Alcoholism) and by CIBEREHD from the Instituto de Salud Carlos III.Peer Reviewe

Cysteine cathepsins control hepatic NF-κB-dependent inflammation via sirtuin-1 regulation

Sirtuin-1 (SIRT1) regulates hepatic metabolism but its contribution to NF-κB-dependent inflammation has been overlooked. Cysteine cathepsins (Cathepsin B or S, CTSB/S) execute specific functions in physiological processes, such as protein degradation, having SIRT1 as a substrate. We investigated the roles of CTSB/S and SIRT1 in the regulation of hepatic inflammation using primary parenchymal and non-parenchymal hepatic cell types and cell lines. In all cells analyzed, CTSB/S inhibition reduces nuclear p65-NF-κB and κB-dependent gene expression after LPS or TNF through enhanced SIRT1 expression. Accordingly, SIRT1 silencing was sufficient to enhance inflammatory gene expression. Importantly, in a dietary mouse model of non-alcoholic steatohepatitis, or in healthy and fibrotic mice after LPS challenge, cathepsins as well as NF-κB-dependent gene expression are activated. Consistent with the prominent role of cathepsin/SIRT1, cysteine cathepsin inhibition limits NF-κB-dependent hepatic inflammation through the regulation of SIRT1 in all in vivo settings, providing a novel anti-inflammatory therapeutic target in liver disease.This study was supported by grants from the Instituto de Salud Carlos III (PI13/00374 to MM), Ministerio de Economía y Competitividad (SAF2015-69944-R to JFC, SAF2013-47246-R to AC, SAF2015-66515-R to AM) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases (US-NIAAA to JFC); and by CIBERehd. AT is a recipient of a FPU fellowship recipient from the Ministerio de Educación, Cultura y Deporte.Peer Reviewe

Regorafenib Alteration of the BCL-xL/MCL-1 Ratio Provides a Therapeutic Opportunity for BH3-Mimetics in Hepatocellular Carcinoma Models

Background: The multikinase inhibitor regorafenib, approved as second-line treatment for hepatocellular carcinoma (HCC) after sorafenib failure, may induce mitochondrial damage. BH3-mimetics, inhibitors of specific BCL-2 proteins, are valuable drugs in cancer therapy to amplify mitochondrial-dependent cell death. Methods: In in vitro and in vivo HCC models, we tested regorafenib's effect on the BCL-2 network and the efficacy of BH3-mimetics on HCC treatment. Results: In hepatoma cell lines and Hep3B liver spheroids, regorafenib cytotoxicity was potentiated by BCL-xL siRNA transfection or pharmacological inhibition (A-1331852), while BCL-2 antagonism had no effect. Mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-3 activation mediated A-1331852/regorafenib-induced cell death. In a patient-derived xenograft (PDX) HCC model, BCL-xL inhibition stimulated regorafenib activity, drastically decreasing tumor growth. Moreover, regorafenib-resistant HepG2 cells displayed increased BCL-xL and reduced MCL-1 expression, while A-1331852 reinstated regorafenib efficacy in vitro and in a xenograft mouse model. Interestingly, BCL-xL levels, associated with poor prognosis in liver and colorectal cancer, and the BCL-xL/MCL-1 ratio were detected as being increased in HCC patients. Conclusion: Regorafenib primes tumor cells to BH3-mimetic-induced cell death, allowing BCL-xL inhibition with A-1331852 or other strategies based on BCL-xL degradation to enhance regorafenib efficacy, offering a novel approach for HCC treatment, particularly for tumors with an elevated BCL-xL/MCL-1 ratio

Antiapoptotic BCL-2 proteins determine Sorafenib/regorafenib resistance and BH3-mimetic efficacy in hepatocellular carcinoma

