Progress in research on neuroprotective mechanism of glucagon-like peptide-1 receptor agonists and analogues in Parkinson's disease / 中国生物制品学杂志

@#Parkinson's disease(PD)is the second common neurodegenerative disease that mostly occurs in middle-aged and elderly people. Currently,Levodopa is the main first-line treatment drug,but the long-term efficacy of patients is not good,and even side effects such as“on-off”phenomenon and orthostatic hypotension occur. Glucagon-like peptide-1receptor agonists(GLP-1RA)and analogues are endogenous peptide hormones that can be released into the blood and enter the central nervous system to exert neuroprotection by crossing the blood-brain barrier. Numerous studies have shown that GLP-1RA can improve movement disorders and restore dopaminergic neuron activity in PD. However,the mechanism of GLP-1RA is not yet fully clear. This paper summarized the mechanism of GLP-1RA and its analogues in improving PD movement disorders and restoring dopaminergic neuron activity,and reviewed the aspects of reducing neuroinflammation,inhibiting oxidative stress,inhibiting apoptosis,regulating mitochondrial morphology,increasing neuronal protrusions,enhancing autophagy,and regulating intestinal flora homeostasis,so as to provide new ideas for research of the mechanisms of PD and development of GLP-1RA-related new drugs.

Demethylenetetrahydroberberine protects dopaminergic neurons in a mouse model of Parkinson's disease / 中国天然药物

Parkinson's disease (PD) is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist. However, the pathogenesis of PD remains undefined, which becomes the main limitation for the development of clinical PD treatment. Demethylenetetrahydroberberine (DMTHB) is a novel derivative of natural product berberine. This study was aimed to explore the neuroprotective effects and pharmacological mechanism of DMTHB on Parkinson's disease using C57BL/6 mice. A PD model of mice was induced by administration of MPTP (20 mg·kg-1) and probenecid (200 mg·kg-1) twice per week for five weeks. The mice were administered with DMTHB daily by gavage at the dose of 5 and 50 mg·kg-1 for one- week prophylactic treatment and five-week theraputic treatment. The therapeutic effects of DMTHB were evaluated by behavior tests (the open field, rotarod and pole tests), immunohistochemical staining of tyrosine hydroxylase (TH), Nissl staining and biochemical assays. The molecular mechanisms of DMTHB on the key biomarkers of PD pathological states were analyzed by Western blot (WB) and qRT-PCR. DMTHB treatment alleviated the behavioral disorder induced by MPTP-probenecid. Nissl staining and TH staining showed that the damage of dopaminergic neurons in the substantia nigra was remarkably suppressed by DMTHB treatment. Western blot results showed that the ratio of Bcl-2/Bax and TH increased, but the level of α-synuclein (α-syn) was remarkably reduced, which indicated that the apoptosis of dopaminergic neurons in mice was significantly reduced. The protein phosphorylation of p-PI3K, p-AKT and p-mTOR also increased about 2-fold, compared with the model group. Furthermore, qRT-PCR results demonstrated that the mRNA levels of pro-inflammatory cytokines, IL-1β and TNF-α, were reduced, but the level of anti-inflammatory cytokine IL-10 increased after DMTHB treatment. Finally, the cellular assay displayed that DMTHB was also a strong antioxidant to protect neuron cell line PC12 by scavenging ROS. In this study, we demonstrated DMTHB alleviates the behavioral disorder and protects dopaminergic neurons through multiple-target effects includubg anti-apoptotic, anti-inflammatory and antioxidant effects.

