Rosa A. González‐Polo

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.

Autophagic cell death is morphologically characterized by an accumulation of autophagic vacuoles. Here, we show that inactivation of LAMP2 by RNA interference or by homologous recombination leads to autophagic vacuolization in nutrient-depleted cells. Cells that lack LAMP2 expression showed an enhanced accumulation of vacuoles carrying the marker LC3, yet a decreased colocalization of LC3 and lysosomes, suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. While a fraction of mitochondria from starved LAMP2-expressing cells colocalized with lysosomal markers, within autophagolysosomes, no such colocalization was found on removal of LAMP2 from the experimental system. Of note, LAMP1 depletion had no such effects and did not aggravate the phenotype induced by LAMP2-specific small interfering RNA. Serum and amino acid-starved LAMP2-negative cells exhibited an accumulation of autophagic vacuoles and then succumbed to cell death with hallmarks of apoptosis such as loss of the mitochondrial transmembrane potential, caspase activation and chromatin condensation. While caspase inhibition retarded cell death, it had no protective effect on mitochondria. Stabilization of mitochondria by overexpression of Bcl-2 or the mitochondrion-targeted cytomegalovirus protein vMIA, however, blocked all signs of apoptosis. Neither caspase inhibition nor mitochondrial stabilization antagonized autophagic vacuolization in LAMP2-deficient cells. Altogether, these data indicate that accumulation of autophagic vacuoles can precede apoptotic cell death. These findings argue against the clear-cut distinction between type 1 (apoptotic) and type 2 (autophagic) cell death.

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.

Mitochondria form close physical contacts with a specialized domain of the endoplasmic reticulum (ER), known as the mitochondria-associated membrane (MAM). This association constitutes a key signaling hub to regulate several fundamental cellular processes. Alterations in ER-mitochondria signaling have pleiotropic effects on a variety of intracellular events resulting in mitochondrial damage, Ca2+ dyshomeostasis, ER stress and defects in lipid metabolism and autophagy. Intriguingly, many of these cellular processes are perturbed in neurodegenerative diseases. Furthermore, increasing evidence highlights that ER-mitochondria signaling contributes to these diseases, including Parkinson's disease (PD). PD is the second most common neurodegenerative disorder, for which effective mechanism-based treatments remain elusive. Several PD-related proteins localize at mitochondria or MAM and have been shown to participate in ER-mitochondria signaling regulation. Likewise, PD-related mutations have been shown to damage this signaling. Could ER-mitochondria associations be the link between pathogenic mechanisms involved in PD, providing a common mechanism? Would this provide a pharmacological target for treating this devastating disease? In this review, we aim to summarize the current knowledge of ER-mitochondria signaling and the recent evidence concerning damage to this signaling in PD.

Abstract Background Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β‐glucocerebrosidase‐1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S ‐GBA1 mutation on cellular homeostasis and vulnerability in a patient‐specific cellular model of PD. Methods We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy‐lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. Results The N370S mutation produced a significant reduction in β‐glucocerebrosidase‐1 protein and enzyme activity and β‐glucocerebrosidase‐1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Conclusions Our results support a connection between the loss of β‐glucocerebrosidase‐1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1 ‐PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1 ‐PD shares common features with lipid‐storage diseases. © 2017 International Parkinson and Movement Disorder Society

Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1′-dimethyl-4,4′-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP+ (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

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.

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.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder. While most PD cases are idiopathic, the known genetic causes of PD are useful to understand common disease mechanisms. Recent data suggests that autophagy is regulated by protein acetylation mediated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. The changes in histone acetylation reported to be involved in PD pathogenesis have prompted this investigation of protein acetylation and HAT and HDAC activities in both idiopathic PD and G2019S leucine-rich repeat kinase 2 (LRRK2) cell cultures. Fibroblasts from PD patients (with or without the G2019S LRRK2 mutation) and control subjects were used to assess the different phenotypes between idiopathic and genetic PD. G2019S LRRK2 mutation displays increased mitophagy due to the activation of class III HDACs whereas idiopathic PD exhibits downregulation of clearance of defective mitochondria. This reduction of mitophagy is accompanied by more reactive oxygen species (ROS). In parallel, the acetylation protein levels of idiopathic and genetic individuals are different due to an upregulation in class I and II HDACs. Despite this upregulation, the total HDAC activity is decreased in idiopathic PD and the total HAT activity does not significantly vary. Mitophagy upregulation is beneficial for reducing the ROS-induced harm in genetic PD. The defective mitophagy in idiopathic PD is inherent to the decrease in class III HDACs. Thus, there is an imbalance between total HATs and HDACs activities in idiopathic PD, which increases cell death. The inhibition of HATs in idiopathic PD cells displays a cytoprotective effect.

