- Email: [email protected]
2.316 6-Hydroxydopamine nigrostriatal lesion modulates alpha-synuclein expression in different rat brain regions
Poster Presentations: Alpha-synuclein and Lewy Bodies 2.313 Alcoholism, alpha-synuclein, and Parkinson disease: A case-control study L. Brighina1° , N.K. Schneider, T.G. Lesnick, M. de Andrade, J.M. Cunningham, M.J. Farrer, S.J. Lincoln, D. Mrazek, W.A. Rocca, D.M. Maraganore 1 Monza, Italy Objective: Variability in the allele length of a dinucleotide repeat sequence (REP1) within the alpha-synuclein gene (SNCA) promoter is associated with Parkinson’s disease (PD) [JAMA 2006; 296: 661–670] and also with alcohol dependence [Hum Mol Genet 2005; 14: 967–971]. Medical diagnosis of alcoholism is inversely associated with PD [Neurology 2000; 14: 1350–1358]. The aim of this study was to determine whether the association of REP1 genotype and PD is independent of alcoholism, whether the association of alcoholism and PD is independent of REP1 genotype, and whether REP1 genotype and alcoholism have joint effects on PD susceptibility. Method: Cases were recruited prospectively from the Department of Neurology of the Mayo Clinic in Rochester, MN, after June 1, 1996. The controls included unaffected siblings of cases and unrelated population controls. We assessed alcohol use using a structured telephone interview. We screened for alcoholism (overt or covert) using the CAGE questionnaire. Genotyping was performed using an ABI 3730 platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined using conditional logistic regression models. Results: Our study included a total of 893 case-control pairs. There was an increased risk of PD with increasing SNCA REP1 bp length (OR 1.18 for each REP1 genotype score unit, 95% CI 1.02−1.35; p = 0.02). There was a trend of decreasing risk of PD with increasing CAGE score (p = 0.0002). The association of REP1 genotype score with PD remained significant after adjusting for CAGE score, and the inverse association of CAGE score with PD remained significant after adjusting for REP1 genotype score. No significant pairwise interactions were observed for REP1 genotype and CAGE scores. Conclusion: Alcoholism (as measured by CAGE score) and REP1 genotype score were associated with PD susceptibility (main effects), but they did not have multiplicative joint effects on PD susceptibility.
2.314 Identification of modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide RNAi T. van Ham1° , K. Thijssen, R. Breitling, R. Hofstra, R. Plasterk, E. Nollen Netherlands
1 Groningen,
Objective: The protein alpha-synuclein is tightly connected to development of Parkinson’s disease: hallmark of PD is the presence of protein inclusions in the brain containing the protein alpha-synuclein. In addition, multiplication of the alpha-synuclein gene has been shown to be causative for PD. We aim to identify genes involved in the formation of alphasynuclein inclusions to provide an understanding of the mechanism of and genetic susceptibility to age-related sporadic PD. Method: We created nematodes (C. elegans) transgenic for human alphasynuclein fused to yellow fluorescent protein to visually track inclusion formation. We used this model in a genome-wide RNAi feeding screen to find genetic inactivations that alter a-synuclein inclusion formation. Results: Alpha-synuclein inclusions develop during aging in the transgenic C. elegans strain. The inclusions contain mostly mobile protein material and also immobile, aggregated protein at old age. Using genome-wide RNAi we identified genes that increase the formation of inclusions when silenced by RNAi. Interestingly, there was a significant overrepresentation of the genes that function in the ER, Golgi and vesicular membranes, when compared to random sets of genes. In addition, we found several genes associated with aging to increase inclusion formation when silenced. Conclusion: We developed a small animal model of alpha-synuclein accumulation, in which age-dependent inclusion formation can be visually
S125
tracked. Using this model we found genes that increase the number of alpha-synuclein inclusions when silenced. The overrepresentation of genes with a role in vesicular trafficking and ER protein quality control functioning in the endomembrane system suggests that these processes are involved in the age-dependent formation of alpha-synuclein inclusions. The genes found may provide an understanding of genetic susceptibility to PD and molecular targets for therapy.