O-GlcNAcylation in Ventral Tegmental Area Dopaminergic Neurons Regulates Motor Learning and the Response to Natural Reward / 神经科学通报·英文版

Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways, and its disturbance has been found in neurodegenerative diseases and metabolic disorders. However, its role in the mesolimbic dopamine (DA) system, especially in the ventral tegmental area (VTA), needs to be elucidated. Here, we found that injection of Thiamet G, an O-GlcNAcase (OGA) inhibitor, in the VTA and nucleus accumbens (NAc) of mice, facilitated neuronal O-GlcNAcylation and decreased the operant response to sucrose as well as the latency to fall in rotarod test. Mice with DAergic neuron-specific knockout of O-GlcNAc transferase (OGT) displayed severe metabolic abnormalities and died within 4-8 weeks after birth. Furthermore, mice specifically overexpressing OGT in DAergic neurons in the VTA had learning defects in the operant response to sucrose, and impaired motor learning in the rotarod test. Instead, overexpression of OGT in GABAergic neurons in the VTA had no effect on these behaviors. These results suggest that protein O-GlcNAcylation of DAergic neurons in the VTA plays an important role in regulating the response to natural reward and motor learning in mice.

Effect of propofol on dopaminergic neurons of mice with Parkinson′s disease / 中华麻醉学杂志

Objective:To evaluate the effect of propofol on dopaminergic neurons of mice with Parkinson′s disease (PD).Methods:Forty-eight pathogen-free healthy male C57BL/6 mice, aged 8-12 weeks, weighing 22-32 g, were divided into 3 groups ( n=16 each) using a random number table method: control group (group C), group PD and propofol group (group Pro). The neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropypridine (MPTP) was intraperitoneally injected for 7 consecutive days in PD and Pro groups, while the equal volume of normal saline was given for 7 consecutive days in group C. At 30 min after intraperitoneal injection of MPTP, propofol 25 mg/kg was intraperitoneally injected in group Pro, while the equal volume of normal saline was given daily in group C and group PD.At day 8 after the end of establishment of the model, gait analysis system experiment and rotarod test were used to record the step distance and retention time.The animals were sacrificed after the behavioral tests, and the brain tissues were removed for the dopamine neuron count in substantia nigra (by immunohistochemistry) and for determination of the expression of alpha-Synuclein (α-Syn) and Tyrosine hydroxylase (TH) in the substantia nigra (by Western blot). Results:Compared with group C, the step distance was significantly decreased, retention time were shortened, the dopamine neuron count in substantia nigra was decreased, the expression of TH was down-regulated, and expression of α-Syn in substantia nigra was up-regulated in group PD ( P<0.05), and no significant change was found in the parameters mentioned above in group Pro ( P>0.05). Compared with group PD, the step distance was significantly increased, retention time were prolonged, the dopamine neuron count in substantia nigra was increased, the expression of TH was up-regulated, and expression of α-Syn in substantia nigra was down-regulated in group Pro ( P<0.05). Conclusion:Propofol has protective effect on dopaminergic neurons of PD mice, and the mechanism may be related to the down-regulation of α-Syn expression in substantia nigra.

Autophagy inhibitor 3-methyladenine could not modulate rotenone neurotoxicity in primary mesencephalic cell culture / El inhibidor de la autofagia 3-metiladenina no logra modular la neurotoxicidad de la rotenona en el cultivo primario de células mesencefálicas

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.

Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.

Effect of benzo(a)pyrene on dopaminergic neurons and α-synuclein in brain and its mechanism involved / 北京大学学报(医学版)

OBJECTIVE@#To analyze the effect of benzopyrene on the decrease of dopaminergic neurons, and the increase and aggregation of α-synuclein, which are the pathological features of Parkinson's disease, and to explore its possible mechanisms.@*METHODS@#Eight-month-old transgenic mice with human SNCA gene were randomly divided into a BaP-exposed group and a control group. BaP and solvent corn oil were injected intraperitoneally to BaP-exposed group and control group respectively, once a day for 60 days. The motor dysfunction of mice was tested by rotarod test. The effects of BaP on the decrease of dopaminergic neurons and increase and aggregation of α-synuclein were observed by immunohistochemistry and Western blot experiments respectively, and the expression of related mRNA was detected by quantitative real-time PCR (qRT-PCR). Twenty genes were tested in the study, mainly related to neurotransmitter transporter (2 genes), neurotransmitter receptor function (10 genes), cellular autophagy (5 genes), and α-synuclein aggregation and degradation (3 genes).@*RESULTS@#After BaP exposure, the movement time of the mice in the rotarod test was significantly reduced (P<0.05). The substantia nigra dopami-nergic neurons in the mice were significantly reduced, which was 62% of the control group (P<0.05), and the expression of α-synuclein in the midbrain increased, which was 1.36 times that of the control group (P<0.05). After BaP exposure, mRNA expressions of 14 genes in the midbrain of the mice were significantly down-regulated (P<0.05). Alpha-synuclein degradation and cell autophagy (5 genes), neuron transporters (2 genes), and neurotransmitter receptor functions (5 genes) were involved. The expression of one gene, Synphilin-1, was significantly up-regulated (P<0.01), which was related to α-synuclein aggregation.@*CONCLUSION@#BaP exposure not only inhibited function of neurotransmitter receptor and dopamine transporter, but also interfered cell autophagy, thereby hindering the degradation of α-synuclein, which could lead to decrease of dopaminergic neurons in substantia nigra and increase and aggregation of α-synuclein in midbrain, as the significant pathology of Parkinson's disease. Therefore, BaP exposure may increase the risk of Parkinson's disease.