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP+ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 microM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.

J. Neurochem . (2009) 109 , 889–898. Abstract The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age‐related neurodegenerative disorders, including Parkinson’s disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ‐1 have been reported to be associated with autosomal recessive, early‐onset parkinsonism. In this paper we established a model system to study the involvement of the DJ‐1 protein in paraquat‐induced autophagy. When human neuroblastoma SH‐SY5Y cells were transfected with DJ‐1‐specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat‐induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3‐methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ‐1 in the autophagic response produced by paraquat, providing evidence for the role of PD‐related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's 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.

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 research of new biomarkers for Parkinson's disease is essential for accurate and precocious diagnosis, as well as for the discovery of new potential disease mechanisms and drug targets. The main objective of this work was to identify metabolic changes that might serve as biomarkers for the diagnosis of this neurodegenerative disorder. For this, we profiled the plasma metabolome from mice with neurotoxin-induced Parkinson's disease as well as from patients with familial or sporadic Parkinson's disease. By using mass spectrometry technology, we analyzed the complete metabolome from healthy volunteers compared to patients with idiopathic or familial (carrying the G2019S or R1441G mutations in the LRRK2 gene) Parkinson's disease, as well as, from mice treated with 6-hydroxydopamine to induce Parkinson disease. Both human and murine Parkinson was accompanied by an increase in plasma levels of unconjugated bile acids (cholic acid, deoxycholic acid and lithocholic acid) and purine base intermediary metabolites, in particular hypoxanthine. The comprehensive metabolomic analysis of plasma from Parkinsonian patients underscores the importance of bile acids and purine metabolism in the pathophysiology of this disease. Therefore, plasma measurements of certain metabolites related to these pathways might contribute to the diagnosis of Parkinson's Disease.

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 Parkinson's 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.

Parkinson's 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 Parkinson's 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.

Abstract The neuropathology associated with Parkinson's disease (PD) is thought to involve excessive production of free radicals, dopamine autoxidation, defects in glutathione peroxidase expression, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and 1‐methyl‐4‐phenylpyridinium ion (MPP + ), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. We show here that the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) increased significantly after treatment of cultured cerebellar granule cells (CGCs) with 50 μM MPP + . Co‐treatment with antioxidants such as ascorbate (ASC), catalase, α‐tocopherol (α‐TOH), coenzyme Q 10 (CoQ 10 ) or superoxide dismutase (SOD) rescued the cells from MPP + ‐induced death. MPP + ‐induced cell death was also abolished by co‐treatment with nitric oxide synthase (NOS) inhibitors such as 7‐nitroindazole (7‐NI), 2‐ethyl‐2‐thiopseudourea hydrobromide (EPTU) or S ‐methylisothiourea sulphate (MPTU). We also tested the protective effects of an iron chelator (deferoxamine mesylate, DFx) and a peroxynitrite scavenger (FeTTPS) and the results lend further support to the view that the free radical cytotoxicity plays an essential role in MPP + ‐induced death in primary cultures of CGC.

During coevolution with their hosts, viruses have "learned" to intercept or to activate the principal signal transducing pathways leading to cell death. A number of proteins from pathophysiologically relevant viruses are targeted to mitochondria and regulate (induce or inhibit) the apoptosis-associated permeabilization of mitochondrial membranes. Such proteins are encoded by human immunodeficiency virus 1, Kaposi's sarcoma-associated herpesvirus, human T-cell leukemia virus-1, hepatitis B virus, cytomegalovirus, and Epstein Barr virus, among others. Within mitochondria, such apoptosis regulators from viral origin can target distinct proteins from the Bcl-2 family and the permeability transition pore complex including the adenine nucleotide translocase, cyclophilin D, the voltage-dependent anion channel, and the peripheral benzodiazepine receptor. Thus, viral proteins can regulate apoptosis at the mitochondrial level by acting on a variety of different targets.

