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3.231 CHEMICALS POSSESSING A NEUROTROPHIN-LIKE ACTIVITY ON DOPAMINERGIC NEURONS IN PRIMARY CULTURE
Wednesday, 14 December 2011 / Parkinsonism and Related Disorders 18S2 (2012) S161–S234
S211
3.230 THE OBSERVATION OF SYNERGISTIC PROTECTION BY GDNF AND E2 ON INJURED DOPAMINERGIC NEURONS VIA ELECTRON MICROSCOPY Z. Shi, M. Wang, L. Li, H. Dong, H. Yu, D. Gao. Neurobiology & Anatomy, Xu Zhou Medical College, Xuzhou, China
3.232 RESTORATION OF THE DOPAMINERGIC ACTIVITY IN THE BRAIN OF PARKINSON’S DISEASE PATIENTS USING SV40-BASED VIRAL GENE DELIVERY VECTORS P. de Haan, W. de Vries, E.C. Wolters. Amarna Therapeutics B.V., Leiden, The Netherlands
Previous studies have suggested that the estrogenic a-receptor (ERa), in injured dopaminergic neurons, accumulates in the cytoplasmic surface of cellular membrane after co-administration of glial cell line-derived neurotrophic factor (GDNF) and 17b-estradiol (E2), and associates with intracellular domain of N-cadherin. Moreover, the phosphorylation of N-cadherin (Tyr-860) determines the binding amount of N-cadherin and ERa. For strengthen the conclusions, we observe the distributions and association of N-cadherin and ERa in cells by electron microscope. Using MN9D cells injured by 6-OHDA, immunofluorescence staining and immune electron microscopy (EM) we show that enzymelabeled Phospho-N-cadherin (Tyr-860) mainly exists in cytoplasmic surface of cellular membrane and gold-labeled ERa mainly exists in cytoplasm without co-administration of GDNF and E2. After treatment with GDNF and E2, most ERa-labeled gold particles transfers to the cytoplasmic surface of cellular membrane and bind with intracellular domain of N-cadherin. These findings further indicate treatment with GDNF and E2 can promote the phosphorylation of N-Cadherin (Tyr-860) and then associate with ERa in injured dopaminergic neurons.
Parkinson’s Disease (PD) is the second most common neurodegenerative disorder. PD progression is associated with alpha-synuclein deposition in brain stem neurons and with chronic stimulation of inflammatory responses in the brain. PD patients are currently treated with medications to increase the levels of dopamine to manage the early motor symptoms (parkinsonism) of the disease. Recently, a number of gene therapy clinical trials have been conducted with the aim to restore the dopaminergic activity in the nigral substance. These approaches alleviate the disease symptoms, but do not cure the disease. In order to prevent further inflammatory damage and to repair the dopaminergic activity in the nigral substance, adeno-associated viral (AAV) vectors were used to deliver neurotrophic factors to the brain stem. These AAV trials revealed that despite the excellent safety profile of AAV vectors humans have a CTL memory for AAV that renders AAV-mediated gene therapies highly inefficient. Replication-incompetent gene delivery vectors derived from the macaque polyomavirus SV40 are safe, non-immunogenic and highly efficient. Furthermore, SV40 vectors efficiently transduce primate brain cells without any sign of immune activation. We have developed an SV40-based gene delivery platform (SVac) that solves the immunity problems associated with AAV and the production problems of SV40 vector particles. In order to repair the dopaminergic activity in the nigral substance and to prevent further inflammatory damage we will construct and produce a series of recombinant SVac vectors encoding neurotrophic factors and test them in the marmoset PD model for their capacity to stop disease progression.
3.231 CHEMICALS POSSESSING A NEUROTROPHIN-LIKE ACTIVITY ON DOPAMINERGIC NEURONS IN PRIMARY CULTURE R. Raisman-Vozari1 , F. Schmidt2 , P. Champy2 , B. Seon-Meniel3 , X. Franck3 , G. Le Douaron3 , B. Figadere ` 4 . 1 Th´erapeutique Exp´erimentale de la neurod´eg´en´erescence, INSERM UMR 975 – CNRS UMR 7225 – CRICM-ICM, Paris, 2 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Facult´e de Pharmacie, 3 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Faculte de Pharmacie, 4 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Chˆ atenay-Malabry, France Neurotrophic factors have been shown to possess strong neuroprotective and neurorestaurative properties in Parkinson’s disease (PD) patients. However the issues to control their delivery into the interest areas of the brain and their surgical administration linked to their unability to cross the blood brain barrier are many drawbacks responsible of undesirable side effects limiting their clinical use. A strategy implying the use of neurotrophic small molecules could provide an interesting alternative avoiding neurotrophin administration and side effects. In an attempt to develop drugs mimicking neurotrophic factors, we have designed and synthesized low molecular weight molecules that exhibit neuroprotective and neuritogenic potential for dopaminergic neurons. A cell-based screening of an in-house quinoline-derived compound collection led to the characterization of compounds exhibiting both activities in the nanomolar range on mesencephalic dopaminergic neurons in spontaneous or 1-methyl4-phenylpyridinium (MPP(+))-induced neurodegeneration. This study provides evidence that rescued neurons possess a functional dopamine transporter and underlines the involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in these processes. Cell-based screening led to the discovery of a potent neurotrophic compound possessing expected physicochemical properties for blood brain barrier penetration as a serious candidate for therapeutic use in PD. In addition, in an animal model of PD, we observed the protection of dopaminergic neurons after chronic treatment with one of our molecules.
