胶质细胞多巴胺反应基因克隆及其特征分析
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摘要
多巴胺（dopamine，DA，化学名3，4-羟基苯丙胺）是中枢神经系统主
要的儿茶酚胺类神经递质，多巴胺参与控制机体运动、行为、情绪、摄食
及内分泌调节等。在中枢神经系统所有的慢作用递质中，DA是长期以来
受到最多关注的神经递质，主要原因是其与神经系统重大医学问题联系最
为紧密。临床上常见疾病如帕金森氏病（Parkinson's disease，PD）是中脑黑
质DA能神经元的选择性退变所致，亨廷顿氏舞蹈病（Huntington's chorea）
是由于纹状体内DA投射神经元的损害，目前所有的精神分裂症
（schizophrenia）治疗药物在本质上主要是以DA受体的拮抗剂起作用，注
意力缺陷/过敏障碍（attention deficit hyperactivity disorder，ADHD）的症状
可用DA能神经调节药物得到极大缓解。药物可卡因、苯丙胺、阿片与尼
古丁、酒精等通过调节DA神经传递而获得成瘾性。人们已越来越清楚地
认识到，DA在人类身心健康中具有非常重要的生理作用。DA的研究也
成为了当今神经科学中的前沿研究领域，为揭示人类思维和精神活动规律
及其有关疾病的奥秘提供了可能的途径。
尽管人们对DA的研究有广泛的兴趣，并且也取得了辉煌的成就，但
目前对DA发生作用的细胞和分子机制仍不清楚。随着近年来细胞或分子
水平研究的广泛开展，发现脑内除神经元以外胶质细胞也是DA作用的重
要靶细胞。事实上，脑组织主要由神经元和胶质细胞共同构成，数量上胶
质细胞与神经元比是10～50：1，胶质细胞占脑体积一半以上，已经表明
胶质细胞在神经系统具有非常重要的功能。最早的实验发现脑组织胶质细
胞的提取物有腺甘酸环化酶相关的DA受体，接着，发现DA可刺激皮质
和纹状体，非脑干来源的培养星形胶质细胞CAMP积聚。进一步应用电
生理和放射性配体结合自显影研究提示胶质细胞具有DA受体。我们应用
pH］DA放射性受体分析法（r。山。reCeptofN11山ng SSS。y，R RARA）研究发现
三个不同部位来源的星形胶质细胞具有5个不同的解离常数，表明胶质细
胞分布广泛而且具有部位异质特性。近期的研究利用分子生物学技术如
PCR和原位杂交，证实培养纹状体胶质细胞表达DI和DZ受体。另外，
胶质细胞具有类似神经元的DA摄取系统。由此可见，胶质细胞是脑内
DA作用的重要靶细胞，并且胶质细胞可能在DA神经系统疾病中具有非
常重要的作用。本研究利用细胞与分子生物学的多种技术方法，克隆与鉴
定系列胶质细胞DA的相关基因，并对部分新基因功能进行初步的特性分
析。一方面，从理论上有利于分析DA作用于星形胶质细胞的分子机制，
了解胶质细胞在＊A神经系统中的生理功能：另一方面，可能获得＊A系
统疾病的相关基因，对揭示帕金森氏病，精神分裂症和药物成瘸等疾病的
病因，提高临床诊断以及治疗水平有重要的应用价值。
本研究首先从大鼠脑组织分离培养星形胶质细胞，以多巴胺DA处理
后提取 mMx，然后结合应用抑制消减杂交侣u以ession subtractlve
hybridization，SSH）和选择性差异筛选（PCR·Select Differential Screening）
方法，分离获得27个DA调节表达cDNA克隆。经Northern杂交验证，
序列测定与生物信息学分析，明确了 14个 DA调节表达的基因，包括神
经递质转运相关的多功能调节蛋白泛素蛋白连接酶Nedd4，调节细胞分泌
活动的内质网钙联结蛋白，能量代谢相关的葡萄糖调节蛋白，DA代谢相
关酶NAD（P）H－酮还原酶和NADH－泛酮氧化还原酶，以及铁蛋白H亚单
位等。实验结果提示＊pA激活了胶质细胞内复杂的信号传递通路，并且
涉及生长因子信号途径、凿体激素信号途径和白介素调节通路之间的相互
交流（cros叶alking卜（）信号传递的靶基因有四类：代谢酶，应激蛋白，
转运蛋白，以及生长发育调节蛋白等。O）其中几个基因变异与神经疾病
密切相关，也提示多巴胺能神经疾病与非多巴胺能神经疾病之间也有某些
共同的机制。
Vlll
人类基因的发现及功能研究，尤其重要疾病的相关基因研究是目前世
界范围的重大课题。多巴胺＊opamine，DA）是中枢神经系统重要的神经递
质，与神经系统重大医学问题联系最为紧密。胶质细胞DA相关基因的克
隆对揭示胶质细胞在DA系统中的重要作用，以及对揭示帕金森病，精神
分裂症和药物成噶等DA系统疾病的病因，提高临床诊断以及治疗水平有
重要的应用价值。本研究采用同源克隆策略川 CIOllillg），采用
DA处理的大鼠星形胶质细胞的消减PCR探针，筛选人类胎脑cDNA噬
菌体文库。通过高质量全长cDNA噬菌体文库的构建，经过严格的杂交
的筛选，挑选了96个阳性克隆，经过测序分析明确了61条DA相关的全
长基因，并且筛选到数个全长的新基因。通过文献查阅与生物信息学分
析，比较全面地了解了DA激活星形胶质细胞的信号传递机制及其相关功
能意义，并且新基因的克隆为进一步的基因功能研究及其开发应用研究奠
定了重要的基础。
在我们筛选到的数条新的人类全长cDNA基因中，依据生物信息学
分析我们选择两
Molecular Cloning and Characterization of Dopamine
Responsive Genes From Astrocytes
Abstract
Dopamine (3,4-dihydroxyphenethylamine; DA) is the predominant catecholamine neurotransmjtter in the mammalian central nervous system. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. Of many slow-acting neurotransmitters, DA has received by far the most attention, mainly because that several pathological conditions have been linked to dysregulation of dopaminergic transmission. It has been found by a number of investigators that glial cells are targets of DA. However, few studies have reported on the molecular cascade associated with receptor activation and DA metabolism in glial cells during dopaminergic