Potential Role of the Mitochondria for the Dermatological Treatment of Papillon-Lefèvre

The Papillon–Lefèvre syndrome (PLS) is a rare autosomal recessive disorder caused by mutations in the Cathepsin C (CTSC) gene, characterized by periodontitis and palmoplantar hyperkeratosis. The main inflammatory deficiencies include oxidative stress and autophagic dysfunction. Mitochondria are the main source of reactive oxygen species; their impaired function is related to skin diseases and periodontitis. The mitochondrial function has been evaluated in PLS and mitochondria have been targeted as a possible treatment for PLS. We show for the first time an important mitochondrial dysfunction associated with increased oxidative damage of mtDNA, reduced CoQ10 and mitochondrial mass and aberrant morphologies of the mitochondria in PLS patients. Mitochondrial dysfunction, determined by oxygen consumption rate (OCR) in PLS fibroblasts, was treated with CoQ10 supplementation, which determined an improvement in OCR and a remission of skin damage in a patient receiving a topical administration of a cream enriched with CoQ10 0.1%. We provide the first evidence of the role of mitochondrial dysfunction and CoQ10 deficiency in the pathophysiology of PLS and a future therapeutic option for PLS.Andalusian regional government (Grupo de Investigación Junta de Andalucía) CTS11

L-arginine ameliorates defective autophagy in GM2 gangliosidoses by mTOR modulation

Aims: Tay–Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay–Sachs and Sandhoff diseases. Results: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. Innovation: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. Conclusions: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

Inhibition of the NLRP3 inflammasome improves lifespan in animal murine model of Hutchinson-Gilford Progeria

Inflammation is a hallmark of aging and accelerated aging syndromes such as Hutchinson-Gilford progeria syndrome (HGPS). In this study, we present evidence of increased expression of the components of the NLRP3 inflammasome pathway in HGPS skin fibroblasts, an outcome that was associated with morphological changes of the nuclei of the cells. Lymphoblasts from HGPS patients also showed increased basal levels of NLRP3 and caspase 1. Consistent with these results, the expression of caspase 1 and Nlrp3, but not of the other inflammasome receptors was higher in the heart and liver of Zmpste2

L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.

AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

Inhibition of the NLRP3 inflammasome improves lifespan in animal murine model of Hutchinson–Gilford Progeria

Inflammation is a hallmark of aging and accelerated aging syndromes such as Hutchinson–Gilford progeria syndrome (HGPS). In this study, we present evidence of increased expression of the components of the NLRP3 inflammasome pathway in HGPS skin fibroblasts, an outcome that was associated with morphological changes of the nuclei of the cells. Lymphoblasts from HGPS patients also showed increased basal levels of NLRP3 and caspase 1. Consistent with these results, the expression of caspase 1 and Nlrp3, but not of the other inflammasome receptors was higher in the heart and liver of Zmpste24−/− mice, which phenocopy the human disease. These data were further corroborated in LmnaG609G/G609G mice, another HGPS animal model. We also showed that pharmacological inhibition of the NLRP3 inflammasome by its selective inhibitor, MCC950, improved cellular phenotype, significantly extended the lifespan of progeroid animals, and reduced inflammasome-dependent inflammation. These findings suggest that inhibition of the NLRP3 inflammasome is a potential therapeutic approach for the treatment of HGPS.Junta de Andalucía PI-0036-201

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.This study was funded by Institute of Health Carlos III (ISCiii) (PI16/00090, PI19/00838 and PI19/01266), Spanish Ministry of Economy and Competitiveness (BFU2016-80006-P), Andalusian Ministry of Economy, Innovation, Science and Employment (BIO-216 and CTS-6264), Andalusian Ministry of Equality, Health and Social Policies (PI-0198-2016) and Valencian Ministry of Education, Culture and Sports (PROMETEO/2019/027). P de la C-O was supported by FPU predoctoral fellowship (FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E N-V was supported by the the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii. We thank the Biomedical Research Network Center for Cardiovascular Diseases (CIBERcv), and the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and co-financed by European Regional Development Fund (ERDF) "A way to achieve Europe" for their financial support