3.233 EFFECT OF MCI-186 ON THE INTRACELLULAR CALCIUM AND MEMBRANE FLUIDITY IN THE RATS WITH PARKINSON’S DISEASE Y. Ming. Jiangsu University, Zhenjiang, China Aim: To determine the neuroprotective effect of MCI-186 through decreasing the concentration of intracellular calcium ([Ca2+ ]i ) and the mean microviscosity (MMV) in the rats with Parkinson’s disease (PD). Methods: Eighty male Wistar rats were randomly divided into 4 groups: normal control group, sham-operated group, PD group, and MCI-186 treatment group. The rats with PD were successfully made with injection of 6-hydroxydopamine (6-OHDA) into the striatum. Tyrosine hydroxylase-positive neurons were respectively detected by immunohistochemistry at the 1st, 7th, 14th and 28th day. At above-mentioned time, [Ca2+ ]i were detected by Fura-2/AM fluorimetry and membrane fluidity was also measured using fluorescence spectrophotometer [neurons were enzymatically isolated and loaded with the fluorescent dye, DPH (1,6-diphenyl1,3,5-hexatriene); MMV was used as index of membrane fluidity]. Results: Compared with the normal control group and the sham operation group, [Ca2+ ]i , and MMV were increased, but the number of tyrosine hydroxylase-positive neurons were decreased in the PD group from the 1st day and changed the highest level at the 14th day (P < 0.01). Compared with PD group, [Ca2+ ]i , MMV were significantly decreased and the number of tyrosine hydroxylasepositive neurons was gradually increased in MCI-186 treatment group from the 7th day to the 1mon (P < 0.01 or P < 0.05). Conclusion: MCI-186 can protect neurons in the rats with PD through decreasing the intracellular calcium concentration and improving the membrane fluidity (that is, decreasing the mean microviscosity), which are probably important anti-oxidative mechanisms of MCI-186.
S211
3.230 THE OBSERVATION OF SYNERGISTIC PROTECTION BY GDNF AND E2 ON INJURED DOPAMINERGIC NEURONS VIA ELECTRON MICROSCOPY Z. Shi, M. Wang, L. Li, H. Dong, H. Yu, D. Gao. Neurobiology & Anatomy, Xu Zhou Medical College, Xuzhou, China
3.232 RESTORATION OF THE DOPAMINERGIC ACTIVITY IN THE BRAIN OF PARKINSON’S DISEASE PATIENTS USING SV40-BASED VIRAL GENE DELIVERY VECTORS P. de Haan, W. de Vries, E.C. Wolters. Amarna Therapeutics B.V., Leiden, The Netherlands
Previous studies have suggested that the estrogenic a-receptor (ERa), in injured dopaminergic neurons, accumulates in the cytoplasmic surface of cellular membrane after co-administration of glial cell line-derived neurotrophic factor (GDNF) and 17b-estradiol (E2), and associates with intracellular domain of N-cadherin. Moreover, the phosphorylation of N-cadherin (Tyr-860) determines the binding amount of N-cadherin and ERa. For strengthen the conclusions, we observe the distributions and association of N-cadherin and ERa in cells by electron microscope. Using MN9D cells injured by 6-OHDA, immunofluorescence staining and immune electron microscopy (EM) we show that enzymelabeled Phospho-N-cadherin (Tyr-860) mainly exists in cytoplasmic surface of cellular membrane and gold-labeled ERa mainly exists in cytoplasm without co-administration of GDNF and E2. After treatment with GDNF and E2, most ERa-labeled gold particles transfers to the cytoplasmic surface of cellular membrane and bind with intracellular domain of N-cadherin. These findings further indicate treatment with GDNF and E2 can promote the phosphorylation of N-Cadherin (Tyr-860) and then associate with ERa in injured dopaminergic neurons.
