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Trk受体亚型的功能。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。对这些激酶缺陷亚型在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，截断的Trk受体可以以显性阴性方式或通过配体隔离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尚不清楚。截断受体胞内结构域的高度序列保守表明其可能与细胞质蛋白和信号传导能力有特定的相互作用。事实上，最近有报道称BDNF通过截断的TrkB T1受体诱导星形胶质细胞钙波的产生。然而，TrkB T1受体与钙动员的分子机制及其生理作用尚不清楚。有趣的是，TrkB T1在16三体（Ts16）唐氏综合征小鼠模型的大脑中过度表达50%，Ts16海马神经元在培养中过早死亡。神经退行性变通常与人类唐氏综合征有关，TrkB T1在阿尔茨海默病患者中也过表达。为了进一步研究TrkBT1在神经元存活中的作用，我们制造了一只特异性缺乏TrkBT1激酶缺陷受体亚型的小鼠。该突变未引起总体表型，可用于纠正Ts16小鼠体内TrkB T1的水平。重要的是，海马神经元来自TrkB T1 -/+；Ts16小鼠在体外可避免Ts16神经元细胞过早死亡（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了对比，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受体水平上。我们现在正在研究TrkB T1表达升高的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ca2 ++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，但焦虑增加与杏仁核基底外侧神经元的神经突长度和复杂性的形态学异常有关。在体内，通过去除一个BDNF等位基因而减少的TrkB信号传导可以被TrkB部分挽救。T1缺失，这是通过改善与BDNF单倍不足相关的攻击性增强和体重增加而揭示的。因此，我们的结果提供了证据，在生理水平上，TrkB。T1受体是TrkB的重要调节因子。体内FL信号。有趣的是，在正常的非应激条件下，这些小鼠似乎没有增加对肿瘤形成的易感性。然而，我们发现来自癌症小鼠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，尽管事实上正常的神经胶质细胞表达高水平的这种受体异构体。因此，我们现在删除TrkB。T1在p53/NF1突变小鼠中的表达，以研究该受体的缺失是否会增加肿瘤易感性。截断的TrkC受体，如TrkC TK-，从未涉及细胞内信号传导。为了鉴定可能结合TrkC TK-高度保守的胞内结构域的蛋白，我们进行了酵母双杂交筛选，并鉴定了一个与TrkC TK-以配体依赖的方式特异性相互作用的衔接蛋白（GRASP/tamalin）。tamalin和TrkC TK-都在大脑中表达，具有重叠的解剖和亚细胞分布。我们还发现NT-3启动TrkCTK-/Tamalin复合物可通过adp核糖化因子6 （ARF6）激活Rac1 GTPase。NT-3结合TrkCTK-/Tamalin诱导ARF6易位到膜上，进而引起膜褶皱和细胞突起的形成。因此，我们已经证明，缺乏激酶活性的截断TrkC受体可以激活特定的细胞内信号通路，该通路将NT-3与神经元发育和可塑性的关键成分联系起来，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的上游激活剂，ARF6是内核体膜运输、内吞作用和细胞表面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Trk受体在肿瘤中的作用的重要问题。虽然Trk受体的表达已经在许多肿瘤类型中被发现（如神经母细胞瘤、髓母细胞瘤、胰腺癌、黑色素瘤等），但很难解释为什么在一些肿瘤中，一种受体的表达与良好的预后相关（如髓母细胞瘤或神经母细胞瘤中的TrkC表达），而在其他肿瘤中，它与不良预后相关（如胰腺癌中的TrkC表达）。几乎没有人注意到在这些肿瘤中表达哪些特异性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。我们的发现提出了一种有趣的可能性，即截断受体单独表达或与酪氨酸激酶同工型相关的表达可能解释了这些肿瘤的一些生长特征。因此，我们计划确定在已建立的肿瘤细胞系中是否存在Trk受体亚型的差异表达。我们还想确定调节特定受体亚型的表达是否能影响肿瘤细胞的增殖和转移潜能。综上所述，我们的研究结果表明，截断的Trk受体影响参与细胞存活、囊泡运输和细胞运动的信号传导。这些都是在病理条件下发生改变的关键细胞生物学过程。因此，我们计划在体外继续对截断的Trk受体活性进行解剖，并利用我们生成的条件小鼠模型进一步扩展我们在体内的分析。