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ABSTRACT: Mammalian protein kinase C-interacting cousin of thioredoxin (PICOT) is a multi-domain mono-thiol glutaredoxin that is involved in several signal transduction pathways and is necessary for cell growth and metastasis. Here, we demonstrate that PICOT is a cleavage substrate of the apoptosis-related protein caspase-3. In vitro cleavage assays indicated that PICOT was specifically cleaved by caspase-3. Similarly, endogenous PICOT was cleaved in cell death responses induced by staurosporine and etoposide. These phenomena were blocked in the presence of a pan-caspase inhibitor. Using site-directed mutagenesis, we identified two putative caspase-3 cleavage sequences in PICOT, DRLD(101)/G and EELD(226)/T. Interestingly, overexpression of either PICOT wild type or the D101A/D226A double point mutant accelerated etoposide-induced activation of caspase-3 whereas siRNA-mediated knockdown of PICOT blocked this phenomenon. Our data raise the possibility that the pro-apoptotic role of PICOT is actively regulated via caspase-3-mediated cleavage.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
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ABSTRACT: Loss-of-function mutations in the PARK7/DJ-1 gene cause early onset autosomal-recessive Parkinson disease. DJ-1 has been implicated in protection of neurons from oxidative stress and in regulation of transcriptional activity. However, whether there is a relationship between the subcellular localization of DJ-1 and its function remains unknown. Therefore, we examined the subcellular localization of DJ-1 during dopaminergic neurodegeneration induced by various insults. Immunoblotting and immunocytochemistry showed that the nuclear pool of DJ-1 dramatically increased in both MN9D dopaminergic neuronal cells and primary cultures of mesencephalic dopaminergic neurons after 6-hydroxydopamine (6-OHDA) treatment. This was paralleled by a corresponding decrease in its cytosolic level, indicating drug-induced nuclear translocation of DJ-1. The same phenomenon was detected in other cell death paradigms induced by pro-oxidants including hydrogen peroxide and cupric chloride. Consequently, cotreatment with the antioxidant N-acetyl-l-cysteine blocked the translocation of DJ-1 into the nucleus. However, mutation at cysteine 106 had no effect on the translocation of DJ-1 into the nucleus, suggesting that reactive oxygen species-mediated downstream signaling and/or modifications other than oxidative modification are involved in its nuclear translocation. Ectopic expression of nucleus localization signal (NLS)-tagged DJ-1 prevented cell death from 6-OHDA. We investigated whether nuclear DJ-1 was involved in transcriptional regulation and found that DJ-1 was localized in promyelocytic leukemia bodies, and this localization increased upon 6-OHDA treatment. We also confirmed that binding of DJ-1 and promyelocytic leukemia bodies indeed increased after 6-OHDA treatment. Consequently, expression levels of acetylated p53 and PUMA were downregulated in cells overexpressing DJ-1 or NLS-tagged DJ-1. Taken together, our data suggest that nuclear translocation of DJ-1 may protect neurons from cell death after oxidative stress.
Free radical biology & medicine 06/2012; 53(4):936-50. · 5.42 Impact Factor
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ABSTRACT: Loss-of-function mutations in the DJ-1/PARK7 gene are responsible for early-onset autosomal-recessive Parkinson's disease. DJ-1 is implicated in the protection of neurons from oxidative stress by scavenging hydrogen peroxide and regulating the transcriptional activity of multiple pathways. Here, we attempted to identify the protein profiles modulated by DJ-1 in MN9D dopaminergic neurons following 6-hydroxydopamine (6-OHDA) treatment. We found that reactive oxygen species (ROS) levels increased in DJ-1-deficient cells that were either untreated or subjected to 6-OHDA treatment. The incidence of apoptosis after 6-OHDA treatment was increased in DJ-1 knockdown cells. Using these cells, we then performed two-dimensional gel electrophoresis in conjunction with mass spectrometry to assess changes in protein profiles before and after 6-OHDA treatment. Several protein spots were positively or negatively altered in DJ-1-deficient cells with or without 6-OHDA. Among the altered proteins, immunoblot analysis confirmed an increase in galectin-7 and a decrease in peroxiredoxin-6 in DJ-1 knockdown cells. Moreover, transcriptional levels of putative p53 target proteins, including selenophosphate synthetase 1 and glycogen phosphorylase, were increased in the DJ-1 knockdown cells. Taken together, our data suggest that increases in pro-apoptotic proteins and decreases in anti-apoptotic proteins render DJ-1 knockdown cells more susceptible to oxidative stress.
