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

다국어 초록 (Multilingual Abstract)

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 (Table of Contents)

• 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