Rubén Gómez-Sánchez

The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson’s disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: Modulation by the Nrf2/Trx axis

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases.

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

ASK1 Overexpression Accelerates Paraquat-Induced Autophagy via Endoplasmic Reticulum Stress

Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ER stress-induced ASK1 activation could play an important role both in neuronal apoptosis and an autophagic response in the pathogenesis of several neurodegenerative diseases, including Parkinsons disease. The mechanism by which ASK1 executes apoptosis and/or autophagy under ER stress is still unclear. We have addressed this question using SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression with or without PQ. These data support the idea that the autophagic response could have a protector role. We found also an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure. These data suggest that the WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. In conclusion, we report that PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1. However, when the toxic stimuli remain, the cell eventually succumbs to apoptosis.

Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinsons disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24 h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3 h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

G2019S LRRK2 mutant fibroblasts from Parkinson’s disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy

Parkinsons disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinsons patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD.

Curcumin exposure induces expression of the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) in rat mesencephalic cells

Turmeric (curry powder), an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound known as curcumin or diferuloylmethane. Curcumin is a widely studied phytochemical with a variety of biological activities. In addition to its anti-inflammatory and antimicrobial/antiviral properties, curcumin is considered as a cancer chemopreventive agent as well as a modulator of gene expression and a potent antioxidant. Since oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra in Parkinsons disease (PD), curcumin has been proposed to have potential therapeutic value for the treatment of neurodegenerative diseases such as PD. Following age, a family history of PD is the most commonly reported risk factor, suggesting a genetic component of the disease in a subgroup of patients. The LRRK2 gene has emerged as the gene most commonly associated with both familial and sporadic PD. Here, we report that exposure of rat mesencephalic cells to curcumin induces the expression of LRRK2 mRNA and protein in a time-dependent manner. The expression of other PD-related genes, such alpha-synuclein and parkin, was not affected by exposure to curcumin, and PTEN-induced putative kinase 1 (PINK1) was not expressed in rat mesencephalic cells. As LRRK2 overexpression is strongly associated with the pathological inclusions found in several neurodegenerative disorders, further studies are needed to evaluate the effects of curcumin as a therapeutic agent for neurodegenerative diseases.

Mitochondria-Associated Membranes (MAMs): Overview and Its Role in Parkinson’s Disease

Mitochondria-associated membranes (MAMs) are structures that regulate physiological functions between endoplasmic reticulum (ER) and mitochondria in order to maintain calcium signaling and mitochondrial biogenesis. Several proteins located in MAMs, including those encoded by PARK genes and some of neurodegeneration-related proteins (huntingtin, presenilin, etc.), ensure this regulation. In this regard, MAM alteration is associated with neurodegenerative diseases such as Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s diseases (HD) and contributes to the appearance of the pathogenesis features, i.e., autophagy dysregulation, mitochondrial dysfunction, oxidative stress, and lately, neuronal death. Moreover,, ER stress and/or damaged mitochondria can be the cause of these disruptions. Therefore, ER-mitochondria contact structure and function are crucial to multiple cellular processes. This review is focused on the molecular interaction between ER and mitochondria indispensable to MAM formation and on MAM alteration-induced etiology of neurodegenerative diseases.

mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.

The MAPK1/3 pathway is essential for the deregulation of autophagy observed in G2019S LRRK2 mutant fibroblasts.

The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway.