Effect of NF1 mutation on choroid plexus function

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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.