Biochemical and Biophysical Research Communications 04/2012; 422(1):8-14. · 2.48 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 04/2012; 287(28):23271-82. · 4.77 Impact Factor
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ABSTRACT: The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson's disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.
Apoptosis 02/2012; · 4.07 Impact Factor
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ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. To discover potential key molecules in this process, we utilized cDNA microarray technology to obtain an expression profile of transcripts in MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine. Using a self-organizing map algorithm, data mining and clustering were combined to identify distinct functional subgroups of genes. We identified alterations in the expression of 81 genes in eight clusters. Among these genes, we verified protein expression patterns of MAP kinase phosphatase 1 and sequestosome 1 using both cell culture and rat brain models of PD. Immunological analyses revealed increased expression levels as well as aggregated distribution patterns of these gene products in 6-hydroxydopamine-treated dopaminergic neurons. In addition to the identification of other proteins that are known to be associated with protein aggregation, our results raise the possibility that a more widespread set of proteins may be associated with the generation of protein aggregates in dying neurons. Further research to determine the functional roles of other altered gene products within the same cluster as well as the seven remaining clusters may provide new insights into the neurodegeneration that underlies PD pathogenesis.
Acta Neurovegetativa 09/2011; 118(11):1585-98. · 2.73 Impact Factor
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ABSTRACT: Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.
Apoptosis 07/2011; 16(11):1087-100. · 4.07 Impact Factor
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ABSTRACT: The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including α- and β-tubulin. Further analysis of α- and β-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of α-tubulin and between residues 216 and 238 of β-tubulin, respectively. We found that an increase in α-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.
Amino Acids 03/2011; 40(3):809-18. · 3.25 Impact Factor
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ABSTRACT: Accumulating evidence has revealed that autophagy may be beneficial for treatment of neurodegenerative diseases through removal of abnormal protein aggregates. However, the critical autophagic events during neurodegeneration remain to be elucidated. Here, we investigated whether prototypic autophagic events occur in the MN9D dopaminergic neuronal cell line upon exposure to N-methyl-4-phenylpyridinium (MPP (+) ), a well-known dopaminergic neurotoxin. MPP (+) treatment induced both morphological and biochemical characteristics of autophagy, such as accumulation of autophagic vacuoles and LC3-II form and decreased p62 levels. Further investigation revealed that these phenomena were largely the consequences of blocked autophagic flux. Following MPP (+) treatment, levels of LC3-II formed and p62 dramatically increased in the Triton X-100-insoluble fraction. Levels of ubiquitinated proteins also increased in this fraction. Further colocalization analyses revealed that the punctated spots positive for both p62 and LC3 were more intense following MPP (+) treatment, suggesting drug-induced enrichment of these two proteins in the insoluble fraction. Intriguingly, reciprocal immunoprecipitation analysis revealed that p62 mainly precipitated with LC3-II form following MPP (+) treatment. Transient transfection of the mutant form of Atg4B, Atg4B (C74A) , which inhibits LC3 processing, dramatically decreased binding between p62 and LC3-II form. Taken together, our results indicate that p62 can be efficiently localized to autophagic compartments via preferential binding with LC3-II form. This colocalization may assist in removal of detergent-insoluble forms of damaged cellular proteins during dopaminergic neurotoxin-induced impairment of autophagic flux.
Autophagy 01/2011; 7(1):51-60. · 7.45 Impact Factor
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ABSTRACT: Recent studies have demonstrated that clioquinol, an antibiotic with an anti-amyloid effect, acts as a zinc ionophore under physiological conditions. Because increases in labile zinc may induce autophagy, we examined whether clioquinol induces autophagy in cultured astrocytes in a zinc-dependent manner. Within 1h of exposure to 0.1-10 μM clioquinol, the levels of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker of autophagy, began to increase in astrocytes. Confocal live-cell imaging of GFP-LC3-transfected astrocytes showed the formation of LC3(+) autophagic vacuoles (AVs), providing a further indication that clioquinol induced autophagy. Addition of 3-methyladenine or small-interfering RNA against autophagy-related gene 6 (ATG6/Beclin-1) blocked clioquinol-induced increases in LC3-II. FluoZin-3 fluorescence microscopy showed that, like the zinc ionophore pyrithione, clioquinol increased intracellular zinc levels in the cytosol and AVs in an extracellular zinc-dependent manner. Zinc chelation with N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) reduced, and addition of zinc increased the levels of LC3-II and LC3(+) puncta, indicating that zinc influx plays a key role therein. Moreover, astrocytes and SH-SY5Y cells expressing mutant huntingtin (mHttQ74) accumulated less aggregates when treated with clioquinol, and this effect was reversed by TPEN. These results indicate that clioquinol-induced autophagy is likely to be physiologically functional. The present study demonstrates that clioquinol induces autophagy in a zinc-dependent manner and contributes to clearance of aggregated proteins in astrocytes and neurons. Hence, in addition to its metal-chelating effect in and around amyloid beta (Aβ) plaques, clioquinol may contribute to the reduction of Aβ loads by activating autophagy by increasing or normalizing intracellular zinc levels in brain cells.
