Investigating SNX13 in Cerebellar Function and Disease

研究 SNX13 在小脑功能和疾病中的作用

• 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
• 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

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 脂质是神经元功能和结构的重要组成部分。相关基因突变 人们越来越认识到神经退行性疾病中脂质代谢紊乱，但 调节神经元脂质代谢的机制尚不清楚。我们和其他人发现了 SNX14， 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 网状（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 SNX14 在神经细胞中与其旁系同源物 SNX13 相互作用。 SNX13 也是一种 ER 驻留蛋白 控制甘油三酯含量、LD 值和溶酶体胆固醇稳态。我们最近确定 三名患有类似于 SCAR20 的新型小脑共济失调的儿童的纯合 SNX13 变异。然而，很少 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 会导致变性。我的首要假设是 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。为了检验这个假设，目标 1 将通过以下方式击倒 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）。成功完成这些目标将是 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。此外，这些 努力将揭示基本的疾病机制，可用于为有针对性的治疗策略提供信息 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。