Exposure of cerebellar granule cells (CGCs) to 1-methyl-4-phenylpyridinium (MPP+) results in apoptotic cell death, which is markedly attenuated by co-treatment of CGCs with the radical scavenger vitamin E. Analysis of free radical production and mitochondrial transmembrane potential (DeltaPsim), using specific fluorescent probes, showed that MPP+ mediates early radical oxygen species (ROS) production without a loss of DeltaPsim. Exposure to MPP+ also produces an early increase in Bad dephosphorylation and translocation of Bax to the mitochondria. These events are accompanied by cytochrome c release from mitochondria to cytosol, which is followed by caspase 3 activation. Exposure of the neurons to vitamin E maintains Bad phosphorylation and attenuates Bax translocation, inhibiting cytochrome c release and caspase activation. MPP+-mediated cytochrome c release is also prevented by allopurinol, suggesting the participation of xanthine oxidase in the process. Our results indicate that free radicals play an active role in the MPP+-induced early events that culminate with cell death.

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work.

Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.

Paraquat (PQ) (1, 1’-dimethyl-4, 4’-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson’s disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.Addendum toInhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic Cell Death in Neuroblastoma SH-SY5Y CellsR.A. González-Polo, M. Niso-Santano, M.A. Ortíz-Ortíz, A. Gómez-Martín, J.M. Morán, L. García-Rubio, J. Francisco-Morcillo, C. Zaragoza , G. Soler and J.M. FuentesToxicological Science 2007; In press

Curcumin, the active compound of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. This agent has been shown to regulate numerous transcription factors, cytokines, protein kinases, adhesion molecules, redox status and enzymes that have been linked to inflammation. While curcumin has been identified as an activator of apoptosis in several cell lines, the mechanism by which it initiates apoptosis, however, remains poorly understood. We considered curcumin from the point of view of its ability to protect against oxidative stress, the latter being one factor strongly implicated in the development of Parkinson's disease. Although the etiology of Parkinson's disease remains unknown, epidemiological studies have linked exposure to pesticides such paraquat to an increased risk of developing the condition. Analysis of the neurotoxic properties of these pesticide compounds has been focused on their ability to induce oxidative stress in neural cells. Given curcumin's capacity to protect against oxidative stress, it has been considered as a potential therapeutic agent for neurodegenerative diseases such as Parkinson's disease that involve an oxidative stress component. In the present report we describe the effect of curcumin in paraquat-mediated apoptosis of N27 mesencepahlic cells. We show that subtoxic concentrations of curcumin sensitize N27 mesencephalic cells to paraquat-mediated apoptosis.

Abstract Paraquat (PQ) (1,1-dimethyl-4,4′-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). In this sense, understanding of the molecular mechanism underlying PQ-induced toxicity to neural cells is important for optimal use as well as for the development of new drugs. To gain insights into PQ-induced neurotoxicity, polymerase chain reaction (PCR) array analysis focused on a panel of apoptosis-related genes was performed using neuroblastoma SH-SY5Y cells. Up to 65 apoptosis-related genes were monitored. Our analysis of apoptotic process through microarray technology showed that in PQ-induced neuroblastoma SH-SY5Y cells, there is a different expression of BIK, CASP3, CASP7, CRADD, DAPK, FAS, and other related genes, in comparison to unstimulated cells. Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases as PD. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in PQ-mediated toxicity of neural cells. The authors would like to thank to Maria Pura Delgado for helpful assistance in the laboratory work. Miguel Angel Ortiz Ortiz is a predoctoral recipient of a fellowship from Second Vice-presidency and Regional Ministry of Economy, Commerce and Innovation, from the Junta of Extremadura. Rosa Ana González Polo is currently recipient of a “Juan de la Cierva” grant from Spanish Ministry of Education and Science. This work was supported by grants PRI06B124 from Junta de Extremadura and PI040828 and PI070400 from FIS‐ISCIII, Spanish Ministry of Health and Consumer Affairs.