Neurobiology of Disease 01/2011; 42(3):242-51. · 5.40 Impact Factor
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ABSTRACT: Anamorsin, also called cytokine-induced apoptosis inhibitor 1 (CIAPIN1), was recently identified to confer resistance to apoptosis induced by growth factor deprivation and to be indispensible for hematopoiesis. Recently, it was demonstrated that anamorsin is also widely distributed in both fetal and adult tissues. In this study, we evaluated the tissue distribution of anamorsin in the central nervous system (CNS) during development. In situ hybridization and immunoblot analyses revealed that anamorsin mRNA and protein were both highly and widely expressed in various regions of the CNS, including the cerebral cortex, hippocampus, midbrain, cerebellum, medulla, and spinal cord. Based on these findings, we examined its cellular localization during drug-induced neurodegeneration in MN9D dopaminergic cells. Both immunocytochemical localization and immunoblot analyses indicated that cytosolic anamorsin was translocated into the nucleus in a time-dependent manner following treatment with a reactive oxygen species (ROS)-inducing drug, 6-hydroxydopamine (6-OHDA). Treatment of cells with the apoptosis-inducing reagent, staurosporine, did not appear to cause translocation of anamorsin into the nucleus. When cells were treated with the nuclear export inhibitor, Leptomycin B, alone or with 6-OHDA, nuclear anamorsin levels increased, indicating that nuclear influx and efflux of anamorsin are regulated by 6-OHDA treatment. In rat brain injected with 6-OHDA, nuclear translocation of anamorsin was identified in certain tyrosine hydroxylase (TH)-positive neurons as well as TH-negative cells. Furthermore, treatment of MN9D cells with hydrogen peroxide or ROS-inducing trace metals caused nuclear translocation of anamorsin. Taken together, our data indicate that nuclear translocation of anamorsin is a ROS-dependent event and may participate in the regulation of transcription of critical molecules during dopaminergic neurodegeneration.
Acta Neurovegetativa 10/2010; 118(3):433-44. · 2.73 Impact Factor
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ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disorder caused by selective degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Although mitochondrial abnormality, oxidative stress and proteasomal dysfunction are recognized as major contributors to the progression of PD, there is a limited understanding of the key molecular events that provoke degeneration of DA neurons. Using a proteomic approach, we attempted to identify profiles of proteins with altered expression levels in rats following unilateral stereotaxic injection of 6-hydroxydopamine into the SNc. Protein expression profiles of these proteins in the substantia nigra and the striatum were made using two-dimensional gel electrophoresis in conjunction with a mass spectrometry. More than 70 identified proteins displayed significant differences in their temporal and spatial expression pattern between experimental and vehicle-operated control groups. Based on the identity of the proteins, we further searched for potential binding partners using biological databases available on the web and constructed a protein interaction network. Among several interconnected proteins in the network, we verified the interaction between prohibitin and the NADH-ubiquinone oxidoreductase 30kDa subunit (NDUFS3 subunit; a mitochondrial complex I subunit) by co-immunoprecipitation. We also confirmed, using immunohistochemical localization, that both prohibitin and the NDUFS3 subunit were increased in the dying DA neurons, suggesting its potential role in regulating mitochondrial function in dying DA neurons. Furthermore, knockdown of prohibitin accelerated 6-hydroxydopamine-induced cell death in SH-SY5Y cells. Our results raise the possibility that interconnected proteins in the network may positively or negatively impact the progression of DA neuronal death.
