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

项目摘要/摘要 纤毛疾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 在不同的器官，包括嗅觉系统。据认为，睫毛病变导致的嗅觉功能障碍 由成熟嗅觉神经元(MOSNs)的多纤毛缺陷引起的 气味检测所需的机械。这一提议提供了证据，证明未成熟的光网络(IOSNs) 具有表达Arl13b的初级纤毛，Ar13b是一种典型的纤毛标记。Arl13b是一种GTP酶，是一种致病因子 纤毛疾病的基因，Joubert综合征(JS)。在知识上存在着一种深刻的鸿沟，因此 OSNs中的Arl13b尚不清楚，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，而且在纤毛外也被发现，并与 轴突发育。很少有研究探索Arl13b在纤毛外神经元和 这些报告显示了不同的影响，表明这可能是神经细胞类型特有的。第一季度初步数据 这一建议表明，OMP启动子下Arl13b的缺失会导致结构变形 以及OSNs在嗅球内突触的肾小球功能。尽管Arl13b被广泛认为是一种 纤毛的典型标记，令人惊讶的是在表达OMP的mOSNs的纤毛中没有检测到它，这表明 Arl13b在纤毛外OSNs中的作用这一提议的中心假设是，在OSN中， Arl13b是嗅球内肾小球正常轴突神经支配所必需的。以下两个 AIMS将检验这一假设。 目的1：检测嗅核Arl13b缺失对嗅球内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的方法 量化轴突树枝和突触的形成以及(2)电生理学测量突触 Ar13b OSN基因缺失小鼠OB内OSNs向其投射神经元的传递 目的2：阐明纤毛Arl13b与胞外Arl13b在慢性肾小球损伤中的作用。 Ar13b在OSN中的损失。为了测试目标2，我将使用不包括纤毛的全局敲入小鼠模型 本规则第13B条的格式。此外，还将进行腺病毒基因替换实验，以评估 纤毛排除形式的Arl13b挽救发育完全的OSNs的肾小球损害。 我的结果将为Arl13b如何控制嗅觉感觉神经元中的轴突突触和 将是第一个显示嗅觉系统中JS突变/缺失的外显性的人。这项研究的发现 该提案将对纤毛疾病引起的嗅觉障碍的机制产生广泛的影响，呼吁 纤毛外纤毛病变蛋白在嗅觉系统中的作用。

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.