Role of Neurotrophins in the Development of the Mammalian Nervous System

神经营养素在哺乳动物神经系统发育中的作用

• 批准号: 8348996
• 负责人: Lino Tessarollo
• 金额: $ 52.5万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348996
• 关键词: ADP-ribosylation factor 6; Accounting; Actins; Adaptor Signaling Protein; Address; Adverse effects; Affect; Affinity; Aggressive behavior; Alleles; Alzheimer' s Disease; Amygdaloid structure; Animal Model; Anxiety; Astrocytes; Attention; Binding; Biological; Biological Process; Brain; Brain-Derived Neurotrophic Factor; Calcium Oscillations; Cell Death; Cell Differentiation process; Cell Line; Cell Survival; Cell surface; Cells; Characteristics; Clinic; Clinical Trials; Complex; Cytoplasmic Protein; Cytoskeleton; Development; Disease; Dissection; Dominant-Negative Mutation; Down Syndrome; Endocytosis; Environment; Excision; Family; Genes; Glioblastoma; Goals; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Human; In Vitro; Knowledge; Length; Ligands; Link; Maintenance; Malignant Neoplasms; Mediating; Membrane; Membrane Protein Traffic; Mind; Molecular; Molecular Mechanisms of Action; Mus; Mutant Strains Mice; Mutation; NF1 gene; NGFR Protein; Neoplasms; Nephroblastoma; Nerve Degeneration; Nervous system structure; Neurites; Neuroblastoma; Neurodegenerative Disorders; Neuroglia; Neuronal Plasticity; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 2; Neurotrophic Tyrosine Kinase Receptor Type 3; Normal Cell; Organism; Outcome; Pancreatic carcinoma; Parkinson Disease; Pathway interactions; Patients; Peptides; Peripheral; Phenotype; Phosphotransferases; Physiological; Predisposition; Process; Production; Property; Prostate carcinoma; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Reporting; Research; Role; Signal Pathway; Signal Transduction; Stress; TP53 gene; Trisomy 16; Tumor Cell Line; Tyrosine Kinase Domain; Vertebrates; Weight Gain; Yeasts; cell motility; endosome membrane; improved; in vitro activity; in vivo; interest; medulloblastoma; melanoma; mouse Trisomy 16; mouse model; neoplastic cell; neuron development; neuronal survival; neurotrophic factor; neurotrophin 4; outcome forecast; overexpression; preclinical study; premature; receptor; release of sequestered calcium ion into cytoplasm; therapeutic target; tumor; tumor growth; yeast two hybrid system

Function of kinase-deficient Trk receptor isoforms. 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 mechanism 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. Interestingly, these mice do not appear to have increased susceptibility to tumor formation, in normal non stressed conditions. However, we found that glioblastoma cell lines derived from a mouse model of cancer, lacking the p53 and NF1 genes, do not express any TrkB.T1 receptors despite the fact that normal glia cells express high levels of this receptor isoform. Therefore, we are now deleting TrkB.T1 in the p53/NF1 mutant mouse to investigate whether loss of this receptor increases tumor susceptibility. Truncated TrkC receptors, such as TrkC TK-, have never been implicated in intracellular signaling. To identify proteins that might bind the highly conserved intracellular domain of TrkC TK-, we conducted a yeast two-hybrid screen and identified an adaptor protein (GRASP/tamalin) that interacts specifically with TrkC TK- in a ligand dependent manner. Both tamalin and TrkC TK- are expressed in the brain with overlapping anatomical and subcellular distribution. We also found that NT-3 initiation of the TrkCTK-/Tamalin complex leads to activation of Rac1 GTPase through the ADP-ribosylation factor 6 (ARF6). NT-3 binding to TrkCTK-/Tamalin induces ARF6 translocation to the membrane, which in turn causes membrane ruffling and formation of cellular protrusions. Thus, we have shown that a truncated TrkC receptor lacking kinase activity can activate a specific intracellular signaling pathway that links NT-3 to key components of neuronal development and plasticity, such as regulation of the actin cytoskeleton and membrane trafficking. Moreover, we have established NT-3 as an unsuspected upstream activator of ARF6, a regulator of endosome membrane trafficking, endocytosis and actin remodeling at the cell surface; processes that are important for cell motility. Our studies showing that TrkC TK- can affect cell motility raise an important question concerning the role of Trk receptors in tumors. While expression of Trk receptors has been found in many tumor types (e.g. neuroblastoma, medulloblastoma, pancreatic carcinoma, melanoma, etc.), it has been difficult to explain how expression of one receptor in some tumors is associated with a favorable outcome (e.g. TrkC expression in medulloblastoma or neuroblastoma) while in other tumors it is linked to a negative prognosis (e.g. TrkC expression in pancreatic carcinoma). Virtually no attention has been paid to which specific Trk receptor isoforms (kinase active or deficient) are expressed in these tumors and whether they confer specific biological properties to neoplastic growth. Our findings raise the intriguing possibility that expression of truncated receptors alone or in association with the tyrosine kinase isoforms may account for some of these tumor growth characteristics. Thus, we plan to determine whether there is differential expression of Trk receptor isoforms among established tumor cell lines. We also want to determine whether modulating the expression of specific receptor isoforms can influence the proliferative and metastatic potential of tumor cells. Taken together, our findings suggest that truncated Trk receptors affect signaling involved in cell survival, vesicular transport and cell motility. These are all key cell biological processes that are altered in pathological conditions. Thus, we plan to continue our dissection of truncated Trk receptor activities in vitro and further extend our analysis in vivo by using the conditional mouse models we have generated.

