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3.140 THE CONTRIBUTION OF THE MYD88-DEPENDENT PATHWAY IN ANIMAL MODELS OF PARKINSONISM
Wednesday, 14 December 2011 / Parkinsonism and Related Disorders 18S2 (2012) S161–S234
Conclusion: The consequences suggested that excess copper has damage effect on astrocytes. The decrease of GSH and the activity of GR may responsible for the injured function of antioxidative of astrocyte. Astrocyte will product more nitric oxide to intensify oxidation and may play a role in the activation of inflammation. 3.139 ALTERATIONS IN MARKERS OF AGING IN SUBSTANTIA NIGRA OF MICE LACKING GLUTATHIONE S-TRANSFERASE PI (GSTPI) Y. Jiao, A. Pani, H. Jang, Y. Dou, J. Smith, S. Richard. Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Aging is a major risk factor in Parkinson’s disease. As the brain ages, there are increased levels of oxidative stress that can alter mitochondrial function, protein aggregation and inflammation. The major antioxidant protein in the brain is glutathione, which functions by binding with electrophiles to a glutathione S-transferase (GST). In DA neurons of the SN, the only GST present is GSTpi. To examine if this protein participates in regulation of markers of ageing we examined a number of parameters including DA neuron number, DA protein levels and inflammatory markers at 4, 12 and 24 months in a WT strain resistant to oxidative stressinduced degeneration (Swiss-Webster, SW) and compared them to mice lacking GSTpi. We found that SW mice did not show any age-induced changes. However, GSTpi-null mice we found a progressive loss of SN DA neurons and striatal DA. We also examined mRNA and protein levels of genes involved in dopamine handling, inflammation, and cell survival. Although no age-related changes were seen in WT mice, we did find age-related alterations in bdnf, gdnf, vegf, il-1b, tnfa, nos2, nox1, vmat2, dat, and sod1 mRNA as well as IL1, IL10, IL15, IP10 protein in mice lacking GSTpi. The lack of age related changes in WT mice might explain why many mouse models that seek to mimic human diseases of ageing do not show phenotypes seen in humans. Since we see these in the GSTpi-null mouse, its use as a background strain in future studies may allow development of better age-related disease models. 3.140 THE CONTRIBUTION OF THE MYD88-DEPENDENT PATHWAY IN ANIMAL MODELS OF PARKINSONISM J. Drouin-Ouellet, M. Cote, C. Gibrat, M. Bousquet, F. Calon, J. Kriz, D. Soulet, F. Cicchetti. Centre de Recherche du CHUL (CHUQ); Laval University, Quebec, QC, Canada MyD88 is the most common adaptor protein implicated in tolllike receptor (TLR) signaling pathways leading to inflammation, a phenomenon suspected to play a critical role in the pathophysiology of Parkinson’s disease (PD) but for which the key players have not clearly been identified. To specifically address this question, we subjected wild type (WT) and MyD88−/− mice to a subacute (7 injections administered over a 5-day period) or acute (3 injections over a 6-hour period) MPTP treatment, a toxin which recreates several pathological features of PD in animals. We have previously reported that the MyD88-dependent pathway is required for MPTP-induced enteric nervous system (ENS) dopaminergic degeneration via infiltrating macrophages, which adopt a pro-repair immunophenotype in MyD88−/− MPTP-treated mice. In the brain, we observed that dopaminergic degeneration following a subacute MPTP regimen, which only induces a mild inflammatory response, is, on the other hand, MyD88-independent. We thus investigated the possible role of the MyD88-dependent pathway in a more severe inflammatory context provided by an acute MPTP treatment. HPLC analyses demonstrated that MyD88−/− mice are as vulnerable to dopamine striatal depletion as WT mice (p < 0.05 and p < 0.01, respectively). This was accompanied by a strong microglial and astrocytic response in the SNpc, which was of similar magnitude both in WT and MyD88−/− mice. Our observations suggest that MPTP-induced dopaminergic degeneration leading to parkinsonism in the CNS is MyD88-independent in two MPTP animal models.