2.315 Identification of potent small molecule inhibitors of alpha-synuclein aggregation in cell culture and by in vitro screening A. Snow1° , L. Esposito, J. Cummings, T. Lake, M. Hudson, F. Cheng, A. Ferree, S. Saha, B. Wolozin 1 Kirkland, USA Objective: A rotenone-induced cell culture model of Parkinson’s disease (PD) was primarily utilized to test the ability of ProteoTech’s small molecule library of compounds (representing new chemical entities) to inhibit aggregation of alpha-synuclein, a major component of PD Lewy bodies. Method: In this cell culture model, A53T mutant alpha-synuclein was overexpressed in human BEM-17 neuroblastoma cells. Cells exposed to 1 or 5uM rotenone accumulated alpha-synuclein aggregates containing a large amount of beta-sheet secondary structure as detected by positive Thioflavin S fluorescence and quantitative image analysis. Results: Treatment of rotenone-treated cells with different novel small molecule compounds identified 4 compounds (referred to as PD-61, 86, 31 and 13) that profoundly reduce the accumulation of Thioflavin-S positive alpha-synuclein aggregates by 87−91%, 73−91%, 40−84% and 57−70%, respectively. A marked reduction of alpha-synuclein aggregation by these compounds was confirmed using Thioflavin T fluorometry, Congo red binding assays and circular dichroism spectroscopy. In addition to their potent alpha-synuclein anti-aggregation properties, these compounds also provided good protection against rotenone-induced neurotoxicity in cell culture and in a C. elegans survival assay. Conclusion: These studies indicate that we have identified unique small molecule compounds that have potent alpha-synuclein anti-aggregation and neuroprotective properties and thus may serve as promising new therapeutics for PD and related disorders. Funded by ProteoTech Inc. and a LEAPS Award from the Michael J. Fox PD Research Foundation.
2.316 6-Hydroxydopamine nigrostriatal lesion modulates alpha-synuclein expression in different rat brain regions M. Perovic1° , A. Mladenovic, S. Ruzdijic, S. Kanazir Serbia and Montenegro
1 Belgrade,
Objective: alpha-Synuclein is considered to be involved in pathogenesis of PD and other neurodegenerative diseases. Its filamentous aggregates are found as principal constituents of Lewy bodies and some others pathological intraneuronal deposits. Due to the localization of alpha-synuclein in presynaptic terminals, its role in synaptic function and plasticity was postulated as well. 6-Hydroxydopamine (6-OHDA) rat model of PD is characterized by acute loss of dopaminergic neurons, but specific alpha-synuclein aggregates were not reported. Therefore, this model could reveal possible role of alpha-synuclein in plasticity given that some compensatory sprouting of dopaminergic neurons exists after period of extensive neuronal death. Method: Adult male Mill-Hill Hooded rats (n = 24) received four-site intrastriatal stereotaxic injection of 6-OHDA or saline (sham operated animals) into the right lateral striatum. Five weeks later, animals were killed by decapitation and the model was verified by tyrosine hydroxylase (TH) imunohistochemistry. Western blot analysis was used to detemine the level of alpha-synuclein protein and confocal microscopy for analysis of alphasynuclein co-localization with TH, synaptophysine, MAP 2 and GAP-43.
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Tuesday, 11 December 2007
Results: Using Western blot analysis, significant increase (p < 0.05) in the level of alpha-synuclein was detected in ipsilateral striatum, mesencephalon and cerebellum by 31%, 49% and 40%, respectively. alphaSynuclein co-localized with TH and synaptophysin, but not with MAP 2, demonstrating it exclusive localization in presynaptic region of dopaminergic neurons. Co-localization of alpha-synuclein with GAP-43 and its role in synaptic plasticity is discussed. Conclusion: Our results further support the involvement of alphasynuclein in synaptic reorganization following irreversible loss of dopaminergic neurons in SNpc and indicate that the process of reorganization is not limited to the nigrostriatal system.