激酶缺陷型 Trk 受体亚型的功能。 TrkB 和 TrkC 编码许多同种型，包括那些缺乏催化酪氨酸激酶结构域的同种型。人们对这些激酶缺陷同种型在 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表明，截短的 Trk 受体可以以显性失活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发生特异性相互作用的潜力。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中钙波的产生。然而，TrkB T1 受体与钙动员的分子机制及其生理作用仍不清楚。有趣的是，TrkB T1 在唐氏综合症 16 三体 (Ts16) 小鼠模型的大脑中过度表达 50%，并且 Ts16 海马神经元在培养物中过早死亡。神经退行性疾病通常与人类唐氏综合症相关，TrkB T1 在阿尔茨海默病患者中也过度表达。为了进一步研究 TrkB T1 在神经元存活中的作用，我们培育了一只专门缺乏 TrkBT1 激酶缺陷型受体亚型的小鼠。该突变不会引起总体表型，可用于纠正 Ts16 小鼠体内 TrkB T1 的水平。重要的是，来自 TrkB T1 -/+ 的海马神经元； Ts16 小鼠在体外逃脱了 Ts16 神经元的过早细胞死亡（Dorsey et al. 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鲜明对比，即神经变性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 TrkB T1 表达升高产生有害影响的分子机制。具体来说，我们正在研究 TrkBT1 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，但表现出与基底外侧杏仁核神经元神经突长度和复杂性形态异常相关的焦虑增加。通过删除一个 BDNF 等位基因而导致的体内 TrkB 信号传导减少可以通过 TrkB.T1 删除来部分挽救，这通过改善与 BDNF 单倍体不足相关的攻击性增强和体重增加来揭示。因此，我们的结果提供了证据，证明在生理水平上，TrkB.T1 受体是体内 TrkB.FL 信号传导的重要调节因子。有趣的是，在正常的非应激条件下，这些小鼠似乎并没有增加对肿瘤形成的易感性。然而，我们发现源自小鼠癌症模型的胶质母细胞瘤细胞系缺乏 p53 和 NF1 基因，不表达任何 TrkB.T1 受体，尽管正常胶质细胞表达高水平的这种受体亚型。因此，我们现在在 p53/NF1 突变小鼠中删除 TrkB.T1，以研究该受体的缺失是否会增加肿瘤易感性。截短的 TrkC 受体，例如 TrkC TK-，从未与细胞内信号传导有关。为了鉴定可能结合 TrkC TK- 高度保守的胞内结构域的蛋白质，我们进行了酵母双杂交筛选，并鉴定了一种接头蛋白 (GRASP/tamalin)，它以配体依赖性方式与 TrkC TK- 特异性相互作用。 tamalin 和 TrkC TK- 均在大脑中表达，具有重叠的解剖和亚细胞分布。我们还发现 TrkCTK-/Tamalin 复合物的 NT-3 启动导致通过 ADP-核糖基化因子 6 (ARF6) 激活 Rac1 GTPase。 NT-3 与 TrkCTK-/Tamalin 结合诱导 ARF6 易位到膜上，进而导致膜皱起和细胞突起的形成。因此，我们发现缺乏激酶活性的截短 TrkC 受体可以激活特定的细胞内信号通路，将 NT-3 与神经元发育和可塑性的关键组成部分联系起来，例如肌动蛋白细胞骨架和膜运输的调节。此外，我们已经确定 NT-3 是 ARF6 的一个未被怀疑的上游激活剂，ARF6 是细胞表面内体膜运输、内吞作用和肌动蛋白重塑的调节剂；对细胞运动很重要的过程。我们的研究表明 TrkC TK- 可以影响细胞运动，这提出了一个关于 Trk 受体在肿瘤中的作用的重要问题。虽然在许多肿瘤类型（例如神经母细胞瘤、髓母细胞瘤、胰腺癌、黑色素瘤等）中发现了 Trk 受体的表达，但很难解释某些肿瘤中一种受体的表达如何与有利的结果相关（例如 TrkC髓母细胞瘤或神经母细胞瘤中的表达），而在其他肿瘤中，它与阴性预后相关（例如胰腺癌中的 TrkC 表达）。事实上，没有人关注这些肿瘤中表达了哪些特定的 Trk 受体亚型（激酶活性或缺陷）以及它们是否赋予肿瘤生长特定的生物学特性。我们的研究结果提出了一个有趣的可能性，即截短受体的单独表达或与酪氨酸激酶亚型相关的表达可能解释了其中一些肿瘤生长特征。因此，我们计划确定已建立的肿瘤细胞系中 Trk 受体亚型是否存在差异表达。我们还想确定调节特定受体亚型的表达是否可以影响肿瘤细胞的增殖和转移潜力。 综上所述，我们的研究结果表明，截短的 Trk 受体会影响细胞存活、囊泡运输和细胞运动相关的信号传导。这些都是在病理条件下发生改变的关键细胞生物过程。因此，我们计划继续在体外剖析截短的 Trk 受体活性，并通过使用我们生成的条件小鼠模型进一步扩展我们的体内分析。