Developmental Action of Estrogen on Cognitive Substrates

描述（由申请人提供）：我们在出生后第7天（P7）7天的野生型（+/+）和雌激素受体-α（ER-α）基因破坏（-/-）（ERKO）小鼠新皮质和子宫中均获得了令人信服的证据，有力地表明存在一种新型的质膜相关的推定ER，既不是ER-α（67 kDa），也不是ER-β（54-64 kDa）。所提出的研究旨在进一步表征这种推定的ER，我将其命名为“ER-X”。“ER-X”是一种约62-63 kDa的蛋白质，其：（a）在P7的纯化的小窝样微结构域中表达和富集，但在成人、新皮层和子宫质膜中不表达和富集;（B）以高亲和力结合3 H-雌二醇，但配体特异性与ER-α和ER-β完全不同;（c）与ER-α的配体结合结构域表现出一定的同源性;（d）似乎介导促分裂原活化蛋白激酶（MAPK）级联的雌激素活化;（e）是发育调节的;和（f）在缺血性中风周围的成人新皮质组织中上调。我们将使用标准的方法在一系列的补充和相关的细胞生物学，生物化学和分子生物学实验中开发野生型和ERKO新皮层，使用完整的组织;器官型培养;和质膜制备。这些方法将包括（i）通过与17 β雌二醇比较的竞争性置换试验，（ii）免疫沉淀和Western印迹;（iii）原位杂交;（iv）免疫组织化学;和（v）cDNA微阵列分析。这些实验旨在确定（1）“ER-X”是否是多配体受体、G蛋白偶联受体或受体酪氨酸激酶;和（2）表征、定量和比较暴露于17 α-和17 β-雌二醇的ER-X调节的基因。这些研究对中枢神经系统发育和认知障碍具有深远的意义。这些结果将为雌激素的发育作用和神经保护作用提供新的信息。 了解雌激素如何影响发育可能有助于解释雌激素对高级认知过程产生影响的发现，雌激素或其缺乏可能是认知性二态性障碍、精神分裂症、特纳综合征和唐氏综合征以及与阿尔茨海默病和帕金森病相关的认知缺陷的危险因素。理解涉及“ER-X”的发育机制对于认知缺陷的治疗方法具有相当大的临床意义，从而为大脑中“ER-X”的药物开发和治疗干预选择性开辟道路。

DESCRIPTION (provided by applicant): We have compelling evidence in both wild-type (+/+) and estrogen receptor-alpha (ER-alpha) gene-disrupted (-/-) (ERKO) postnatal day- (P7) 7 mouse neocortex and uterus that points strongly to the existence of a novel, plasma-membrane-associated, putative ER that is neither ER-alpha (67 kDa) nor ER-beta (54-64 kDa). The proposed studies are designed to further characterize this putative ER, which I have designated "ER-X." "ER-X" is an about 62-63 kDa protein that: (a) is expressed and enriched in purified caveolar-like microdomains of P7, but not adult, neocortical and uterine plasma-membranes; (b) binds 3H-estradiol with high affinity but with ligand specificities radically different from ER-alpha and ER-beta; (c) exhibits some homology with the ligand binding domain of ER-alpha; (d) appears to mediate estrogen activation of the Mitogen-Activated Protein Kinase (MAPK) cascade; (e) is developmentally regulated; and (f) is up-regulated in adult neocortical tissue surrounding an ischemic stroke. We will use standard methods in a series of complementary and correlative cell biological, biochemical, and molecular biological experiments in developing wild-type and ERKO neocortex, using intact tissue; organotypic cultures; and plasma membrane preparations. These methods will include (i) competitive displacement assays, by comparison with 17betaestradiol, (ii) immunoprecipitation and Western blotting; (iii) in situ hybridization; (iv) immunohistochemistry; and (v) cDNA microarray analysis. The experiments are designed to determine (1) Whether "ER-X" is a multiligand receptor; a G-protein coupled receptor or a receptor tyrosine kinase; and (2) To characterize, quantify and compare the genes regulated by ER-X exposure to 17alpha- and 17beta-estradiol. These studies have profound implications for CNS development and cognitive disorders. The results will provide new information regarding the developmental actions and neuroprotective effects of estrogen. Understanding how estrogen influences development may help explain findings that estrogen exerts effects on higher order cognitive processes, and that estrogen or its deficiency may be risk factors for the development of the sexually dimorphic disorders of cognition; schizophrenia; Turner's and Down's Syndromes; and cognitive deficits associated with AIzheimer's and Parkinson's diseases. Understanding developmental mechanisms involving "ER-X" have considerable clinical significance for therapeutic approaches to cognitive deficits, leading the way to drug development and therapeutic intervention selective for "ER-X" in the brain.