neuronal activity.In this study, a series of cellular and molecular biological technichies were applied to isolate and identify the DA responsive genes from glial cells.Analysis of DA responsive genes should provides a better understanding of the functional impact of glial cells in dopaminergic transmission, and the complex cellular and molecular mechanism of the disorder of dopaminergic transmission including Parkinson’s disease, schizophrenia, and drug addiction etc.
In the present study, we have investigated the influence of DA on gene transcription in primary glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes primarily cultured from rat cerebral tissues in either standard media or treated with DA.PCR-select differential screening was used to further verify
the differentially expressed cDNA clones, and positive clones' were
sequenced and the mRNAs were re-examined on Northern blots. Fourteen
sequences were identified, among which l1 were homologous to known
genes, 3 were homologous to expressed sequence tags (ESTs). The analysis
of all these identified sequences suggested that comp1ex intracellu1ar
signa1ing, involving cross talks with growth factor pathway, steroid
hormone pathway, and/or interferon-regulated 2-5A pathway, is induced by
DA in astrocytes. The target genes of the signaling pathway were fOund to
fall into fOur groups, including metabolic enzymes, stress proteins, transfer
proteins, and growth regulation proteins. In addition, several genes have
been established their relationship with specific neurodegenerative diseases,
showing that there is an over1ap in pathogenic mechanisms of those
diseases.
Molecular cloning and functional analysis of human genes especialy
those human disease genes are important biological tasks at present. DA is
the predominant catecholamine neurotransmitter in the mammalian central
nervous system, and several pathological conditions have been linked to
dysregulation of dopaminergic transmission. In the present study, we have
isolated a series of full-length cDNAs of DA responsive genes from huamn
brain using a homologous cloning strategy.A high-quality cDNA x phage
library with good representation of full-length cDNAs was constructed by
Smart technique.Two runs of hybridization with SSH-PCR products of DA
resonsive cDNA fragmnets (see Part one) as probe were performed, and 96
positive clones were selected and sequenced, 6l candidate DA responsive
genes were isolated,of which 4 new full-length cDNAs were found and
submitted to GeneBank.Analysis of these genes suggested that the complex
molecular cascade associated with receptor activation and DA metabolism
in glial cells during dopaminergic neuronal activity and the complex
IV
cellular and molecular mechanism of the disorder of dopaminergic
transmission.Some nove1 genes isolated laid a fOundation fOr further
functional research and development of theraPy for dopaminergic diseases.
