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The neurotrophic factors pathways on dopaminergic neuron and astrocyte in animal model of Parkinson’s disease

남진한 (Nam, Jin Han, 경희대학교 일반대학원)

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초록 moremore
Part 1 The expression of human ras homolog enriched in brain with a S16H mutation [hRheb(S16H)] in adult dopaminergic (DA) neurons protects the nigrostriatal DA projection in the 6-hydroxydopamine (6-OHDA)-treated animal model of Parkinson’s disease (PD). However, the mechanism is still unclear and...
Part 1 The expression of human ras homolog enriched in brain with a S16H mutation [hRheb(S16H)] in adult dopaminergic (DA) neurons protects the nigrostriatal DA projection in the 6-hydroxydopamine (6-OHDA)-treated animal model of Parkinson’s disease (PD). However, the mechanism is still unclear and little is known about the induction of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by hRheb-activated intracellular signaling pathways in adult neurons. Here, we show that transduction of nigral DA neurons with hRheb(S16H) significantly increases the levels of phospho-cAMP response element binding protein (p-CREB), GDNF and BDNF in DA neurons, and the increased levels are attenuated by rapamycin, a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1). Moreover, the protective effects of hRheb (S16H) against 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity are attenuated by treatment with specific neutralizing antibodies for GDNF and BDNF. These results show that activation of hRheb/mTORC1 signaling pathway could impart to DA neurons the important ability to continuously produce GDNF and BDNF as therapeutic agents against PD. Part 2 CNTF is expressed within astrocyte of SNpc in MPP+ injected Parkinson’s animal model. And, TRPV1 exogenous agonist, capsaicin is enhanced CNTF expression within astrocyte results capsaicin prevents degeneration of nigrostriatal pathway and amphetamine-induced ipsilateral rotation behavior in MPP+ lesion Parkinson’s animal models. However, CNTF regulation mechanism is uncovered in astrocyte. Here we shown, Capsaicin regulates CNTF and mTOR downstream signal, such as p-p70S6K in mesencephalon human and rat astrocyte in vitro. And, in vivo studies, Capsaicin treated MPP+ lesion Parkinson’s animal models, p-p70S6K phosphorylation levels enhanced within astrocyte. Also, CNTF expression levels increased in CAP treated MPP+ lesion Parkinson’s animal models. These results show that capsaicin prevents degeneration of nigrostriatal pathway and amphetamine-induced rotation by TRPV1-mTOR pathway regulates CNTF in MPP+ lesion Parkinson’s animal models.
목차 moremore
I. Introduction . 1
1 Parkinsons disease (PD) 1
1.1 Pathogenesis of Parkinsons disease 1
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I. Introduction . 1
1 Parkinsons disease (PD) 1
1.1 Pathogenesis of Parkinsons disease 1
2 Animal model of Parkinsons disease 2
2.1 MPTP / MPP+ / 6-OHDA animal models (synthetic neurotoxin) 2
2.2 Paraquat/Rotenone (Herbicide/Pesticide) 3
2.3 Genetic factors based models of PD 3
3 Dopaminergic neuron 4
4 Microglia in Parkinsons disease 4
5 Astrocyte in Parkinsons disease 5
6 Therapeutic strategies for PD 5
6.1 Gene Therapy 6
6.2 Gene Therapy in dopaminergic neuron 6
7 Neurotrophic factors (NTFs) 7
7.1 GDNF 7
7.2 BDNF 8
7.3 CNTF 8
8 Mammalian target of rapamycin (mTOR) 9
8.1 Physiological characteristics of mTORC1 9
8.2 mTOR in human 10
8.3 Role of mTOR in CNS 10
8.4 Role of mTORC1 in neuron 11
8.5 Role of mTORC1 in astrocyte 11
8.6 mTOR in neurodegenerative disease 11
9 Transient Receptor Potential Vanilloid subtype 1 (TRPV1) 12
9.1 TRPV1 in human 12
9.2 Role of TRPV1 in CNS 12
9.3 TRPV1 in neuron 13
9.4 TRPV1 in glial cells 13
9.5 TRPV1 in neurodegenerative disease 13
II. Materials and Methods 15
1. Animals 15
2. Stereotaxic injection 15
3. Capsaicin and Rapamycin injection Animals 15
4. Adeno-Association viruses production 15
5. AAV injection 16
6. BDNF and GDNF Neutralizing antibody- injection 16
7. Lenti-virus injection 17
8. Tissue preparation and Immunohistochemistry 17
9. Double-immunofluorescence of rat 18
10. Western blot analysis 18
11. MTT assay 19
12. image J analysis 19
13. Amphetamine induced rotation test 19
14. Mesencephalon astrocyte culture 20
15. Stereological estimation . 20
16. Cell size analysis 20
17. Statistical analysis 21
III. Results 26
Aim of study for Part 1 27
1. AAV-GFP or hRheb(S16H) transduction on SNpc neuron in adult rat 29
2. AAV-hRheb(S16H) induced activation on mTORC1 signaling in the SN 34
3. Trophic effect of AAV-hRheb(S16H) in dopaminergic neuron in vivo 37
4. AAV-hRheb(S16H) stimulates the expression of GDNF and BDNF in dopaminergic neuron 40
5. Induction of GDNF and BDNF by activation of the hRheb(S16H)/mTORC 1signaling pathway in DA neurons in vivo 43
6. Induction of GDNF and BDNF by activation of the hRheb(S16H) / mTORC 1/ CREB signaling pathway in DA neurons in vivo 46
7. Neuroprotective effects of hRheb(S16H) in the unilateral MPP+ neurotoxin model of PD 49
8. AAV-hRheb(S16H)-induced GDNF and BDNF contribute to neuroprotection in the MPP+ model of PD 54

Aim of study for Part 2 62
1. TRPV1, p70S6K and CNTF expression in astrocyte of SN in vivo 63
2. Lenti virus-control-shRNA transduction on astrocyte in SNpc of rat brains in vivo 66
3. TRPV1 shRNA decrease p-70S6K and CNTF expression on mesencephalon astrocyte in MPP+ injected rat brain 69
4. Total-p70S6K shRNA decrease CNTF expression on mesencephalon astrocyte in MPP+ injected rat brain 76
5. Capsaicin rescues nigrostriatal DA neurons from MPP+ toxicity 81
6. Capsaicin enhances p-p70S6K expression in astrocyte in vivo 84
7. Capsaicin enhances CNTF expression in astrocyte in vivo 87
8. Capsaicin enhances mTOR downstream signal and CNTF in human and rat mesencephalon astrocyte in vitro 90
9. TRPV1-mTOR-CNTF pathway in astrocyte of MPP+ injected SNpc1 95
IV. Discussions 97
V. Summary 102
VI. Bibliography 104