Effect of NF1 mutation on choroid plexus function

描述（由申请方提供）：1型神经纤维瘤病患者存在一系列中枢神经系统（CNS）发育缺陷，包括脑室扩大、皮质和海马分层紊乱、脑中线结构扩大、星形细胞增多、星形细胞瘤和视神经胶质瘤。我们研究的长期目标是了解Nf 1缺乏如何导致这些结构缺陷，这些结构缺陷与学习，认知和神经功能障碍相关。先前的工作表明，小鼠脑祖细胞中的条件性Nf 1敲除表型模仿了一些，但不是所有这些方面，表明缺失的因素。环境中的Nf 1缺乏加剧了Nf 1损失的细胞自主效应。因此，我们决定研究环境减少的Nf 1是否会影响大脑发育。脑干细胞和祖细胞浸泡在脑脊液（CSF）中，脑脊液（CSF）提供由附近的脉络丛（CP）产生的生长/信号因子。以前的研究没有检查Nf 1缺陷对CP功能和CSF组成的影响，因为迄今为止进行的条件性敲除不影响CP。假设：CP中Nf 1的缺失和导致的Ras过度活跃导致CSF产生改变，从而导致大脑发育异常。我们将在两个具体目标中解决以下问题：1。减少Nf 1会改变CP功能吗？和2.降低CP中的Nf 1会导致大脑发育异常吗？这些问题将使用体内和体外模型来解决。我们将通过将从发育早期（在他莫昔芬对照下）在CP中特异性表达cre的转甲状腺素蛋白-cre-ERT小鼠系Tg（Ttr-cre/Esr 1 *）1Vco与floxed Nf 1小鼠系Nf 1 tm 1 Par杂交来产生诱导型条件性敲除。所得Ttr-cre/Esr 1; Nf 1 fl/fl Nf 1耗竭小鼠的对照是Ttr-cre/Esr 1; Nf 1 +/+同窝仔。我们还将在体内使用子宫内递送的慢病毒载体与Nf 1 shRNA相对于乱序和空载体对照在发育中的CP中进行Nf 1的遗传性敲低。将通过比较基因表达来检查Nf 1消耗后的CP功能，以鉴定改变的分泌因子。将检查CSF的组成，重点是CP产生的影响脑祖细胞的生长因子，如BMP 7、IGF 2和中期因子，以及通过CP转录组分析揭示的新因子。将评估Nf 1缺乏对脑发育的影响，使用我们实验室非常熟悉的技术定量心室大小，脑祖细胞增殖，神经元和神经胶质细胞的形成，皮质分层，细胞死亡和肿瘤形成的发生率。我们的合作者Kevin Pumiglia博士是Nf 1功能的专家，Norman Saunders博士是开发哺乳动物CP功能的专家，他们将提供建议和指导。如果这一假设得到支持，它将揭示CP作为1型神经纤维瘤病治疗的潜在靶点：因为它是一个相对孤立的结构，细胞中的基因表达可以被修饰，正如我们在初步研究中所显示的那样。这可能有助于减少加重1型神经纤维瘤病的环境因素，从而减缓CNS疾病的进展。 公共卫生相关性：患有1型神经纤维瘤病的患者具有与学习、认知和神经问题相关的脑缺陷。在这项研究中，我们打算研究大脑如何在这种疾病的动物模型中形成。具体来说，我们将询问是否在大脑中的一个重要结构，脉络丛，产生脑脊液，在与神经纤维瘤病1型相关的脑缺陷的发展中发挥关键作用。如果是这样，那么脉络丛可以作为治疗的靶点，以帮助使导致相关发育缺陷的脑环境因素中的畸变正常化，并可能改善1型神经纤维瘤病患者的异常神经功能或肿瘤生长。

DESCRIPTION (provided by applicant): Patients with neurofibromatosis type 1 have a range of central nervous system (CNS) developmental defects, including enlarged ventricles, perturbed cortical and hippocampal layering, enlarged brain midline structures, astrocytosis, astrocytomas and optic nerve gliomas. The long-term goal of our studies is to understand how Nf1 deficiency causes these structural defects, which are correlated with learning, cognitive and neurological impairments. Prior work has shown that conditional Nf1 knockout in mouse brain progenitor cells phenocopies some, but not all of these aspects, suggesting missing factors. Nf1 deficiency in the environment exacerbates cell-autonomous effects of Nf1 loss. Hence, we decided to investigate whether environmental reduced Nf1 could impact brain development. Brain stem cells and progenitor cells are bathed in cerebrospinal fluid (CSF) that provides growth/signaling factors produced by the nearby choroid plexus (CP). Prior studies did not examine the result of Nf1 deficiency on CP function and CSF composition because the conditional knockouts performed to date did not affect the CP. Hypothesis: Loss of Nf1 and resulting hyperactive Ras in the CP results in altered CSF production leading to abnormal brain development. We will address the following questions in two specific aims:1. Does reducing Nf1 alter CP function? and 2. Does reducing Nf1 in the CP lead to abnormal brain development? These questions will be approached using in vivo and in vitro models. We will generate an inducible conditional knockout by crossing the Transthyretin-cre-ERT mouse line Tg(Ttr- cre/Esr1*)1Vco which expresses cre specifically in the CP from early times in development (and under tamoxifen control) to the floxed Nf1 mouse line Nf1tm1Par. Controls for the resulting Ttr-cre/Esr1; Nf1fl/fl Nf1 depleted mice are the Ttr-cre/Esr1; Nf1+/+ littermates. We will also perform heritable knockdown of Nf1 in the developing CP in vivo using in utero delivered lentiviral vectors with Nf1 shRNA versus scrambled and empty vector controls. CP function after Nf1 depletion will be examined by comparing gene expression to identify altered secreted factors. The composition of the CSF will be examined, focusing on CP-generated growth factors that impact brain progenitor cells such as BMP7, IGF2 and Midkine, and novel factors revealed through the CP transcriptome analysis. The effect of Nf1 deficiency on brain development will be assessed, quantifying ventricular size, brain progenitor proliferation, the formation of neuronal and glial cells, cortical layering, the incidence of cell death and tumor formation, using techniques highly familiar to our lab. Our collaborators Dr. Kevin Pumiglia, a specialist in Nf1 function, and Dr. Norman Saunders, an expert in developing mammalian CP function, will provide advice and guidance. If the hypothesis is supported, it would reveal the CP as a potential target of neurofibromatosis type 1 therapy: as it is a relatively isolated structure, gene expression in the cells can be modified, as we have shown in preliminary studies. This could help reduce environmental factors that exacerbate neurofibromatosis type 1 and thus attenuate progression of the disease in the CNS. PUBLIC HEALTH RELEVANCE: Patients with neurofibromatosis type 1 have brain defects that are associated with learning, cognitive and neurological problems. In this study, we intend to examine how the brain forms in animal models of this disease. Specifically, we will ask whether an important structure in the brain, the choroid plexus, which generates cerebrospinal fluid, plays a key role in the development of brain defects associated with neurofibromatosis type 1. If so, then the choroid plexus could be targeted for therapy to help normalize aberrations in brain environmental factors that contribute to the associated developmental defects, and potentially ameliorate abnormal neurological function or tumor growth in neurofibromatosis type 1 patients.