MN9D Cells Research Articles

Alteration in the sphingolipid metabolism leads to activation of the apoptotic cascade in the MPTP induced mouse model of Parkinson's disease

Parkinson's disease is a degenerative disorder of the central nervous system. It results from the death of dopaminergic neurons in the substantia nigra of the mid brain. Sphingolipids are important for neuronal survival. The sphingolipid metabolic pathway is regulated by two enzymes, namely Sphk1 and Sphk2. Sphk1 phosphorylates sphingosine leading to the formation of S1P which is essential for the survival of the neurons. The present study has demonstrated that both SPHK1 and its isoform SPHK2 in dopaminergic neurons of the substantia nigra of the brain showed a significant decrease in the MPTP‐induced mouse model of Parkinson's disease. This decrease was confirmed in both tissue samples and in the MN9D cell line treated with MPP+. Furthermore inhibition of the isoforms using dimethylsphingosine (DMS) leads to apoptosis of the neurons which was analysed using flow cytometry. Moreover Caspase 3 seems to increase significantly in both the MPP+ and DMS treated groups. The present results suggest that, the alteration of sphingolipid metabolic pathway may be a cause for the death of dopaminergic neurons in the MPTP induced mouse model of Parkinson's disease. Supported by grant no: MOE 2009‐T2–1‐061 from the Ministry of Education, Singapore.

Nuclear Import and Export Signals Control the Subcellular Localization of Nurr1 Protein in Response to Oxidative Stress*

Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.

RESP18 is Involved in the Cytotoxicity of Dopaminergic Neurotoxins in MN9D Cells

RESP18 (Regulated endocrine-specific protein, 18 kDa) was first identified as a dopaminergic drugs-regulated intermediate pituitary transcript. RESP18 protein is a unique endoplasmic reticulum (ER) resident protein. Its functions in the brain especially in the nervous system disorders remain unknown. ER stress (ERS) has been proved to be one of the important pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Here, we explored the association of RESP18 and ERS in cell models of PD. Dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP⁺), 6-hydroxydopamine (6-OHDA), and rotenone evoked dramatic MN9D cell death. The transcriptional expressions of RESP18 and two ERS markers--binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) manifested differential changes in MN9D cells treated with MPP⁺, 6-OHDA, and rotenone. The RESP18 protein levels increased in MPP⁺ and 6-OHDA-treated cells, but did not change in the cells treated with rotenone, while the protein levels of ER molecular chaperone heat shock protein 90 kDa beta member 1/glucose-regulated protein 94 (HSP90B1/GRP94) and BiP in the cells were up-regulated by MPP⁺ and 6-OHDA, respectively. Salubrinal, an ERS inhibitor, significantly reduced MPP⁺ and 6-OHDA-induced cell death. Moreover, ERS inducer--thapsigargin and tunicamycin, decreased the expression of RESP18, which is different from the changes of BiP, GRP94, and CHOP. Silencing RESP18 expression with Lenti-shRNA alleviated MPP⁺-induced cell death, while over-expression of RESP18 resulted in aggravated cell death induced by MPP⁺ and 6-OHDA. Taken together, our results suggest that RESP18 is involved in the cytotoxicity of dopaminergic neurotoxins.

Cutting Edge: IL-13Rα1 Expression in Dopaminergic Neurons Contributes to Their Oxidative Stress–Mediated Loss following Chronic Peripheral Treatment with Lipopolysaccharide

Inflammation and its mediators, including cytokines and reactive oxygen species, are thought to contribute to neurodegeneration. In the mouse brain, we found that IL-13Rα1 was expressed in the dopaminergic (DA) neurons of the substantia nigra pars compacta, which are preferentially lost in human Parkinson's disease. Mice deficient for Il13ra1 exhibited resistance to loss of DA neurons in a model of chronic peripheral inflammation using bacterial LPS. IL-13, as well as IL-4, potentiated the cytotoxic effects of t-butyl hydroperoxide and hydrogen peroxide on mouse DA MN9D cells. Collectively, our data indicate that expression of IL-13Rα1 on DA neurons can increase their susceptibility to oxidative stress-mediated damage, thereby contributing to their preferential loss. In humans, Il13ra1 lies on the X chromosome within the PARK12 locus of susceptibility to Parkinson's disease, suggesting that IL-13Rα1 may have a role in the pathogenesis of this neurodegenerative disease.

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3-kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.

