Investigating SNX13 in Cerebellar Function and Disease

项目总结 脂质是神经元功能和结构的重要组成部分。与基因突变相关的基因突变 神经退行性疾病中脂代谢紊乱的认识越来越多，但 调节神经元脂代谢的机制还知之甚少。我们和其他人发现了SNX14，一种 脊髓小脑性共济失调(SCAR20)相关的SNX蛋白作为内质调节因子 网状(ER)-脂滴(LD)生物发生，脂肪酸减饱和，和小脑脂质动态平衡。我有过 发现SNX14与其类似物SNX13在神经细胞中相互作用。SNX13也是内质网驻留蛋白 它控制甘油三酯含量、LD数和溶酶体胆固醇动态平衡。我们最近确认了 三名患有类似于SCAR20的新的小脑性共济失调的儿童的SNX13纯合子变异。然而，几乎没有 已知SNX13在神经元中作用的基本分子机制，以及如何耗竭SNX13 Snx13导致退行性变。我的主要假设是SNX13在神经元脂质稳态中发挥作用， 这对小脑功能和生存至关重要。为了验证这一假设，目标1将通过以下方式摧毁Snx13 腺相关病毒介导的Snx13 sgRNAs对dCAS9-KRAb小鼠体内表达的影响 SNX13对小脑完整性和运动行为的影响。AIM 2将利用基因组编辑的人类多能干细胞 衍生神经元培养以确定SNX13患者突变对条件下LD动态平衡的影响 细胞应激(例如，过量脂肪酸和兴奋性毒性)。这些目标的成功实现将是 为未来研究神经元LD的动态平衡奠定了重要基础，同时为 我在CRISPR/CAS9方法学、干细胞生物学和行为学方面接受了出色的培训。此外，这些 将努力揭示基本的疾病机制，这些机制可用于指导有针对性的治疗策略 携带SNX13或SNX14突变的患者，包括其他相关的神经退行性和代谢性疾病。 拟议的培训计划由华盛顿大学的奈亚拉·秋津博士和贝弗利·戴维森博士赞助。 宾夕法尼亚州和费城儿童医院。这项建议提供了非凡的培训体验 这将扩大我在细胞神经生物学方面的技术专长，并培养我在科学方面的专业技能 沟通、指导和领导力，所有这些都将促进我成为一名领导者的职业目标 神经变性领域的研究人员。

PROJECT SUMMARY Lipids are an essential component of neuronal function and structure. Genetic mutations associated with disturbed lipid metabolism in neurodegenerative disorders is becoming increasingly recognized, yet the mechanisms that regulate neuronal lipid metabolism is poorly understood. We and others have found SNX14, a sorting-nexin (SNX) protein associated with a spinocerebellar ataxia (SCAR20), to be a regulator of endoplasmic reticulum (ER)-lipid droplet (LD) biogenesis, fatty acid desaturation, and cerebellar lipid homeostasis. I have discovered that SNX14 interacts with its paralog, SNX13, in neural cells. SNX13 is also an ER resident protein that controls triglyceride content, LD numbers, and lysosomal cholesterol homeostasis. We recently identified homozygous SNX13 variants in three children with a novel cerebellar ataxia similar to SCAR20. However, little is known regarding the basic molecular mechanisms behind SNX13 function in neurons, and how depletion of Snx13 leads to degeneration. My overarching hypothesis is that SNX13 functions in neuronal lipid homeostasis, which is critical for cerebellar function and survival. To test this hypothesis, Aim 1 will knock down Snx13 by AAV-mediated in vivo delivery of Snx13 sgRNAs to dCAS9-KRAB expressing mice to investigate the role of Snx13 in cerebellar integrity and motor behavior. Aim 2 will leverage genome edited human pluripotent stem cell derived neuronal cultures to define the impact of SNX13 patient mutations on LD homeostasis under conditions of cellular stress (e.g., excess fatty acids and excitotoxicity). Successful completion of these aims will be an important foundation for future studies investigating neuronal LD homeostasis, while simultaneously providing me with outstanding training in CRISPR/Cas9 methodologies, stem cell biology, and behavior. Further, these efforts will uncover basic disease mechanisms that can be used to inform targeted therapeutic strategies for patients with SNX13 or SNX14 mutations, including other relevant neurodegenerative and metabolic disorders. The proposed training plan is sponsored by Dr. Naiara Akizu and Dr. Beverly Davidson at the University of Pennsylvania and Children’s Hospital of Philadelphia. This proposal provides a phenomenal training experience that will expand my technical expertise in cellular neurobiology, and cultivate my professional skills in science communication, mentoring, and leadership, all of which will facilitate my career goals of becoming a leading researcher in the field of neurodegeneration.