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 Parkinson's 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.

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.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. This neuronal loss, inherent to age, is related to exposure to environmental toxins and/or a genetic predisposition. PD-induced cell death has been studied thoroughly, but its characterization remains elusive. To date, several types of cell death, including apoptosis, autophagy-induced cell death, and necrosis, have been implicated in PD progression. In this study, we evaluated necroptosis, which is a programmed type of necrosis, in primary fibroblasts from PD patients with and without the G2019S leucine-rich repeat kinase 2 (LRRK2) mutation and in rotenone-treated cells (SH-SY5Y and fibroblasts). The results showed that programmed necrosis was not activated in the cells of PD patients, but it was activated in cells exposed to rotenone. Necrostatin-1 (Nec-1), an inhibitor of the necroptosis pathway, prevented rotenone-induced necroptosis in PD models. However, Nec-1 affected mitochondrial morphology and failed to protect mitochondria against rotenone toxicity. Therefore, despite the inhibition of rotenone-mediated necroptosis, PD models were susceptible to the effects of both Nec-1 and rotenone.

Phenolic compounds derived from olive oil have beneficial health properties against cancer, neurodegenerative, and metabolic diseases. Therefore, there are discrepancies in their impact on mitochondrial function that result in changes in oxidative capacity, mitochondrial respiration, and energetic demands. This review focuses on the versatile role of oleuropein, a potent antioxidant that regulates the AMPK/SIRT1/mTOR pathway to modulate autophagy/mitophagy and maintain metabolic homeostasis.

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.

The identification of Parkinson's disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S-LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder.

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD) and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP+) results in cell death. We have studied the implication of various membrane transporter systems on MPP+ neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP+ toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP+ entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP+ exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP+-induced cell death.

When neural cells are exposed to paraquat, nitric oxide generation increases primarily due to an increase in the expression of the inducible isoform of nitric oxide synthase. The nitric oxide generated has controversial actions in paraquat exposure, as both protective and harmful effects have been described previously. While the actions mediated by nitric oxide in neural cells have been well described, there is evidence that nitric oxide may also be an important modulator of the expression of several genes during paraquat exposure. To better understand the actions of nitric oxide and its potential role in paraquat-induced gene expression, we examined changes in GCH1, ARG1, ARG2, NOS1, NOS2, NOS3, NOSTRIN, NOSIP, NOS1AP, RASD1, DYNLL1, GUCY1A3, DDAH1, DDAH2 and CYGB genes whose expression is controlled by or involved in signaling by the second messenger nitric oxide, in rat mesencephalic cells after 3, 6, 12 and 24 h of paraquat exposure. A qPCR strategy targeting these genes was developed using a SYBR green I-based method. The mRNA levels of all the genes studied were differentially regulated during exposure. These results demonstrate that nitric oxide-related genes are regulated following paraquat exposure of mesencephalic cells and provide the basis for further studies exploring the physiological and functional significance of nitric oxide-sensitive genes in paraquat-mediated neurotoxicity.

The pathogenesis of neurodegenerative diseases involves altered activity of proteolytic systems and accumulation of protein aggregates. Autophagy is an intracellular process in which damaged organelles and long-lived proteins are degraded and recycled for maintaining normal cellular homeostasis. Disruption of autophagic activity in neurons leads to modify the cellular homeostasis, causing deficient elimination of abnormal and toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons to clear abnormal protein aggregates and survive. This review aims to give an overview of some of the main modulators of autophagy that are currently being studied as possible alternatives in the search of therapies that slow the progression of neurodegenerative diseases, which are incurable to date. Keywords: Autophagy, Natural substances, Neurodegeneration, Protection.

Abstract Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson’s disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster . This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.