Sorafenib, systemic treatment for advanced hepatocellular carcinoma (HCC), and regorafenib, novel second line treatment after sorafenib failure, have efficacy limited by evasive mechanisms of acquired-drug resistance. BCL-2 proteins participate in the response to tyrosine kinase inhibitors; however, their role in HCC therapy with sorafenib/regorafenib remains uncertain. BH3-mimetic ABT-263 (navitoclax) enhanced sorafenib activity, inducing cell death via a mitochondrial caspase-dependent mechanism, after BCL-xL/BCL-2 inhibition. Sorafenib-resistant hepatoma cells (HepG2R and Hep3BR) exhibited altered mRNA expression of BCL-2 and other anti-apoptotic family members, such as MCL-1, priming drug-resistant cancer cells to death by BH3-mimetics. ABT-263 restored sorafenib efficacy in sorafenib-resistant cell lines and HCC mouse models. Moreover, in mice xenografts from patient-derived BCLC9 cells, better tumor response to sorafenib was associated to higher changes in the BCL-2 mRNA pattern. HCC non-treated patients displayed altered BCL-2, MCL-1 and BCL-xL mRNA levels respect to adjacent non-tumoral biopsies and an increased BCL-2/MCL-1 ratio, predictive of navitoclax efficacy. Moreover, regorafenib administration also modified the BCL-2/MCL-1 ratio and navitoclax sensitized hepatoma cells to regorafenib by a mitochondrial caspase-dependent mechanism. In conclusion, sorafenib/regorafenib response is determined by BCL-2 proteins, while increased BCL-2/MCL-1 ratio in HCC sensitizes drug resistant-tumors against ABT-263 co-administration. Thus, changes in the BCL-2 profile, altered in HCC patients, could help to follow-up sorafenib efficacy, allowing patient selection for combined therapy with BH3-mimetics or early switch them to second line therapy

Regulación por colesterol de la respuesta celular frente al estrés oxidativo mitocondrial: implicaciones patológicas

Trabajo presentado en el XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona, España, del 5 al 8 de septiembre de 2011Peer Reviewe

Mitochondrial oxidative and nitrosative stress as a therapeutic target in diseases

Mitochondria are fundamental to life. In addition to providing energy, they constitute crucial nodes in multiple signaling pathways that can ultimately decide cell fate. Under physiological conditions, 0.4 to 4% of oxygen consumed by mitochondria is incompletely reduced and leads to the generation of reactive oxygen species (ROS). These chemical moieties, which at low levels are instrumental for several physiological processes and facilitate adaptation to stress via signaling, in excess can be harmful not only to the organelle itself, but also may affect the whole-cell function. Mitochondria have different ways to counter oxidative/nitrosative damage, including a variety of antioxidants, both enzymatic and non-enzymatic mechanisms, of which mitochondrial GSH (mGSH) plays a key role, as described in detail in the review by Marí et al. [1] in this Special Issue

Mitochondrial cholesterol loading exacerbates amyloid β peptide-induced inflammation and neurotoxicity

The role of cholesterol in Alzheimer’s disease (AD) has been linked to the generation of toxic amyloid β peptides (Aβ). Using genetic mouse models of cholesterol loading, we examined whether mitochondrial cholesterol regulates Aβ neurotoxicity and AD pathology. Isolated mitochondria from brain or cortical neurons of transgenic mice overexpressing SREBP-2 (sterol regulatory element binding protein 2) or NPC1 (Niemann-Pick type C1) knock-out mice exhibited mitochondrial cholesterol accumulation, mitochondrial glutathione (mGSH) depletion and increased susceptibility to Aβ1– 42-induced oxidative stress and release of apoptogenic proteins. Similar findings were observed in pharmacologically GSH-restricted rat brain mitochondria, while selective mGSH depletion sensitized human neuronal and glial cell lines to Aβ1–42-mediated cell death. Intracerebroventricular human Aβ delivery colocalized with mitochondria resulting in oxidative stress, neuroinflammation and neuronal damage that were enhanced in Tg-SREBP-2 mice and prevented upon mGSH recovery by GSH ethyl ester coinfusion, with a similar protection observed by intraperitoneal administration of GSH ethyl ester. Finally, APP/PS1 (amyloid precursor protein/presenilin 1) mice, a transgenic AD mouse model, exhibited mitochondrial cholesterol loading and mGSH depletion. Thus, mitochondrial cholesterol accumulation emerges as a novel pathogenic factor in AD by modulating Aβ toxicity via mGSH regulation; strategies boosting the particular pool of mGSH may be of relevance to slow down disease progression.This work was supported in part by the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica Grants SAF2005-03923 and SAF2006-06780, by Research Center for Liver and Pancreatic Diseases Grant P50 AA 11999 funded by the National Institute on Alcohol Abuse and Alcoholism, and by the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas supported by the Instituto de Salud Carlos III. A.C. is supported by the Ramon y Cajal Research Program (Ministerio de Educación y Ciencias).Peer reviewe

APP/PS1 mice overexpressing SREBP-2 exhibit combined Aß accumulation and tau pathology underlying Alzheimer's disease