Protective Effects of Gallic Acid on the MPTP-Mediated Dopaminergic Neurons Damage in Parkinson's Disease / 中国药学杂志

OBJECTIVE: To study the neuroprotective effects of gallic acid in animal models of Parkinson's disease (PD) and its related molecular mechanisms. METHODS: Thirty-six mice were randomly divided into control group, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) group and gallic acid group. Mice in MPTP group and gallic acid group were given an intraperitoneal injection of 30 mg•kg-1 MPTP daily for 7 d. The mice in control group were given the same amount of normal saline at the same time. The mice in gallic acid group received oral administration of 200 mg•kg-1 gallic acid daily from the first day, gallic acid was administered continuously for 14 d, and mice in MPTP group and control group received the same amount of normal saline. After 7 d of drug administration, the behavioral function was evaluated by rod climbing test and suspension test. The expression of tyrosine hydroxylase(TH) in the substantia nigra and striatum were detected by immunohistochemistry. The number of apoptotic neurons in the substantia nigra were measured by TUNEL assay. SIRT3 mRNA levels were analyzed by qRT-PCR, and protein expression levels were detected by Western blot. RESULTS: Compared with the control group, the ability of motor coordination weakened, the TH expression levels decreased in the substantia nigra and striatum, the number of apoptotic neurons in the substantia nigra significantly increased, the SIRT3 mRNA and protein levels in the substantia nigra obviously declined, and the SOD2 protein expression also dramatically reduced in the MPTP group, the differences between the groups were all statistically significant (P<0.05). After treatment with gallic acid, the ability of motor coordination enhanced, the TH expression level elevated, the number of apoptotic neurons markedly reduced, SIRT3 mRNA and protein levels increased, and SOD2 protein expression also up-regulated in the gallic acid group. Compared with the MPTP group, the differences were all statistically significant (P<0.05). CONCLUSION: Gallic acid can improve the behavioral dysfunction and inhibit dopaminergic neurons damage in PD mice. The mechanism of action may be related to the up-regulation of SIRT3 gene expression.

Role of sirt3 in molecular mechanism of melatonin protecting dopaminergic neurons in Parkinson' s disease / 中国药理学通报

