Elucidation of the molecular mechanisms and physiological significance of the structural stabilization of neural networks by neurotrophins

本研究的目的是通过分析脑源性神经营养因子（BDNF）受体TrkB的亚型来阐明神经网络稳定的分子机制和生理意义。为了实现这一目的，首先，我制备了表达能够干扰TrkB亚型表达的TrkB亚型siRNA的慢病毒载体。另外，为了检测T1在蛋白质水平上的敲减是否发生，制备了抗T1抗体，得到了T1的优良抗体。接下来，为了确立向胎儿脑室注射慢病毒载体的方法，在各种条件下进行了载体的注射。然而，注射后胚胎的发育并没有很好地进行。另一方面，当用载体感染P7的新皮层神经元或新皮层神经元的原代培养物时，发现T1 siRNA病毒载体感染的神经元树突变长，神经元树突分支点增多，明确GABA能神经元是由第1层抑制性神经元祖细胞产生的（L1-INP细胞）（Ohira等人，Nature Neuroscience 2010）。在哺乳动物CNS中存在两种TrkB受体亚型。全长同种型TK+是典型的酪氨酸激酶受体。相反，截短的同种型T1在胞内结构域中具有同种型特异性的11个氨基酸。有趣的是，T1在几乎所有的L1-INP细胞中表达，而表达TK+的L1-INP细胞的比例很低，这表明T1在L1-INP细胞的增殖和分化中起作用。

The aim of this present study is the clarification of the molecular mechanisms and physiological significance of the stabilization of neural networks by analyzing the subtypes of brain-derived neurotrophic factor (BDNF) receptor TrkB. To achieve this purpose, first of all, I made the lentivirus vectors which expressed siRNAs of TrkB subtypes that were able to interfere with the expression of TrkB subtypes. In addition, the anti-T1 antibody was made to detect whether the knockdown of T1 at the protein level occurred, and I obtained an excellent antibody for T1.Next, to establish an injection method of the lentivirus vectors into the cerebral ventricles of the fetals, I performed injections of the vectors at various conditions. However, the developments of the embryos after the injection have not advanced well. On the other hand, when the neocortical neurons at P7 or the primary cultures of neocortical neurons were infected with the vectors, I found that the dendritic processes of neurons infected with T1 siRNA virus vectors became long and the number of the branching points of neuronal processes increased.We have clarified that GABAergic neurons are generated from layer 1 inhibitory neuron progenitor cells (L1-INP cell) by ischemia (Ohira et al., Nature Neuroscience 2010). There are two TrkB receptor subtypes in the mammalian CNS. The full-length isoform TK+ is a typical tyrosine kinase receptor. In contrast, the truncated isoform T1 possesses the isoform specific 11 amino acids in the intracellular domain. Interestingly, T1 was expressed in almost all L1-INP cells, whereas the ratio of L1-INP cells expressing TK+ was low, suggesting that T1 plays a role in the proliferation and differentiation of L1-INP cells.