Mechanism of Sonic hedgehog-induced axon growth and guidance

这项建议的目的是了解蛋白质Sonic Hedgehog (Shh)，以浓度依赖的方式调节视网膜神经节细胞轴突的生长和引导。 具体地说，将研究Shh结合的受体(S)，以确定是否对两者的反应都较低 而高浓度Shh是由一个或两个不同的受体介导的，这已经被 报道了轴突对引导因子WNT3的浓度依赖反应。A联席- 将使用标记的Shh蛋白进行免疫沉淀实验，以确定所有候选 Shh与轴突上的受体结合。轴突上候选受体的表达将被研究 然后进行功能研究，以确定轴突的生长反应是否需要受体 嘘。与Shh信号蛋白Smoothens(Smo)相互作用的蛋白质也将被研究。它 已经证明，SMO在低和高水平的轴突生长反应的转导中都是必需的。 浓度和Smo结合蛋白是在其他系统中传递刺猬信号所必需的。 为了确定轴突中的结合伙伴，使用抗Smo抗体和 将以Smo胞质尾部为诱饵进行酵母双杂交筛选。检查嘘 在受体和下行信号水平上的信号将使我们更好地理解 Shh以浓度依赖的方式影响轴突生长的分子机制 一般而言，轴突引导因子如何引导轴突生长。 总体而言，拟议的研究与NINDS的使命有关，因为它将导致更好的 了解神经回路是如何在发育过程中建立的，这一过程对所有 神经功能。此外，它还可能有助于开发脊髓损伤的治疗方法，其中 轴突受到不可修复的损害，并导致对可能导致的神经紊乱的更好理解 由异常回路形成引起，包括癫痫和自闭症。 了解在发育过程中调节轴突生长的机制将对 正在开发脊髓损伤的治疗方法，其中神经连接受损，对其有 目前还没有治愈方法。此外，有人建议，一些神经障碍，包括自闭症和 癫痫，可能是大脑发育过程中异常的神经回路形成所致。因此，研究如何 轴突生长将对确定某些神经疾病的原因和开发治疗方法至关重要 适用于患有衰弱脊髓损伤的人。

The objective of this proposal is to understand the mechanism by which the protein, Sonic hedgehog (Shh), acts in a concentration-dependent manner to regulate retinal ganglion cell axon growth and guidance. Specifically, the receptor(s) to which Shh binds will be investigated to determine if the response to both low and high concentrations of Shh is mediated by one receptor or two different receptors, which has been reported for the concentration-dependent response of axons to the guidance factor, Wnt3. A co- immunoprecipitation experiment will be performed using tagged Shh protein to identify all candidate receptors to which Shh binds on axons. Expression of candidate receptors on axons will be investigated followed by functional studies to determine if the receptorsare required for the growth response of axons to Shh. Proteins that interact with the Shh signaling protein, Smoothened (Smo), will also be investigated. It has been shown that Smo is required for transducing the axon growth responses at both low and high concentrations and Smo binding proteins are required for transducing the hedgehog signal in other systems. To identify binding partners in axons, co-immunoprecipitation experiments using an anti-Smo antibody and a yeast two-hybrid screen using the cytoplasmic tail of Smo as bait will be performed. Examining Shh signaling at both the receptor and downstreamsignaling levels will lead to a better understanding of the molecular mechanisms by which Shh affects axon growth in a concentration-dependent manner and, more generally, how axon guidance factors may direct axongrowth. Overall, the proposed research relates to the mission of the NINDS in that it will lead to a better understanding of how the neural circuitry is established during development, a process that is critical for all neurological functions. Also, it may aid in the development of therapies for spinal cord injuries, in which axons are irreparably damaged, and lead to a better understanding of neurological disorders that may result from abnormal circuit formation, including epilepsy and autism. Understanding the mechanisms that regulate axon growth during development will be important in developing therapies for spinal cord injuries, in which nerve connections are damaged and for which there is currently no cure. Also, it has been suggested that some neurological disorders, including autism and epilepsy, may result from abnormal circuit formation during brain development. Therefore, researching how axons grow will be critical for identifying the causes of some neurological disorders and in developing a cure for people with debilitating spinal cord injuries.