Neurochemistry International 08/2010; 57(1):16-32. · 2.86 Impact Factor
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ABSTRACT: Spinal cord injury (SCI) induces massive cell death, leading to permanent neurological disability. No satisfactory treatment is currently available. Ghrelin, a gastric hormone, is known to stimulate GH release from the hypothalamus and pituitary gland. Here, we report that ghrelin administration improves functional recovery after SCI in part by inhibiting apoptosis of neurons and oligodendrocytes. Ghrelin was not detected in normal, uninjured spinal cords, but spinal cord neurons and oligodendrocytes expressed the ghrelin receptor. Ghrelin significantly inhibited apoptotic cell death of neurons and oligodendrocytes, release of mitochondrial cytochrome c, and activation of caspase-3 after moderate contusion SCI. Ghrelin also significantly increased the level of phosphorylated ERK but decreased the level of phosphorylated p38MAPK. In addition, ghrelin increased the level of ERK-dependent brain-derived neurotrophic factor expression and decreased the level of pronerve growth factor expression. Furthermore, the neuroprotective effects of ghrelin were mediated through the ghrelin receptor. Finally, ghrelin significantly improved functional recovery and reduced the size of the lesion volume and the loss of axons and myelin after injury. These results suggest that ghrelin may represent a potential therapeutic agent after acute SCI in humans.
Endocrinology 05/2010; 151(8):3815-26. · 4.46 Impact Factor
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ABSTRACT: Calbindin-D28K protects against apoptotic and necrotic cell death; these effects have been attributed to its ability to buffer calcium. In this study, we investigated the mechanisms underlying the neuroprotective effects of calbindin-D28K in staurosporine (STS)-induced apoptosis and 1-methyl-4-phenylpyridinium (MPP(+))-induced necrosis. Treatment of the dopaminergic neuronal cell line MN9D with STS or MPP(+) induced cell death that was associated with increased levels of free intracellular calcium. However, only MPP(+)-induced death was inhibited by co-treatment of the cells with a calcium chelator or a sodium/calcium antiporter inhibitor. Overexpression of calbindin-D28K prevented MPP(+)-induced MN9D cell death, which occurs in the absence of any detectable caspase activation. These pro-survival effects of calbindin-D28K were associated with the inhibition of calcium-mediated calpain activation, as determined by processing of Bax. Overexpression of calbindin-D28K also blocked STS-induced MN9D death. However, this effect was accompanied by the inhibition of capase-3 cleavage, poly(ADP-ribose)polymerase cleavage, and caspase activity. These findings suggest that calbindin-D28K protects against both types of cell death by inhibiting caspase- or calcium-mediated death signaling pathway.
Biochemical and Biophysical Research Communications 07/2008; 371(1):127-31. · 2.48 Impact Factor
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Young Mook Lee,
Seong H Park,
Dong-Ik Shin,
Jee-Yeon Hwang,
Bokyung Park,
Yun-Jong Park,
Tae H Lee,
Ho Z Chae,
Byung K Jin,
Tae H Oh, Young J Oh
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ABSTRACT: The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.
Journal of Biological Chemistry 05/2008; 283(15):9986-98. · 4.77 Impact Factor
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ABSTRACT: Spinal cord injury (SCI) causes a permanent neurological disability, and no satisfactory treatment is currently available. After SCI, pro-nerve growth factor (proNGF) is known to play a pivotal role in apoptosis of oligodendrocytes, but the cell types producing proNGF and the signaling pathways involved in proNGF production are primarily unknown. Here, we show that minocycline improves functional recovery after SCI in part by reducing apoptosis of oligodendrocytes via inhibition of proNGF production in microglia. After SCI, the stress-responsive p38 mitogen-activated protein kinase (p38MAPK) was activated only in microglia, and proNGF was produced by microglia via the p38MAPK-mediated pathway. Minocycline treatment significantly reduced proNGF production in microglia in vitro and in vivo by inhibition of the phosphorylation of p38MAPK. Furthermore, minocycline treatment inhibited p75 neurotrophin receptor expression and RhoA activation after injury. Finally, minocycline treatment inhibited oligodendrocyte death and improved functional recovery after SCI. These results suggest that minocycline may represent a potential therapeutic agent for acute SCI in humans.
