Project description:The anti-epileptic drug zonisamide is reported to exert beneficial effects in patients with Parkinson's disease. To elucidate the pathophysiological mechanisms underlying the anti-parkinsonism effects of zonisamide, we examined the effect of zonisamide co-administered with levodopa in the striata of rats with 6-hydoroxydopamine hemiparkinsonism by using a DNA microarray for genome-wide gene expression profiling. We found that the expression of some genes related to metabolism and nervous system development and function were upregulated by zonisamide; expression of these genes was downregulated by levodopa. Furthermore, many genes related to the immune system and inflammation were downregulated by zonisamide, and their expression was upregulated by levodopa. These results indicate that zonisamide has a protective effect when co-administered with levodopa. Rats were anesthetized with pentobarbital 240 mg/kg, i.p. and unilateral lesions of the left medial forebrain bundle were made via injection of 8 mg 6-OHDA hydrobromide in 4 ml of sterile saline containing 0.01% ascorbic acid. Motor disturbance was assessed by counting the number of full rotations per min in a cylindrical container of M-OM-^F30 cm diameter at 10-min intervals for the first 60 min after METH (3 mg/kg, i.p.) administration [3]. Behavioral screening was carried out after 2-3 weeks of recovery and animals that performed at least 7 turns/min during METH challenge were certified as hemiparkinsonism rats and included in the study. Seven days after METH challenge, hemiparkinsonism rats were treated with saline, L-DOPA (100 mg/kg, i.p.), or L-DOPA (100 mg/kg, i.p.) and ZNS (50 mg/kg, i.p.) once per day for 7 days. L-DOPA was injected 30 min after benserazide hydrochloride (a peripheral decarboxylase inhibitor). ZNS was injected 30 min after L-DOPA injection. The 4 experimental groups were classified as follows: the non-lesioned side of saline-treated 6-OHDA-injected rats (NL), the lesioned side of saline-treated 6-OHDA-injected rats (SL), the lesioned side of L-DOPA-treated 6-OHDA-injected rats (DL), and the lesioned side of L-DOPA and ZNS-treated 6-OHDA-injected rats (ZDL). Three animals were assigned to each group.

Project description:Methamphetamine (METH) is a widely abused illicit amphetamine that causes acute biochemical and molecular changes in the brain. However, there are very few studies that have investigated the long-term effects of a single METH exposure in adult animals. The purpose of this study was thus to determine the consequences of a single injection of METH (10) mg/kg) on the biochemical and transcriptional effects of a second challenge injection of the drug (2.5 mg/kg) given one month later. Towards that end, we measured monoamine levels and gene expression by microarray and quantitative PCR analyses in the nucleus accumbens (NAc) of 4 groups of rats: saline-pretreated and saline-challenged (SS); saline-pretreated and METH-challenged (SM); METH-pretreated and saline-challenged (MS); and METH-pretreated and METH-challenged (MM). The rats were euthanized 2 hours after the acute challenge injection and NAc samples were harvested. The acute METH challenge caused increases in NAc DA and HVA levels in both the SM and MM groups. Microarray analyses revealed that an injection of METH (2.5 mg/kg) produced acute changes (1.8-fold; p < 0.01) in the expression of 418 (358 up-, 60 downregulated) genes including several immediate early genes (IEGs) such as Arc, c-fos, and fosB in the SM group. Several neuropeptides including Cart, Crf (Crh), and vasopressin were upregulated in that group. Injection of the higher dose of METH (10 mg/kg) did not influence monoamine levels but caused changes in 510 (345 up-, 165 downregulated) transcripts measured one month later (MS group). This list included Cart and Crf but not any IEGs. The MM group showed changes in 774 (572 up-, 202 downregulated) genes that also included vasopressin, Cart, and Crf whose expression was enhanced by the second METH injection. Quantitative PCR confirmed the METH-induced changes in the expression of the neuropeptides. We also confirmed the METH-induced potentiation of Crf expression in the METH-pretreated group. These observations suggest that a single injection of a moderate dose of METH can produce long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crf expression also suggest that a single injection of a moderate METH dose can cause prolonged dysregulation in the endogenous stress system in the rat brain.

Project description:The therapeutic benefits of L–3,4–dihydroxyphenylalanine (L-DOPA) in Parkinson’s disease (PD) patients severely diminishes with the onset of L-DOPA-induced dyskinesia (LID), a debilitating motor side effect. LID is mainly due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions. However, the molecular mechanisms that promote LID remain unclear. Here, we have reported that increased striatal RasGRP1 (also known as CalDAG-GEF-II) is instrumentally linked to the development of LID in a 6-hydroxydopamine (6-OHDA) lesioned mouse model of PD. L-DOPA treatment rapidly upregulated RasGRP1 in the dopamine-1 receptor positive neurons in the dorsal striatum. RasGRP1 deleted mice (RasGRP1–/–) had drastically diminished LID, and RasGRP1–/– mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediated L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway. RasGRP1 bind directly with and acts 2 as a guanine nucleotide exchange (GEF) for Ras-homolog-enriched in the brain (Rheb), a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase 10a (Pde10a), Pde2a, catechol-o-methyltransferase (Comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), as downstream regulators of RasGRP1 that are linked to LID vulnerability. Moreover, we found that RASGRP1 protein is also upregulated predominantly in the striatum of MPTP-lesioned macaque treated with L-DOPA, emphasizing the translational potential of this protein. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of LID in the dorsal striatum. Pharmacological or gene-depletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing LID in PD patients.

