Developmental Action of Estrogen on Cognitive Substrates

描述(由申请人提供)：我们有令人信服的证据在野生型(+/+)和雌激素受体-α(ER-α)基因中断(-/-)(Erko)出生后一天-(P7)7小鼠新皮质和子宫，强烈表明存在一种新的，质膜相关的推测ER，既不是ER-α(67 KDa)，也不是ER-β(54-kDa)。拟议的研究旨在进一步描述这种假定的ER，我将其命名为“ER-X”。ER-X是一种约62-63 kDa的蛋白质，它：(A)在P7的纯化的空泡样微域中表达和丰富，但不在成人、新皮质和子宫质膜中表达；(B)以高亲和力结合3H-雌二醇，但具有与ER-α和ER-β完全不同的配体特异性；(C)与ER-α的配体结合区域显示出一些同源性；(D)似乎介导了丝裂原激活的蛋白激酶(MAPK)级联的雌激素激活；(E)受发育调节；以及(F)在缺血性卒中周围的成人新皮质组织中上调。我们将在一系列互补和相关的细胞生物学、生化和分子生物学实验中使用标准方法，利用完整组织、器官培养和质膜制剂开发野生型和Erko新皮质。这些方法将包括(I)竞争置换分析，(Ii)免疫沉淀和Western blotting；(Iii)原位杂交；(Iv)免疫组织化学；(V)cDNA微阵列分析。这些实验旨在确定(1)ER-X是多配体受体、G蛋白偶联受体还是受体酪氨酸激酶；以及(2)表征、量化和比较ER-X暴露于17α和17β-雌二醇所调控的基因。这些研究对中枢神经系统发育和认知障碍有着深远的影响。这一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这一发现，即雌激素对更高级别的认知过程产生影响，雌激素或雌激素缺乏可能是导致认知的性二态障碍、精神分裂症、特纳和唐氏综合症以及与艾兹海默氏症和帕金森氏症相关的认知缺陷的风险因素。了解与“ER-X”相关的发育机制对于认知障碍的治疗方法具有相当大的临床意义，为大脑中选择“ER-X”的药物开发和治疗干预开辟了道路。

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.