Analysis of Brain-derived Neurotrophic Factor Signaling in Spinal Muscular Atrophy

脊髓性肌萎缩症（SMA）的主要临床特征是前角细胞变性和肌肉去神经支配，在脊髓性肌萎缩症小鼠模型中得到了很好的体现。SMA 1型小鼠模型的初级运动神经元表现出轴突伸长改变，同时在较小的生长锥中肌动蛋白mRNA和蛋白水平降低。分化缺陷对应于神经末梢的兴奋性受损，这是由于n型特定电压门控钙通道积累减少所致（Cav2.2）。这些数据提出了一个问题，即对于运动神经元的分化和成熟至关重要的生长因子受体及其相应的信号通路的定位是否在缺smn的运动神经元中受到影响。在第一种方法中，我们可以清楚地表明，被称为原肌球蛋白受体激酶B （TrkB）的脑源性神经营养因子（BDNF）受体的水平在smn缺陷运动神经元的生长锥中降低。基于这一观察，我们希望详细研究BDNF/TrkB信号如何对应于受影响的肌动蛋白细胞骨架、兴奋性和smn缺乏运动神经元的神经传递。该项目将分为三个部分。在第一部分中，我们将重点关注对照和缺smn运动神经元生长锥上TrkB易位的时间过程，以了解肌动蛋白动力学如何干扰TrkB向神经肌肉终板突触前腔室细胞表面的转移。我们从前期研究中得知，在缺smn的生长锥中，phospho-TrkB水平显著降低，在我们项目的第二部分，我们将试图找出TrkB下游的哪些信号通路（MAK激酶或Akt/mTOR通路）支持控制运动神经元中突触前的适当分化和成熟，以及哪些信号通路在缺smn的运动神经元中受到影响。总的来说，这些途径是否直接导致突触囊泡释放概率的改变，或者是否需要新合成的蛋白质或肌动蛋白细胞骨架的快速变化，这是一个悬而未决的问题。最后，对smn缺陷运动神经元中受影响的BDNF/TrkB信号的详细分析将通过关注肌动蛋白转录物的细胞内拯救和蛋白水平的实验来完成，以了解smn缺陷运动神经元中有缺陷的BDNF/TrkB信号、兴奋性干扰和分化缺陷是否可以通过适当形成肌动蛋白细胞骨架来补偿。因此，我们的预期结果对于更好地理解SMA的病理生理非常重要，其中突触前兴奋性、肌动蛋白动力学和神经传递受到影响，并被认为是导致早期发病的原因。

Degeneration of anterior horn cells and denervation of muscles are the main clinical characteristics of spinal muscular atrophy (SMA) and well represented in SMA mouse models. Primary motoneurons from SMA type I mouse models exhibit altered axon elongation combined with reduced actin mRNA and protein levels in smaller growth cones. The differentiation defects correspond to impaired excitability at the nerve terminals due to reduced accumulation of N-type specific voltage-gated calcium channels (Cav2.2). These data raise the question whether the localization of e.g. growth factor receptors and their corresponding signaling pathways, which are important for differentiation and maturation of motoneurons, is affected in Smn-deficient motoneurons.In a first approach we could clearly show that the level of the Brain-derived Neurotrophic Factor (BDNF) receptor termed Tropomyosin-receptor-kinase B (TrkB) is reduced in growth cones of Smn-deficient motoneurons. Based on this observation we would like to investigate in detail how BDNF/TrkB signaling corresponds to affected actin cytoskeleton, excitability and neurotransmission in Smn-deficient motoneurons. The project shall be structured in three parts. In the first part we will focus on the time course of TrkB translocation at the growth cone in control and Smn-deficient motoneurons to understand how actin dynamics interfere with shifting TrkB to the cell surface in the presynaptic compartment of the neuromuscular endplate. As we know from our preliminary studies that the level of phospho-TrkB is significantly reduced in Smn-deficient growth cones, in the second part of our project we will try to figure out which signaling pathways downstream of TrkB (MAK kinase or Akt/mTOR pathway) support proper differentiation and maturation of presynapses in control motoneurons and which are affected in Smn-deficient motoneurons. It is in general an open question whether these pathways directly lead to altered release probability of synaptic vesicles or if newly synthesized proteins or rapid changes in the actin cytoskeleton are needed. Finally, the detailed analysis of affected BDNF/TrkB signaling in Smn-deficient motoneurons will be completed by experiments that focus on the intracellular rescue of actin transcript and proteins levels to understand whether defective BDNF/TrkB signaling, excitability disturbances and differentiation defects in Smn-deficient motoneurons can be compensated by proper formation of the actin cytoskeleton. Our expected results are thus important for a better understanding of the pathophysiology of SMA, in which presynaptic excitability, actin dynamics and neurotransmission are affected and thought to cause early events in the etiopathology.