6-OHDA Treatment Research Articles

Montelukast treatment protects nigral dopaminergic neurons against microglial activation in the 6-hydroxydopamine mouse model of Parkinson's disease.

Although the main cause of degeneration of the nigrostriatal dopaminergic (DA) projection in Parkinson's disease (PD) is still controversial, many reports suggest that excessive inflammatory responses mediated by activated microglia can induce neurotoxicity in the nigrostriatal DA system in vivo. Montelukast, which plays an anti-inflammatory role, is used to treat patients with asthma. In addition, recent studies have reported that its administration could reduce neuroinflammatory activities, showing beneficial effects against various neuropathological conditions. These results suggest that montelukast may be a useful drug to alleviate inflammatory responses in PD, even though there are no reports showing its beneficial effects against neurotoxicity in the nigrostriatal DA system. In the present study, our results showed that treatment with montelukast could protect DA neurons against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity and its administration significantly attenuated the production of neurotoxic cytokines such as tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) from activated microglia in the substantia nigra (SN) and striatum following 6-OHDA treatment. Therefore, we suggest that montelukast can be used as a potential inhibitor of microglial activation to protect DA neurons in the adult brain against PD.

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. Conclusion: CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway.

Leucine-rich repeat kinase 2 exacerbates neuronal cytotoxicity through phosphorylation of histone deacetylase 3 and histone deacetylation.

Parkinson's disease (PD) is characterized by slow, progressive degeneration of dopaminergic neurons in the substantia nigra. The cause of neuronal death in PD is largely unknown, but several genetic loci, including leucine-rich repeat kinase 2 (LRRK2), have been identified. LRRK2 has guanosine triphosphatase (GTPase) and kinase activities, and mutations in LRRK2 are the major cause of autosomal-dominant familial PD. Histone deacetylases (HDACs) remove acetyl groups from lysine residues on histone tails, promoting transcriptional repression via condensation of chromatin. Here, we demonstrate that LRRK2 binds to and directly phosphorylates HDAC3 at Ser-424, thereby stimulating HDAC activity. Specifically, LRRK2 promoted the deacetylation of Lys-5 and Lys-12 on histone H4, causing repression of gene transcription. Moreover, LRRK2 stimulated nuclear translocation of HDAC3 via the phoshorylation of karyopherin subunit α2 and α6. HDAC3 phosphorylation and its nuclear translocation were increased in response to 6-hydroxydopamine (6-OHDA) treatment. LRRK2 also inhibited myocyte-specific enhancer factor 2D activity, which is required for neuronal survival. LRRK2 ultimately promoted 6-OHDA-induced cell death via positive modulation of HDAC3. These findings suggest that LRRK2 affects epigenetic histone modification and neuronal survival by facilitating HDAC3 activity and regulating its localization.

Mesencephalic astrocyte-derived neurotrophic factor reduces cell apoptosis via upregulating GRP78 in SH-SY5Y cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects dopaminergic neurons from damage. In this study, we used MTT, immunohistochemistry, and TUNEL staining to investigate the protective effect of MANF in SH-SY5Y cells treated with 6-OHDA or overexpressed α-synuclein. Cleaved caspase-3 levels significantly increased in cells treated with 6-OHDA or overexpressed α-synuclein. 6-OHDA or α-synuclein overexpression that induced cleaved caspase-3 levels to increase was reduced by MANF treatment. In addition, MANF treatment upregulated GRP78 expressions in cells treated with 6-OHDA or overexpressed α-synuclein, and RNAi knockdown for GRP78 could block the MANF induced cell survival from 6-OHDA treatment. Furthermore, GRP78 overexpression inhibited 6-OHDA-induced apoptosis. Our data suggest that MANF inhibits apoptosis induced by 6-OHDA and overexpressed α-synuclein in SH-SY5Y cells via upregulating GRP78 in the transcriptional pattern.