2.317 Regulation of the iron-responsive proteins amyloid precursor protein and alpha-synuclein by green tea polyphenol (−)-epigallocatechin-3-gallate L. Reznichenko1° , T. Amit, O. Weinreb, M.B.H. Youdim, S.A. Mandel Israel
1 Haifa,
Objective: Brain iron dysregulation has been implicated in the pathophysiology of Parkinson’s (PD) and Alzheimer’s (AD) diseases. The objective of this study is to analyze the effect of EGCG [(−)-epigallocatechin3-gallate)], believed to induce neuroprotection via its antioxidant, iron chelating, and antiapoptotic activities, on the expression of proteins, carrying an iron-responsive element (IRE) in their 5 -UTR mRNAs. Method: Cell culture models: SH-SY5Y neuroblastoma and CHO cells, over-expressing the “Swedish” mutated amyloid precursor protein (APP) (CHO/DNL). Assays: plasmid transfection, luciferase reporter transcription, immunoprecipitation. In vivo model: MPTP-induced parkinsonism in mice. Immunohistochemistry in the substancia nigra (SN). Results: 1. EGCG dose-dependently (1−10 mM) increased transferrin receptor (TfR) protein and mRNA levels in the human dopaminergic SHSY5Y neuroblastoma cells. The Alzheimer’s APP, which is also regulated by iron, was reduced by EGCG without altering APP mRNA levels, suggesting a posttranscriptional action. Indeed, EGCG suppressed the translation of a luciferase reporter gene fused to the APP-5 -mRNA UTR, encompassing the APP IRE. The finding that Fe2 SO4 reversed EGCG action on APP and TfR proteins, reinforces the likelihood that these effects are mediated through modulation of the intracellular iron pool. 2. EGCG reduced toxic b-amyloid peptides generation in CHO/DNL cells. 3. MPTP caused a significant up-regulation of a-synuclein immunolabeling in the tyrosine hydroxylase-containing neurons of the SN, while this effect was prevented by EGCG. Conclusion: The recent discovery of an IRE in the 5 -UTR mRNAs of a-synuclein and APP shed a new light on the potential use of iron chelators, such as EGCG, desferrioxamine and the novel bifunctional MAO-A and B iron chelator, M30, as neuroprotective compounds for PD and AD via post-translational regulation of APP and a-synuclein production.
2.318 The effects of melanin on alpha-synuclein overexpressing dopaminergic cells J. Li1° 1 Tianjin,
China, People’s Republic of
Objective: The objective of this study was to investigate the correlation of melanin and a-synuclein in human dopaminergic cell line. Method: Transfected SH-SY5Y cell line was constructed by Lipofectamine assay. Normal SH-SY5Y cell line and cell line with stably overexpressed a-synuclein was chosen in the following experiments. Both cell lines were treated with Fenton Reagent (FR) and/or dopamine melanin (DAM). Cells viability, cytotoxicity and oxidative status was detected by measuring living cells with trepan blue assay, lactate dehydrogenase (LDH) activity and lipid peroxidation. Results: Each FR and DAM themselves resulted more cell death in a-synuclein over-expressed cell line than normal SH-SY5Y cell line. LDH
and LPO activities were significantly enhanced in a-synuclein cell line with FR and/or DAM treatment. DAM did not protect both cell lines from oxidative stress. Conclusion: The interaction of DAM with a-synuclein may induce neuron degeneration, especially when they exposed to oxidative stress. This may imply the more vulnerable neurons in substantia nigra of Parkinson’s disease. 2.319 Phosphorylation of tau and alpha-synuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer’s Disease, and in Parkinson’s disease and related alpha-synucleinopathies G. Muntan´e1° , E. Dalf´o, A. Mart´ınez, I. Ferrer de Llobregat, Spain
1 L’Hospitalet
Objective: Phosphorylation of tau and phosphorylation of a-synuclein are crucial abnormalities in Alzheimer’s disease (AD) and a-synucleinopathies (Parkinson’s disease: PD, and Dementia with Lewy bodies: DLB), respectively. The present study is focused on modifications of tau and a-synuclein in the frontal cortex in AD and a-synucleinopathies. Method: The presence and distribution of phospho-tau and phosphosynuclein was examined by sub-fractionation, gel electrophoresis and Western blotting in the frontal cortex of cases with AD, PD, DLB pure form and common form, and in age-matched controls. Results: Phospho-tauSer396 has been found in synaptic-enriched fractions in AD frontal cortex at entorhinal/transentorhinal, limbic and neocortical stages, thus suggesting early