Aim To investigate the role of SIRT3 in the molecular mechanism of melatonin protecting do-paminergic neurons in Parkinson' s disease ( PD). Methods Forty-eight mice were randomly divided into control group, model group and treatment group. The mice in treatment group received intraperitoneal injection of melatonin (10 mg • kg"1) and MPTP (30 mg • kg ~1). The mice in model group only received intraperitoneal injection of MPTP (30 mg • kg~1 ) , and the mice in control group received the same a-mount of normal saline. Melatonin was administered continuously for 14 days. The expressions of TH and lba-1 in substantia nigra were analyzed by immunohis-tochemistry. The levels of oxidative stress ( ROS, MDA, SOD) and inflammatory factors (TNF-a, IL-lp) in the midbrain were measured by ELISA. SIRT3 mRNA level was analyzed by qRT-PCR, and protein expression level was detected by immunocytochemistry assay and Western blot. Results Compared to control group, the TH expression decreased and Iba-1 expression increased in the substantia nigra, the oxidative stress and inflammatory injury in the midbrain were significantly enhanced, the SIRT3 mRNA and protein levels in the substantia nigra obviously declined, the SOD2 protein expression was also dramatically reduced, and the iNOS protein expression was elevated in model group; the differences between the groups were all statistically significant ( P < 0. 05 ). After treatment with melatonin, the TH expression increased, Iba-1 expression decreased, oxidative stress and inflammatory injury markedly decreased, SIRT3 mRNA and protein levels were elevated, SOD2 protein expression was up-regulated, and iNOS protein expression was down-regulated in treatment group. Compared to model group, the differences were all statistically significant ( P < 0.05). Conclusions Melatonin can counteract the damage of dopaminergic neurons by up-regulating the expression of SIRT3 in PD animal model. Its mechanisms of action are related to inhibiting microglia activation, and alleviating oxidative stress and inflammation injury.

Construction of mutant NDUFS7 plasmid and its effects on neural cells / 中国药科大学学报

@#To investigate the effects of NDUFS7 gene mutation on neurons, the mutant plasmid of pcDNA3.1(+)-NDUFS7 Q208STOP was constructed and transfected into differentiated SH-SY5Y cells. The effect of transfecting mutant plasmid on the viability of dopaminergic neural cells was detected by MTT assay. The effect of transfection of mutant plasmid on apoptosis was detected by Annexin Ⅴ-FITC/PI staining followed by flow cytometry assay. The changes in the expression levels of apoptosis-related proteins Bax and Bcl-2 in cells after transfection of mutant plasmid were detected by Western blot. The effects of transfection of mutant plasmid on the mitochondrial membrane potential in differentiated SH-SY5Y cells and the intervention effect of antioxidant Trolox were examined using JC-1 fluorescent probe. The intervention effect of Trolox on the apoptosis of differentiated SH-SY5Y cells transfected with mutant plasmid was detected by PI/Hoechst staining. The results showed that the subunit mutation of mitochondrial complex I in dopaminergic neurons could lead to decreased neuronal viability and increased apoptosis, while antioxidants could alleviate the abnormal mitochondrial membrane potential and apoptosis caused by transfection of mutant plasmids, suggesting that transfection of mutant plasmid of NDUFS7 gene could lead to apoptosis by causing abnormal mitochondrial function in dopaminergic neurons.

Neuroprotective Effects of Low-molecular-weight Chondroitin Sulfate on Dopaminergic Neurons in MPTP-induced Parkinson ’s Disease Model Mice / 中国药房

OBJECTIVE： To observe neuroprotective effects of low-molecular-weight chondroitin sulfate （CS） on dopaminergic neurons in Parkinson’s disease （PD） mice model induced by 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （MPTP）. METHODS： C57BL/6 mice were randomly divided into control group， MPTP injury group， low-molecular-weight CS low-dose and high-dose groups （100， 400 mg/kg）. Control group and MPTP injury group were given constant volume of normal saline intragstrically， administration groups were given relevant medicine intragastrically， once a day， for consecutive 17 d. Since 11th day after medication， except for control group， other groups were given MPTP solution （20 mg/kg） intraperitoneally to induce PD model， once a day， consecutive 5 d. After last medication， behavioral changes of mice （10 mice in each group） were evaluated by rotary rod fatigue tester. The damage of dopamine neurons （the percentage of TH positive cell and the percentage of fluorescence intensity） in substantia nigra of mice （3 mice in each group） was detected by immunohistochemistry and immunofluorescence. The content of dopamine in striatum was determined by HPLC （6 mice in each group）. The changes of oxidant stress indexes （SOD， GSH-Px， MDA） in substantia nigra of mice were determined by chemical colorimetry （6 mice in each group）. RESULTS： Compared with control group， retention time of mice on rotating rods was shortened significantly in MPTP injury group； TH positive cells of substantia nigra were decreased significantly， fluorescence intensity was obviously weakened； the percentage of positive cells and fluorescence intensity， the content of dopamine in striatum， the activities of SOD and GSH-Px in substantia nigra were decreased significantly， while the content of MDA was increased significantly （P＜0.01）. Compared with MPTP injury group， retention time of mice on the rotating rods was prolonged significantly in low-molecular-weight CS groups， the number of TH positive cells was increased significantly in substantia nigra and fluorescence intensity was increased significantly； the percentage of positive cells， the percentage of fluorescence intensity and the content of dopamine in striatum were increased significantly， while above indexes of high-dose group were significantly longer or higher than those of low-dose group （P＜0.05 or P＜0.01）. The activities of SOD and GSH-Px in substantia nigra were increased significantly in low-molecular-weight CS groups， while the content of MDA in substantia nigra was decreased significantly in low-molecular-weight CS high-dose group （P＜0.05 or P＜0.01）. CONCLUSIONS： Prophylactic administration of low-molecular-weight CS can relieve the damage of dopaminergic neurons in substantia nigra of PD model mice induced by MPTP in a dose-dependent manner， and increase the secretion of dopamine in striatum. The effect may be related to the inhibition of lipid peroxidation and the enhancement of antioxidant capacity of tissues.