Blocking Sympathetic Nervous System Reverses Partially Stroke-Induced Immunosuppression but does not Aggravate Functional Outcome After Experimental Stroke in Rats.

Stoke results in activation of the sympathetic nervous system (SNS), inducing systemic immunosuppression. However, the potential mechanisms underlying stroke-induced immunosuppression remain unclear. Here, we determined the SNS effects on functional outcome and explored the interactions among SNS, β-arrestin2 and nuclear factor-κB (NF-κB) after experimental stroke in rats. In the current study, stroke was induced by a transient middle cerebral artery occlusion (MCAO) in rats, and SNS activity was inhibited by intraperitoneal injection of 6-hydroxydopamine HBr (6-OHDA). 7.0T Micro-MRI and Longa score were employed to assess the functional outcome after stroke. Flow cytometry and ELISA assay were used to measure the expression of MHC class II, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Western blot was conducted to analyze β-arrestin2 and NF-κB protein expression levels after experimental stroke. We found significantly increased infarct volumes and functional impairment after MCAO at different post-surgery time points, which were not aggravated by 6-OHDA treatment. SNS blockade partially reversed the expression of MHC class II after stroke over time, as well as TNF-α and IFN-γ levels in lipopolysaccharide-stimulated macrophages in vitro. Treatment of MCAO rats with SNS-inhibitor significantly diminished NF-κB activation and enhanced β-arrestin2 expression after stroke. This study suggests that pharmacological SNS inhibition dose not aggravate functional outcome after stroke. Stroke-induced immunosuppression may be involved in the SNS-β-arrestin2-NF-κB pathway.

L-DOPA Reverses the Increased Free Amino Acids Tissue Levels Induced by Dopamine Depletion and Rises GABA and Tyrosine in the Striatum.

Perturbations in the cerebral levels of various amino acids are associated with neurological disorders, and previous studies have suggested that such alterations have a role in the motor and non-motor symptoms of Parkinson's disease. However, the direct effects of chronic L-DOPA treatment, that produces dyskinesia, on neural tissue amino acid concentrations have not been explored in detail. To evaluate whether striatal amino acid concentrations are altered in peak dose dyskinesia, 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian mice were treated chronically with L-DOPA and tissue amino acid concentrations were assessed by HPLC analysis. These experiments revealed that neither 6-OHDA nor L-DOPA treatment are able to alter glutamate in the striatum. However, glutamine increases after 6-OHDA and returns back to normal levels with L-DOPA treatment, suggesting increased striatal glutamatergic transmission with lack of dopamine. In addition, glycine and taurine levels are increased following dopamine denervation and restored to normal levels by L-DOPA. Interestingly, dyskinetic animals showed increased levels of GABA and tyrosine, while aspartate striatal tissue levels are not altered. Overall, our results indicate that chronic L-DOPA treatment, besides normalizing the altered levels of some amino acids after 6-OHDA, robustly increases striatal GABA and tyrosine levels which may in turn contribute to the development of L-DOPA-induced dyskinesia.

Comparative Analysis of the Effects of Neurotrophic Factors CDNF and GDNF in a Nonhuman Primate Model of Parkinson's Disease.

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson’s disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesioned monkeys and analyzed the amino acid sequence of marmoset CDNF. The severity of 6-OHDA lesions and treatment effects were monitored in vivo using 123I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of 123I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF.

Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration.