IL6 Protects MN9D Cells and Midbrain Dopaminergic Neurons from MPP+-Induced Neurodegeneration

The degeneration of midbrain dopaminergic (mDA) neurons is the hallmark of Parkinson's disease (PD), and several in vivo and in vitro models have been established to resemble the processes occurring during disease progression. One of the most commonly used disease models for PD is the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively kills mDA neurons when applied systemically. In vivo, MPTP intoxication is accompanied by a strong microglia response which is characterised by the release of inflammatory molecules such as tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL6) that are believed to further drive inflammation-mediated degeneration of mDA neurons. Here, we addressed the question whether primary ventral mDA neurons and MN9D cells release cytokines in vitro and how these cytokine profiles change after treatment with MPP(+). Our results demonstrate that both culture models show different cytokine profiles under control conditions indicating that comparisons between both models should be made very carefully. Moreover, MN9D cells released high levels of IL6 and IP10/CXCL10, both of which were down regulated after treatment with MPP(+). MN9D-derived IL6 seems to be important for MN9D survival since neutralisation of endogenous IL6 resulted in degeneration of MN9D cells. Moreover, recombinant IL6 was able to rescue MN9D cells and primary mDA neuron cultures from MPP(+)-induced neurotoxicity, underlining the neuroprotective properties of IL6.

Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

RGS10 exerts a neuroprotective role through the PKA/c‐AMP response‐element (CREB) pathway in dopaminergic neuron‐like cells

Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacologic approaches, we identified protein kinase A and the downstream phospho-cAMP response element-binding signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress.

Dopaminochrome induces caspase‐independent apoptosis in the mesencephalic cell line, MN9D

Parkinson's disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.

N-acetylcysteine Protects against Apoptosis through Modulation of Group I Metabotropic Glutamate Receptor Activity

The activation of group I metabotropic glutamate receptor (group I mGlus) has been shown to produce neuroprotective or neurotoxic effects. In this study, we investigated the effects of N-acetylcysteine (NAC), a precursor of the antioxidant glutathione, on group I mGlus activation in apoptosis of glial C6 and MN9D cell lines, and a rat model of Parkinson's disease (PD). We demonstrated that NAC protected against apoptosis through modulation of group I mGlus activity. In glial C6 cells, NAC promoted phosphorylation of ERK induced by (s)-3,5- dihydroxy-phenylglycine (DHPG), an agonist of group I mGlus. NAC enhanced the group I mGlus-mediated protection from staurosporine (STS)-induced apoptosis following DHPG treatment. Moreover, in rotenone-treated MN9D cells and PD rat model, NAC protected against group I mGlus-induced toxicity by compromising the decrease in phosphorylation of ERK, phosphorylation or expression level of TH. Furthermore, the results showed that NAC prohibited the level of ROS and oxidation of cellular GSH/GSSG (Eh) accompanied by activated group I mGlus in the experimental models. Our results suggest that NAC might act as a regulator of group I mGlus-mediated activities in both neuroprotection and neurotoxicity via reducing the oxidative stress, eventually to protect cell survival. The study also suggests that NAC might be a potential therapeutics targeting for group I mGlus activation in the treatment of PD.

Hypoxia regulation of ATP13A2 (PARK9) gene transcription

Parkinson's disease (PD) is the second most common neurodegenerative disorders with a variable combination of motor and non-motor symptoms. Mutations in several genes including ATP13A2 (PARK9) are reported to be associated with PD. The underlying mechanism of PD is not well defined, however, both genetic and environmental causes contribute to it. ATP13A2 gene locates in chromosome 1 and contains 29 exons encoding for a protein of 1180 amino acids with 10 transmembrane domains. Abnormal gene expression has been implicated in neurodegenerative disorders. The transcriptional regulation of the ATP13A2 gene is unknown. In this report, we cloned and functionally characterized the human ATP13A2 gene promoter. We showed that the promoter region of the human ATP13A2 gene contains hypoxia response elements which can bind to transcription factor hypoxia-inducible factor 1α (HIF-1α). Hypoxia up-regulated ATP13A2 transcription via HIF-1α in HEK293 and dopaminergic MN9D cells. Our study indicates that hypoxia signaling plays a very important role in the regulation of human ATP13A2 gene expression. Further study is needed to determine the role of hypoxia in the pathogenesis of PD and its interaction with other PD causative genes, which will provide insights to the role of hypoxia and dysregulation of gene expression in Parkinson's disease.

N-cadherin-ERα-Src signal models mediate the synergistic potentiation of activation of PI3K/Akt signal pathway in injured dopaminergic neurons by GDNF and E2

Background Accumulating evidence indicates that glial cell linederived neurotrophic factor (GDNF) synergizes with 17b-estradiol (E2) could protect dopaminergic neurons. However, the mechanisms have not yet been elucidated. Based on the fact that either E2 or GDNF can activate the intracellular PI3K/Akt signal pathway, we hypothesize that the synergic protection of dopaminergic neurons exerted by E2 and GDNF is ascribed to enhancing the activation of the cellular PI3K/Akt signal pathway in a certain way. Method We studied the potential mechanism under the synergistic protective effects of E2 and GDNF on dopaminergic neurons using the MN9D cell line. The MN9D cells were treated with 6-OHDA before incubating with either E2 or GDNF or both. Endogenous AKT phosphorylation and precise underlying mechanistic studies were revealed using co-immunoprecipitation(co-IP), western blot and immuno-fluorescent staining. Result