Interleukin-4 Contributes to Degeneration of Dopamine Neurons in the Lipopolysaccharide-treated Substantia Nigra in vivo

The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN) in vivo of lipopolysaccharide (LPS)-treated rat. Tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral DA neurons at 3 and 7 day post-LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 day and remained elevated at 7 day post-LPS. IL-4 immunoreactivity was detected exclusively in microglia. IL-4 neutralizing antibody (NA) significantly increased survival of DA neurons in LPS-treated SN in vivo by inhibiting microglial activation and production of proinflammatory mediator such as IL-1β as assessed by immunihistochemical, RT-PCR and ELISA analysis, respectively. Accompanying neuroprotection are IL-4NA effects on decreased disruption of blood-brain barrier and astrocytes. The present data suggest that endogenously expressed IL-4 from reactive microglia may be involved in the neuropathological processes of degeneration of DA neurons occurring in Parkinson's disease.

The role of the AMPK-mTOR pathway in paraquat-induce autophagy in PC12 cells / 中华劳动卫生职业病杂志

Objective@#To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) .@*Methods@#The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 μmol/L PQ for 24 hours, and the cells were induced by 300 μmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 μmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3Ⅱ, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot.@*Results@#Compared with the control group, the cell survival rate of the 100, 200, 300, 400 μmol/L PQ group decreased significantly, and showed a dose-dependent pattern (P<0.05) . The survival rate of cells treated with 300 μmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) (P<0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 μmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased (P<0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased (P<0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3Ⅱand Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher (P<0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300μmol/L PQ-treatd groups, with statistically significant difference (P<0.05) .@*Conclusion@#AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.

Research on effect of midbrain VTA dopaminergic neurons in general anesthesia / 重庆医学

Objective To investigate the effect of dopaminergic neurons of midbrain ventral tegmental area(VTA) in general anesthesia.Methods Forty adult healthy male SD rats were randomly divided into lesion group (n=20) and control group (n=20).The lesion group was given the bilateral infusion of specific dopaminergic neuron injury agent 6-OHDA in midbrain lateral VTA,while the control group received the same volume of normal saline at the same areas.The time of loss of righting reflex (LORR)loss and recovery of righting reflex(RORR)at postoperative 2 week were observed in each group.Results Compared with the control group,the LORR time in the lesion group was shortened and the RORR time was significantly prolonged under propofol-induced anesthesia (P＜0.05).However,the LORR time under the isoflurane anesthesia had no statistically significant difference between the two groups(P＞0.05),while the RORR time in the lesion group was increased(P＜0.05).Conclusion Dopaminergic neurons in midbrain VTA might play different roles in the induction and recovery of different general anesthetics.