MiRNA-331-5p expression level is altered in neuronal cell under elevated oxidative stress

Event Abstract Back to Event MiRNA-331-5p expression level is altered in neuronal cell under elevated oxidative stress Pei Ling Yeo1, Soi Moi Chye2, Rhun Yian Koh2, Kenny G. Voon3 and Khuen Yen Ng4, 5* 1 International Medical University, Postgraduate Studies and Research, Malaysia 2 International Medical University, Department of Human Biology, Malaysia 3 International Medical University, Department of Pathology, Malaysia 4 Monash University Malaysia, Jeffrey Cheah School of Medicine and Health Sciences, Malaysia 5 Monash University Malaysia, School of Pharmacy, Malaysia Parkinson’s Diseases (PD) is a debilitating neurodegenerative disorder causing movement and neurological disabilities. Apoptotic death of the dopaminergic neurons in the substantia nigra pars compacta region of midbrain of PD patients are known to be the devastating events following the buildup of oxidative stress. MicroRNA (miRNA) is a class of small, non-coding RNA which plays important roles in regulating gene expression post-transcriptionally. Increasing evidence suggested that miRNA profile might be altered in PD. A recent investigation reported that miRNA-331-5p was significantly elevated (~ 22 folds of that in controls) in the blood plasma of PD patients. However, no study has been conducted to understand the pathological involvement of this miRNA in PD. Thus, this study aims to investigate whether levels of miRNA-331-5p in neuronal cells are affected under high oxidative stress. In this study, neurotoxin, 6-hydroxydopamine (6-OHDA) was added to SH-SY5Y cells for 6, 12, and 24 hours to induce oxidative stress. Cell viability was assessed with MTT assay whereas levels of reactive oxygen species (ROS) was quantified with DCFH-DA fluorescent probe staining. Expression of miRNA-331-5p in the cells was quantified using quantitative Real-time PCR. Result shows that 6-OHDA treatment caused a time-dependent increased in ROS generation most pronounced in 100 and 200 µM (up to 6 folds) treatment groups. 6-OHDA treatment also resulted in a time-dependent decreased of cell viability to 7% across all concentration tested. Subsequently, miRNA expression level showed significant up-regulation with 12 and 24 hours treatment groups. This increase in miRNA-331-5p level is correlated to the increase in ROS level and neuronal cell death. In conclusion, these findings suggest that miRNA-331-5p might be involved in the pathogenesis of PD as its expression is altered under elevated oxidative stress. Keywords: Oxidative Stress, miRNA, Pathogenesis, Parkinson’s disease, neurodegeneration Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Yeo P, Chye S, Koh R, Voon KG and Ng K (2016). MiRNA-331-5p expression level is altered in neuronal cell under elevated oxidative stress. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00119 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Khuen Yen Ng, Monash University Malaysia, Jeffrey Cheah School of Medicine and Health Sciences, Bandar Sunway, Selangor, Malaysia, ng.khuen.yen@monash.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Pei Ling Yeo Soi Moi Chye Rhun Yian Koh Kenny G Voon Khuen Yen Ng Google Pei Ling Yeo Soi Moi Chye Rhun Yian Koh Kenny G Voon Khuen Yen Ng Google Scholar Pei Ling Yeo Soi Moi Chye Rhun Yian Koh Kenny G Voon Khuen Yen Ng PubMed Pei Ling Yeo Soi Moi Chye Rhun Yian Koh Kenny G Voon Khuen Yen Ng Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Neuroprotective Effects of Tanshinone I Against 6-OHDA-Induced Oxidative Stress in Cellular and Mouse Model of Parkinson's Disease Through Upregulating Nrf2.

In this study, we investigated whether tanshinone I (T-I) has therapeutic effects in cellular and animal model of Parkinson's disease (PD), and explore its possible mechanism. For this purpose, human neuroblastoma SH-SY5Y cells were cultured and exposed to 100 μM 6-hydroxydopamine (6-OHDA) in the absence or presence of T-I (1, 2.5 and 5 μM). The results revealed that 6-OHDA-induced cell death was reduced by T-I pretreatment as measured by MTT assay, lactate dehydrogenase release and flow cytomety analysis of cell apoptosis. The increase in the reactive oxygen species caused by 6-OHDA treatment was also attenuated by T-I in SH-SY5Y cells. T-I pretreatment was also shown to result in an increase in nuclear factor erythroid-2-related factor 2 (Nrf2) protein levels and its transcriptional activity as well as the upregulation of Nrf2-dependent genes encoding the antioxidant enzymes heme oxygenase-1, glutathione cysteine ligase regulatory subunit and glutathione cysteine ligase modulatory subunit in SH-SY5Y cells. Moreover, in the in vivo experiment, T-I treatment significantly attenuated 6-OHDA-induced striatal oxidative stress and ameliorated dopaminergic neurotoxicity in 6-OHDA-lesioned mice, as evidenced by western blot analysis of tyrosine hydroxylase (TH) and TH immunostaining of dopaminergic neurons in the substantia nigra and the striatum. Taken together, the results suggest that T-I may be beneficial for the treatment of neurodegenerative diseases like PD.

