Investigating SNX13 in Cerebellar Function and Disease

研究 SNX13 在小脑功能和疾病中的作用

基本信息

批准号：
10750029
负责人：
Vanessa Breanne Sanchez
金额：
$ 4.77万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-18 至 2025-09-17
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10750029
关键词：
Address Affect Ataxia Behavior Bilateral Biogenesis Biological Biological Process Brain CRISPR interference CRISPR screen CRISPR/Cas technology Cells Cellular Neurobiology Cellular Stress Cerebellar Ataxia Cerebellar Diseases Cerebellum Child Cholesterol Cholesterol Homeostasis Communication Confocal Microscopy DNA Sequence Alteration Data Dependovirus Development Disease Embryo Endoplasmic Reticulum Energy Metabolism Fatty Acids Foundations Future Goals Guide RNA Harvest Histologic Homeostasis Human Image Impairment In Situ Hybridization Induced pluripotent stem cell derived neurons Injections Leadership Lipids Mediating Mentors Metabolic Metabolic Diseases Methodology Methods Molecular Motor Motor Skills Mus Mutation N-Methylaspartate Nerve Degeneration Neurobiology Neurodegenerative Disorders Neuroglia Neurons Oleic Acids Oxidative Stress Pathogenicity Patients Pediatric Hospitals Pennsylvania Philadelphia Production Proteins Recombinant adeno-associated virus (rAAV)Regulation Research Research Personnel Role Science Site Spinocerebellar Ataxias Stains Structure System Technical Expertise Techniques Testing Training Transgenic Organisms Triglycerides Universities Variant Visual Work acid stress behavior test career cognitive skill excitotoxicity experience genome editing genome-wide human pluripotent stem cell in vivo insight knock-down lipid metabolism motor behavior mouse model neonate neuron component novel paralogous gene preservation promoter protein function skill acquisition skills sorting nexins stem cell biology stressor targeted treatment translational neuroscience

项目摘要

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.

项目概要脂质是与基因突变相关的神经功能和结构的重要组成部分。人们越来越认识到神经退行性疾病中脂质代谢紊乱，但我们和其他人发现了 SNX14，一种调节神经元脂质代谢的机制。与脊髓小脑共济失调 (SCAR20) 相关的分选连接蛋白 (SNX) 蛋白，是内质的调节剂我有网状（ER）-脂滴（LD）生物发生、脂肪酸去饱和和小脑脂质稳态。发现 SNX14 与其旁系同源物 SNX13 在神经细胞中相互作用，SNX13 也是一种 ER 驻留蛋白。我们最近发现了控制甘油三酯含量、LD 值和溶酶体胆固醇稳态的因素。三个患有类似于 SCAR20 的新型小脑共济失调的儿童中存在纯合 SNX13 变异。众所周知，SNX13 在神经元中发挥作用的基本分子机制，以及如何耗尽 Snx13 会导致退化，我的总体假设是 SNX13 在神经元脂质稳态中发挥作用。这对于小脑功能和生存至关重要。为了检验这一假设，Aim 1 将通过以下方式敲低 Snx13。 AAV 介导的 Snx13 sgRNA 体内递送至 dCAS9-KRAB 表达小鼠，以研究其作用 Snx13 在小脑完整性和运动行为中的作用目标 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 SNX13 患者突变对条件下 LD 稳态的影响细胞应激（例如，过量的脂肪酸和兴奋性毒性）的成功完成将是一个成功的目标。为未来研究神经元LD稳态的重要基础，同时提供我在 CRISPR/Cas9 方法、干细胞生物学和行为方面接受过出色的培训。将揭示基本的疾病机制，这些机制可用于为以下疾病提供针对性的治疗策略：患有 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。拟议的培训计划由美国大学的 Naiara Akizu 博士和 Beverly Davidson 博士赞助宾夕法尼亚州和费城儿童医院该提案提供了非凡的培训体验。这将扩展我在细胞神经生物学方面的技术专业知识，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领导者的职业目标神经退行性疾病领域的研究员。