行政院國家科學委員會專題研究計畫 成果報告
MPTP 傷害紋狀體黑質區徑路及網膜系統引發免疫發炎反 應之機制研究(3/3)
計畫類別： 個別型計畫
計畫編號： NSC91-2320-B-006-058-
執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立成功大學解剖學科(所)
計畫主持人： 陳淑姿
報告類型： 完整報告
報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢
中 華 民 國 92 年 5 月 12 日
行政院國家科學委員會專題研究計畫成果報告
MPTP傷害紋狀體黑質區徑路及視網膜系統引發免疫發炎反應之機制 研究
Studies on the inflammatory reaction in the nigrostriatal pathway and retinal system following MPTP intoxication (3/3)
計劃類別：個別型計劃
計劃編號：NSC 91-2320-B-006-058
執行期限：91年 8月 1日 至 92 年 7月 31日 主 持 人：陳 淑 姿 成功大學醫學院 解剖學科
一、中文摘要
巴金森氏症為常見之神經退化性疾病，主要病因是紋狀體-黑質區路徑中 多巴胺神經細胞病變造成。由於MPTP對多巴胺細胞之傷害有專一性，研究人員 常以此藥物注射實驗動物，做為探討巴金森氏症病因與治療之研究模式。已知 巴金森氏症患者眼球有病變現象，而實驗動物MPTP毒性可堆積並傷害視網膜細 胞，我們的實驗因此以藥物注射作體腔，比較MPTP對紋狀體黑質區徑路與網膜 多巴胺神經細胞(連絡網膜節細胞與光接受體)的影響，神經膠細胞群改變的情形 以及細胞素之表現。
實驗結果，藥物注射後之視網膜細胞群結構以Nissl染色無法看出有無明顯 改變，若是以免疫細胞化學法觀察則可發現，含多巴胺之神經細胞並無大量減 少，網膜神經膠細胞有增加現象，細胞素TNFá, IL-1â免疫反應性增加，證實網 膜細胞受到MPTP之毒害並引發免疫發炎作用。紋狀體黑質區徑路神經細胞大量 死亡，被活化之膠細胞也有增加現象，包含星狀及小膠細胞之反應性有明顯變 化，但是細胞素TNFá, IL-1â免疫反應性卻不明顯，網膜系統與黑質區/紋狀體路 徑間之多巴胺神經細胞對MPTP 之反應不同，雖然多巴胺細胞群結構及功能不 同為一重要因素，但是兩者之神經膠細胞反應與細胞生物素作用似乎決定神經
組織是否潰變死亡。
關鍵詞：巴金森氏症、細胞死亡、Bcl-2、神經膠細胞、細胞生物素、視網膜、
紋狀體-黑質區路徑
一、英文摘要
(I) To determine the roles of different members of the family of B cell lymphoma proto-oncogene (Bcl-2) in relation to neurotoxin-induced neuronal degeneration, the pattern of the expression of a number of molecules of the Bcl-2 family was studied immuno- cytochemically in the retinas of C57BL/6J mice after intraperitoneal injection of 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three days to 12 weeks following MPTP treatment, a detectable reduction of tyrosine hydroxylase immunoreactivity in the amacrine cells was observed with an increase of Bcl-2 expression in the Müller glial cells, and a
de novo expression of Bad and Bax in the
retinal ganglion cells, optic nerve fibers and plexiform layers. In contrast, a slight decrease of Bcl-xL immunoreactivity in the retinal ganglion cells was observed while Bcl-2 xS/L immunoreactivity was increased slightly in the retinas of MPTP-treated mice as compared with that of the controls. In animals that received MPTP injection, an increase in immunostaining of GFAP, glutamine synthase and Mac-1 (CD11b) in astrocytes, Müller cells and microglia was invariably observed, indicating an activation or dysfunction of retinal glial cells. These findings are consistent with the current view that glial dysfunction is important in mediating the cytotoxic effect of a variety of neurotoxic molecules including MPTP and that different members of Bcl-2 family may have different roles as far as neuronal degeneration or neuroprotection is concerned.(published on Neurochem. Res. 2003, 28(6): 805-814)
(II) 1-methyl-4-phenylpyridinium ion (MPP+), a toxic metabolite of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine, produces a selective destruction of nigrostriatal dopaminergic neurons and a similar movement disorder of Parkinson’s disease in human, primate, and rodent. However, the toxic effect of MPP+ to glia cells is unknown. In this study, we have demonstrated MPP+ (10-5~10-3 M) induced human U373 glioblastoma cell death in
a dose dependent manner at 48 h after treatment. The MPP+-induced apoptotic cell death was further confirmed by DAPI stain and MC540 stain as well as caspase 3 activation.
Co-treatment of melatonin, known as an antioxidant secreted by pineal gland, significantly prevents cell apoptosis and caspase 3 activation caused by MPP+ treatment.
