A role of ARL13B in the control of OSN innervation of the olfactory bulb

ARL13B 在控制嗅球 OSN 神经支配中的作用

• 批准号: 10535529
• 负责人: Julien Habif
• 金额: $ 4.15万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2023-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10535529
• 关键词: Affect; Axon; COVID-19; Cells; Cilia; Coupled; Data; Defect; Deletion Mutation; Detection; Development; Disease; Electrophysiology (science); Excision; Failure; Fluorescence Microscopy; Functional disorder; Gene Expression; Genes; Genetic Diseases; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Impairment; Induced Mutation; Joubert syndrome; Knock-in Mouse; Knockout Mice; Knowledge; Literature; Measures; Modeling; Mus; Mutation; Neurites; Neurons; Odors; Olfactory Pathways; Olfactory dysfunction; Organ; Patients; Penetrance; Penetration; Persons; Phenotype; Point Mutation; Population; Prevalence; Proteins; Reporting; Research; Role; Signal Transduction; Smell Perception; Structure; Synapses; Synaptic Transmission; Testing; Time; body system; cell type; ciliopathy; experimental study; gene replacement; insight; mouse model; nerve supply; novel; olfactory bulb; olfactory sensory neurons; promoter; protein transport; synaptogenesis

PROJECT SUMMARY/ABSTRACT Ciliopathies are a class of inherited disorders induced by mutations of ciliary genes and manifest in dysfunction in various organs, including the olfactory system. It is believed that ciliopathy induced olfactory dysfunction is caused by defects in the multi-cilia of mature olfactory sensory neurons (mOSNs) which possess the machinery necessary for odorant detection. This proposal provides evidence that immature OSNs (iOSNs) possess primary cilia that express ARL13B, a canonical ciliary marker. ARL13B is a GTPase and a causative gene for the ciliopathy, Joubert syndrome (JS). There is a profound gap in knowledge whereby the role of ARL13B in OSNs is unknown and it is unclear if JS patients suffer from smell impairment. Intriguingly, ARL13B not only localizes to cilia but was also discovered outside of the cilium and implicated in neurite development. Few studies have explored the function of ARL13B outside of the cilium in neurons and the reports show variable effects, suggesting that this may be neuronal cell-type specific. Preliminary data in this proposal show that the deletion of ARL13B under the OMP promoter causes deformations in the structure and function of glomeruli where OSNs synapse in the olfactory bulb. Although ARL13B is widely considered a canonical marker of cilia, it is surprisingly not detected in the cilia of mOSNs which express OMP, suggesting a role of ARL13B in OSNs outside of the cilium. The central hypothesis of this proposal is that in OSNs, extraciliary ARL13B is integral for proper axon innervation of glomeruli in the olfactory bulb. The following two aims will test this hypothesis. Aim 1: Measure the effects of ARL13B loss in OSNs on axon innervation to glomeruli in the olfactory bulb. Experiments proposed in Aim 1 will use (1) adenoviral gene expression coupled with fluorescence microscopy to quantify axonal arborization and synapse formation as well as (2) electrophysiology to measure synaptic transmission from OSNs to their projection neurons in the OB of Arl13b OSN null mice. Aim 2: Elucidate the contribution of ciliary versus extraciliary ARL13B on the glomerular defects induced by the loss of ARL13B in OSNs. To test Aim 2, I will use a global knock-in mouse model with a ciliary excluded form of ARL13B. Also, adenoviral gene replacement experiments will be conducted to assess the ability of the ciliary excluded form of ARL13B to rescue the glomerular impairments in fully developed OSNs. My results will provide novel insight into how ARL13B controls axon synapse in olfactory sensory neurons and would be the first to show the penetrance of JS mutations/deletions in the olfactory system. The findings of this proposal will have broad implications for mechanisms of ciliopathy induced olfactory dysfunction, calling for the consideration of roles for ciliopathy proteins outside of the cilium in the olfactory system.

项目概要/摘要 纤毛病是一类由纤毛基因突变引起的遗传性疾病，表现为功能障碍 分布于各个器官，包括嗅觉系统。据认为，纤毛病引起的嗅觉功能障碍是 由成熟嗅觉感觉神经元 (mOSN) 的多纤毛缺陷引起，该神经元具有 气味检测所需的机械。该提案提供了不成熟的 OSN（iOSN）的证据 拥有表达 ARL13B（一种典型的纤毛标记）的初级纤毛。 ARL13B 是一种 GTP 酶，也是一种致病因子 纤毛病、朱伯特综合征（JS）的基因。知识上存在巨大差距，因此 OSN 中的 ARL13B 未知，也不清楚 JS 患者是否患有嗅觉障碍。 有趣的是，ARL13B 不仅定位于纤毛，还在纤毛外部被发现并与 神经突发育。很少有研究探讨 ARL13B 在神经元和纤毛之外的功能 报告显示了不同的影响，表明这可能是神经元细胞类型特异性的。初步数据在 该提议表明，OMP 启动子下 ARL13B 的缺失会导致结构变形 以及 OSN 在嗅球中突触的肾小球的功能。尽管 ARL13B 被广泛认为是 纤毛的典型标志物，令人惊讶的是在表达 OMP 的 mOSN 的纤毛中没有检测到它，这表明 ARL13B 在纤毛外 OSN 中的作用。该提案的中心假设是在 OSN 中， 纤外 ARL13B 对于嗅球中肾小球的适当轴突神经支配是不可或缺的。下面两个 目标将检验这一假设。 目标 1：测量 OSN 中 ARL13B 缺失对嗅球中肾小球轴突神经支配的影响。 目标 1 中提出的实验将使用 (1) 腺病毒基因表达与荧光显微镜相结合 量化轴突分枝和突触形成以及 (2) 电生理学测量突触 在 Arl13b OSN 缺失小鼠的 OB 中，从 OSN 到投射神经元的传输。 目标 2：阐明睫状 ARL13B 与睫状外 ARL13B 对由以下因素引起的肾小球缺陷的影响 OSN 中 ARL13B 的丢失。为了测试 Aim 2，我将使用排除了纤毛的全局敲入小鼠模型 ARL13B 的形式。此外，还将进行腺病毒基因替换实验，以评估腺病毒基因替换的能力。 ARL13B 的纤毛排除形式可挽救完全发育的 OSN 中的肾小球损伤。 我的结果将为 ARL13B 如何控制嗅觉感觉神经元中的轴突突触提供新的见解 将是第一个显示嗅觉系统中 JS 突变/缺失的外显率的人。本次调查结果 该提案将对纤毛病引起的嗅觉功能障碍的机制产生广泛的影响，呼吁 考虑嗅觉系统中纤毛之外的纤毛病蛋白的作用。