Role of Neurotrophins in the Development of the Mammalian Nervous System

TrkB和TrkC编码许多同种型，包括缺乏催化酪氨酸激酶结构域的那些。关于这些激酶缺陷型在Trk信号传导中的功能知之甚少。体外研究和我们自己的体内研究已经表明，截短的Trk受体可以以显性负性方式或通过配体螯合抑制激酶活性受体亚型的功能。然而，这种活动的生理相关性仍不清楚。截短受体的细胞内结构域的高度序列保守性表明与细胞质蛋白和信号传导能力的特异性相互作用的潜力。事实上，最近已经报道BDNF通过截短的TrkB T1受体诱导星形胶质细胞中钙波的产生。然而，TrkB T1受体与钙动员及其生理作用的分子机制仍不清楚。有趣的是，TrkB T1在唐氏综合征的16三体（Ts 16）小鼠模型的大脑中过表达50%，并且Ts 16海马神经元在培养中过早死亡。神经变性通常与人类唐氏综合征相关，TrkB T1也在阿尔茨海默病患者中过表达。为了进一步研究TrkB T1在神经元存活中的作用，我们产生了一种缺乏特异性TrkBT 1激酶缺陷型受体亚型的小鼠。该突变不引起总体表型，并且可用于校正Ts 16小鼠体内的TrkB T1水平。重要的是，TrkB T1 -/+; Ts 16小鼠的海马神经元在体外逃脱了Ts 16神经元的过早细胞死亡（多尔西等人，2006）。这是一个非常令人兴奋的结果，因为它与早期的假设形成对比，即神经退行性变是由于神经营养因子供应不足而发生的。相反，我们的研究表明，细胞死亡和存活的调节可以发生在Trk受体水平。我们现在正在研究导致TrkB. T1水平失调的分子机制，以及TrkB T1表达升高的不利影响。具体而言，我们正在解决TrkBT 1对全长TrkB受体活性和细胞内Ca++水平调节的影响。在这方面，我们发现TrkB.T1缺陷小鼠发育正常，但表现出焦虑增加，这与基底外侧杏仁核神经元的神经突的长度和复杂性的形态异常有关。通过去除一个BDNF等位基因而减少的TrkB信号传导可以通过TrkB.T1缺失而部分挽救，这通过改善与BDNF单倍不足相关的增强的攻击性和体重增加来揭示。因此，我们的研究结果提供了证据，在生理水平上，TrkB. T1受体是体内TrkB.FL信号传导的重要调节剂。此外，我们最近发现Rbfox 1（一种与智力残疾、癫痫和自闭症相关的RNA结合蛋白）的上调选择性地增加了海马TrkB.T1亚型的表达。在生理上，增加Rbfox 1损害BDNF依赖的LTP，这可以通过遗传恢复TrkB.T1水平来挽救。Rbfox 1上调与TrkB.T1同种型表达特异性增加相关的Rbfox 1基因的RNA-seq分析也表明，受Rbfox 1功能获得影响的基因与受Rbfox 1缺失影响的基因惊人地不同。这些发现不仅将TrkB确定为Rbfox 1病理生理学的主要靶点，而且还表明Rbfox 1功能的获得或丧失调节不同的遗传景观。我实验室的另一个重要研究领域是集中在细胞外和细胞内区域中Trk受体复合物结构域的功能的体内解剖，以鉴定调节受体活化和在特定细胞类型和神经元回路中的功能的机制和/或蛋白质。到目前为止，我们已经表明，在TrkA的质膜区域（KFG）的三个氨基酸的胞内结构域调节受体的泛素化和功能。在这个小鼠模型中，我们发现了基底前脑胆碱能区TrkA信号在恐惧反应和恐惧表达调节中的新功能。我们的发现对接受癌症治疗的患者也有深远的影响。表达TrkA的基底前脑胆碱能神经元调节恐惧回路并可能影响PTSD的发展，这一发现提出了一个问题，即用泛Trk抑制剂治疗癌症患者是否会使他们患PTSD的风险更高。目前，我们已经开始在最近产生的具有这种突变的小鼠模型中对TrkB受体的胞外区中的富含亮氨酸的重复（LRR）结构域的体内功能进行表征。由于LRR存在于许多神经细胞粘附分子中，并与轴突导向、靶向选择、突触形成和突触发生性粘附有关，我们希望这项工作将有助于我们确定TrkB在复杂行为调节中的分子机制。

TrkB and TrkC encode a number of isoforms, including those that lack the catalytic tyrosine kinase domain. Little is known about the function of these kinase deficient isoforms in Trk signaling. In vitro studies, and our own in vivo studies, have shown that truncated Trk receptors can inhibit the function of kinase-active receptor isoforms in a dominant-negative manner or by ligand sequestration. The physiological relevance of this activity is, however, still unclear. The high degree of sequence conservation of the intracellular domains of truncated receptors suggests the potential for specific interactions with cytoplasmic proteins and signaling capabilities. Indeed, it has been reported recently that BDNF induces the production of calcium waves in astroglia through the truncated TrkB T1 receptor. However, the molecular mechanism(s) linking the TrkB T1 receptor to calcium mobilization and its physiological role is still unknown. Interestingly, TrkB T1 is 50% overexpressed in the brain of the trisomy 16 (Ts16) mouse model of Down syndrome and Ts16 hippocampal neurons die prematurely in culture. Neurodegeneration is commonly associated with Down syndrome in humans and TrkB T1 is also overexpressed in Alzheimer's patients. To further investigate the role of TrkB T1 in neuronal survival, we generated a mouse lacking specifically the TrkBT1 kinase-deficient receptor isoform. This mutation caused no gross phenotype and could be used to correct the levels of TrkB T1 in Ts16 mice in vivo. Importantly, hippocampal neurons from TrkB T1 -/+; Ts16 mice escaped the premature cell death of Ts16 neurons in vitro (Dorsey et al. 2006). This is a very exciting result because it contrasts with earlier hypotheses that