Two important novel genes, a putative GTPase DRP and a LIM
domain transcription factor DATl, were selected fOr further studies based
on bioinfOrmatic ana1ysis. In this study, the rat full-length DRP a
要的儿茶酚胺类神经递质，多巴胺参与控制机体运动、行为、情绪、摄食
及内分泌调节等。在中枢神经系统所有的慢作用递质中，DA是长期以来
受到最多关注的神经递质，主要原因是其与神经系统重大医学问题联系最
为紧密。临床上常见疾病如帕金森氏病（Parkinson's disease，PD）是中脑黑
质DA能神经元的选择性退变所致，亨廷顿氏舞蹈病（Huntington's chorea）
是由于纹状体内DA投射神经元的损害，目前所有的精神分裂症
（schizophrenia）治疗药物在本质上主要是以DA受体的拮抗剂起作用，注
意力缺陷/过敏障碍（attention deficit hyperactivity disorder，ADHD）的症状
可用DA能神经调节药物得到极大缓解。药物可卡因、苯丙胺、阿片与尼
古丁、酒精等通过调节DA神经传递而获得成瘾性。人们已越来越清楚地
认识到，DA在人类身心健康中具有非常重要的生理作用。DA的研究也
成为了当今神经科学中的前沿研究领域，为揭示人类思维和精神活动规律
及其有关疾病的奥秘提供了可能的途径。
尽管人们对DA的研究有广泛的兴趣，并且也取得了辉煌的成就，但
目前对DA发生作用的细胞和分子机制仍不清楚。随着近年来细胞或分子
水平研究的广泛开展，发现脑内除神经元以外胶质细胞也是DA作用的重
要靶细胞。事实上，脑组织主要由神经元和胶质细胞共同构成，数量上胶
质细胞与神经元比是10～50：1，胶质细胞占脑体积一半以上，已经表明
胶质细胞在神经系统具有非常重要的功能。最早的实验发现脑组织胶质细
胞的提取物有腺甘酸环化酶相关的DA受体，接着，发现DA可刺激皮质
和纹状体，非脑干来源的培养星形胶质细胞CAMP积聚。进一步应用电
生理和放射性配体结合自显影研究提示胶质细胞具有DA受体。我们应用
pH］DA放射性受体分析法（r。山。reCeptofN11山ng SSS。y，R RARA）研究发现
三个不同部位来源的星形胶质细胞具有5个不同的解离常数，表明胶质细
胞分布广泛而且具有部位异质特性。近期的研究利用分子生物学技术如
PCR和原位杂交，证实培养纹状体胶质细胞表达DI和DZ受体。另外，
胶质细胞具有类似神经元的DA摄取系统。由此可见，胶质细胞是脑内
DA作用的重要靶细胞，并且胶质细胞可能在DA神经系统疾病中具有非
常重要的作用。本研究利用细胞与分子生物学的多种技术方法，克隆与鉴
定系列胶质细胞DA的相关基因，并对部分新基因功能进行初步的特性分
析。一方面，从理论上有利于分析DA作用于星形胶质细胞的分子机制，
了解胶质细胞在＊A神经系统中的生理功能：另一方面，可能获得＊A系
统疾病的相关基因，对揭示帕金森氏病，精神分裂症和药物成瘸等疾病的
病因，提高临床诊断以及治疗水平有重要的应用价值。
本研究首先从大鼠脑组织分离培养星形胶质细胞，以多巴胺DA处理
后提取 mMx，然后结合应用抑制消减杂交侣u以ession subtractlve
hybridization，SSH）和选择性差异筛选（PCR·Select Differential Screening）
方法，分离获得27个DA调节表达cDNA克隆。经Northern杂交验证，
序列测定与生物信息学分析，明确了 14个 DA调节表达的基因，包括神
经递质转运相关的多功能调节蛋白泛素蛋白连接酶Nedd4，调节细胞分泌
活动的内质网钙联结蛋白，能量代谢相关的葡萄糖调节蛋白，DA代谢相
关酶NAD（P）H－酮还原酶和NADH－泛酮氧化还原酶，以及铁蛋白H亚单
位等。实验结果提示＊pA激活了胶质细胞内复杂的信号传递通路，并且
涉及生长因子信号途径、凿体激素信号途径和白介素调节通路之间的相互
交流（cros叶alking卜（）信号传递的靶基因有四类：代谢酶，应激蛋白，
转运蛋白，以及生长发育调节蛋白等。O）其中几个基因变异与神经疾病
密切相关，也提示多巴胺能神经疾病与非多巴胺能神经疾病之间也有某些
共同的机制。
Vlll
人类基因的发现及功能研究，尤其重要疾病的相关基因研究是目前世
界范围的重大课题。多巴胺＊opamine，DA）是中枢神经系统重要的神经递
质，与神经系统重大医学问题联系最为紧密。胶质细胞DA相关基因的克
隆对揭示胶质细胞在DA系统中的重要作用，以及对揭示帕金森病，精神
分裂症和药物成噶等DA系统疾病的病因，提高临床诊断以及治疗水平有
重要的应用价值。本研究采用同源克隆策略川 CIOllillg），采用
DA处理的大鼠星形胶质细胞的消减PCR探针，筛选人类胎脑cDNA噬
菌体文库。通过高质量全长cDNA噬菌体文库的构建，经过严格的杂交
的筛选，挑选了96个阳性克隆，经过测序分析明确了61条DA相关的全
长基因，并且筛选到数个全长的新基因。通过文献查阅与生物信息学分
析，比较全面地了解了DA激活星形胶质细胞的信号传递机制及其相关功
能意义，并且新基因的克隆为进一步的基因功能研究及其开发应用研究奠
定了重要的基础。
在我们筛选到的数条新的人类全长cDNA基因中，依据生物信息学
分析我们选择两