Parkinson’s Disease (PD) is the second most common neurodegenerative disorder. PD progression is associated with alpha-synuclein deposition in brain stem neurons and with chronic stimulation of inflammatory responses in the brain. PD patients are currently treated with medications to increase the levels of dopamine to manage the early motor symptoms (parkinsonism) of the disease. Recently, a number of gene therapy clinical trials have been conducted with the aim to restore the dopaminergic activity in the nigral substance. These approaches alleviate the disease symptoms, but do not cure the disease. In order to prevent further inflammatory damage and to repair the dopaminergic activity in the nigral substance, adeno-associated viral (AAV) vectors were used to deliver neurotrophic factors to the brain stem. These AAV trials revealed that despite the excellent safety profile of AAV vectors humans have a CTL memory for AAV that renders AAV-mediated gene therapies highly inefficient. Replication-incompetent gene delivery vectors derived from the macaque polyomavirus SV40 are safe, non-immunogenic and highly efficient. Furthermore, SV40 vectors efficiently transduce primate brain cells without any sign of immune activation. We have developed an SV40-based gene delivery platform (SVac) that solves the immunity problems associated with AAV and the production problems of SV40 vector particles. In order to repair the dopaminergic activity in the nigral substance and to prevent further inflammatory damage we will construct and produce a series of recombinant SVac vectors encoding neurotrophic factors and test them in the marmoset PD model for their capacity to stop disease progression.
3.231 CHEMICALS POSSESSING A NEUROTROPHIN-LIKE ACTIVITY ON DOPAMINERGIC NEURONS IN PRIMARY CULTURE R. Raisman-Vozari1 , F. Schmidt2 , P. Champy2 , B. Seon-Meniel3 , X. Franck3 , G. Le Douaron3 , B. Figadere ` 4 . 1 Th´erapeutique Exp´erimentale de la neurod´eg´en´erescence, INSERM UMR 975 – CNRS UMR 7225 – CRICM-ICM, Paris, 2 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Facult´e de Pharmacie, 3 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Faculte de Pharmacie, 4 Laboratoire de Pharmacognosie, CNRS Universit´e Paris-Sud 11, Chˆ atenay-Malabry, France Neurotrophic factors have been shown to possess strong neuroprotective and neurorestaurative properties in Parkinson’s disease (PD) patients. However the issues to control their delivery into the interest areas of the brain and their surgical administration linked to their unability to cross the blood brain barrier are many drawbacks responsible of undesirable side effects limiting their clinical use. A strategy implying the use of neurotrophic small molecules could provide an interesting alternative avoiding neurotrophin administration and side effects. In an attempt to develop drugs mimicking neurotrophic factors, we have designed and synthesized low molecular weight molecules that exhibit neuroprotective and neuritogenic potential for dopaminergic neurons. A cell-based screening of an in-house quinoline-derived compound collection led to the characterization of compounds exhibiting both activities in the nanomolar range on mesencephalic dopaminergic neurons in spontaneous or 1-methyl4-phenylpyridinium (MPP(+))-induced neurodegeneration. This study provides evidence that rescued neurons possess a functional dopamine transporter and underlines the involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in these processes. Cell-based screening led to the discovery of a potent neurotrophic compound possessing expected physicochemical properties for blood brain barrier penetration as a serious candidate for therapeutic use in PD. In addition, in an animal model of PD, we observed the protection of dopaminergic neurons after chronic treatment with one of our molecules.
3.233 EFFECT OF MCI-186 ON THE INTRACELLULAR CALCIUM AND MEMBRANE FLUIDITY IN THE RATS WITH PARKINSON’S DISEASE Y. Ming. Jiangsu University, Zhenjiang, China Aim: To determine the neuroprotective effect of MCI-186 through decreasing the concentration of intracellular calcium ([Ca2+ ]i ) and the mean microviscosity (MMV) in the rats with Parkinson’s disease (PD). Methods: Eighty male Wistar rats were randomly divided into 4 groups: normal control group, sham-operated group, PD group, and MCI-186 treatment group. The rats with PD were successfully made with injection of 6-hydroxydopamine (6-OHDA) into the striatum. Tyrosine hydroxylase-positive neurons were respectively detected by immunohistochemistry at the 1st, 7th, 14th and 28th day. At above-mentioned time, [Ca2+ ]i were detected by Fura-2/AM fluorimetry and membrane fluidity was also measured using fluorescence spectrophotometer [neurons were enzymatically isolated and loaded with the fluorescent dye, DPH (1,6-diphenyl1,3,5-hexatriene); MMV was used as index of membrane fluidity]. Results: Compared with the normal control group and the sham operation group, [Ca2+ ]i , and MMV were increased, but the number of tyrosine hydroxylase-positive neurons were decreased in the PD group from the 1st day and changed the highest level at the 14th day (P < 0.01). Compared with PD group, [Ca2+ ]i , MMV were significantly decreased and the number of tyrosine hydroxylasepositive neurons was gradually increased in MCI-186 treatment group from the 7th day to the 1mon (P < 0.01 or P < 0.05). Conclusion: MCI-186 can protect neurons in the rats with PD through decreasing the intracellular calcium concentration and improving the membrane fluidity (that is, decreasing the mean microviscosity), which are probably important anti-oxidative mechanisms of MCI-186.