Role of Neurotrophins in the Development of the Mammalian Nervous System

TrkB和TrkC编码许多异构体，包括那些缺乏催化酪氨酸激酶结构域的异构体。目前对这些激酶缺失亚型在Trk信号转导中的作用知之甚少。体外研究和我们自己的体内研究表明，截短的Trk受体可以显性-负性方式或通过配体隔离来抑制激酶活性受体亚型的功能。然而，这一活动的生理相关性仍不清楚。截短受体胞内区的高度序列保守性表明有可能与细胞质蛋白和信号转导能力发生特定的相互作用。事实上，最近有报道称，BDNF通过截短的TrkB T1受体诱导星形胶质细胞产生钙波。然而，TrkBT1受体与钙动员的分子机制(S)及其生理作用尚不清楚。有趣的是，TrkB T1在唐氏综合症16三体(TS16)小鼠模型的大脑中有50%的过度表达，TS16海马神经元在培养中过早死亡。在人类中，神经退行性变通常与唐氏综合症有关，而TrkB T1在阿尔茨海默病患者中也过度表达。为了进一步研究TrkBT1在神经元存活中的作用，我们培育了一只缺乏TrkBT1激酶缺陷受体亚型的小鼠。该突变不引起大体表型，可用于体内校正TS16小鼠TrkB T1的水平。重要的是，来自TrkB T1-/+；TS16小鼠的海马神经元在体外避免了TS16神经元的细胞过早死亡(Dorsey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了鲜明对比，即神经退行性变是由于神经营养因子供应不足造成的。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受体的水平上。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表达升高的潜在有害影响。具体地说，我们正在研究TrkBT1对全长TrkB受体活性和细胞内钙水平调节的影响。在这方面，我们发现TrkB.T1缺陷小鼠发育正常，但表现出更多的焦虑，与杏仁基底外侧核神经元突起的长度和复杂性的形态异常有关。在体内，通过去除一个BDNF等位基因而导致的TrkB信号的减少可以被TrkB.T1缺失部分挽救，这表现为与BDNF单倍性不足相关的增强的攻击性和体重增加的改善。因此，我们的结果提供了证据，在生理水平上，TrkB.T1受体是体内TrkB.FL信号的重要调节因子。此外，我们最近发现，与智能障碍、癫痫和自闭症相关的RNA结合蛋白RBFOX1的上调，选择性地增加了海马TrkB.T1亚型的表达。从生理上讲，增加的RBFOX1损害了BDNF依赖的LTP，这可以通过基因恢复TrkB.T1水平来挽救。对RBFOX1上调伴随TrkB.T1亚型表达特异性增加的海马区的RNA-SEQ分析也表明，受RBFOX1功能获得影响的基因与受RBFOX1缺失影响的基因惊人地不同。这些发现不仅证实了TrkB是RBFOX1病理生理学的主要靶点，而且也表明RBFOX1功能的获得或丧失调节着不同的遗传格局。我的实验室的另一个重要研究领域是在体内解剖Trk受体复合体结构域在细胞外和细胞内的功能，以确定调节特定细胞类型和神经元回路中受体激活和功能的机制和/或蛋白质。到目前为止，我们已经证明TrkA(KFG)膜旁区域的一个三个氨基酸的胞内区调节受体的泛素化和功能。通过这个小鼠模型，我们在基底前脑胆碱能区发现了TrkA信号在调节恐惧反应和恐惧表达中的一个新功能。我们的发现对接受癌症治疗的患者也有深远的影响。TrkA表达的基底前脑胆碱能神经元调节恐惧回路，并可能影响创伤后应激障碍的发展，这一发现提出了一个问题，即使用泛Trk抑制剂治疗癌症患者是否会使他们患上创伤后应激障碍的风险更高。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表征TrkB受体胞外区富含亮氨酸重复序列(LRR)结构域的体内功能。由于LRRs存在于许多神经细胞黏附分子中，并与轴突引导、靶点选择、突触形成和突触形成有关，我们希望这项工作将有助于我们了解TrkB在复杂行为调控中的分子机制。

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.