Modulation of lysosomal function for the treatment of Batten disease

项目摘要/摘要 神经性蜡样脂褐素增多症(NCLS或巴顿病)是最具破坏性的遗传性疾病之一 也是美国儿童神经退行性变的最常见原因。目前没有 治愈这些疾病，治疗在很大程度上仍然是支持性的。NCLS的特点是进步性 溶酶体内未降解的细胞物质的堆积；这种堆积被认为是缺陷造成的 在自噬-溶酶体途径中，但本身可能有助于发病。我们的数据显示，缺乏 幼年巴顿病蛋白CLN3的一部分会损害溶酶体酶亚群的成熟，并且 海藻糖介导的TFEB的激活改善了自噬-溶酶体途径的主要调节因子 幼年巴顿病(JNCL)小鼠模型的疾病负担。我们建议研究新的机制 TFEB激活可能导致JNCL和其他神经退行性疾病的翻译应用 由溶酶体介导的细胞清除缺陷引起的。首先，我们将检验海藻糖的假设- 诱导的溶酶体增强纠正JNCL小鼠溶酶体酶的缺陷成熟(目标1)。 我们将通过使用敲入基因进行蛋白质组学分析，在体内测试这一假说 我们建立的Lamp1FLAG小鼠品系能够有效地从小鼠组织中分离溶酶体。第二，我们将 测试假设，减少或抑制Akt，我们已经确定的TFEB的一种激酶抑制剂，将 降低JNCL小鼠神经病理改变(目标2)。我们将通过使用两个互补的 方法：在遗传学上，通过使用Akt1/-小鼠，在药理学上，通过使用Akt药物抑制剂 目前正在进行临床开发。第三，我们将检验协同药理激活的假设 通过调制两个正交通路的TFEB将导致更大的自噬增强- 溶酶体系统和更好地减少JNCL的病理特征比单独使用任何一种策略(目标3)。这 假设是基于我们的发现，非受体酪氨酸激酶，Src是一个必要的因素 激活TFEB的另一种激酶抑制因子mTORC1。这些研究将开创药理激活的先河。 TFEB在神经退行性疾病模型中的作用。如果成功，这项研究将提供一个强大的范例 TFEB的激活，可能为巴顿病的临床治疗奠定基础，并有可能， 自噬-溶酶体途径受损引起的额外的神经退行性储存障碍。

PROJECT SUMMARY/ABSTRACT Neuronal ceroid lipofuscinoses (NCLs or Batten disease) are among the most devastating inherited disorders of childhood and the most common cause of neurodegeneration in children in the U.S. There is currently no cure for these disorders, and treatments remain largely supportive. NCLs are characterized by the progressive intralysosomal accumulation of undegraded cellular material; this accumulation is thought to result from defects in the autophagy-lysosomal pathway, but could itself contribute to pathogenesis. Our data show that deficiency of the juvenile Batten disease protein, CLN3, impairs maturation of a subset of lysosomal enzymes and that trehalose-mediated activation of TFEB, a master regulator of the autophagy-lysosomal pathway, ameliorates disease burden in a mouse model of juvenile Batten disease (JNCL). We propose to study novel mechanisms of TFEB activation that could lead to translational application for JNCL and other neurodegenerative disorders caused by defects in lysosome-mediated cellular clearance. First, we will test the hypothesis that trehalose- induced lysosomal enhancement corrects defective maturation of lysosomal enzymes in JNCL mice (Aim 1). We will test this hypothesis in vivo by conducting proteomics analyses based on the use of a knock-in Lamp1FLAG mouse line we have generated to efficiently isolate lysosomes from mouse tissues. Second, we will test the hypothesis that reduction or inhibition of Akt, a kinase inhibitor of TFEB we have identified, will decrease neuropathology of JNCL mice (Aim 2). We will reduce Akt activity by using two complementary approaches: genetically, by using Akt1-/- mice, and pharmacologically, by using an Akt drug inhibitor that is currently in clinical development. Third, we will test the hypothesis that synergistic pharmacological activation of TFEB by modulation of two orthogonal pathways will result in a greater enhancement of the autophagy- lysosomal system and better reduction of JNCL pathological hallmarks than either strategy alone (Aim 3). This hypothesis is based on our findings that the non-receptor tyrosine kinase, Src, is an essential factor for activation of mTORC1, another kinase inhibitor of TFEB. These studies will pioneer pharmacological activation of TFEB in a model of neurodegenerative disorder. If successful, this study will provide a powerful paradigm of TFEB activation that could lay the foundation for the clinical treatment of Batten disease and, potentially, additional neurodegenerative storage disorders caused by impairment of the autophagy-lysosomal pathway.