Molecular Regulation of TIMP-2 in Neuron Cell Cycle

TIMP-2在神经元细胞周期中的分子调控

• 批准号: 7090036
• 负责人: DIANE M JAWORSKI
• 金额: $ 34.32万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090036
• 关键词: axon; bromodeoxyuridine; cell cycle; cell differentiation; dendrites; extracellular matrix; genetic regulation; genetically modified animals; integrins; laboratory mouse; metalloendopeptidases; nerve stem cell; neuronal guidance; neurons; newborn animals; radioassay; tissue /cell culture; tissue inhibitor of metalloproteinases; axon; bromodeoxyuridine; cell cycle; cell differentiation; dendrites; extracellular matrix; genetic regulation; genetically modified animals; integrins; laboratory mouse; metalloendopeptidases; nerve stem cell; neuronal guidance; neurons; newborn animals; radioassay; tissue /cell culture; tissue inhibitor of metalloproteinases

DESCRIPTION (provided by applicant): Information about the developmental regulation of the extracellular matrix in the CNS is incomplete and the function of proteins such as the tissue inhibitor of matrix metalloproteinases (TIMPS) is poorly understood. We recently characterized the expression of the four known TIMPs during CNS development in vivo. The expression of TIMP-2 by post-mitotic neurons correlates with neuronal differentiation. We demonstrate herein that TIMP-2 induces cell cycle arrest and neuronal differentiation in a cell autonomous manner. Strikingly, TIMP-2 's effect was independent of its MMP-inhibitory activity. The punctate labeling of TIMP-2 on the cell surface combined with the interaction of TIMP-2 with alpha3 beta1 integrin suggests that TIMP-2 may exert its MMP-independent activities via integrins. The persistence of nestin-positive progenitors in the neocortical ventricular zone and the reduced neurite length of Timp-2 -null neurons suggest that neuronal differentiation is delayed in the absence of TIMP-2. This is the first report to detail the mechanism of TIMP-2 action in neuronal ceils and demonstrates a novel function for TIMP- 2 in neurons independent of MMP inhibition. Primary cultured Timp-2 -/- and wild-type cerebral cortical neurons will be used to test the hypothesis that TIMP- 2 plays a role in neuronal differentiation independent of MMP-inhibitory activity via interaction with integrins. In aim 1, the number, location and affinity of TIMP-2 receptors will be determined and the Timp-2 null neurite length phenotype will be rescued by exogenous application of TIMP-2 protein with and without MMP-inhibitory activity. In aim 2, the integrin(s) to which TIMP-2 binds will be identified and competitive binding assays will be performed to block the phenotypic rescue by TIMP-2. Aim 3 will determine whether Timp-2 deletion alters cortical progenitor cell cycle parameters or laminar fate in vivo using two BrdU injection paradigms. Unlike somatic cells, neurons irreversibly withdraw from the cell cycle and permanently remain quiescent. The mechanisms responsible for the maintenance of neuronal quiescence are poorly understood. The identification of molecules responsible for neuronal quiescence has significant implications for the ability of the adult brain to generate new neurons in response injury as well as in neurological deficits such as Alzheimer's and Parkinson's disease.

描述（由申请人提供）：有关中枢神经系统中细胞外基质发育调控的信息是不完整的，并且蛋白质（例如基质金属蛋白酶的组织抑制剂（TIMP））的功能知之甚少。我们最近表征了体内CNS发育过程中四个已知TIMP的表达。有丝分裂后神经元的TIMP-2表达与神经元分化相关。我们在此证明TIMP-2以细胞自主方式诱导细胞周期停滞和神经元分化。令人惊讶的是，TIMP-2的效应与其MMP抑制活性无关。 TIMP-2在细胞表面上的点状标记结合了TIMP-2与alpha3 beta1整合素的相互作用，这表明TIMP-2可以通过整合素通过整合素发挥其与MMP无关的活性。 Nestin阳性祖细胞在新皮质心室区域和TIMP-2无效神经元的神经突长度的持久性表明，在没有TIMP-2的情况下，神经元分化会延迟。这是详细详细介绍神经元天花板中TIMP-2作用机理的第一份报告，并证明了TIMP-2在与MMP抑制无关的神经元中的新功能。 原发性培养的TIMP-2 - / - 和野生型脑皮质神经元将用于检验以下假设，即TIMP-2通过与整合素相互作用而独立于MMP抑制活性在神经元分化中起作用。在AIM 1中，将确定TIMP-2受体的数量，位置和亲和力，TIMP-2 NULL神经突长度表型将通过有或没有MMP抑制活性的TIMP-2蛋白的外源应用来挽救。在AIM 2中，将确定TIMP-2结合的整联蛋白，并将进行竞争性结合测定，以阻止TIMP-2的表型救助。 AIM 3将确定TIMP-2缺失是否使用两个BRDU注入范式在体内改变皮质祖细胞周期参数或层流命运。 与体细胞不同，神经元不可逆转地退出细胞周期，并永久保持静止。对神经元静止的维持的机制知之甚少。负责神经元静脉的分子的鉴定对成年大脑在反应损伤以及阿尔茨海默氏病和帕金森氏病等神经系统缺陷方面产生新的神经元的能力具有重要意义。