S199
However, the results obtained in the ENS suggest potential new target sites for the treatment of peripheral inflammation. 3.141 NURR1 DETERMINES THE PREFERENTIAL DEGENERATION OF MIDBRAIN DOPAMINERGIC NEURONS IN A PARKINSON’S DISEASE MOUSE MODEL X. Lin1,2 , L. Parisiadou2 , G. Liu2 , J. Yu2 , H. Cai2 . 1 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 2 National Institute on Aging, NIH, Bethesda, MD, USA Parkinson’s disease (PD) is pathologically characterized by a preferential loss of midbrain dopaminergic neurons (MDNs); however, the underlying pathogenic mechanism remains elusive. a-synuclein (a-syn) plays a prominent role in the pathogenesis of PD. To gain the mechanistic insights into the a-syn-induced MDN degeneration, we modeled the PD-related a-syn A53T missense mutation in mice by selectively expressing the transgene in the MDNs. Here we show the mutant mice developed profound motor disabilities and robust MDN degeneration, resembling the key clinical and pathological phenotypes of PD. Moreover, in examining the mechanism of a-syn-dependent dopaminergic deficiency, we found that a-syn suppressed the expression and function of nuclear receptor related protein 1 (Nurr1), a crucial regulator for both the development and maintenance of MDNs. Conversely, inhibition of proteasome-dependent degradation of Nurr1 ameliorated the a-syn-induced loss of MDNs. Therefore, our study identifies Nurr1 as a critical determinant of a-syn-mediated preferential loss of MDNs in PD. 3.142 CAENORHABDITIS ELEGANS AS A MODEL SYSTEM OF PARKINSON DISEASE FOR HIGH-CONTENT SCREENING S. Lehtonen, M. Oksanen, M. Lakso, G. Wong, M. Courtney, J. Koistinaho. Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland Conservation between Caenorhabditis elegans (C. elegans) and mammals in genomic, biosynthetic and metabolic pathways as well as the advantages of observing dopaminergic neurons morphology in vivo and the ease of genetic manipulation make C. elegans an excellent model organism for Parkinson’s disease. While humans have billions of neurons, the microscopic roundworm C. elegans has precisely 302, of which only eight produce dopamine (DA) in hermaphrodites. Here we introduce an MPP(+)-based model for high-content screenings using nematode C. elegans (Pdat1::GFP). 96-well plates containing a 40-ml suspension with a final bacterial density at OD600 of 2, 10 ml of MPP+ and anti-PD chemical compounds, and average of 20 animals per well are imaged with a BD pathway 855 High-Content Imager with 10x objective (Olympus), acquiring 25 contiguous fields per well under control of Attovision software (BD). The image montages are then segmented by Attovision software in two phases, first to define the clusters of bright GFP-positive DA neurons within the heads of the worms, and second to identify individual GFP-positive neurons while excluding fragments of neurons or shrunken neurons. This segmentation algorithm was found to efficiently detect worm heads with intact healthy GFP-positive neurons. The sum of GFP fluorescence intensity from all defined head regions containing the GFP-positive neurons is calculated from each montage, as this was found to be the most sensitive and consistent parameter we tested as an indicator of MPP+-induced neurodegeneration. Our data suggest that C. elegans MPP+ model can be used for the quantitative evaluation of anti-PD drugs.
Conclusion: The consequences suggested that excess copper has damage effect on astrocytes. The decrease of GSH and the activity of GR may responsible for the injured function of antioxidative of astrocyte. Astrocyte will product more nitric oxide to intensify oxidation and may play a role in the activation of inflammation. 3.139 ALTERATIONS IN MARKERS OF AGING IN SUBSTANTIA NIGRA OF MICE LACKING GLUTATHIONE S-TRANSFERASE PI (GSTPI) Y. Jiao, A. Pani, H. Jang, Y. Dou, J. Smith, S. Richard. Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA Aging is a major risk factor in Parkinson’s disease. As the brain ages, there are increased levels of oxidative stress that can alter mitochondrial function, protein aggregation and inflammation. The major antioxidant protein in the brain is glutathione, which functions by binding with electrophiles to a glutathione S-transferase (GST). In DA neurons of the SN, the only GST present is GSTpi. To examine if this protein participates in regulation of markers of ageing we examined a number of parameters including DA neuron number, DA protein levels and inflammatory markers at 4, 12 and 24 months in a WT strain resistant to oxidative stressinduced degeneration (Swiss-Webster, SW) and compared them to mice lacking GSTpi. We found that SW mice did not show any age-induced changes. However, GSTpi-null mice we found a progressive loss of SN DA neurons and striatal DA. We also examined mRNA and protein levels of genes involved in dopamine handling, inflammation, and cell survival. Although no age-related changes were seen in WT mice, we did find age-related alterations in bdnf, gdnf, vegf, il-1b, tnfa, nos2, nox1, vmat2, dat, and sod1 mRNA as well as IL1, IL10, IL15, IP10 protein in mice lacking GSTpi. The lack of age related changes in WT mice might explain why many mouse models that seek to mimic human diseases of ageing do not show phenotypes seen in humans. Since we see these in the GSTpi-null mouse, its use as a background strain in future studies may allow development of better age-related disease models. 