Journal of Neuroscience 08/2007; 27(29):7751-61. · 7.11 Impact Factor
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ABSTRACT: The neuroprotective effect of mitochondrial isocitrate dehydrogenase (IDPm), an enzyme involved in the reduction of NADP(+) to NADPH and the supply of glutathione (GSH) in mitochondria, was examined using SH-SY5Y cells overexpressing IDPm (S1). S1 cells showed higher NADPH and GSH levels than vector transfectant (V) cells and were more resistant to staurosporine-induced cell death than controls. Staurosporine-induced cytochrome c release, caspase-3 activation, and production of reactive oxygen species (ROS) were significantly attenuated in S1 cells as compared to V cells and reduced by antioxidants, trolox and GSH-ethyl ester (GSH-EE). Staurosporine-induced the release of Mcl-1 from mitochondria that formed a complex with Bim. Mcl-1 was then cleaved to a shortened form in a caspase-3 dependent manner; its release was attenuated far more in S1 than in V cells after staurosporine treatment. Finally, the staurosporine-induced decrease in mitochondrial membrane potential (Deltapsi(m)) was correlated with the time of mitochondrial Mcl-1 release; the loss of Deltapsi(m) was attenuated significantly in S1 cells as compared to that in V cells. These results suggest that the neuroprotective effect of IDPm may result from increases in NADPH and GSH levels in the mitochondria. This, in turn, inhibits mitochondrial ROS production after cytochrome c release, which seems to be mediated through Mcl-1 release.
Journal of Neuroscience Research 02/2007; 85(1):139-52. · 2.74 Impact Factor
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ABSTRACT: This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.
Glia 09/2005; 51(2):98-110. · 4.82 Impact Factor
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ABSTRACT: Although alpha-synuclein is the main structural component of the insoluble filaments that form Lewy bodies in Parkinson disease (PD), its physiological function and exact role in neuronal death remain poorly understood. In the present study, we examined the possible functional relationship between alpha-synuclein and several forms of matrix metalloproteinases (MMPs) in the human dopaminergic neuroblastoma (SK-N-BE) cell line. When SK-N-BE cells were transiently transfected with alpha-synuclein, it was secreted into the extracellular culture media, concomitantly with a significant decrease in cell viability. Also the addition of nitric oxide-generating compounds to the cells caused the secreted alpha-synuclein to be digested, producing a small fragment whose size was similar to that of the fragment generated during the incubation of alpha-synuclein with various MMPs in vitro. Among several forms of MMPs, alpha-synuclein was cleaved most efficiently by MMP-3, and MALDI-TOF mass spectra analysis showed that alpha-synuclein is cleaved from its C-terminal end with at least four cleavage sites within the non-Abeta component of AD amyloid sequence. Compared with the intact form, the protein aggregation of alpha-synuclein was remarkably facilitated in the presence of the proteolytic fragments, and the fragment-induced aggregates showed more toxic effect on cell viability. Moreover, the levels of MMP-3 were also found to be increased significantly in the rat PD brain model produced by the cerebral injection of 6-hydroxydopamine into the substantia nigra. The present study suggests that the extracellularly secreted alpha-synuclein could be processed via the activation of MMP-3 in a selective manner.
Journal of Biological Chemistry 08/2005; 280(26):25216-24. · 4.77 Impact Factor
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ABSTRACT: Recent studies indicate that activation of stress-activated protein kinases may be implicated in a broad range of biological activities including differentiation. To directly examine whether stress-activated protein kinases are involved in neuronal differentiation, we utilized retinoic acid-induced and spontaneous models of neurite outgrowth in dopaminergic neurons. Here, we show that retinoic acid-induced neurite outgrowth in MN9D dopaminergic neuronal cells was accompanied by activation of c-Jun N-terminal kinase but not p38. Consequently, cotreatment with a specific inhibitor of c-Jun N-terminal kinase or overexpression of c-Jun N-terminal kinase-binding domain of c-Jun N-terminal kinase-interacting protein-1 blocked retinoic acid-induced neurite outgrowth. In primary cultures of dopaminergic neurons, the extent of neurite outgrowth increased spontaneously in a time-dependent manner. When these cultures were treated with a specific inhibitor of c-Jun N-terminal kinase, the total extent of neurites, the primary neurite length and the number of neurites per cell were suppressed significantly. Thus, our data indicate that the c-Jun N-terminal kinase signal seems to play an important role during morphological differentiation in cultured dopaminergic neurons.
Neuroreport 06/2005; 16(8):823-8. · 1.66 Impact Factor