Indirubin-3-Oxime Effectively Prevents 6OHDA-Induced Neurotoxicity in PC12 Cells via Activating MEF2D Through the Inhibition of GSK3β.

Indirubin-3-oxime (I3O), a synthetic derivative of indirubin, was originally designed as potent inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3β (GSK3β) for leukemia therapy. In the current study, we have shown, for the first time, that I3O prevented 6-hydroxydopamine (6OHDA)-induced neuronal apoptosis and intracellular reactive oxygen species accumulation in PC12 cells in a concentration-dependent manner. GSK3β inhibitors but not CDK5 inhibitors reduced the neurotoxicity induced by 6OHDA. Moreover, the activation of GSK3β was observed after 6OHDA treatment. Furthermore, 6OHDA substantially decreased the transcriptional activity of myocyte enhancer factor 2D (MEF2D), a transcription factor that plays an important role in dopaminergic neuron survival, and reduced nuclear localized MEF2D expression. Interestingly, indirubin-3-oxime and GSK3β inhibitors prevented 6OHDA-induced dysregulation of MEF2D. In addition, short hairpin RNA-mediated decrease of MEF2D expression significantly abolished the neuroprotective effects of indirubin-3-oxime. Collectively, our results strongly suggested that indirubin-3-oxime prevented 6OHDA-induced neurotoxicity via activating MEF2D, possibly through the inhibition of GSK3β. In view of the capability of indirubin-3-oxime to cross the blood-brain barrier, our findings further indicated that indirubin-3-oxime might be a novel drug candidate for neurodegenerative disorders, including Parkinson's disease in particular.

Lymphocytes reduce nigrostriatal deficits in the 6-hydroxydopamine mouse model of Parkinson's disease.

Neuroinflammation is a well-known neuropathological feature of Parkinson's disease (PD), but it remains controversial whether it is causal or consequential to neurodegeneration. While the role of microglia in the pathogenesis has been thoroughly investigated in human and different rodent models, data concerning the impact of the adaptive immune system on the pathogenesis of PD are still rare, although lymphocyte populations were found in brain tissue of PD patients and have been implicated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurodegeneration in mice. To test the hypothesis that the adaptive immune system contributes to the progression of PD in the murine 6-hydroxydopamine (6-OHDA) model, we performed unilateral 6-OHDA injection into the medial forebrain bundle and compared wild-type mice with recombination activating gene-1 deficient mice (RAG-1(-/-)), that lack mature lymphocytes. After 6-OHDA injection, immune-deficient mice moved significantly slower and less often than wild-type mice. Rotarod analysis displayed a shorter latency to fall in RAG-1(-/-) mice. Immunohistochemical analysis in wild-type mice demonstrated a higher CD8+ T cell density in the ipsilesional striatum compared to sham-operated animals. Cell counts of tyrosine hydroxylase positive dopaminergic neurons of the substantia nigra in immune compromised mice were significantly reduced compared to wild-type mice. Wild type bone marrow reconstitution into RAG-1(-/-) recipients rescued the clinical deterioration as well as the neurodegeneration in RAG-1(-/-) deficient recipients ameliorated clinical symptoms and neurodegeneration after 6-OHDA treatment. Our data indicate that lymphocytes reduce the clinical and neuropathological impact of 6-OHDA lesioning and thus may play a protective role in this toxic mouse model of PD.

