Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.España, Ministerio de Sanidad FIS PI13/00129España, Ministerio de Economía y Competitividad SAF2013-44021-R and CTQ2010-15848España, Junta de Andalucía CTS-5725 and FQM-146

Coenzyme Q10 therapy

For a number of years, coenzyme Q10 (CoQ10) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in blood plasma, and extensively investigated its antioxidant role. These 2 functions constitute the basis for supporting the clinical use of CoQ10. Also, at the inner mitochondrial membrane level, CoQ10 is recognized as an obligatory cofactor for the function of uncoupling proteins and a modulator of the mitochondrial transition pore. Furthermore, recent data indicate that CoQ 10 affects the expression of genes involved in human cell signaling, metabolism and transport, and some of the effects of CoQ10 supplementation may be due to this property. CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, aging-related oxidative stress and carcinogenesis processes, and also statin treatment. Many neurodegenerative disorders, diabetes, cancer, and muscular and cardiovascular diseases have been associated with low CoQ10 levels as well as different ataxias and encephalomyopathies. CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral administration of CoQ10 is a frequent antioxidant strategy in many diseases that may provide a significant symptomatic benefit.This work was supported by grants (FIS PI10/00543, FIS EC08/00076) from the Ministerio de Sanidad, Spain, and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea); Servicio Andaluz de Salud-Junta de Andalucía (SAS 111242); Proyecto de Investigación de Excelencia de la Junta de Andalucía (CTS-5725); and by AEPMI (Asociación de Enfermos de Patología Mitocondrial), FEEL (Fundación Española de Enfermedades Lisosomales) and ALBA Andalucía (Federación Andaluza de Fibromialgia y Fatiga Crónica).Peer Reviewe

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License.Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.This work was supported by FIS PI10/00543 grant, FIS EC08/00076 grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS 111242 grant, Servicio Andaluz de Salud Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, and by AEPMI (Asociación de Enfermos de Patología Mitocondrial).Peer reviewe

Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease

Background Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal β-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. Results Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. Conclusion These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.info:eu-repo/semantics/publishedVersio

Stabilization of apoptotic cells: generation of zombie cells

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.This work was supported by FIS PI10/00543 grant, Ministerio de Sanidad, Spain, and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS 111242 grant, Servicio Andaluz de Salud-Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, BFU2012-38208 and by AEPMI (Asociación de Enfermos de Patología Mitocondrial).Peer Reviewe

Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells

Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.This work was supported by FIS PI13/00129 grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, AEPMI (Asociación de Enfermos de Patología Mitocondrial) and ENACH (Asociación de Enfermedades Neurodegenerativas por Acumulación Cerebral de Hierro).Peer Reviewe

Cytoskeleton Rearrangements during the Execution Phase of Apoptosis

Apoptosis is a regulated energy‐dependent process for the elimination of unnecessary or damaged cells during embryonic development, tissue homeostasis and many pathological conditions. Apoptosis is characterized by specific morphological and biochemical features in which caspase activation has a pivotal role. During apoptosis, cells undergo characteristic morphological reorganizations in which the cytoskeleton participates actively. Traditionally, this cytoskeleton rearrangement has been assigned mainly to actinomyosin ring contraction, with microtubule and intermediate filaments both reported to be depolymerized at early stages of apoptosis. However, recent results have shown that microtubules are reformed during the execution phase of apoptosis forming an apoptotic microtubule network (AMN). Current hypothesis proposes that AMN is required to maintain plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disruption provokes apoptotic cell collapse, secondary necrosis and the subsequent release of toxic molecules which can damage surrounding cells and promote inflammation. Therefore, AMN formation in physiological or pathological apoptosis is essential for tissue homeostasis

Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as ¿-spectrin, paxilin, focal adhesion kinase and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.This work was supported by FIS PI10/00543 Grant, FIS EC08/00076 Grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS 111242 Grant, Servicio Andaluz de Salud-Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, and by Asociación de Enfermos de Patología Mitocondrial (AEPMI).Peer Reviewe

The Apoptotic Microtubule Network During the Execution Phase of Apoptosis

Apoptosis is a regulated energy-dependent process of cell death characterized by specific morphological and biochemical features in which caspase activation has a central role. During apoptosis, cells undergo characteristic morphological rearrangements in which the cytoskeleton participates actively. From a historical point of view, this reorganization has been assigned mainly to actinomyosin ring contraction with microtubule and intermediate filaments, both reported to be depolymerized at early stages of apoptosis. However, recent results have shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis, forming an apoptotic microtubule network (AMN). AMN is closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon.” Apoptotic microtubules’ reorganization has been reported in many cell types and under many apoptotic inducers. Recently, it has been proposed that AMN is essential for preserving plasma membrane permeability and cell morphology during the execution phase of apoptosis. Apoptotic microtubules’ depolymerization leads cells to secondary necrosis and the release of toxic intracellular contents that can harm surrounding cells and initiate inflammation. Therefore, microtubules’ reorganization in physiological apoptosis during development and in the adult organism or in pathological apoptosis induced by anticancer treatments or chronic inflammation is essential for tissue homeostasis, preventing cell damage and inflammation