tau phosphorylation at the synapses. Phospho-tauSer396 is also found in synaptic-enriched fractions in the frontal cortex in PD and DLB pure and common forms, thus suggesting increased tau phosphorylation at the synapses in these a-synucleinopathies. Increased phosphorylated a-synucleinSer129 levels are observed in the synaptic-enriched fractions of the frontal cortex in PD and DLB pure and common forms, and in advanced stages of AD. Tau phosphorylation at Ser396 has also been found in synaptic-enriched fractions in 12-month-old transgenic mice bearing the A53T a-synuclein mutation. Conclusion: It can be suggested that synapses are targets of abnormal tau and a-synuclein phosphorylation in both groups of diseases. These observations support the concept of an interface between AD and a-synucleinopathies. 2.320 Protein expression levels and activity of the glial glutamate transporters in the substantia nigra pars reticulata and striatum of 6-hydroxydopamine hemi-Parkinson rats A. Massie1° , B. Mertens, L. Arckens, S. Sarre, Y. Michotte Belgium
1 Brussels,
Objective: One of the mediators of neurodegeneration in Parkinson’s disease is glutamate excitotoxicity. Increased activity of the subthalamic nucleus has been observed in animal models and may result in increased glutamate release in substantia nigra pars reticulata (SNr). Since extracellular glutamate concentrations are determined by glutamate reuptake from the extracellular space, we studied expression and activity of the glial glutamate transporters, GLAST and GLT-1, in SNr and striatum of hemi-Parkinson rats with 6-hydroxydopamine (6-OHDA) medial forebrain bundle lesions, at different times post-lesion. Method: We used Western blotting to study the expression levels of GLAST and GLT-1. To investigate whether a change in expression level is linked to a change in glutamate reuptake activity, we performed in vivo microdialysis experiments during which we perfused the probe with L-transpyrrolidine-3,4-dicarboxylate (L-PDC, 1mM) or dihydrokainate (DHK, 1mM), two inhibitors of the glutamate transporters. Results: For both transporters we observed differences in expression level between SNr of the lesioned and non-lesioned side of the hemiParkinson rat, depending on the survival time after lesioning. In the striatum
2.314 Identification of modifiers of alpha-synuclein inclusion in a C. elegans model by genome-wide RNAi T. van Ham1° , K. Thijssen, R. Breitling, R. Hofstra, R. Plasterk, E. Nollen Netherlands
1 Groningen,
Objective: The protein alpha-synuclein is tightly connected to development of Parkinson’s disease: hallmark of PD is the presence of protein inclusions in the brain containing the protein alpha-synuclein. In addition, multiplication of the alpha-synuclein gene has been shown to be causative for PD. We aim to identify genes involved in the formation of alphasynuclein inclusions to provide an understanding of the mechanism of and genetic susceptibility to age-related sporadic PD. Method: We created nematodes (C. elegans) transgenic for human alphasynuclein fused to yellow fluorescent protein to visually track inclusion formation. We used this model in a genome-wide RNAi feeding screen to find genetic inactivations that alter a-synuclein inclusion formation. Results: Alpha-synuclein inclusions develop during aging in the transgenic C. elegans strain. The inclusions contain mostly mobile protein material and also immobile, aggregated protein at old age. Using genome-wide RNAi we identified genes that increase the formation of inclusions when silenced by RNAi. Interestingly, there was a significant overrepresentation of the genes that function in the ER, Golgi and vesicular membranes, when compared to random sets of genes. In addition, we found several genes associated with aging to increase inclusion formation when silenced. Conclusion: We developed a small animal model of alpha-synuclein accumulation, in which age-dependent inclusion formation can be visually
S125
tracked. Using this model we found genes that increase the number of alpha-synuclein inclusions when silenced. The overrepresentation of genes with a role in vesicular trafficking and ER protein quality control functioning in the endomembrane system suggests that these processes are involved in the age-dependent formation of alpha-synuclein inclusions. The genes found may provide an understanding of genetic susceptibility to PD and molecular targets for therapy.
