Endo-lysosomal mechanisms and treatment in atypical cerebellar neurodevelopment

项目总结 克里斯汀森综合征(CS)是一种单基因、X连锁的神经系统疾病，由 NA/H交换器6(NHE6)，为研究神经发育和 神经变性(Gilfian等人，2008年)。这是最常见的X连锁疾病之一(Tarpey等人)。2009年) 并且值得注意的是小脑病理，如浦肯野细胞丢失(Garbern等人)。2010年，克里斯汀森等人。(1999年) 和小脑萎缩(PesCosolido等人)。2014年)。NHE6基因突变如何引起小脑相关病理 是未知的。通过确定小脑发育异常的细胞机制，新颖的 针对进行性小脑症状的治疗药物可以进行测试。我们的初步证据 概述了NHE6突变小鼠的进行性浦肯野细胞丢失，运动功能障碍已在 这些小鼠(Stromme等人)2011年，Sikora等人。2015年)。我们之前的研究表明，NHE6的丢失会导致 内体过度酸化，导致神经元分枝和BDNF信号减少(欧阳等人 艾尔2013)。此外，CS小鼠模型已显示出内体-溶酶体功能缺陷 (Stromme等人)2011年)，表明自噬功能障碍是NHE6突变的潜在机制。 浦肯野细胞退化可由自噬缺陷引起(Hara等人。2006)。因此，我们假设 NHE6突变导致内体过度酸化和自噬增强，导致浦肯野细胞 导致小脑发育异常和变性。我们还将测试这些缺陷的假设 在我们的小鼠模型中，IGF-1在体外和体内的治疗可能逆转这种作用。我们将解决这些问题 通过以下具体目标进行假设。在第一个目标中，我们将证明过量的 NHE6突变型浦肯野细胞低pH内膜与浦肯野细胞发育异常相关 这可以通过体外药物来挽救或恶化。在第二个目标中，我们将阐明一种机制，包括 体内外NHE6缺失型浦肯野细胞自噬增强和mTOR活性降低可能是 经IGF-1治疗后逆转。我们还将研究IGF-1治疗是否可以改善运动 NHE6突变小鼠的协调缺陷。我们将利用一只NHE6基因缺失的雄性小鼠的小脑切片 检测内溶酶体膜、浦肯野细胞发育、自噬、mTOR活性和IGF的模型 治疗。从这个项目中获得的信息将提供对小脑的机械理解。 克里斯汀森综合征的病理学，可能还有其他与小脑相关的神经疾病 症状。此外，这项研究可能确定一种FDA批准的药物IGF，作为一种潜在的治疗方法 伴有小脑功能障碍的神经性疾病。

PROJECT SUMMARY Christianson syndrome (CS) is a monogenic, X-linked neurologic disorder caused by changes in the Na+/H+ exchanger 6 (NHE6) that provides an opportunity to study both neurodevelopment and neurodegeneration (Gilfillan et al 2008). It is one of the most common X-linked disorders (Tarpey et al. 2009) and is notable for cerebellar pathology such as Purkinje cell loss (Garbern et al. 2010, Christianson et al. 1999) and cerebellar atrophy (Pescosolido et al. 2014). How NHE6 mutations cause cerebellar-associated pathology is unknown. By identifying the cellular mechanism underlying abnormal cerebellar development, novel therapeutic agents targeting progressive cerebellar symptoms can be tested. Our preliminary evidence recapitulates progressive Purkinje cell loss in NHE6 mutant mice, and motor dysfunction has been identified in these mice (Stromme et al. 2011, Sikora et al. 2015). Our previous research has shown loss of NHE6 results in endosomal overacidification, which leads to decreased neuronal arborization and BDNF signaling (Ouyang et al. 2013). Furthermore, a CS mouse model has shown to have deficits in endosomal-lysosomal functioning (Stromme et al. 2011), suggesting autophagy dysfunction as a potential mechanism in NHE6 mutations. Purkinje cell degeneration can be caused by autophagy deficits (Hara et al. 2006). Therefore, we hypothesize that NHE6 mutations cause endosomal overacidification and enhanced autophagy resulting in Purkinje cell that leads to abnormal cerebellar development and degeneration. We will also test the hypothesis these defects may be reversed by IGF-1 treatment in vitro and in vivo in our mouse model. We will address these hypotheses through the following Specific Aims. In the first aim we will demonstrate that an over-abundance of low pH endo-membranes in NHE6 mutant Purkinje cells is associated with abnormal Purkinje cell development that can be rescued or worsened by drugs in vitro. In the second aim we will elucidate a mechanism involving enhanced autophagy and decreased mTOR activity in NHE6-null Purkinje cells in vitro and in vivo that may be reversed by IGF-1 treatment. We will also examine whether IGF-1 treatment may ameliorate motor coordination deficits in NHE6 mutant mice. We will utilize cerebellar slices from an NHE6 null male mouse model to examine endo-lysosomal membranes, Purkinje cell development, autophagy, mTOR activity and IGF treatment. The information obtained from this project will provide a mechanistic understanding of cerebellar pathology in Christianson syndrome and, possibly, other neurologic disorders with cerebellar-related symptoms. Furthermore, this research may identify an FDA-approved drug, IGF, as a potential therapeutic for neurologic disorders with cerebellar dysfunction.