Moreover, the time profile of H2O2 production determined by specific florescent probe of DCFH was biphasic that it transiently decreased at 6 h and followed by a significant increase at 36 h and 48 h. On the contrary, O2•- production determined by DEH florescent probe presented a persistent increase from a beginning at 24 h to 48 h after MPP+ treatment. MPP+ markedly lowered the level of glutathione (GSH) and elevated oxidative stress indicated by a high ratio of GSSG (oxidized glutathione)/GSH in a time dependent manner which started at 6 h. GSH depletion by diethyl maleate, but not by buthionine sulfoximine, caused glia cell death and enhanced the MPP+ toxicity. The MPP+-induced oxidative stress, including the free radical production and the destruction of GSH antioxidant defense system, were dramatically attenuated by melatonin co-treatment. Therefore, our results demonstrated that protective effect of melatonin for glia cells is attributed to effectively preventing free radical overproduction and GSH dysfunction induced by MPP+ treatment. (Poster presentation on
Apoptosis 2003 in
Luxembourg)Key words: neurotoxin, neuroprotection, Bcl-2, glutathione, cytokines, dopaminergic neuron, retina, nigrostriatal pathway, Parkinson’s disease
RESULTS
I. Structural analysis following MPTP i.p. injection
While only a small group of apoptotic cells was observed at the striatum and substantia nigra (SN) several hours to weeks postinjection, tyrosine hydrolase (TH)
immunoreactivity, a marker for dopaminergic neurons, showed a clear sign of severe degeneration in the SN 3 d to 2 mo after MPTP intoxication, as compared with that of the controls. In the retina with MPTP treatment, the number of TH-positive neurons in the inner nuclear layer was not change, although the staining intensity in the neurites was reduced.
II. Distribution of GFAP, Mac-1 and glial associated proteins
In the control animals faint to moderate immunoreactivity was observed in the striatal and nigral neurons, while GFAP, Mac-1 (CD11b), vimentin and GS, markers for astrocytes and microglia, was highly expressed in the nigrostriatal pathway. The cellular number and staining intensity were increased significantly 1 to 7 days later. The increase of activated glial cells may indicate an inflammatory reaction as revealed by the activity of E. Coli M4 and the altered vasopermeability in the injured brain. In the present study, several hours to days after MPTP administration, immunoreactivities of CD markers and certain cytokines appeared. For example, IL-1â was faintly expressed in the retina, however, glia-like processes became IL-1â positive postinjection. A colocalization of this cytokine and GFAP was also observed. However, although glial activation was obvious in the injured nigrostriatal pathway, very weak immunoreactivity for IL-1â, TNFá and other cytokines was found. The different reaction to MPTP intoxication in two systems may indicate that there are different regulatory gene products to protect neurons from toxic effect.
III. Bcl-2 family in the normal and injured retinas and nigrostriatal pathway
As previous studies, Bcl-2 was primarily distributed in the Müller glial processes in the normal retina. However, the staining intensity was decreased slightly 3h to 7 d postinjury. Bad, Bcl-x and Hrk immunoreactivities were mainly present in the retinal ganglion cells and nerve fiber layers of normal tissues, whereas the expression was downregulated by the MPP+ intoxication. Bax was expressed in the nerve fiber layer in
the normal and injured retinas. For A1 and Bcl-w, only faint immunoreactivity was found in both groups. Interestingly, Bid was expressed in the inner/outer segments of photoreceptors and outer limiting membrane, resembling the outer part of Müller glial processes. The pattern was not altered following MPP+ intravitreal injection.
IV. Glial response in the MPP
+-lesioned brain and retina
GFAP-immunoreactive cells (astrocytes) were evenly distributed in the nigrostriatal pathway of intact brain, however, a massive loss of GFAP-positive cells in the injected region was observed within a few days after lesion. On the other hand, OX-42-reactive cells were upregulated within 1 to 4 h after MPP+ administration and expressed throughout the ipsilateral striatum within a few days. The increase of OX-42 expression reduced 1 mo postinjury as previous described for Bcl-2 and APP. In the SN a few hours to days subsequent to MPP+ injection, the staining intensity increased and the ramified processes of OX-42-positive cells became much thicker, representing characteristic features of activated microglia. In the retinal system, the expression of OX-42 and GFAP was not changed postlesion. However, the staining intensity of GS, a marker for Müller cell body and processes, was decreased significantly several hours to days after intoxication.
CONCLUSIONS
1. This study indicates that intrastriatal injection of the neurotoxin has direct cytotoxic effect on neurons and glia, which are accompanied by the alteration of Bcl-2 and associated proteins in injured nigrostriatal pathway and retinal system.
2. Since Bcl-2 immunostaining within AD and PD brain tissues was shown to be increased with disease severity, their expressions may have a role in compensatory responses under PD/AD conditions, perhaps offering a neuroprotective mechanism to the remaining neurons.
3. The ultrastructure of lesioned nigrostriatal has also examined, indicating that the MPTP administration not only affects brain-blood-barrier induced but also induces neuronal cell death.
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