2.315 Identification of potent small molecule inhibitors of alpha-synuclein aggregation in cell culture and by in vitro screening A. Snow1° , L. Esposito, J. Cummings, T. Lake, M. Hudson, F. Cheng, A. Ferree, S. Saha, B. Wolozin 1 Kirkland, USA Objective: A rotenone-induced cell culture model of Parkinson’s disease (PD) was primarily utilized to test the ability of ProteoTech’s small molecule library of compounds (representing new chemical entities) to inhibit aggregation of alpha-synuclein, a major component of PD Lewy bodies. Method: In this cell culture model, A53T mutant alpha-synuclein was overexpressed in human BEM-17 neuroblastoma cells. Cells exposed to 1 or 5uM rotenone accumulated alpha-synuclein aggregates containing a large amount of beta-sheet secondary structure as detected by positive Thioflavin S fluorescence and quantitative image analysis. Results: Treatment of rotenone-treated cells with different novel small molecule compounds identified 4 compounds (referred to as PD-61, 86, 31 and 13) that profoundly reduce the accumulation of Thioflavin-S positive alpha-synuclein aggregates by 87−91%, 73−91%, 40−84% and 57−70%, respectively. A marked reduction of alpha-synuclein aggregation by these compounds was confirmed using Thioflavin T fluorometry, Congo red binding assays and circular dichroism spectroscopy. In addition to their potent alpha-synuclein anti-aggregation properties, these compounds also provided good protection against rotenone-induced neurotoxicity in cell culture and in a C. elegans survival assay. Conclusion: These studies indicate that we have identified unique small molecule compounds that have potent alpha-synuclein anti-aggregation and neuroprotective properties and thus may serve as promising new therapeutics for PD and related disorders. Funded by ProteoTech Inc. and a LEAPS Award from the Michael J. Fox PD Research Foundation.
2.316 6-Hydroxydopamine nigrostriatal lesion modulates alpha-synuclein expression in different rat brain regions M. Perovic1° , A. Mladenovic, S. Ruzdijic, S. Kanazir Serbia and Montenegro
1 Belgrade,
Objective: alpha-Synuclein is considered to be involved in pathogenesis of PD and other neurodegenerative diseases. Its filamentous aggregates are found as principal constituents of Lewy bodies and some others pathological intraneuronal deposits. Due to the localization of alpha-synuclein in presynaptic terminals, its role in synaptic function and plasticity was postulated as well. 6-Hydroxydopamine (6-OHDA) rat model of PD is characterized by acute loss of dopaminergic neurons, but specific alpha-synuclein aggregates were not reported. Therefore, this model could reveal possible role of alpha-synuclein in plasticity given that some compensatory sprouting of dopaminergic neurons exists after period of extensive neuronal death. Method: Adult male Mill-Hill Hooded rats (n = 24) received four-site intrastriatal stereotaxic injection of 6-OHDA or saline (sham operated animals) into the right lateral striatum. Five weeks later, animals were killed by decapitation and the model was verified by tyrosine hydroxylase (TH) imunohistochemistry. Western blot analysis was used to detemine the level of alpha-synuclein protein and confocal microscopy for analysis of alphasynuclein co-localization with TH, synaptophysine, MAP 2 and GAP-43.