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

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the agedependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD+ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.Andalusian regional government; Consejería de Salud de la Junta de Andalucia, Grant/ Award Number: PI‐0036‐2014; Ministerio de economía y competitividad, Grant/Award Number: SAF2017‐84494‐C2‐1‐

Inhibition of the NLRP3 inflammasome prevents ovarian aging

Inflammation is a hallmark of aging and is negatively affecting female fertility. In this study, we evaluate the role of the NLRP3 inflammasome in ovarian aging and female fertility. Age-dependent increased expression of NLRP3 in the ovary was observed in WT mice during reproductive aging. High expression of NLRP3, caspase-1, and IL-1β was also observed in granulosa cells from patients with ovarian insufficiency. Ablation of NLRP3 improved the survival and pregnancy rates and increased anti-Müllerian hormone levels and autophagy rates in ovaries. Deficiency of NLRP3 also reduced serum FSH and estradiol levels. Consistent with these results, pharmacological inhibition of NLRP3 using a direct NLRP3 inhibitor, MCC950, improved fertility in female mice to levels comparable to those of Nlrp3−/− mice. These results suggest that the NLRP3 inflammasome is implicated in the age-dependent loss of female fertility and position this inflammasome as a potential new therapeutic target for the treatment of infertility

Autophagy dysfunctions and lysosomal permeabilization in Tay-Sachs and Sandhoff diseases