Project description:Methamphetamine (METH) is a widely abused illicit amphetamine that causes acute biochemical and molecular changes in the brain. However, there are very few studies that have investigated the long-term effects of a single METH exposure in adult animals. The purpose of this study was thus to determine the consequences of a single injection of METH (10) mg/kg) on the biochemical and transcriptional effects of a second challenge injection of the drug (2.5 mg/kg) given one month later. Towards that end, we measured monoamine levels and gene expression by microarray and quantitative PCR analyses in the nucleus accumbens (NAc) of 4 groups of rats: saline-pretreated and saline-challenged (SS); saline-pretreated and METH-challenged (SM); METH-pretreated and saline-challenged (MS); and METH-pretreated and METH-challenged (MM). The rats were euthanized 2 hours after the acute challenge injection and NAc samples were harvested. The acute METH challenge caused increases in NAc DA and HVA levels in both the SM and MM groups. Microarray analyses revealed that an injection of METH (2.5 mg/kg) produced acute changes (1.8-fold; p < 0.01) in the expression of 418 (358 up-, 60 downregulated) genes including several immediate early genes (IEGs) such as Arc, c-fos, and fosB in the SM group. Several neuropeptides including Cart, Crf (Crh), and vasopressin were upregulated in that group. Injection of the higher dose of METH (10 mg/kg) did not influence monoamine levels but caused changes in 510 (345 up-, 165 downregulated) transcripts measured one month later (MS group). This list included Cart and Crf but not any IEGs. The MM group showed changes in 774 (572 up-, 202 downregulated) genes that also included vasopressin, Cart, and Crf whose expression was enhanced by the second METH injection. Quantitative PCR confirmed the METH-induced changes in the expression of the neuropeptides. We also confirmed the METH-induced potentiation of Crf expression in the METH-pretreated group. These observations suggest that a single injection of a moderate dose of METH can produce long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crf expression also suggest that a single injection of a moderate METH dose can cause prolonged dysregulation in the endogenous stress system in the rat brain. 24 samples. SS (4), SM (6), MS (7), MM (7)

Project description:This study addresses the molecular mechanisms underlying the action of subthalamic nucleus high frequency stimulation (STN-HFS) in the treatment of ParkinsonM-bM-^@M-^Ys disease and its interaction with levoDOPA (L-DOPA), focusing on the striatum. The objectives were 1) to identify the molecular signature of STN-HFS action at striatal level, associated with its efficient antiparkinsonian action, and 2) to investigate the molecular substrates of the interaction between the two treatments in order to evidence possible genes involved in dyskinesia. Striatal gene expression profile was assessed in rats with nigral DOPAmine neuron lesion, either treated or not, using agilent microarrays and qPCR verification. The treatments consisted in anti-akinetic STN-HFS (5 days), chronic L-DOPA treatment inducing dyskinesia (LIDs) or the combination of the two treatments that exacerbated LIDs. STN-HFS modulated 71 genes with functional or biochemical annotation, including genes sharing the GO terms regulation of growth, regulation of apoptosis, extracellular region. Ttr, Igf2, Sostdc1 and Nr4A3 (Nor-1), are among the 5 genes showing the highest specific upregulation. Down-regulated genes include Prkcd, Sirt5 and Bbc3. These results show that genes involved in neuroprotection and/or neurogenesis are key components of STN-HFS action in the striatum. STN-HFS and LDOPA treatment share very few common gene regulation features suggesting that the molecular substrates underlying their striatal action are mostly different. In addition to genes already reported to be associated with LIDs (Pdyn, Trh, Grm4/mGlu4, Cnr1/CB1), the comparison between DOPA and DOPA/STN-HFS identifies immunity-related genes: C1s, Rt1-Da and Irf7a, as potential players in L-DOPA side effects. Total RNA was extracted from striatal tissue from four groups of 3 animals bearing 6-hydroxyDOPAmine (6-OHDA)-induced lesion of the nigrostriatal DA pathway: lesion alone without any subsequent treatment (L), L-DOPA treatment for 19 days (D), STN-HFS for 5 days (S) and combination of L-DOPA and STN-HFS (DS).

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Transcriptome profiling in 6-hydroxydopamine hemiparkinsonian rats reveals methamphetamine-mediated dopamine-independent changes in striatal gene expression.

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