Molecular Cloning and Characterization of Dopamine
Responsive Genes From Astrocytes
Abstract
Dopamine (3,4-dihydroxyphenethylamine; DA) is the predominant catecholamine neurotransmjtter in the mammalian central nervous system. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. Of many slow-acting neurotransmitters, DA has received by far the most attention, mainly because that several pathological conditions have been linked to dysregulation of dopaminergic transmission. It has been found by a number of investigators that glial cells are targets of DA. However, few studies have reported on the molecular cascade associated with receptor activation and DA metabolism in glial cells during dopaminergic neuronal activity.In this study, a series of cellular and molecular biological technichies were applied to isolate and identify the DA responsive genes from glial cells.Analysis of DA responsive genes should provides a better understanding of the functional impact of glial cells in dopaminergic transmission, and the complex cellular and molecular mechanism of the disorder of dopaminergic transmission including Parkinson’s disease, schizophrenia, and drug addiction etc.
In the present study, we have investigated the influence of DA on gene transcription in primary glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes primarily cultured from rat cerebral tissues in either standard media or treated with DA.PCR-select differential screening was used to further verify
the differentially expressed cDNA clones, and positive clones' were
sequenced and the mRNAs were re-examined on Northern blots. Fourteen
sequences were identified, among which l1 were homologous to known
genes, 3 were homologous to expressed sequence tags (ESTs). The analysis
of all these identified sequences suggested that comp1ex intracellu1ar
signa1ing, involving cross talks with growth factor pathway, steroid
hormone pathway, and/or interferon-regulated 2-5A pathway, is induced by
DA in astrocytes. The target genes of the signaling pathway were fOund to
fall into fOur groups, including metabolic enzymes, stress proteins, transfer
proteins, and growth regulation proteins. In addition, several genes have
been established their relationship with specific neurodegenerative diseases,
showing that there is an over1ap in pathogenic mechanisms of those
diseases.