Protective effect of a novel herbmedicine, Hepad, on apoptosis of SH-SY5Y cells and a rat model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. In this study, we investigated the effects of a novel herb formula, Hepad, on PD. Dose-dependent treatment with 1-methyl-4-phenylpyridinium (MPP+) decreased the viability of SH-SY5Y cells, and Hepad inhibited the toxic effect of MPP+. Hepad blocked the production of reactive oxygen species (ROS) induced by MPP+ in SH-SY5Y cells, and suppressed the activation of caspase 9 and caspase 3 due to MPP+. A rat model of PD was generated by 6-hydroxydopamine (6-OHDA) injection into the left medial forebrain bundle (MFB) of SD rats. In D-amphetamine sulfate-induced rotational behavioral tests, Hepad administration attenuated circling behavior relative to the 6-OHDA-treated disease group. In addition, Hepad treatment significantly increased the tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta (SNpc) that had decreased in response to 6-OHDA treatment (P<0.05). OX-6 expression, which indicates the presence of microglial cells, decreased significantly after treatment of Hepad in contrast to the 6-OHDA-treated disease group (P<0.05). These results indicate that Hepad may be a useful neuroprotective material for the treatment of neurodegenerative disorders such as PD.

Synthetic phenylethanoid glycoside derivatives as potent neuroprotective agents

Several phenylethanoid glycoside derivatives were designed and synthesized. Most of the synthetic compounds showed significant neuroprotective effects, including antioxidative and anti-apoptotic properties. Specifically, target compounds displayed potent effects against various toxicities such as H2O2 and 6-hydroxydopamine (6-OHDA) in PC12 cells. Among the synthetic derivatives, three compounds (5, 6, 8) exhibited much superior activities to the marketed drug Edaravone. The compounds were able to prevent the 6-OHDA-induced damage in PC12 cells in a dose-dependent manner. The anti-apoptotic effects could be observed via cell morphological changes. Moreover, the compounds significantly reduced the intracellular ROS increase resulting from 6-OHDA treatment. The preliminary structure–activity relationships were also explored. Compounds 5, 6, 8 may hold the potential as promising neuroprotective agents and new lead compounds for the treatment of neurodegenerative diseases or cerebral ischemia.

Characterization of nociceptive response to chemical, mechanical, and thermal stimuli in adolescent rats with neonatal dopamine depletion

Rats with dopamine depletion caused by 6-hydroxydopamine (6-OHDA) treatment during adulthood and the neonatal period exhibit akinetic motor activity and spontaneous motor hyperactivity during adolescence, respectively, indicating that the behavioral effects of dopamine depletion depend on the period of lesion development. Dopamine depletion during adulthood induces hyperalgesic response to mechanical, thermal, and/or chemical stimuli, whereas the effects of neonatal dopamine depletion on nociceptive response in adolescent rats are yet to be examined. The latter aspect was addressed in this study, and behavioral responses were examined using von-Frey, tail flick, and formalin tests. The formalin test revealed that rats with neonatal dopamine depletion exhibited a significant increase in nociceptive response during interphase (6–15min post formalin injection) and phase 2 (16–75min post formalin injection). This increase in nociceptive response to the formalin injection was not reversed by pretreatment with methamphetamine, which ameliorates motor hyperactivity observed in adolescent rats with neonatal 6-OHDA treatment. The von-Frey filament and tail flick tests failed to reveal significant differences in withdrawal thresholds between neonatal 6-OHDA-treated and vehicle-treated rats. The spinal neuronal response to the formalin injection into the rat hind paw was also examined through immunohistochemical analysis of c-Fos protein. Significantly increased numbers of c-Fos-immunoreactive cells were observed in laminae I–II and V–VI of the ipsilateral spinal cord to the site of the formalin injection in rats with neonatal dopamine depletion compared with vehicle-treated rats. These results suggest that the dopaminergic neural system plays a crucial role in the development of a neural network for tonic pain, including the spinal neural circuit for nociceptive transmission, and that the mechanism underlying hyperalgesia to tonic pain is not always consistent with that of spontaneous motor hyperactivity induced by neonatal dopamine depletion.