KEAP1 is a cytoplasmic protein that functions as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 leads to an accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, we investigated whether Keap1 deficiency affects the endosome-lysosomal pathway. We used KEAP1-deficient mouse embryonic fibroblasts (MEFs) and combined Western blot analysis and fluorescence microscopy with fluorometric and pulse chase assays to analyze the levels of lysosomal-endosomal proteins, lysosomal function, and autophagy activity. We found that the loss of keap1 downregulated the protein levels and activity of the cathepsin D enzyme. Moreover, KEAP1 deficiency caused lysosomal alterations accompanied by an accumulation of autophagosomes. Our study demonstrates that KEAP1 deficiency increases nondegradative lysosomes and identifies a new role for KEAP1 in lysosomal function that may have therapeutic implications.

A significant loss in ATP levels was found in cerebellar granule cells with 1-methyl-4-phenylpyridinium. Exposure of cerebellar granule cells to low concentrations of 1-methyl-4-phenylpyridinium (100 microM) resulted in a time and dose-dependent decreases in ATP levels and cell death. This neurotoxin caused inhibition of the enzymatic activity of NADH-dehydrogenase of mitochondrial complex I and consequent impairment of mitochondrial electronic transport with a reduction in the depletion of cytosolic NAD(+) levels. Activation of lactate dehydrogenase activity (detected by the increase of the lactate in the culture medium) partially reduced this depletion. Addition of glucose but not pyruvate to the culture medium protected 1-methyl-4-phenylpyridinium-induced cell death. These results suggest the 1-methyl-4-phenylpyridinium causes impairment of cellular energy metabolism with a major dependence on glycolysis as a source of energy. This fact could also explain the partial neuroprotection observed by glucose.

Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user’s laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier.

The exposure of immortalized rat neuroblast cells to MPP(+) and paraquat results in cell death. Heat shock pre-treatment prior to the addition of MPP(+) and paraquat significantly reduced cell death and led to an increase in the synthesis of Hsp 27 and Hsp70 proteins. Quercetin inhibits the synthesis of heat shock proteins (Hsp) and prevents their protective effect, which suggests that this protection was dependent on the Hsps synthesis. These data indicate that heat shock protects cells from the toxic effect of MPP(+) and paraquat. These results and the structural similarity between paraquat and MPP(+) support the role of paraquat as a putative risk factor in the etiology of Parkinson's disease.

Efavirenz (EFV), a first generation non-nucleoside analogue reverse transcriptase inhibitor widely employed in combination antiretroviral therapy regimens over the last 20 years, has been associated with a wide range of neuropsychiatric effects and has also been linked with HIV-associated neurocognitive disorder (HAND). EFV has been reported to alter mitochondrial dysfunction and bioenergetics in different cell types, including astrocytes. Here, we analyzed whether this mitochondrial effect is associated with alterations in autophagy and, more specifically, mitophagy. U251-MG cells were exposed to EFV (10 and 25 μM; 24 h) and the effect was compared with that of CCCP - an uncoupler of the mitochondrial membrane potential and widely-employed in vitro inducer of mitophagy – and those of the known pharmacological stressors rotenone and thapsigargin, selected due to reported similarities with EFV. EFV induces autophagy with functional autophagic flux despite the accumulated p62/SQSTM1. However, it fails to activate canonical mitophagy (according to mitochondrial mass and expression of mitophagy-related proteins). The fact that EFV-exposed cells display decreased levels of TOM20, an outer mitochondrial membrane protein, together with the association of TOM20 with autophagosomes (LC3), points to an alternative form of mitochondrial degradation. Moreover, the perinuclear mitochondrial cluster in EFV-treated cells differs from that displayed with CCCP. Also, in EFV-treated cells, p62 was associated with mitochondria, which may be related to the mito-protective function of this autophagic protein. In conclusion, these findings add to the existing knowledge of the EFV-triggered mitochondrial interference, a mechanism that may be implicated in the adverse CNS events observed in the clinics.

Pompe disease or glycogen storage disease type II (OMIM: 232300) is a lysosomal storage disorder resulting from a partial or total lack of acid alphaglucosidase, which may produce muscle weakness, gait abnormalities, or even death by respiratory failure. In the last decade, autophagy has been proposed as a mechanism involved in the severity of symptoms related to this disorder and as a potential therapeutic target to alleviate disease progression. This review summarizes the relationship between autophagy and Pompe disease, including what information has been recently discovered and what remains unclear. Keywords: Autophagy, Glycogen synthase, LC3, Lysosomes, Pompe disease, Recombinant human GAA.