Molecular cloning and functional analysis of human genes especialy
those human disease genes are important biological tasks at present. DA is
the predominant catecholamine neurotransmitter in the mammalian central
nervous system, and several pathological conditions have been linked to
dysregulation of dopaminergic transmission. In the present study, we have
isolated a series of full-length cDNAs of DA responsive genes from huamn
brain using a homologous cloning strategy.A high-quality cDNA x phage
library with good representation of full-length cDNAs was constructed by
Smart technique.Two runs of hybridization with SSH-PCR products of DA
resonsive cDNA fragmnets (see Part one) as probe were performed, and 96
positive clones were selected and sequenced, 6l candidate DA responsive
genes were isolated,of which 4 new full-length cDNAs were found and
submitted to GeneBank.Analysis of these genes suggested that the complex
molecular cascade associated with receptor activation and DA metabolism
in glial cells during dopaminergic neuronal activity and the complex
IV
cellular and molecular mechanism of the disorder of dopaminergic
transmission.Some nove1 genes isolated laid a fOundation fOr further
functional research and development of theraPy for dopaminergic diseases.
Two important novel genes, a putative GTPase DRP and a LIM
domain transcription factor DATl, were selected fOr further studies based
on bioinfOrmatic ana1ysis. In this study, the rat full-length DRP a
引文
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19． Papa S, Sardanelli AM, Scacco S, Technikova-Dobrova Z. cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction. FEBS Lett 444 (1999) 245-9．
20． Bergeron JJ, Brenner MB, Thomas DY, and Williams DB.Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci 19 (1994) 124-128．
21． Massa SM, Longo FM, Zuo J, Wang S, Chen J, Sharp FR.Cloning of rat grp75, an hsp70-family member, and its expression in normal and ischemic brain. J Neurosci Res 40 (1995) 807-819．
22． Plant PJ, Yeder H, Staub 0, ET al. The C2 domain of the ubiquitin protein ligase nedd4 mediates Ca2+-dependent plasma membrane localization. J Biol Chem 272(1997) 32329-32336．
23． Lowenstein EJ, Daly RJ, Batzer AG. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70(1992) 431-42．
24． Roderick HL, Lechleiter JD, Camacho P. Cytosolic phosphorylation of calnexin controls intracellular Ca (2+) oscillations via an interaction with SERCA2b. J Cell Biol 149 (2000) 1235-48．
25． Verkhratsky A, and Kettenmann H. Calcium signalling in glial cells. Trends Neurosci. 1996; 19: 346-352
26． Lopez-Ilasaca M. Signaling from G-protein-coupled receptors-to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol 1998; 56(3) : 269-77
27． Luo Y, Kokkonen GC, Hattori A, Chrest FJ, Roth GS. Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2L cells. Brain Res 850(1999) 21-38．
28． Shah GN, Li J, Schneiderjohn P, Mooradian AD. Cloning and characterization of a complementary DNA for a thyroid hormone-responsive protein in mature rat cerebral tissue. Biochem J 327 (1997) 617-623．
29． Gomes FC, Maia CG, de Menezes JR, Neto VM. Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation. Glia 25 (1999) 247-255．
30． Bahouth SW. Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein alpha-subunits. Biochem J 307 (1995) 831-841．
31． Bisbal, C, Martinand, C, Silhol, M, Lebleu B, Salehzada T. Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway. J. Biol Chem 270 (1995) 13308-13317．
32． Frohman EM, van den Noort S, Gupta S. Astrocytes and intracerebral immune responses. J Clin Immunol 9 (1989) 1-9．
33． Smythies J, Galzigna L.The oxidative metabolism of cateholamines in the brain: a review. Biochimica Biophsica Acta 1380 (1998) 159-162．
34． Stokes AH, Hastings TG, Vrana KE.Cytotoxic and genotoxic potential of dopamine. J Neurosci Res 55 (1999) 659-665．