FLZ protects dopaminergic neuron through activating protein kinase B/mammalian target of rapamycin pathway and inhibiting RTP801 expression in Parkinson's disease models

The pathogenesis of Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons in substantia nigra (SNpc). FLZ, a novel synthetic squamosamide derivative from a Chinese herb, has been shown to have neuroprotective effects in experimental Parkinson's disease (PD) models. However, it is still unclear whether FLZ protects against PD through regulating the function of dopaminergic system. In this study, we carried out a set of in vitro and in vivo experiments to address these questions. Oral administration of FLZ significantly improved motor dysfunction of mice challenged by MPTP. The beneficial effects of FLZ on motor behavior attributed to the elevation of dopamine level in striatum, tyrosine hydroxylase (TH)-positive cells, and TH activity in the middle brain of mouse. Mechanism study showed that treatment of FLZ increased the phosphorylation of activating protein kinase B (Akt) and mammalian target of rapamycin (mTOR). Using LY294002 to block phosphoinositide 3-kinases (PI3K)/Akt signaling pathway prevented the phosphorylation of mTOR and attenuated the neuroprotection of FLZ in MN9D cells challenged by MPP+. In addition, FLZ reduced the expression of RTP801, an important protein in PD, in mice and cells intoxicated by MPTP/MPP+. Taken together, these results revealed a novel role that FLZ elevated TH expression and activity in dopaminergic neuron through activation of Akt/mTOR survival pathway and inhibition of RTP801 in MPTP/MPP+-induced PD models. The data also provided evidence that FLZ had potent neuroprotecive effects and might become a new promising anti-PD drug.

Low concentrations of methamphetamine can protect dopaminergic cells against a larger oxidative stress injury: mechanistic study.

Mild stress can protect against a larger insult, a phenomenon termed preconditioning or tolerance. To determine if a low intensity stressor could also protect cells against intense oxidative stress in a model of dopamine deficiency associated with Parkinson disease, we used methamphetamine to provide a mild, preconditioning stress, 6-hydroxydopamine (6-OHDA) as a source of potentially toxic oxidative stress, and MN9D cells as a model of dopamine neurons. We observed that prior exposure to subtoxic concentrations of methamphetamine protected these cells against 6-OHDA toxicity, whereas higher concentrations of methamphetamine exacerbated it. The protection by methamphetamine was accompanied by decreased uptake of both [3H] dopamine and 6-OHDA into the cells, which may have accounted for some of the apparent protection. However, a number of other effects of methamphetamine exposure suggest that the drug also affected basic cellular survival mechanisms. First, although methamphetamine preconditioning decreased basal pERK1/2 and pAkt levels, it enhanced the 6-OHDA-induced increase in these phosphokinases. Second, the apparent increase in pERK1/2 activity was accompanied by increased pMEK1/2 levels and decreased activity of protein phosphatase 2. Third, methamphetamine upregulated the pro-survival protein Bcl-2. Our results suggest that exposure to low concentrations of methamphetamine cause a number of changes in dopamine cells, some of which result in a decrease in their vulnerability to subsequent oxidative stress. These observations may provide insights into the development of new therapies for prevention or treatment of PD.

Calbindin-D28K inhibits apoptosis in dopaminergic neurons by activation of the PI3-kinase-Akt signaling pathway

Calbindin-D28k (CaBP) has a neuroprotective effect on dopaminergic (DA) neurons in several models of Parkinson's disease. We used the DA cell line MN9D to explore the mechanisms underlying CaBP-mediated protection against the neurotoxin 6-hydroxydopamine (6-OHDA) of DA neurons. In MN9D cells that were transfected with the expression vector pcDNA3-CB containing CaBP cDNA, the expression level of CaBP was significantly increased. After treating with 6-OHDA, a significant decrease in the apoptosis rate of the transfected MN9D cells was noted, as well as an obvious increase in the expression of phosphorylation of Akt (p-Akt); however, no significant change in the expression of total Akt or phospho-p100 (p-p100) occurred after this treatment. After treatment with wortmannin, an inhibitor of the PI3-kinase-Akt (PI-3K/Akt) signal pathway, an increase in the expression level of CaBP was observed, but there were no other obvious changes of the experimental index mentioned previously in the groups transfected with pcDNA3-CB. These studies suggest that CaBP has a significant role in protecting DA cells against the apoptosis induced by 6-OHDA—through PI-3K/Akt signaling pathway—where the non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway might have no relevance.

