Endo-lysosomal mechanisms and treatment in atypical cerebellar neurodevelopment

项目摘要 Christianson综合征（CS）是一种单基因的X连锁神经系统疾病， Na+/H+交换器6（NHE 6）为研究神经发育和 神经变性（Gilfillan et al 2008）。它是最常见的X连锁疾病之一（Tarpey et al. 2009） 值得注意的是小脑病理学，如浦肯野细胞丢失（Garbern et al. 2010，Christianson et al. 1999） 和小脑萎缩（Pescosolido et al. 2014）。NHE 6突变如何导致小脑相关病理 不明通过确定小脑发育异常的细胞机制， 可以测试靶向进行性小脑症状的治疗剂。我们的初步证据 概括了NHE 6突变小鼠中进行性浦肯野细胞丢失，并且在小鼠中发现了运动功能障碍 这些小鼠（Stromme et al. 2011，Sikora et al. 2015）。我们以前的研究表明，NHE 6的损失导致 内体过度酸化，导致神经元分支和BDNF信号传导减少（Oyuang et al. 2013年）。此外，CS小鼠模型已显示内体-溶酶体功能缺陷 （Stromme et al. 2011），表明自噬功能障碍是NHE 6突变的潜在机制。 浦肯野细胞变性可由自噬缺陷引起（Hara et al. 2006）。因此，我们假设 NHE 6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退化。我们还将测试这些缺陷的假设 在我们的小鼠模型中，可以通过体外和体内IGF-1治疗来逆转。我们将解决这些问题 通过以下具体目标的假设。在第一个目标中，我们将证明， NHE 6突变型浦肯野细胞的低pH内膜与异常浦肯野细胞发育相关 可以通过体外药物挽救或恶化。在第二个目标中，我们将阐明一种机制， 在体外和体内NHE 6缺失的浦肯野细胞中增强的自噬和降低的mTOR活性， 通过IGF-1治疗逆转。我们还将研究IGF-1治疗是否可以改善运动功能， NHE 6突变小鼠的协调缺陷。我们将利用来自NHE 6缺失雄性小鼠的小脑切片 用于检查内溶酶体膜、浦肯野细胞发育、自噬、mTOR活性和IGF的模型 治疗从这个项目获得的信息将提供一个小脑机制的理解 Christianson综合征的病理学，可能还有其他小脑相关的神经系统疾病 症状此外，这项研究可能会确定一种FDA批准的药物，IGF，作为一种潜在的治疗， 小脑功能障碍的神经系统疾病。

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.