S126
Tuesday, 11 December 2007
Results: Using Western blot analysis, significant increase (p < 0.05) in the level of alpha-synuclein was detected in ipsilateral striatum, mesencephalon and cerebellum by 31%, 49% and 40%, respectively. alphaSynuclein co-localized with TH and synaptophysin, but not with MAP 2, demonstrating it exclusive localization in presynaptic region of dopaminergic neurons. Co-localization of alpha-synuclein with GAP-43 and its role in synaptic plasticity is discussed. Conclusion: Our results further support the involvement of alphasynuclein in synaptic reorganization following irreversible loss of dopaminergic neurons in SNpc and indicate that the process of reorganization is not limited to the nigrostriatal system.
2.317 Regulation of the iron-responsive proteins amyloid precursor protein and alpha-synuclein by green tea polyphenol (−)-epigallocatechin-3-gallate L. Reznichenko1° , T. Amit, O. Weinreb, M.B.H. Youdim, S.A. Mandel Israel
1 Haifa,
Objective: Brain iron dysregulation has been implicated in the pathophysiology of Parkinson’s (PD) and Alzheimer’s (AD) diseases. The objective of this study is to analyze the effect of EGCG [(−)-epigallocatechin3-gallate)], believed to induce neuroprotection via its antioxidant, iron chelating, and antiapoptotic activities, on the expression of proteins, carrying an iron-responsive element (IRE) in their 5 -UTR mRNAs. Method: Cell culture models: SH-SY5Y neuroblastoma and CHO cells, over-expressing the “Swedish” mutated amyloid precursor protein (APP) (CHO/DNL). Assays: plasmid transfection, luciferase reporter transcription, immunoprecipitation. In vivo model: MPTP-induced parkinsonism in mice. Immunohistochemistry in the substancia nigra (SN). Results: 1. EGCG dose-dependently (1−10 mM) increased transferrin receptor (TfR) protein and mRNA levels in the human dopaminergic SHSY5Y neuroblastoma cells. The Alzheimer’s APP, which is also regulated by iron, was reduced by EGCG without altering APP mRNA levels, suggesting a posttranscriptional action. Indeed, EGCG suppressed the translation of a luciferase reporter gene fused to the APP-5 -mRNA UTR, encompassing the APP IRE. The finding that Fe2 SO4 reversed EGCG action on APP and TfR proteins, reinforces the likelihood that these effects are mediated through modulation of the intracellular iron pool. 2. EGCG reduced toxic b-amyloid peptides generation in CHO/DNL cells. 3. MPTP caused a significant up-regulation of a-synuclein immunolabeling in the tyrosine hydroxylase-containing neurons of the SN, while this effect was prevented by EGCG. Conclusion: The recent discovery of an IRE in the 5 -UTR mRNAs of a-synuclein and APP shed a new light on the potential use of iron chelators, such as EGCG, desferrioxamine and the novel bifunctional MAO-A and B iron chelator, M30, as neuroprotective compounds for PD and AD via post-translational regulation of APP and a-synuclein production.