neurodegeneration occurs due to insufficient supply of neurotrophic factors. Rather, our studies suggest that modulation of cell death and survival can occur at the level of the Trk receptor. We are now investigating the molecular mechanisms leading to the dysregulation of TrkB.T1 levels and underlying the detrimental effect of elevated TrkB T1 expression. Specifically, we are addressing both the effects of TrkBT1 on the activity of the full-length TrkB receptor and on the intracellular regulation of Ca++ levels. In this respect we have found that TrkB.T1 deficient mice develop normally but show increased anxiety in association with morphological abnormalities in the length and complexity of neurites of neurons in the basolateral amygdala. In vivo reduction of TrkB signaling by removal of one BDNF allele could be partially rescued by TrkB.T1 deletion, which was revealed by an amelioration of the enhanced aggression and weight gain associated to BDNF haploinsufficiency. Thus, our results provide evidence that at the physiological level, TrkB.T1 receptors are important regulators of TrkB.FL signaling in vivo. In addition, we have recently found that upregulation of Rbfox1, an RNA binding protein associated with intellectual disability, epilepsy and autism, increases selectively hippocampal TrkB.T1 isoform expression. Physiologically, increased Rbfox1 impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels. RNA-seq analysis of hippocampi with upregulation of Rbfox1 in conjunction with the specific increase of TrkB.T1 isoform expression also shows that the genes affected by Rbfox1 gain of function are surprisingly different from those influenced by Rbfox1 deletion. These findings not only identify TrkB as a major target of Rbfox1 pathophysiology but also suggest that gain or loss of function of Rbfox1 regulate different genetic landscapes. Another important area of research of my laboratory is focused on the in vivo dissection of the function of the domains of Trk receptor complex structure in both the extracellular and intracellular regions to identify mechanisms and/or proteins that regulate receptor activation and function in specific cell types and neuronal circuitries. So far, we have shown that a three amino-acid intracellular domain in the juxtamembrane region of TrkA (KFG) regulates the receptor ubiquitination and function. With this mouse model we identified a new function of TrkA signaling in the basal forebrain cholinergic region in the regulation of fear response and fear expression. Our findings also have profound implications for patients undergoing cancer therapy. The finding that TrkA expressing Basal Forebrain Cholinergic neurons regulate fear circuitries and may influence development of PTSD raises the question of whether treatment of cancer patients with pan-Trk inhibitors put them at higher risk of developing PTSD. Currently, we have begun the characterization of the in vivo function of the leucine rich repeat (LRR) domain in the extracellular region of the TrkB receptors in a recently generated mouse model with such mutation. Since LRRs are found in many neural cell-adhesion molecules and are implicated in axon guidance, target selection, synapse formation and in synaptogenic adhesions we expect that this work will help us identify the molecular mechanism underlying TrkB function in the regulation of complex behaviors.