Effect of NF1 mutation on choroid plexus function

描述(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，包括脑室扩大、皮质和海马层紊乱、大脑中线结构扩大、星形细胞增多症、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缺乏如何导致这些结构性缺陷，这些缺陷与学习、认知和神经损伤相关。先前的工作已经表明，小鼠脑前体细胞中的条件性NF1基因敲除可以复制这些方面的某些方面，但不是全部，这表明缺少这些因素。环境中NF1的缺乏加剧了NF1缺失对细胞自主的影响。因此，我们决定调查环境减少的NF1是否会影响大脑发育。脑干细胞和祖细胞沐浴在脑脊液(CSF)中，脑脊液提供附近脉络丛(CP)产生的生长/信号因子。以前的研究没有检查NF1缺乏对CP功能和脑脊液成分的影响，因为迄今为止进行的条件基因敲除并不影响CP。假设：Nf1的丢失和由此导致的CP中过度活跃的RAS会导致脑脊液产生变化，导致大脑发育异常。我们将在两个具体目标上解决以下问题：1.减少NF1是否会改变CP的功能？Cp中Nf1的减少是否导致脑发育异常？这些问题将通过体内和体外模型来解决。我们将通过将从发育早期(在他莫昔芬控制下)在CP中特异表达cre的Transhyretin-cre-ERT鼠系Tg(Ttr-cre/ESR1*)1Vco与Flosed NF1鼠系Nf1tm1Par杂交，产生可诱导的条件基因敲除。由此产生的Ttr-cre/ESR1；Nf1f1/f1 NF1耗竭小鼠的对照是Ttr-cre/ESR1；Nf1/Litterates。我们还将在体内发育中的CP中使用带有Nf1 shRNA的宫内递送慢病毒载体与扰乱和空载体对照进行可遗传的敲除Nf1。NF1耗尽后的CP功能将通过比较基因表达来确定变化的分泌因子。将检查脑脊液的组成，重点是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素。NF1缺乏对大脑发育的影响将被评估，使用我们实验室非常熟悉的技术，量化脑室大小、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NF1功能专家Kevin Pumiglia博士和哺乳动物CP功能开发专家Norman Saunders博士将提供建议和指导。如果这一假说得到支持，它将揭示CP作为1型神经纤维瘤病治疗的潜在靶点：由于它是一个相对孤立的结构，细胞中的基因表达可以被修改，正如我们在初步研究中所表明的那样。这可能有助于减少加重1型神经纤维瘤病的环境因素，从而减缓疾病在中枢神经系统的进展。 公共卫生相关性：1型神经纤维瘤病患者的大脑缺陷与学习、认知和神经问题有关。在这项研究中，我们打算研究这种疾病的动物模型中大脑是如何形成的。具体地说，我们将询问大脑中产生脑脊液的重要结构脉络丛是否在与1型神经纤维瘤病相关的脑缺陷的发生中发挥关键作用。如果是这样，那么脉络丛可能成为治疗的靶点，以帮助正常化导致相关发育缺陷的脑环境因素的异常，并潜在地改善1型神经纤维瘤病患者的神经功能异常或肿瘤生长。

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.