O'Connor Timothy P, Cockburn Katie, Wang Wenyan, Tapia Lucia, Currie Erin, Bamji Shernaz X
Department of Cellular & Physiological Sciences & the Brain Research Centre, University of British Columbia, Vancouver, Canada.
Neural Dev. 2009 May 23;4:18. doi: 10.1186/1749-8104-4-18.
Semaphorins are known to play an important role in axon guidance and growth by triggering dynamic rearrangements of the actin cytoskeleton in the neuronal growth cone. Intriguingly, some of these guidance molecules are persistently expressed after axonal pathfinding and target recognition are completed. Although their function at these later stages is poorly understood, recent findings suggest a role for these proteins in regulating synaptic connections.
Here we demonstrate that semaphorin 5B (Sema5B) regulates the elimination of synaptic connections in cultured hippocampal neurons. We show that Sema5B is proteolytically processed in neonatal brains and primary hippocampal cultures, resulting in the secretion of Sema5B fragments that include the biologically active semaphorin domain. Overexpression of full-length Sema5B in hippocampal neurons reduces synapse number while expression of a Sema5B construct lacking the semaphorin domain has no effect. Moreover, bath application with the proteolytically processed, secreted fragments containing the semaphorin domain of Sema5B, results in a rapid elimination of synaptic connections as demonstrated by time-lapse imaging. Conversely, depletion of endogenous Sema5B using RNA interference results in a significant increase in synapse number as well as a significant increase in the size of presynaptic and postsynaptic compartments.
Our results demonstrate that in addition to its role as a guidance cue, Sema5B regulates the development and maintenance of synapse size and number in hippocampal neurons. In addition, proteolytic cleavage of Sema5B results in the release of a potentially diffusible semaphorin domain that is a necessary component for its biological function in the regulation of synapse morphology.
已知信号素在轴突导向和生长中发挥重要作用，通过触发神经元生长锥中肌动蛋白细胞骨架的动态重排来实现。有趣的是，其中一些导向分子在轴突寻路和靶标识别完成后仍持续表达。尽管它们在这些后期阶段的功能尚不清楚，但最近的研究结果表明这些蛋白质在调节突触连接中发挥作用。
在此我们证明信号素5B（Sema5B）调节培养的海马神经元中突触连接的消除。我们表明Sema5B在新生大脑和原代海马培养物中进行蛋白水解加工，导致分泌包含生物活性信号素结构域的Sema5B片段。海马神经元中全长Sema5B的过表达减少了突触数量，而缺乏信号素结构域的Sema5B构建体的表达则没有影响。此外，用含有Sema5B信号素结构域的蛋白水解加工后的分泌片段进行浴应用，通过延时成像表明会导致突触连接的快速消除。相反，使用RNA干扰耗尽内源性Sema5B会导致突触数量显著增加以及突触前和突触后区室大小显著增加。
我们的结果表明，除了作为导向信号的作用外，Sema5B还调节海马神经元中突触大小和数量的发育和维持。此外，Sema5B的蛋白水解切割导致释放潜在可扩散的信号素结构域，这是其在调节突触形态的生物学功能中的必要组成部分。