3.140 THE CONTRIBUTION OF THE MYD88-DEPENDENT PATHWAY IN ANIMAL MODELS OF PARKINSONISM J. Drouin-Ouellet, M. Cote, C. Gibrat, M. Bousquet, F. Calon, J. Kriz, D. Soulet, F. Cicchetti. Centre de Recherche du CHUL (CHUQ); Laval University, Quebec, QC, Canada MyD88 is the most common adaptor protein implicated in tolllike receptor (TLR) signaling pathways leading to inflammation, a phenomenon suspected to play a critical role in the pathophysiology of Parkinson’s disease (PD) but for which the key players have not clearly been identified. To specifically address this question, we subjected wild type (WT) and MyD88−/− mice to a subacute (7 injections administered over a 5-day period) or acute (3 injections over a 6-hour period) MPTP treatment, a toxin which recreates several pathological features of PD in animals. We have previously reported that the MyD88-dependent pathway is required for MPTP-induced enteric nervous system (ENS) dopaminergic degeneration via infiltrating macrophages, which adopt a pro-repair immunophenotype in MyD88−/− MPTP-treated mice. In the brain, we observed that dopaminergic degeneration following a subacute MPTP regimen, which only induces a mild inflammatory response, is, on the other hand, MyD88-independent. We thus investigated the possible role of the MyD88-dependent pathway in a more severe inflammatory context provided by an acute MPTP treatment. HPLC analyses demonstrated that MyD88−/− mice are as vulnerable to dopamine striatal depletion as WT mice (p < 0.05 and p < 0.01, respectively). This was accompanied by a strong microglial and astrocytic response in the SNpc, which was of similar magnitude both in WT and MyD88−/− mice. Our observations suggest that MPTP-induced dopaminergic degeneration leading to parkinsonism in the CNS is MyD88-independent in two MPTP animal models.
S199
However, the results obtained in the ENS suggest potential new target sites for the treatment of peripheral inflammation. 3.141 NURR1 DETERMINES THE PREFERENTIAL DEGENERATION OF MIDBRAIN DOPAMINERGIC NEURONS IN A PARKINSON’S DISEASE MOUSE MODEL X. Lin1,2 , L. Parisiadou2 , G. Liu2 , J. Yu2 , H. Cai2 . 1 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 2 National Institute on Aging, NIH, Bethesda, MD, USA Parkinson’s disease (PD) is pathologically characterized by a preferential loss of midbrain dopaminergic neurons (MDNs); however, the underlying pathogenic mechanism remains elusive. a-synuclein (a-syn) plays a prominent role in the pathogenesis of PD. To gain the mechanistic insights into the a-syn-induced MDN degeneration, we modeled the PD-related a-syn A53T missense mutation in mice by selectively expressing the transgene in the MDNs. Here we show the mutant mice developed profound motor disabilities and robust MDN degeneration, resembling the key clinical and pathological phenotypes of PD. Moreover, in examining the mechanism of a-syn-dependent dopaminergic deficiency, we found that a-syn suppressed the expression and function of nuclear receptor related protein 1 (Nurr1), a crucial regulator for both the development and maintenance of MDNs. Conversely, inhibition of proteasome-dependent degradation of Nurr1 ameliorated the a-syn-induced loss of MDNs. Therefore, our study identifies Nurr1 as a critical determinant of a-syn-mediated preferential loss of MDNs in PD. 3.142 CAENORHABDITIS ELEGANS AS A MODEL SYSTEM OF PARKINSON DISEASE FOR HIGH-CONTENT SCREENING S. Lehtonen, M. Oksanen, M. Lakso, G. Wong, M. Courtney, J. Koistinaho. Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland Conservation between Caenorhabditis elegans (C. elegans) and mammals in genomic, biosynthetic and metabolic pathways as well as the advantages of observing dopaminergic neurons morphology in vivo and the ease of genetic manipulation make C. elegans an excellent model organism for Parkinson’s disease. While humans have billions of neurons, the microscopic roundworm C. elegans has precisely 302, of which only eight produce dopamine (DA) in hermaphrodites. Here we introduce an MPP(+)-based model for high-content screenings using nematode C. elegans (Pdat1::GFP). 96-well plates containing a 40-ml suspension with a final bacterial density at OD600 of 2, 10 ml of MPP+ and anti-PD chemical compounds, and average of 20 animals per well are imaged with a BD pathway 855 High-Content Imager with 10x objective (Olympus), acquiring 25 contiguous fields per well under control of Attovision software (BD). The image montages are then segmented by Attovision software in two phases, first to define the clusters of bright GFP-positive DA neurons within the heads of the worms, and second to identify individual GFP-positive neurons while excluding fragments of neurons or shrunken neurons. This segmentation algorithm was found to efficiently detect worm heads with intact healthy GFP-positive neurons. The sum of GFP fluorescence intensity from all defined head regions containing the GFP-positive neurons is calculated from each montage, as this was found to be the most sensitive and consistent parameter we tested as an indicator of MPP+-induced neurodegeneration. Our data suggest that C. elegans MPP+ model can be used for the quantitative evaluation of anti-PD drugs.