Sonic Hedghog Signaling in Cerebellum Development and Medulloblastoma

描述(申请人提供)：小脑是一个大脑结构，负责整合和协调来自多个来源的神经输入，以保持平衡和平衡。这是一种有趣的大脑结构，因为它是在出生后发育的。小脑内对出生后正常发育至关重要的细胞类型之一是小脑颗粒神经元前体(CGNP)。在小鼠生命的头两周，这些细胞经历了一个快速扩张期。CGNPs未能退出这种高增殖计划被认为是导致髓母细胞瘤形成的原因，髓母细胞瘤是儿童脑肿瘤中最常见的形式。髓母细胞瘤对传统的癌症治疗相对敏感，包括手术、化疗和放射治疗。然而，长期存活的人往往遭受终身发育、行为和认知障碍的困扰。这些毁灭性的副作用突显了了解髓母细胞瘤基本机制的必要性，因此可以开发出新的治疗方法，在不损害发育中的大脑的情况下专门针对肿瘤细胞。这个项目的长期目标是确定Sonic Hedgehog和IGF信号通路之间的串扰节点，这两个信号通路都与髓母细胞瘤有关。对这些途径的更多了解可能会导致小分子疗法的发展，减少这些年轻患者对放射和化疗的需求。为了实现这一目标，我们开发了一个包含各种技术和模型的跨学科提案。我们的初步数据表明，Shh信号增加了培养的CGNP中IRS1蛋白水平，而不是IRS1 mRNA。IRS1蛋白是IGF受体下游的一种直接转录因子。在目标1中，我们将检验这一假说，即CGNPs中的Shh增殖信号通过上调IRS1蛋白来积极调节IGF途径的活性。我们将研究IRS1蛋白周转以及多聚体分析，包括微阵列和质谱分析。在目标2中，我们将使用逆转录病毒介导的IRS1过表达和慢病毒递送的shRNA来确定IRS1对于Shh介导的CGNP在培养中的增殖是否必要/充分。在目标3中，我们将通过分析在小脑中结构性或条件性缺失IRS1的小鼠的CGNP增殖和小脑发育来确定IRS1在体内的作用。结合MSKCC小鼠遗传学核心设备，我们将培育出小脑中有条件缺失IRS1的小鼠。我们将通过培养CGNP和用CRE处理去除IRS1来分析CGNP在体内和体外增殖的作用。

DESCRIPTION (provided by applicant): The cerebellum is a brain structure responsible for integrating and coordinating neurological inputs from several sources in order to maintain equilibrium and balance. It is an interesting brain structure in that it develops after birth. One of the cell types within the cerebellum that is critical for proper postnatal development are cerebellar granule neuron precursors (CGNP). During the first two weeks of life in mice these cells undergo a rapid expansion phase. Failure of CGNPs to exit this high proliferate program has been suggested to lead to medulloblastoma formation, the most common form of pediatric brain tumor. Medulloblastoma is relatively responsive to traditional cancer treatments, including surgery, chemotherapy and radiation. However long-term survivors often suffer from life-long developmental, behavioral, and cognitive disturbances. These devastating side effects underscore the need to understand the basic mechanisms underlying medulloblastoma, so novel treatments can be developed to specifically target tumor cells without damaging the developing brain. The long-term objectives of this project is to identify nodes of cross-talk between Sonic hedgehog and IGF signaling pathways both of which have been implicated in medulloblastoma. Greater understanding of these pathways may lead to the development of small molecule treatments reducing the need for radiation and chemotherapy in these young patients. In order to accomplish this goal we have developed an interdisciplinary proposal incorporating various techniques and models. Our preliminary data indicate that Shh signaling increases IRS1 protein levels, a scafold directly downstream of the IGF receptor, but not IRS1 mRNA in cultured CGNPs. In aim 1 we will test the hypothesis that Shh proliferative signaling in CGNPs positively regulates IGF pathway activity through up-regulation of IRS1 protein. We will look at IRS1 protein turnover as well as polysome analysis including microarray and mass spectrometry analysis. In aim 2 we will use retroviral mediated over-expression of IRS1 and lentivirally-delivered shRNAto determine if IRS1 is necessary/sufficient for Shh mediated CGNP proliferation in culture. In aim 3 we will determine the role of IRS1 in vivo by analyzing CGNP proliferation and cerebella development in mice lacking IRS1 either constitutively or conditionally in the cerebellum. In conjunction with MSKCC Mouse Genetics core facility we will develop mice conditionally lacking IRS1 in the cerebellum. We will analyze the role of CGNP proliferation using in vivo as well as in vitro by culturing CGNPs and treating with Cre to remove IRS1.