Talipexole is a non-ergot dopamine (DA) agonist that has been used in the treatment of Parkinson's disease. In the present study, we examined the effect of talipexole on paraquat (PQ)-induced N27 cell death and the intracellular pathways involved in this effect. Pretreatment of N27 cells with talipexole (1mM) resulted in significant protection against paraquat-induced cell death. In N27 cells, talipexole inhibited paraquat-induced apoptotic hallmarks such as cytochrome c release, caspase-3 activation, chromatin condensation and externalization of phosphatidilserine. Talipexole pretreatment prevents the reduction in the anti-apoptotic Bcl-x(L) protein and increases in the pro-apoptotic form of Bak and p-Bad, both induced by PQ. Finally, we also observed that talipexole abrogates the activation of cell death pathways JNK1/2 and p38 produced by PQ, and increases the phosphorylated (active) forms of the pro-survival pathways ERK1/2 and Akt. These results reveal that talipexole exerts a neuroprotective effect in a mesencephalic cell line exposed to the neurotoxin PQ, which is related to the etiology of Parkinson's disease.

Parkinson's disease is the second common neurodegenerative disorder, after Alzheimer's disease. It is a clinical syndrome characterized by loss of dopamine-generating cells in the substancia nigra, a region of the midbrain. The etiology of Parkinson's disease has long been through to involve both genetic and environmental factors. Mutations in the leucine-rich repeat kinase 2 gene cause late-onset Parkinson's disease with a clinical appearance indistinguishable from Parkinson's disease idiopathic. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damage proteins and cytoplasmic organelles. This degradative process has been associated with cellular dysfunction in neurodegenerative processes including Parkinson's disease. We discuss the role of leucine-rich repeat kinase 2 in autophagy, and how the deregulations of this degradative mechanism in cells can be implicated in the Parkinson's disease etiology.

Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the gene encoding the huntingtin protein. Although the precise mechanism by which neuronal degeneration occurs is still unclear, several elements are important to its development: (1) altered gene expression and protein synthesis, (2) mitochondrial damage and (3) improper regulation of the autophagy programme. In this issue of British Journal of Pharmacology, Galindo and co-workers provide the first evidence for a role of the mitochondrial permeability transition pore (mPTP) in mitochondrial fragmentation and autophagy activation. In a model of cell death induced by 3-nitropropionic acid (3-NP) in human neural cells, the authors describe clear functions for mPTP and Bax, but not the mitochondrial fusion/fission machinery, mitochondrial fragmentation and autophagy (mitophagy). This commentary summarises the significance of this relationship and suggests several points for future development.

Autophagy is the major cellular process of degradation and is modulated by several signaling pathways. Phosphatidylinositol 3-kinase (PtdIns3K) class III (Vps34) and PtdIns3K class I regulate the autophagy pathway positively and negatively, respectively. Both classes of PtdIns3K participate in the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P), which plays a crucial role in autophagosome biogenesis and membrane traffic. PtdIns3P is a membrane phospholipid that is associated with endogenous FYVE domain-containing proteins. Indeed, such interactions facilitate autophagosome fusion with lysosomes and subsequent cargo degradation. During starvation-induced autophagy, the expression of FYVE domain-containing proteins increases, and their binding to PtdIns3P is strengthened. Nonetheless, not all FYVE domain proteins are related to the induction of autophagy. This method report presents the quantification of PtdIns3P synthesis by using cells either transiently transfected with or stably expressing FYVE-dsRed.

The etiology of Parkinson's disease (PD) is not completely defined, although environmental factors (for example, exposure to the herbicide paraquat [PQ]) and genetic susceptibility (such as DJ-1 mutations that have been associated with an autosomal-recessive form of early-onset PD) have been demonstrated to contribute. Alterations in macroautophagy have been described in the pathogenesis of this neurodegenerative disease. We have established a model system to study the involvement of the DJ-1 protein in PQ-induced autophagy. When we transfected cells exposed to PQ with DJ-1–specific siRNA, we observed an inhibition of the autophagic events induced by the herbicide, as well as sensitization additive with PQ-induced apoptotic cell death and exacerbation of this cell death in the presence of the autophagy inhibitor 3-methyladenine. These results suggest, for the first time, an active role for DJ-1 in the autophagic response produced by PQ, opening the door to new strategies for PD therapy.