2.318 The effects of melanin on alpha-synuclein overexpressing dopaminergic cells J. Li1° 1 Tianjin,
China, People’s Republic of
Objective: The objective of this study was to investigate the correlation of melanin and a-synuclein in human dopaminergic cell line. Method: Transfected SH-SY5Y cell line was constructed by Lipofectamine assay. Normal SH-SY5Y cell line and cell line with stably overexpressed a-synuclein was chosen in the following experiments. Both cell lines were treated with Fenton Reagent (FR) and/or dopamine melanin (DAM). Cells viability, cytotoxicity and oxidative status was detected by measuring living cells with trepan blue assay, lactate dehydrogenase (LDH) activity and lipid peroxidation. Results: Each FR and DAM themselves resulted more cell death in a-synuclein over-expressed cell line than normal SH-SY5Y cell line. LDH
and LPO activities were significantly enhanced in a-synuclein cell line with FR and/or DAM treatment. DAM did not protect both cell lines from oxidative stress. Conclusion: The interaction of DAM with a-synuclein may induce neuron degeneration, especially when they exposed to oxidative stress. This may imply the more vulnerable neurons in substantia nigra of Parkinson’s disease. 2.319 Phosphorylation of tau and alpha-synuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer’s Disease, and in Parkinson’s disease and related alpha-synucleinopathies G. Muntan´e1° , E. Dalf´o, A. Mart´ınez, I. Ferrer de Llobregat, Spain
1 L’Hospitalet
Objective: Phosphorylation of tau and phosphorylation of a-synuclein are crucial abnormalities in Alzheimer’s disease (AD) and a-synucleinopathies (Parkinson’s disease: PD, and Dementia with Lewy bodies: DLB), respectively. The present study is focused on modifications of tau and a-synuclein in the frontal cortex in AD and a-synucleinopathies. Method: The presence and distribution of phospho-tau and phosphosynuclein was examined by sub-fractionation, gel electrophoresis and Western blotting in the frontal cortex of cases with AD, PD, DLB pure form and common form, and in age-matched controls. Results: Phospho-tauSer396 has been found in synaptic-enriched fractions in AD frontal cortex at entorhinal/transentorhinal, limbic and neocortical stages, thus suggesting early tau phosphorylation at the synapses. Phospho-tauSer396 is also found in synaptic-enriched fractions in the frontal cortex in PD and DLB pure and common forms, thus suggesting increased tau phosphorylation at the synapses in these a-synucleinopathies. Increased phosphorylated a-synucleinSer129 levels are observed in the synaptic-enriched fractions of the frontal cortex in PD and DLB pure and common forms, and in advanced stages of AD. Tau phosphorylation at Ser396 has also been found in synaptic-enriched fractions in 12-month-old transgenic mice bearing the A53T a-synuclein mutation. Conclusion: It can be suggested that synapses are targets of abnormal tau and a-synuclein phosphorylation in both groups of diseases. These observations support the concept of an interface between AD and a-synucleinopathies. 2.320 Protein expression levels and activity of the glial glutamate transporters in the substantia nigra pars reticulata and striatum of 6-hydroxydopamine hemi-Parkinson rats A. Massie1° , B. Mertens, L. Arckens, S. Sarre, Y. Michotte Belgium
1 Brussels,
Objective: One of the mediators of neurodegeneration in Parkinson’s disease is glutamate excitotoxicity. Increased activity of the subthalamic nucleus has been observed in animal models and may result in increased glutamate release in substantia nigra pars reticulata (SNr). Since extracellular glutamate concentrations are determined by glutamate reuptake from the extracellular space, we studied expression and activity of the glial glutamate transporters, GLAST and GLT-1, in SNr and striatum of hemi-Parkinson rats with 6-hydroxydopamine (6-OHDA) medial forebrain bundle lesions, at different times post-lesion. Method: We used Western blotting to study the expression levels of GLAST and GLT-1. To investigate whether a change in expression level is linked to a change in glutamate reuptake activity, we performed in vivo microdialysis experiments during which we perfused the probe with L-transpyrrolidine-3,4-dicarboxylate (L-PDC, 1mM) or dihydrokainate (DHK, 1mM), two inhibitors of the glutamate transporters. Results: For both transporters we observed differences in expression level between SNr of the lesioned and non-lesioned side of the hemiParkinson rat, depending on the survival time after lesioning. In the striatum