Allicin Protects PC12 Cells Against 6-OHDA-Induced Oxidative Stress and Mitochondrial Dysfunction via Regulating Mitochondrial Dynamics

Background: Parkinson disease (PD) is a common adult-onset neurodegenerative disorder, and PD related neuronal injury is associated with oxidative stress and mitochondrial dysfunction. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti-oxidative and anti-apoptotic activities in in vitro and in vivo studies. Methods: The present study aimed to investigate the potential protective role of allicin in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA) in PC12 cells. The protective effects were measured by cell viability, decreased lactate dehydrogenase (LDH) release and flow cytometry, and the anti-oxidative activity was determined by reactive oxygen species (ROS) generation, lipid peroxidation and the endogenous antioxidant enzyme activities. Mitochondrial function in PC12 cells was detected by mitochondrial membrane potential (MMP) collapse, cytochrome c release, mitochondrial ATP synthesis, and the mitochondrial Ca²⁺ buffering capacity. To investigate the potential mechanism, we also measured the expression of mitochondrial biogenesis factors, mitochondrial morphological dynamic changes, as well as detected mitochondrial dynamic proteins by western blot. Results: We found that allicin treatment significant increased cell viability, and decreased LDH release and apoptotic cell death after 6-OHDA exposure. Allicin also inhibited ROS generation, reduced lipid peroxidation and preserved the endogenous antioxidant enzyme activities. These protective effects were associated with suppressed mitochondrial dysfunction, as evidenced by decreased MMP collapse and cytochrome c release, preserved mitochondrial ATP synthesis, and the promotion of mitochondrial Ca²⁺ buffering capacity. In addition, allicin significantly enhanced mitochondrial biogenesis and prevented fragmentation of mitochondrial network after 6-OHDA treatment. The results of western blot analysis showed that the 6-OHDA induced decrease in the expression of optic atrophy type 1 (Opa-1), increase in mitochondrial fission 1 (Fis-1) and dynamin-related protein 1 (Drp-1) were all partially revised by allicin. Conclusion: In summary, our data strongly suggested that allicin treatment can exert protective effects against PD related neuronal injury through inhibiting oxidative stress and mitochondrial dysfunction with dynamic changes.

EGCG Protects against 6-OHDA-Induced Neurotoxicity in a Cell Culture Model.

Background. Parkinson's disease (PD) is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression. Objective. To test the protective effect of (−)-epigallocatechin-3-gallate (EGCG) against 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity by regulating iron metabolism in N27 cells. Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulatory gene and protein expression were measured by RT-PCR and Western blotting. Intracellular iron uptake was measured using 55Fe. The EGCG protection was further tested in primary mesencephalic dopaminergic neurons by immunocytochemistry. Results. EGCG protected against 6-OHDA-induced cell toxicity. 6-OHDA treatment significantly (p < 0.05) increased divalent metal transporter-1 (DMT1) and hepcidin and decreased ferroportin 1 (Fpn1) level, whereas pretreatment with EGCG counteracted the effects. The increased 55Fe (by 96%, p < 0.01) cell uptake confirmed the iron burden by 6-OHDA and was reduced by EGCG by 27% (p < 0.05), supporting the DMT1 results. Pretreatment with EGCG and 6-OHDA significantly increased (p < 0.0001) TH+ cell count (~3-fold) and neurite length (~12-fold) compared to 6-OHDA alone in primary mesencephalic neurons. Conclusions. Pretreatment with EGCG protected against 6-OHDA-induced neurotoxicity by regulating genes and proteins involved in brain iron homeostasis, especially modulating hepcidin levels.