Optogenetic Rescue of Locomotor Dysfunction and Dopaminergic Degeneration Caused by Alpha-Synuclein and EKO Genes

α-Synuclein (α-Syn) is a small presynaptic protein and its mutant forms (e.g. A53T) are known to be directly associated with Parkinson's disease (PD). Pathophysiological mechanisms underlying α-Syn-mediated neurodegeneration in PD still remain to be explored. However, several studies strongly support that overexpression of mutant α-Syn causes reduced release of dopamine (DA) in the brain, and contributes to motor deficits in PD. Using a favorable genetic model Drosophila larva, we examined whether reduced DA release is enough to induce key PD symptoms (i.e. locomotion deficiency and DA neurodegeneration), mimicking a PD gene α-Syn. In order to reduce DA release, we expressed electrical knockout (EKO) gene in DA neurons, which is known to make neurons hypo-excitable. EKO led to a decrease in a DA neuronal marker signal (i.e., TH – tyrosine hydroxylase) and locomotion deficits in Drosophila larva. In contrast, acute and prolonged exposure to blue light (BL, 470 nm) was sufficient to activate channelrhodopsin 2 (ChR2) and rescue PD symptoms caused by both α-Syn and EKO. We believe this is for the first time to confirm that locomotion defects by a genetic PD factor such as α-Syn can be rescued by increasing DA neuronal excitability with an optogenetic approach. Our findings strongly support that PD is a failure of DA synaptic transmission, which can be rescued by optogenetic activation of ChR2.

L-calcium channel involving the generation and maintenance of bursting firing in rat substantia nigra pars compacta dopaminergic neurons / 中华神经科杂志

Objective To evaluate the role of calcium channel in the mechanism of the generation and maintenance of bursting firing of substantia nigra pars compacta (SNc) dopaminergic neurons in rats.Methods Using the patch clamp technique,we observed the firing pattern switching features after adding 10 μmol/L N-methyl-D-aspartic acid (NMDA),compared the changes of whole-calcium current and L-type calcium current with or without NMDA,and analyzed the correlation between the generation of burst firing and L-type calcium channel activation.Results After NMDA treatment,the firing pattern of SNc dopaminergic neurons changed to burst firing,which was compromised by a charastistic high plateau potential and series of action potential on it.The current density of L-type calcium current increased significantly after adding NMDA,which,from (2.86 ±0.26) pA/pF (n =28),significantly increased to (3.75 ± 0.18) pA/pF (n =34 ; t =7.52,P =0.002 8).The high plateau potential was almost abolished with the application of verapamil,a specific antagonist of L-type calcium channel.Consiusion NMDA could induce the firing pattern changed to burst firing in SNc dopaminergic neurons,while L-type calcium channel contributes to the process of generation and maintenance of burst firing.

Inhibition of Cdk5/p25 by TFP5 protects dopaminergic neurons in MPTP-induced mouse model of Parkinson's disease / 中华行为医学与脑科学杂志

Objective To study the neuroprotective role of TFP5 in a MPTP-induced mouse model of Parkinson's disease (PD).Methods C57BL/6 mice were used as experimental animals.Briefly, 5 consecutive days of intraperitoneal injection of 25 mg/Kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was applied to induce mouse PD model.The mice were randomized into 5 groups including control group,model group, scrambled TFP5 peptide (Scb) group, TFP5 group and roscovitine group.On the 7th day after the first injection of MPTP,behavior tests were performed, and then western blot method was employed to detect the expression of p25 and phosphorylated MEF2D in substantia nigra.Tyrosine hydroxylase (TH) immunohistochemical staining was performed to observe the apoptosis of dopaminergic neurons in substantia nigra pars compacta (SNpc) 28 days after the first injection of MPTP.Results MPTP increased the expression of p25 (0.48±0.10 vs 0.26±0.02, P＜0.05) and phosphorylated MEF2D (0.81±0.10 vs 0.22±0.02, P＜0.05) in substantia nigra, but decreased the number of dopaminergic neurons in SNpc (348.67±24.40 vs 463.29± 19.61, P＜0.05),resulting in motor impairment in the model mice (P＜0.05).Intraperitoneal injection of 30mg/Kg of TFP5 for 3 days effectively reduced the excessive phosphorylation of MEF2D (0.25 ± 0.12 vs 0.81 ± 0.10, P＜ 0.05) in substantia nigra, rescued dopaminergic neuron reduction of SNpc (422.92±8.41 vs 348.67±24.40, P＜0.05), and improved the motor ability of the model mice (P ＜0.05).Roscovitine exerted almost same neuroprotective role as TFP5 ,while Scb had no protective effect.Conclusion TFP5 can rescue MPTP-induced damage of dopaminergic neurons in substantia nigra, and thus improve motor impairment of model mice,which may be mediated by the inhibition of Cdk5/p25 activity.

