Investigating SNX13 in Cerebellar Function and Disease

项目摘要 脂质是神经元功能和结构的重要组成部分。基因突变与 神经退行性疾病中脂质代谢紊乱正日益被认识到， 调节神经元脂质代谢的机制知之甚少。我们和其他人发现了SNX 14， 与脊髓小脑性共济失调（SCAR 20）相关的分选-连接蛋白（SNX）蛋白，作为内质网的调节剂， 内质网（ER）-脂滴（LD）生物发生、脂肪酸去饱和和小脑脂质稳态。我有 发现SNX 14在神经细胞中与它的蛋白SNX 13相互作用。SNX 13也是ER驻留蛋白 其控制甘油三酯含量、LD数和溶酶体胆固醇稳态。我们最近发现 三名患有与SCAR 20相似的新型小脑共济失调的儿童中的纯合子SNX 13变体。不过小 关于SNX 13在神经元中的功能背后的基本分子机制以及SNX 13的消耗如何是已知的。 Snx 13导致退化。我的首要假设是SNX 13在神经元脂质稳态中起作用， 这对小脑的功能和存活至关重要。为了验证这一假设，Aim 1将通过以下方式击倒Snx 13： AAV介导的Snx 13 sgRNA向表达dCAS 9-KRAB的小鼠的体内递送以研究Snx 13 sgRNA在表达dCAS 9-KRAB的小鼠中的作用。 小脑完整性和运动行为中的snx 13。Aim 2将利用基因组编辑的人类多能干细胞 衍生的神经元培养物，以确定SNX 13患者突变对LD稳态的影响， 细胞应激（例如，过量脂肪酸和兴奋性毒性）。成功实现这些目标将是 为未来研究神经元LD稳态的研究奠定了重要基础，同时提供了 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 这些努力将揭示基本的疾病机制，可用于为靶向治疗策略提供信息， SNX 13或SNX 14突变患者，包括其他相关神经退行性和代谢性疾病。 拟议的培训计划是由博士Naiara Akizu和博士贝弗利戴维森在大学的赞助。 宾夕法尼亚州和费城儿童医院。这个建议提供了一个非凡的培训经验 这将扩大我在细胞神经生物学方面的技术专长，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的职业目标 神经退化领域的研究者

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.