Neuroprotection by scorpion venom heat resistant peptide in 6-hydroxydopamine rat model of early-stage Parkinson's disease.

Neuroprotective effect of scorpion venom on Parkinson's disease (PD) has already been reported. The present study was aimed to investigate whether scorpion venom heat resistant peptide (SVHRP) could attenuate ultrastructural abnormalities in mitochondria and oxidative stress in midbrain neurons of early-stage PD model. The early-stage PD model was established by injecting 6-hydroxydopamine (6-OHDA) (20 μg/3 μL normal saline with 0.1% ascorbic acid) into the striatum of Sprague Dawley (SD) rats unilaterally. The rats were intraperitoneally administered with SVHRP (0.05 mg/kg per day) or vehicle (saline) for 1 week. Two weeks after 6-OHDA treatment, the rats received behavior tests for validation of model. Three weeks after 6-OHDA injection, the immunoreactivity of dopaminergic neurons were detected by immunohistochemistry staining, and the ultrastructure of neuronal mitochondria in midbrain was observed by electron microscope. In the meantime, the activities of monoamine oxidase-B (MAO-B), superoxide dismutase (SOD) and content of malondialdehyde (MDA) in the mitochondria of the midbrain neurons, as well as the inhibitory ability of hydroxyl free radical and the antioxidant ability in the serum, were measured by corresponding kits. The results showed that 6-OHDA reduced the optical density of dopaminergic neurons, induced damage of mitochondrial ultrastructure of midbrain neurons, decreased SOD activity, increased MAO-B activity and MDA content, and reduced the antioxidant ability of the serum. SVHRP significantly reversed the previous harmful effects of 6-OHDA in early-stage PD model. These findings indicate that SVHRP may contribute to neuroprotection by preventing biochemical and ultrastructure damage changes which occur during early-stage PD.

Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra.

BackgroundThe anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson’s disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat.MethodsAt day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls.ResultsIn the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels.ConclusionOur results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson’s disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0209-0) contains supplementary material, which is available to authorized users.

Dimethyl fumarate attenuates 6-OHDA-induced neurotoxicity in SH-SY5Y cells and in animal model of Parkinson’s disease by enhancing Nrf2 activity

Oxidative stress is central to the pathology of several neurodegenerative diseases, including Parkinson’s disease (PD), and therapeutics designed to enhance antioxidant potential could have clinical value. In this study, we investigated whether dimethyl fumarate (DMF) has therapeutic effects in cellular and animal model of PD, and explore the role of nuclear transcription factor related to NF-E2 (Nrf2) in this process. Treatment of animals and dopaminergic SH-SY5Y cells with DMF resulted in increased nuclear levels of active Nrf2, with subsequent upregulation of antioxidant target genes. The cytotoxicity of 6-hydroxydopamine (6-OHDA) was reduced by pre-treatment with DMF in SH-SY5Y cells. The increase in the reactive oxygen species caused by 6-OHDA treatment was also attenuated by DMF in SH-SY5Y cells. The neuroprotective effects of DMF against 6-OHDA neurotoxicity were dependent on Nrf2, since treatment with Nrf2 siRNA failed to block against 6-OHDA neurotoxicity and induce Nrf2-dependent cytoprotective genes in SH-SY5Y cells. In vivo, DMF oral administration was shown to upregulate mRNA and protein levels of Nrf2 and Nrf2-regulated cytoprotective genes, attenuate 6-OHDA induced striatal oxidative stress and inflammation in C57BL/6 mice. Moreover, DMF ameliorated dopaminergic neurotoxicity in 6-OHDA-induced PD animal models as evidenced by amelioration of locomotor dysfunction, loss in striatal dopamine, and reductions in dopaminergic neurons in the substantia nigra and striatum. Taken together, these data strongly suggest that DMF may be beneficial for the treatment of neurodegenerative diseases like PD.