Effect of NF1 mutation on choroid plexus function

描述（由申请人提供）：1型神经纤维瘤病患者有一系列中枢神经系统（CNS）发育缺陷，包括脑室增大、皮层和海马分层紊乱、脑中线结构增大、星形细胞增生、星形细胞瘤和视神经胶质瘤。我们研究的长期目标是了解Nf1缺乏如何导致这些与学习、认知和神经损伤相关的结构缺陷。先前的研究表明，小鼠脑祖细胞中的条件性Nf1敲除会表现出这些方面的一些，但不是全部，这表明缺失了一些因素。环境中Nf1缺乏加剧了Nf1丢失的细胞自主效应。因此，我们决定调查环境中Nf1的减少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，脑脊液提供附近脉络膜丛（CP）产生的生长/信号因子。先前的研究没有检查Nf1缺乏对脑脊液功能和脑脊液组成的影响，因为迄今为止进行的条件敲除并未影响脑脊液。假设：Nf1的缺失和脑脊液中Ras的过度活跃导致脑脊液产生改变，从而导致大脑发育异常。我们将在两个具体目标下解决以下问题：降低Nf1是否会改变CP功能？和2。降低脑膜中Nf1是否会导致大脑发育异常？这些问题将通过体内和体外模型来解决。我们将通过将在发育早期（在他莫昔芬控制下）特异性表达cre的transthyrat -cre- ert小鼠系Tg(Ttr- cre/Esr1*)1Vco与封闭的Nf1小鼠系Nf1tm1Par杂交，产生可诱导的条件敲除。结果tr-cre/Esr1的控制；Nf1fl/fl Nf1缺失小鼠为trr -cre/Esr1；Nf1 + / +同窝出生的。我们还将在体内使用子宫内递送的带有Nf1 shRNA的慢病毒载体与混乱和空载体对照，在发育中的CP中进行Nf1的遗传敲除。Nf1耗尽后的CP功能将通过比较基因表达来确定改变的分泌因子来检查。脑脊液的组成将被检查，重点关注CP产生的影响脑祖细胞的生长因子，如BMP7、IGF2和Midkine，以及通过CP转录组分析揭示的新因子。Nf1缺乏对大脑发育的影响将被评估，量化脑室大小、脑祖细胞增殖、神经元和胶质细胞的形成、皮层分层、细胞死亡发生率和肿瘤形成，使用我们实验室非常熟悉的技术。我们的合作者，Nf1功能专家Kevin Pumiglia博士和哺乳动物CP功能研究专家Norman Saunders博士将提供建议和指导。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：正如我们在初步研究中所显示的，由于它是一个相对孤立的结构，可以改变细胞中的基因表达。这可能有助于减少加剧1型神经纤维瘤病的环境因素，从而减缓中枢神经系统疾病的进展。

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.