35． Bindoli A, Rigobello MP, Galzigna L. Reduction of adrenochrome by rat liver and brain DT-diaphorase. Free Radic. Rec. Comms. 8 (1990) 295-298．
36． Segura-Aguilar J. Peroxidase activity of liver microsomal vitamin D 25-hydroxylase and cytochrome P450 1A2 catalyzes 25-hydroxylation of vitamin D3 and oxidation of dopamine to aminochrome. Biochem Mol Med. 58(1996) 122-129．
37． Damier P, Hirsch EC, Zhang P, et al. Glutathione peroxidase, glial cells and Parkinson's disease. Neuroscience 52 (1993) 1-6．
38． Anan T, Nagata Y, Koga H, et al: Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes. Genes Cells 3 (1998) 751-763．
39． Murray MT, White K, and Munro HN.Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression. Proc Natl Acad Sci USA 84 (1987) 7438-7442．
40． Malecki EA, Devenyi AG, Beard JL, Connor JR. Existing and emerging mechanisms for transport of iron and manganese to the brain. J Neurosci Res 56 (1999) 113-122．
41． Satyal SH, Chen D, Fox SG, Kramer JM, Morimoto RI Negative regulation of the heat shock transcriptional response by HSBP1． Genes Dev 12 (1998) 1962-1974．
42． Lundgren SE, Callahan CA, Thor S, Thomas JB. Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development 121(1995) 1769-1773．
43． Shawlot W, Behringer RR. Requirement for Liml in head-organizer function. Nature 374(1995) 425-430．
44． Drukarch B, van Muiswinkel FL. Drug treatment of Parkinson's disease. Time for phase II. Biochem Pharmacol 59 (2000) 1023-1031．
45． Gerlach M, Ben-Shachar D, Riederer P, Youdim MB. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J Neurochem 63 (1994) 793-807．
46． Lott IT. Down's syndrome, aging, and Alzheimer's disease: a clinical review. Ann N Y Acad Sci 396 (1982) 15-27．
47． Schapira AH. Human complex I defects in neurodegenerative diseases. Biochim Biophys Acta 1364 (1998) 261-270．
48． Schapira AH. Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia. Biochim Biophys Acta 1410 (1999) 159-70．
49． Whatley SA, Curti D, Das Gupta F, Ferrier IN, Jones S, Taylor C, Marchbanks RM. Superoxide, neuroleptics and the uniquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients. Mol Psychiatry 3 (1998) 227-237．
50． McShea A, Zelasko DA, Gerst JL, Smith MA. Signal transduction abnormalities in Alzheimer's disease: evidence of a pathogenic stimuli. Brain Res 815 (1999) 237-42．
2． Greengard P, Allen PB, and Nairn AC.Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron 23 (1999) 435-447．
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14． Inazu M, Kubota N, Takeda H, Zhang J, Kiuchi Y, Oguchi K, Matsumiya T. Pharmacological characterization of dopamine transport in cultured rat astrocytes. Life Sci 64 (1999) 2239-45．
15． Diatchenko L, Lau YC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Gurskaya N, Sverdlov ED and Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93 (1996) 6025-6030．
16． Stein V, Thies OD, Hofmann M.A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acid Res. 25 (1997) 2598-2602．
17． McCarthy KD, De Vellis J (1980) Prepartion of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890-902
18． Reddy PH, Stockburger E, Gillevet P, Tagle DA. Mapping and characterization of novel (CAG) n repeat cDNAs from adult human brain derived by the oligo capture method. Genomics 46(1997) 174-82．
19． Papa S, Sardanelli AM, Scacco S, Technikova-Dobrova Z. cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction. FEBS Lett 444 (1999) 245-9．
20． Bergeron JJ, Brenner MB, Thomas DY, and Williams DB.Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem Sci 19 (1994) 124-128．
21． Massa SM, Longo FM, Zuo J, Wang S, Chen J, Sharp FR.Cloning of rat grp75, an hsp70-family member, and its expression in normal and ischemic brain. J Neurosci Res 40 (1995) 807-819．
22． Plant PJ, Yeder H, Staub 0, ET al. The C2 domain of the ubiquitin protein ligase nedd4 mediates Ca2+-dependent plasma membrane localization. J Biol Chem 272(1997) 32329-32336．
23． Lowenstein EJ, Daly RJ, Batzer AG. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70(1992) 431-42．
