Development of a novel rodent model of hypothalamic hamartoma and epilepsy

摘要：下丘脑错构瘤（HH）是一种非癌性生长的紊乱细胞， 在胚胎发育过程中，由于音刺猬（SHH）通路基因的体细胞突变， 发展HH与现有抗癫痫药物难以控制的痉挛性发作（GS）相关 药物，HH患者也会发展出更严重的耐药性癫痫发作类型， 继发性癫痫因此，存在开发新疗法来治疗糖尿病的显著临床需求。 由HH直接导致的和/或在以下情况下发生的使人衰弱的耐药性癫痫发作 继发性癫痫完全没有动物模型来概括所产生的表型 是为什么新的药理学治疗的发现和开发已经被 受限目前的R21提案试图通过产生用于治疗的HH的新小鼠模型来解决这个问题。 用于治疗发现。这一目标将通过利用他莫昔芬诱导的Cre-Lox重组来实现 独立表达和测试两个病因相关的体细胞突变SHH信号分子在 高峰期下丘脑细胞增殖，并检查所产生的下丘脑解剖，基因 表达和生理学。由于已知SHH信号的激活促进细胞增殖， Aim 1将独立检测表达SmoM 2（一种组成性激活形式）的影响， Smoothened GPCR的），这将产生SHH信号传导的细胞自主上调， 受到影响的下丘脑细胞Aim 2将独立检查表达a SHH途径转录因子GLI 3（GLI 3 T）的截短版本，已在切除的HH中显示 组织中GLI 3 T的表达充当组成型阻遏物，并且预期下调SHH信号传导。 在有限数量的细胞中SHH信号的下调可能与我们的假设不相冲突， SHH信号的上调是HH发展的原因，因为正如最近 研究表明，在发育中的下丘脑细胞嵌合体中SHH信号的缺失可以导致细胞非- 邻近野生型细胞中SHH信号传导的自主上调。因此，本R21提案将利用 现有的小鼠品系，以直接测试HH的发展和随后的癫痫发作表型 可以由体细胞突变引起，所述体细胞突变产生细胞自主或非自主上调， 下丘脑发育过程中的SHH信号传导。因此，这项工作有可能为该领域提供 这是HH的第一个动物模型，并为未来的工作奠定了基础，以确定新的治疗方法， 治疗难治性癫痫发作，并检查继发性癫痫发生的机制。

ABSTRACT: Hypothalamic Hamartomas (HH) are noncancerous growths of disorganized cells and are believed to result as a consequence of somatic mutations in sonic hedgehog (SHH) pathway genes during embryonic development. HH are associated with gelastic seizures (GS) that are difficult to control with existing anti-seizure drugs, and people with HH also go on to develop even more severe pharmacoresistant seizure types due to secondary epileptogenesis. Thus, there is a significant clinical need to develop new therapies to treat the debilitating pharmacoresistant seizures that result as a direct consequence of HH and/or that develop following secondary epileptogenesis. A complete absence of animal models that recapitulate the phenotypes resulting from HH is a significant reason why discovery and development of novel pharmacological treatments has been hindered. The present R21 proposal seeks to solve this problem by generating a novel mouse model of HH for use in therapy discovery. This goal will be accomplished by utilizing tamoxifen-inducible Cre-Lox recombination to independently express and test two etiologically relevant somatic mutations in SHH signaling molecules during periods of peak hypothalamic cellular proliferation and examine resultant hypothalamic anatomy, gene expression, and physiology in these mice. Since activation of SHH signaling is known to promote cell proliferation, Aim 1 will independently examine the effects of expressing SmoM2 (a constitutively activated form of the Smoothened GPCR) that will produce a cell autonomous upregulation of SHH signaling in a mosaic of affected cells in the developing hypothalamus. Aim 2 will independently examine the effects of expressing a truncated version of the SHH pathway transcription factor GLI3 (GLI3T) that has been shown in resected HH tissues. Expression of GLI3T acts as a constitutive repressor and is expected to downregulate SHH signaling. Downregulation of SHH signaling in a limited number of cells may not conflict with our hypothesis that an overall upregulation of SHH signaling is responsible for the development of HH because, as it has recently been demonstrated, a loss of SHH signaling in a mosaic of developing hypothalamic cells can cause a cell non- autonomous upregulation of SHH signaling in neighboring wild-type cells. Therefore, this R21 proposal will utilize existing mouse lines to directly test the hypothesis that development of HH and consequent seizure phenotypes can result from somatic mutations that produce either a cell autonomous or non-autonomous upregulation in SHH signaling during hypothalamic development. Thus, this work has the potential to provide the field with the first animal model of HH and sets the stage for future work to determine novel therapeutic approaches for the treatment of refractory seizures and to examine the mechanisms underlying secondary epileptogenesis.