Autophagy is an essential intracellular process involved in survival, differentiation, development and cellular homeostasis. This catabolic mechanism supplies to the cell nutrients and energy to do its vital functions and gets rid of non-needed cellular components such as lipids, misfolded proteins and damage organelles. It consists on a multistep process highly regulated, conducted by autophagy-related proteins (ATGs), which starts by the formation of a vesicle which engulfs the material to be degraded to form an autophagosome following by the fusion to the lysosome. It contains the hydrolytic enzymes which are involve in the degradation of the cargo in the presence of an acidic environment (Levine & Kroemer, 2008). Dysfunction in the autophagy flux has been implicated in the progression of several diseases such as neurodegeneration, cancer and immune diseases, even in the normal ageing process. It is known that cell inability to degrade the cargo may lead to a defective autophagosome-lysosome fusion and thus, accumulation of autophagosome with non-degraded material inside the cells. This is the molecular base of a set of pathological conditions, known as lysosome storage disorder (LSD). Lysosomal dysfunction in most of these diseases is associated with impaired autophagic flux and autophagosome-lysosome fusion and a secondary accumulation of autophagy substrates such as SQSTM1/p62 and damage mitochondria. The key regulator of autophagy is mTOR which is involved in protein, lipid and nucleotide synthesis, lysosomal biogenesis, transcription, cytoskeletal rearrangements, energy metabolism, cell proliferation and survival. The presence of nutrients, growth factors and hypoxia induce mTOR promoting protein synthesis and cell growth and repressing autophagy. (Villanueva-Paz et al., 2016), (Munson & Ganley, 2015). Tay-Sachs and Sandhoff are two rare inherit LSDs which affect the nervous system and are characterized by early neuronal cell death and progressive neurodegeneration. They are caused by a mutation in a gene which encodes for the α (Tay-Sachs) and β (Sandhoff) subunit for the lysosome enzyme β-hexosaminidase A (HexA). This is the unique enzyme which is able to degrade a type of glycosphingolipids known as GM2 ganglioside. Consequently, there is an accumulation of them leading to cell death (Schuchman & Simonaro, 2013). The severity and progression of the diseases depend on the activity level of the dysfunctional enzyme and can be presented in infantile, juvenile and adult form with progressive neurodegeneration, hypotension, dysphagia, spasms, eye movement abnormalities, etc (Munson & Ganley, 2015). Nowadays, there is not an effective treatment beyond the palliative care. Although many mutations have been described, the molecular and cellular implications are still unknown. In the recent study, working with patient fibroblasts, it have been found low levels of mTOR pathway activation with the subsequent decreasing of protein synthesis and high levels of autophagy. However, it has been determined that the autophagy flux is impaired, with the accumulation of autophagosome with non-degrade material leading to the lysosomal permeabilization. Treatment of patient fibroblasts with L-arginine showed a partial recovery of cellular pathological alterations.La autofagia es un proceso intracelular esencial para la supervivencia, diferenciación, desarrollo y homeostasis celular. Este mecanismo catabólico provee a las células de energía y nutrientes necesarios para la realización de sus funciones vitales y la eliminación de componentes innecesarios para la célula como son lípidos, proteínas mal plegadas o agregadas y organelas dañadas. La autofagia es un proceso guiado por una serie de proteínas asociadas a la autofagia (ATGs), el cual comienza con la formación de una vesícula que engloba el material a degradar formando el autofagosoma. A continuación, tienen lugar la fusión entre el autofagosoma y el lisosoma el cual contiene las enzimas (hidrolasas) y el pH acido para la eliminación del cargo (Levine & Kroemer, 2008). La disfunción en la autofagia está implicada en la progresión de muchas enfermedades como neurodegeneración, cáncer, enfermedades inmunes e incluso en el envejecimiento. Una de las causas de la incapacidad de la degradación del cargo es consecuencia del fallo en la fusión del autofagosoma con el lisosoma que conlleva a la acumulación intracelular de material no degradado. Esto constituye la base de una serie de condiciones patológicas llamadas enfermedades de acumulación lisosomal (LSDs). La disfunción en el lisosoma en la mayoría de estas enfermedades está asociada a un defecto en la autofagia, así como en la fusión del autofagosoma y lisosoma, daño mitocondrial y acumulación de sustratos de la autofagia como SQSTM1/p62. El regulador clave de este proceso es la ruta mTOR la cual está implicada en la síntesis de proteínas, lípidos y nucleótidos, biogénesis lisosomal, trascripción, reordenamiento del citoesqueleto, metabolismo energético, proliferación celular y supervivencia. La presencia de nutrientes, factores de crecimiento o hipoxia inducen mTOR dando lugar al aumento en la síntesis proteica y crecimiento celular, así como con la inhibición de la autofagia (Villanueva-Paz et al., 2016), (Munson & Ganley, 2015). Tay-Sachs y Sandhoff son dos enfermedades raras hereditarias de acumulación lisosomal (LSD) que afecta al sistema nervioso y se caracterizan por muerte neuronal temprana y neurodegeneración progresiva. Están causadas por mutaciones en el gen que codifica la subunidad α (Tay-Sachs) y la subunidad β (Sandhoff) de la enzima β-Hexosaminidasa A (HexA). Esta enzima es la única capaz de degradar un tipo de glicoesfingolípidos conocidos como gangliósidos GM2. Como consecutiva, hay una acumulación intracelular que lleva a la muerte celular (Schuchman & Simonaro, 2013). La severidad y la progresión de estas enfermedades dependen del nivel de actividad de la HexA y pueden ser presentadas en forma infantil, juvenil o adulta. Entre sus síntomas destacan la hipotensión, disfagia, espasmos, movimiento anormal de los ojos y progresiva neurodegeneración (Munson & Ganley, 2015). Actualmente no existe ninguna cura o tratamiento efectivo para estas enfermedades, solo cuidados paliativos. Aunque se han descrito muchas mutaciones asociadas, las implicaciones celulares y moleculares siguen siendo desconocidas. En este estudio, trabajando a partir de fibroblastos de pacientes, se han descrito bajos niveles de activación de mTOR con la subsecuente disminución de la síntesis proteica y altos niveles de autofagia. Sin embargo, esta autofagia lejos de ser positiva en estas enfermedades sufre un bloqueo en el flujo con la acumulación de autofagosoma con material no degradado lo cual lleva a la aparición de permeabilización lisosoma. El tratamiento de las células de los pacientes con L-arginina mostró una parcial recuperación de las alteraciones celulares patológicas