Sorting Protein-related Receptor SorLA Controls Regulated Secretion of Glial Cell Line-derived Neurotrophic Factor

Glial cell line-derived neurotrophic factor (GDNF), after secreted from cells, plays a critical role in central and peripheral neuron survival and function. The secretion of GDNF can be either constitutive or regulated by physiological stimuli; however, the detailed mechanism driving GDNF secretion is still unknown. Here, we report that sorting protein-related receptor with A-type repeats (SorLA), a member of the mammal Vps10p domain receptor, interacts with GDNF and is localized to GDNF-containing vesicles. Overexpression of SorLA significantly increases, and knockdown of SorLA by siRNA decreases, the regulated secretion of GDNF in PC12 and MN9D cells but has no effect on GDNF constitutive secretion. In addition, overexpression of a truncated form of SorLA also impairs GDNF-regulated secretion. Finally, we found that the prodomain of GDNF mediates the interaction of GDNF with SorLA under acidic conditions. Moreover, overexpression of SorLA could enhance the regulated secretion of the GDNF prodomain-GFP fusion protein, suggesting that the prodomain of GDNF is responsible for its regulated secretion. Together, these findings will advance our understanding of the molecular mechanism underlying GDNF-regulated secretion.

Regulation of microglia effector functions by tumor necrosis factor signaling.

The exact biological role of the cytokine tumor necrosis factor (TNF) in the central nervous system (CNS) is not well understood; but overproduction of TNF by activated microglia has been implicated in neuronal death, suggesting that TNF inhibition in the CNS may be a viable neuroprotective strategy. We investigated the role of TNF signaling in regulation of microglia effector functions using molecular, cellular, and functional analyses of postnatal and adult microglia populations in the CNS. No differences were found by flow cytometric analyses in the basal activation state between TNF-null and wild-type mice. Although TNF-null microglia displayed an atypical morphology with cytoplasmic vacuoles in response to stimulation with lipopolysaccharide (LPS), the phagocytic response of TNF-null microglia to Escherichia coli particles in vitro was normal and there were no signs of enhanced caspase 3 activation or apoptosis. Functionally, conditioned media from LPS-stimulated TNF-null microglia was found to have significantly reduced levels of IL-10, IL-6, IL-1β, IL-12, and CXCL1 relative to wild-type microglia and exerted no cytotoxic effects on neurally differentiated dopaminergic (DA) MN9D cells. In contrast, incubation of wild-type microglia with TNF inhibitors selectively depleted the levels of soluble TNF and its cytotoxicity on MN9D cells. To distinguish whether reduced cytotoxicity by LPS-activated TNF-null microglia could be attributed to deficient autocrine TNF signaling, we employed primary microglia deficient in one or both TNF receptors (TNFR1 and TNFR2) in co-culture with MN9D cells and found that neither receptor is required to elicit LPS-evoked TNF production and cytotoxicity on DA cells.

Neuroprotective Effects of Vanillyl Alcohol in Gastrodia elata Blume Through Suppression of Oxidative Stress and Anti-Apoptotic Activity in Toxin-Induced Dopaminergic MN9D Cells

Gastrodia elata Blume (GE) has long been used in oriental countries as a traditional herbal medicine to relieve symptoms associated with neurological ailments such as vertigo, general paralysis and epilepsy. In this study, we have investigated the effects of GE extracts and its major bioactive components on 1-methyl-4-phenylpyridinium (MPP+)-treated MN9D dopaminergic cells, a classic in vitro model for Parkinson’s disease (PD). We found that vanillyl alcohol effectively inhibited the cytotoxicity and improved cell viability in MPP+-induced MN9D dopaminergic cells. The underlying mechanisms of vanillyl alcohol action were also studied. Vanillyl alcohol attenuated the elevation of reactive oxygen species (ROS) levels, decreased in the Bax/Bcl-2 ratio and poly (ADP-ribose) polymerase proteolysis. These results indicate that vanillyl alcohol protected dopaminergic MN9D cells against MPP+-induced apoptosis by relieving oxidative stress and modulating the apoptotic process and is therefore a potential candidate for treatment of neurodegenerative diseases such as Parkinson’s disease.

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.

Silencing of PINK1 induces mitophagy via mitochondrial permeability transition in dopaminergic MN9D cells

Accumulation of dysfunctional Mitochondria has been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, have been identified in early-onset forms of PD. Recent data showed that the loss of PINK1 function led to oxidative stress, mitochondrial damage and autophagic elimination of damaged mitochondria. But the precise mechanism of autophagy induced by loss of PINK1 is unclear. In this study, we found that in mouse dopaminergic MN9D cells, down-regulation of PINK1 by RNA interference resulted in induction of mitochondrial autophagy (mitophagy), abnormal mitochondrial morphology, partial loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Mitophagy in these cells was associated with the up-regulation of autophagy activator Beclin 1 and opening of mitochondrial permeability transition (MPT) pore. These findings suggest that PINK1 may regulate mitophagy through controlling MPT pore opening and general autophagy regulators.