El pdf del artículo es la versión post-print.Current evidence indicates that excess brain cholesterol regulates amyloid-β (Aβ) deposition, which in turn can regulate cholesterol homeostasis. Moreover, Aβ neurotoxicity is potentiated, in part, by mitochondrial glutathione (mGSH) depletion. To better understand the relationship between alterations in cholesterol homeostasis and Alzheimer's disease (AD), we generated a triple transgenic mice featuring sterol regulatory element-binding protein-2 (SREBP-2) overexpression in combination with APPswe/PS1ΔE9 mutations (APP/PS1) to examine key biochemical and functional characteristics of AD. Unlike APP/PS1 mice, APP/PS1/SREBP-2 mice exhibited early mitochondrial cholesterol loading and mGSH depletion. Moreover, β-secretase activation and Aβ accumulation, correlating with oxidative damage and neuroinflammation, were accelerated in APP/PS1/SREBP-2 mice compared with APP/PS1 mice. Triple transgenic mice displayed increased synaptotoxicity reflected by loss of synaptophysin and neuronal death, resulting in early object-recognition memory impairment associated with deficits in spatial memory. Interestingly, tau pathology was present in APP/PS1/SREBP-2 mice, manifested by increased tau hyperphosphorylation and cleavage, activation of tau kinases and neurofibrillary tangle (NFT) formation without expression of mutated tau. Importantly, in vivo treatment with the cell permeable GSH ethyl ester, which restored mGSH levels in APP/PS1/SREBP-2 mice, partially prevented the activation of tau kinases, reduced abnormal tau aggregation and Aβ deposition, resulting in attenuated synaptic degeneration. Taken together, these results show that cholesterol-mediated mGSH depletion is a key event in AD progression, accelerating the onset of key neuropathological hallmarks of the disease. Thus, therapeutic approaches to recover mGSH may represent a relevant strategy in the treatment of AD.This work was supported by Plan Nacional de I+ D+I (SAF2010-03923 to A.C. and SAF2009-11417 and SAF2012-34831 to J.C.F-C); by Centro de Investigación Biomédica en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD); by Instituto de Salud Carlos III; by Research Center for Liver and Pancreatic Diseases, NIAAA/NIH (P50-AA-11999 to J.C. F-C) and by Marato TV3. E.B.C. has a FPI fellowship from Ministerio de Economía y Competitividad.Peer Reviewe

Mitochondrial GSH depletion sensitizes human neuroblastoma cells to beta-amyloid peptide-induced oxidative stress and apoptotic cell death

Comunicación presentada en la Alzheimer's Association 10th International Conference on Alzheimer's Disease and Related Disorders, celebrada del 15 al 20 de julio de 2006 en Madrid (España)The pathogenesis of Alzheimer disease is not completely understood at present, although the generation of toxic beta-amyloid peptide (Aβ) is thought to play a prominent role. One of the cytotoxic effects of the Aβ is reactive oxygen species (ROS) overgeneration. Although, the exact mechanisms of this process are not well defined, emerging evidence points to mitochondria as a source for Aβ-induced ROS generation. Moreover, disturbances in cholesterol homeostasis promote the formation and deposition of Aβ and the progression of AD.Peer Reviewe

Mitochondria, cholesterol and amyloid β peptide: a dangerous trio in Alzheimer disease

The molecular mechanisms of Alzheimer’s disease (AD) are not fully understood. Extensive evidence from experimental models has involved the overgeneration and accumulation of toxic amyloid β peptides (Aβ) in the onset and progression of the disease. The amyloidogenic processing of amyloid precursor protein into pathogenic Aβ fragments is thought to occur in specific domains of the plasma membrane and favored by cholesterol enrichment. Intracellular Aβ accumulation is known to induce oxidative stress, predominantly via mitochondria targeting of toxic Aβ. Recent evidence using mouse models of cholesterol loading has demonstrated that the specific mitochondrial cholesterol pool sensitizes neurons to Aβ-induced oxidant cell death and caspase-independent apoptosis due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. mGSH replenishment by permeable precursors such as glutathione ethyl ester protected against Aβ-mediated neurotoxicity and inflammation. Thus, these novel data expand the pathogenic role of cholesterol in AD indicating that in addition to fostering Aβ generation, mitochondrial cholesterol determines Aβ neurotoxicity via mGSH regulation.This work was supported in part by the Plan Nacional de I+D, grants SAF2008-04974 and SAF2006-06780, by the Research Center for Liver and Pancreatic Diseases Grant P50AA 11999, funded by the U.S. National Institute on Alcohol Abuse and Alcoholism, and by the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), supported by the Instituto de Salud Carlos III.Peer reviewe