Mechanism of Sonic hedgehog-induced axon growth and guidance

描述（由申请人提供）：本提案的目的是了解蛋白质Sonic hedgehog （Shh）以浓度依赖性方式调节视网膜神经节细胞轴突生长和引导的机制。具体来说，我们将研究Shh结合的受体，以确定对低浓度和高浓度Shh的反应是由一个受体还是两个不同的受体介导的，这已经被报道为轴突对引导因子Wnt3的浓度依赖性反应。将使用标记Shh蛋白进行共免疫沉淀实验，以鉴定Shh在轴突上结合的所有候选受体。候选受体在轴突上的表达将被研究，随后将进行功能研究，以确定这些受体是否需要轴突对Shh的生长反应。与Shh信号蛋白相互作用的蛋白Smoothened （Smo）也将被研究。研究表明，在低浓度和高浓度下，轴突生长反应的转导都需要Smo，而在其他系统中，hedgehog信号的转导也需要Smo结合蛋白。为了确定轴突上的结合伙伴，将使用抗Smo抗体和酵母双杂交筛选进行共免疫沉淀实验，以Smo的细胞质尾部为诱饵。在受体和下游信号水平上检查Shh信号将有助于更好地理解Shh以浓度依赖的方式影响轴突生长的分子机制，更一般地说，轴突引导因子如何指导轴突生长。总的来说，拟议的研究与NINDS的使命有关，因为它将使人们更好地了解神经回路在发育过程中是如何建立的，这一过程对所有神经功能都至关重要。此外，它可能有助于开发脊髓损伤的治疗方法，其中轴突不可修复地受损，并导致更好地了解可能由异常电路形成导致的神经系统疾病，包括癫痫和自闭症。了解发育过程中调节轴突生长的机制对于开发脊髓损伤的治疗方法非常重要，脊髓损伤中神经连接受损，目前尚无治愈方法。此外，一些神经系统疾病，包括自闭症和癫痫，可能是由于大脑发育过程中异常的电路形成造成的。因此，研究轴突是如何生长的，对于确定某些神经系统疾病的原因，以及为患有衰弱性脊髓损伤的人开发治疗方法至关重要。

DESCRIPTION (provided by applicant): 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 receptors are 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 downstream signaling 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 axon growth. 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.