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

前角细胞变性和肌肉失神经是脊髓性肌萎缩症(SMA)的主要临床特征，并在SMA小鼠模型中得到很好的表现。SMA I型小鼠模型的初级运动神经元在较小的生长锥体中表现出轴突延长和肌动蛋白mRNA和蛋白水平的降低。分化缺陷对应于神经末梢由于N型特异性电压门控钙通道积累减少而导致的兴奋性受损(Cav2.2)。这些数据提出了一个问题，例如生长因子受体及其相应的信号通路，对运动神经元的分化和成熟至关重要，是否在SMN缺陷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，在SMN缺陷的运动神经元的生长锥体中，脑源性神经营养因子(BDNF)受体的水平降低，称为原肌球蛋白受体-激酶B(TrkB)。基于这一观察，我们想要详细研究BDNF/TrkB信号如何与SMN缺陷运动神经元中受影响的肌动蛋白细胞骨架、兴奋性和神经传递相对应。该项目将分为三个部分。在第一部分中，我们将关注对照运动神经元和SMN缺陷运动神经元中TrkB在生长锥处移位的时间进程，以了解肌动蛋白动力学如何干扰TrkB移位到神经肌肉终板突触前隔区的细胞表面。由于我们从我们的初步研究中知道，在SMN缺乏的生长锥体中，磷酸化TrkB的水平显著降低，在项目的第二部分，我们将试图弄清楚TrkB下游的哪些信号通路(MAK激酶或Akt/mTOR通路)支持对照运动神经元中突触前的适当分化和成熟，以及哪些受到SMN缺陷运动神经元的影响。一般来说，这些途径是否直接导致突触小泡释放概率的改变，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实验完成，重点是细胞内肌动蛋白转录和蛋白水平的挽救，以了解SMN缺陷运动神经元中BDNF/TrkB信号缺陷、兴奋性障碍和分化缺陷是否可以通过肌动蛋白细胞骨架的正确形成来补偿。因此，我们的预期结果对于更好地理解SMA的病理生理学具有重要意义，在SMA中，突触前兴奋性、肌动蛋白动力学和神经传递受到影响，并被认为是病因中的早期事件。

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.