24． Roderick HL, Lechleiter JD, Camacho P. Cytosolic phosphorylation of calnexin controls intracellular Ca (2+) oscillations via an interaction with SERCA2b. J Cell Biol 149 (2000) 1235-48．
25． Verkhratsky A, and Kettenmann H. Calcium signalling in glial cells. Trends Neurosci. 1996; 19: 346-352
26． Lopez-Ilasaca M. Signaling from G-protein-coupled receptors-to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol 1998; 56(3) : 269-77
27． Luo Y, Kokkonen GC, Hattori A, Chrest FJ, Roth GS. Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2L cells. Brain Res 850(1999) 21-38．
28． Shah GN, Li J, Schneiderjohn P, Mooradian AD. Cloning and characterization of a complementary DNA for a thyroid hormone-responsive protein in mature rat cerebral tissue. Biochem J 327 (1997) 617-623．
29． Gomes FC, Maia CG, de Menezes JR, Neto VM. Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation. Glia 25 (1999) 247-255．
30． Bahouth SW. Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein alpha-subunits. Biochem J 307 (1995) 831-841．
31． Bisbal, C, Martinand, C, Silhol, M, Lebleu B, Salehzada T. Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway. J. Biol Chem 270 (1995) 13308-13317．
32． Frohman EM, van den Noort S, Gupta S. Astrocytes and intracerebral immune responses. J Clin Immunol 9 (1989) 1-9．
33． Smythies J, Galzigna L.The oxidative metabolism of cateholamines in the brain: a review. Biochimica Biophsica Acta 1380 (1998) 159-162．
34． Stokes AH, Hastings TG, Vrana KE.Cytotoxic and genotoxic potential of dopamine. J Neurosci Res 55 (1999) 659-665．
35． Bindoli A, Rigobello MP, Galzigna L. Reduction of adrenochrome by rat liver and brain DT-diaphorase. Free Radic. Rec. Comms. 8 (1990) 295-298．
36． Segura-Aguilar J. Peroxidase activity of liver microsomal vitamin D 25-hydroxylase and cytochrome P450 1A2 catalyzes 25-hydroxylation of vitamin D3 and oxidation of dopamine to aminochrome. Biochem Mol Med. 58(1996) 122-129．
37． Damier P, Hirsch EC, Zhang P, et al. Glutathione peroxidase, glial cells and Parkinson's disease. Neuroscience 52 (1993) 1-6．
38． Anan T, Nagata Y, Koga H, et al: Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes. Genes Cells 3 (1998) 751-763．
39． Murray MT, White K, and Munro HN.Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression. Proc Natl Acad Sci USA 84 (1987) 7438-7442．
40． Malecki EA, Devenyi AG, Beard JL, Connor JR. Existing and emerging mechanisms for transport of iron and manganese to the brain. J Neurosci Res 56 (1999) 113-122．
41． Satyal SH, Chen D, Fox SG, Kramer JM, Morimoto RI Negative regulation of the heat shock transcriptional response by HSBP1． Genes Dev 12 (1998) 1962-1974．
42． Lundgren SE, Callahan CA, Thor S, Thomas JB. Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development 121(1995) 1769-1773．
43． Shawlot W, Behringer RR. Requirement for Liml in head-organizer function. Nature 374(1995) 425-430．
44． Drukarch B, van Muiswinkel FL. Drug treatment of Parkinson's disease. Time for phase II. Biochem Pharmacol 59 (2000) 1023-1031．
45． Gerlach M, Ben-Shachar D, Riederer P, Youdim MB. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J Neurochem 63 (1994) 793-807．
46． Lott IT. Down's syndrome, aging, and Alzheimer's disease: a clinical review. Ann N Y Acad Sci 396 (1982) 15-27．
47． Schapira AH. Human complex I defects in neurodegenerative diseases. Biochim Biophys Acta 1364 (1998) 261-270．
48． Schapira AH. Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia. Biochim Biophys Acta 1410 (1999) 159-70．
49． Whatley SA, Curti D, Das Gupta F, Ferrier IN, Jones S, Taylor C, Marchbanks RM. Superoxide, neuroleptics and the uniquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients. Mol Psychiatry 3 (1998) 227-237．
50． McShea A, Zelasko DA, Gerst JL, Smith MA. Signal transduction abnormalities in Alzheimer's disease: evidence of a pathogenic stimuli. Brain Res 815 (1999) 237-42．