Direct Differentiation of Adult Ocular Progenitors into Striatal Dopaminergic Neurons

Parkinson's disease, characterized by motor dysfunction due to the loss of nigrostriatal dopaminergic neurons, is one of the most prevalent age-related neurodegenerative disorders. Given there is no current cure, the stem cell approach has emerged as a viable therapeutic option to replace the dopaminergic neurons that are progressively lost to the disease. The success of the approach is likely to depend upon accessible, renewable, immune compatible, and non-tumorigenic sources of neural progenitors from which stable dopaminergic neurons can be generated efficaciously. Here, we demonstrate that neural progenitors derived from limbus, a regenerative and accessible ocular tissue, represent a safe source of dopaminergic neurons. When the limbus-derived neural progenitors were subjected to a well-established protocol of directed differentiation under the influence of Shh and FGF8, they acquired the biochemical and functional phenotype of dopaminergic neurons that included the ability to synthesize dopamine. Their intrastriatal transplantation in the rat model of hemi-Parkinsonism was associated with a reduction in the amphetamine-induced rotation. No tumor formation was observed 6 weeks post-transplantation. Together, these observations posit limbus-derived neural progenitors as an accessible and safe source of dopaminergic neurons for a potential autologous ex-vivo stem cell approach to Parkinson's disease.

Atorvastatin protects GABAergic and dopaminergic neurons in the nigrostriatal system in an experimental rat model of transient focal cerebral ischemia / La atorvastatina protege las neuronas gabérgicas y dopaminérgicas del sistema nigroestriatal en un modelo experimental de isquemia cerebral focal transitoria en ratas

Introduction: Cerebral ischemia is the third leading cause of death and the primary cause of permanent disability worldwide. Atorvastatin is a promising drug with neuroprotective effects that may be useful for the treatment of stroke. However, the effects of atorvastatin on specific neuronal populations within the nigrostriatal system following cerebral ischemia are unknown. Objective: To evaluate the effects of atorvastatin on dopaminergic and GABAergic neuronal populations in exofocal brain regions in a model of transient occlusion of the middle cerebral artery. Materials and methods: Twenty-eight male eight-week-old Wistar rats were used in this study. Both sham and ischemic rats were treated with atorvastatin (10 mg/kg) or carboxymethylcellulose (placebo) by gavage at 6, 24, 48 and 72 hours post-reperfusion. We analyzed the immunoreactivity of glutamic acid decarboxylase and tyrosine hydroxylase in the globus pallidus, caudate putamen and substantia nigra. Results: We observed neurological damage and cell loss in the caudate putamen following ischemia. We also found an increase in tyrosine hydroxylase immunoreactivity in the medial globus pallidus and substantia nigra reticulata, as well as a decrease in glutamic acid decarboxylase immunoreactivity in the lateral globus pallidus in ischemic animals treated with a placebo. However, atorvastatin treatment was able to reverse these effects, significantly decreasing tyrosine hydroxylase levels in the medial globus pallidus and substantia nigra reticulata and significantly increasing glutamic acid decarboxylase levels in the lateral globus pallidus. Conclusion: Our data suggest that post-ischemia treatment with atorvastatin can have neuro-protective effects in exofocal regions far from the ischemic core by modulating the GABAergic and dopaminergic neuronal populations in the nigrostriatal system, which could be useful for preventing neurological disorders.