PD (Parkinson's disease) is a neurodegenerative disorder caused by loss of dopamine-generating cells in the substantia nigra. The implication of genetic factors in the aetiology of PD has an essential importance in our understanding of the development of the disease. Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene cause late-onset PD with a clinical appearance indistinguishable from idiopathic PD. Moreover, LRRK2 has been associated with the process of autophagy regulation. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damaged proteins and cytoplasmic organelles. In the present paper, we discuss the role of LRRK2 in autophagy, and the importance of this relationship in the development of nigral degeneration in PD.

Autophagy is an intracellular catabolic mechanism mediated by lysosomes, which is responsible for most of the degradation and recycling of cytoplasmic components and intracellular dysfunctional or damaged organelles. Increasing evidences suggest that autophagic deregulation causes accumulation of abnormal proteins or damaged organelles, which is a characteristic of chronic neurodegenerative conditions, such as Parkinson's disease (PD), a multifactorial disorder, which is neuropathologically characterized by age-dependent neurodegeneration of dopaminergic neurons in the midbrain. Indeed, promoting the clearance of aggregate-prone proteins via pharmacological induction of autophagy has proved to be an useful mechanism for protecting cells against the toxic effects of these proteins in the context of neurodegenerative diseases and protecting neurons from apoptosis. This special issue is composed of seven excellent reviews addressing the analysis of different models of Parkinsonism in which autophagy plays a key role.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3' untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand-receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by mitochondrial dysfunction, oxidative stress, and later neuronal death. Several genetics and environmental factors have been implicated in the PD pathogenesis. 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. The deregulation of autophagic process in nerve cells is thought to be a possible cause of PD. G2019S mutant fibroblasts exhibited higher autophagic activity that can trigger cell death. In this sense, G2019S mutant cells displayed increased apoptosis hallmarks and enough susceptibility to MPP+ (1-methyl-4-phenylpyridinium). G2019S LRRK2 heightens the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) reduced the enhanced autophagy suggesting that the G2019S mutation induces autophagy via MEK/ERK pathway. Further, the inhibition of this exacerbated autophagy reduces the sensitivity remarked in G2019S mutant cells.

This report describes a laboratory exercise that was incorporated into a Cell Biology and Molecular Biology advanced course. The exercise was made for a class size with eight students and was designed to reinforce the understanding of basic molecular biology techniques. Students used the techniques of reverse transcription and arginase activity measurement as well as nitric oxide determination to discover whether two specific genes were expressed by cytokine-stimulated dendritic cells. The experiment served as the basis for discussing the importance of differential gene expression inside the eukaryotic cell and the importance of cytokines in the immune system.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a dysregulation of cellular degradation system. An accumulation of misfolded proteins has been founded in the brains of parkinsonian patients, causing neuroinflammation and oxidative stress, and leading to a progressive neurodegeneration. Autophagy plays an important role in the progression of PD. In this chapter, we analyze the relationship of different types of autophagy (microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy) with the oxidative stress and with several proteins involved in PD, showing deregulation of these degradative processes when these proteins are mutated. Also, we show a possible therapeutic alternative based on autophagy inducers that might be a potential drug for PD treatment.

LRRK2 mutations have been described as a common cause of Parkinson's disease (PD) in patients from northern Spain. Here we investigated the prevalence of these mutations in a cohort of Spanish PD patients (n = 96) from Extremadura, a region in southwestern Spain.To evaluate the rate of the G2019S and R1441G/C/H LRKK2 mutations in PD patients and healthy controls (n = 163).Here we show that the G2019S mutation is present at a low prevalence in our Spanish cohort, while the R1441G/C/H mutation, which has been reported to be common in northern Spain, was not observed in the PD patients or in the controls.LRRK2 mutations do not appear to be a common cause of Parkinson's disease in Extremadura, Spain.