Introducción. La isquemia cerebral es la tercera causa de muerte y la primera de discapacidad permanente en el mundo. La atorvastatina es un fármaco neuroprotector prometedor para el tratamiento de la apoplejía; sin embargo, su acción sobre las poblaciones neuronales del sistema nigroestriatal después de la isquemia aún se desconoce. Objetivo. Evaluar el efecto de la atorvastatina sobre poblaciones gabérgicas y dopaminérgicas en regiones exofocales en un modelo de oclusión transitoria de la arteria cerebral media. Materiales y métodos. Se utilizaron 28 ratas Wistar macho de ocho semanas de edad. Los ejemplares con isquemia simulada y los ejemplares sometidos a isquemia fueron tratados con atorvastatina (10 mg/kg) y carboximetilcelulosa (placebo) administrados por medio de sonda a las 6, 24, 48 y 72 horas después de la reperfusión. Se analizó la inmunorreacción de la descarboxilasa del ácido glutámico y de la tirosina hidroxilasa en el globo pálido, el putamen caudado y la sustancia negra. Resultados. Los datos confirmaron el daño neurológico y la pérdida celular en el putamen caudado. Se incrementó la inmunorreacción de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata , disminuyendo la inmunorreacción de la descarboxilasa del ácido glutámico en el globo pálido lateral de los animales isquémicos tratados con placebo; sin embargo, el tratamiento con atorvastatina pudo revertirla, lo que logró una disminución significativa de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata y aumentando los niveles de descarboxilasa del ácido glutámico en el globo pálido lateral. Conclusión. Nuestros datos sugieren que la atorvastatina en el tratamiento posterior a la isquemia ejerce neuroprotección en las zonas exofocales, modulando las poblaciones neuronales gabérgicas y dopaminérgicas del sistema nigroestriatal, lo que podría prevenir trastornos neurológicos.

Functional Role of Parkin against Oxidative Stress in Neural Cells

BACKGROUND: Parkinson disease (PD) is caused by selective cell death of dopaminergic neurons in the substantia nigra. An early onset form of PD, autosomal recessive juvenile parkinsonism has been associated with a mutation in the parkin gene. The function of parkin is known to remove misfolding proteins and protect cell death. We aimed to investigate the role of parkin against oxidative stress in neuronal cells. METHODS: Parkin knockout embryonic stem cells (PKO ES cells) were differentiated into neurons by adherent monolayer culture method. Oxidative stress was induced by the treatment of 1-methyl-4-phenylpyridinium (MPP+) in neurons derived from wild type and PKO ES cells, and cell viability was examined by MTT assay. After exposure to MPP+, Tuj1-positive cell population was compared between PKO and wild type cells by fluorescence activated cell sorter (FACS) analysis. The activated caspase3 protein level was also measured by Western blot analysis, FACS and immunocytochemistry. RESULTS: There was no difference in the efficiency of neuronal differentiation between wild type and PKO ES cells. After exposure to MPP+, no significant differences were found in cell viability and Tuj1-positive cell population between the two groups determined by MTT assay and FACS analysis, respectively. The activated caspase3 protein levels examined by Western blot analysis, FACS and immunocytochemistry were not changed in PKO cells compared with those of wild type cells after MPP+ treatment. CONCLUSION: These results suggest that PKO neuronal cells including dopaminergic neurons are not sensitive to caspase3-dependent cell death pathway during the response against MPP+-induced oxidative stress.

Medaka Fish Parkinson's Disease Model

The teleost fish has been widely used in creating neurodegenerative models. Here we describe the teleost medaka fish Parkinson's disease (PD) models we developed using toxin treatment and genetic engineering. 1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors and tunicamycin treatment in our model fish replicated some salient features of PD: selective dopamine cell loss and reduced spontaneous movement with the last three toxins producing inclusion bodies ubiquitously in the brain. Despite the ubiquitous distribution of the inclusion bodies, the middle diencephalic dopaminergic neurons were particularly vulnerable to these toxins, supporting the idea that this dopamine cluster is similar to the human substantia nigra. PTEN-induced putative kinase 1 (PINK1) homozygous mutants also showed reduced spontaneous swimming movements. These data indicate that medaka fish can serve as a new model animal of PD. In this review we summarize our previous data and discuss future prospects.