CYTOSKELETAL TARGETS OF NEURONAL SIGNALING

该建议涉及神经元细胞骨架的分子控制 蛋白质的细胞外信号。 了解神经元 细胞骨架的调节是重要的，因为几种疾病的过程， 包括阿尔茨海默病和精神分裂症被认为会影响 细胞骨架，从而改变神经元功能。 在生长过程中， 发育和突触发生时，神经元的形态受到控制 由生长因子和神经递质等局部环境因素决定。 成年神经元的微观结构也被认为是修改， 对突触或神经体液活动的反应。 我们建议研究 神经元形态可塑性的细胞基础， 调节微管的磷酸化状态和功能- 相关蛋白MAP 2。 MAP 2被认为在 树枝状形态的发展和维持，通过其 与微管和其他细胞骨架元件的相互作用。 多 已知蛋白激酶和蛋白磷酸酶修饰MAP 2 磷酸化和MAP 2的功能，但很少有人知道， MAP 2在体内磷酸化的特性。 我们以前的研究 揭示了一个信号，它改变了完整的MAP 2磷酸化， 兴奋性氨基酸受体的激活。 MAP 2成为 由于N-甲基-D- 天冬氨酸受体激活 这种影响似乎涉及一部小说 NMDA受体的信号传导途径。 在拟议的研究中， 我们将定义调节这些信号的信号转导途径。 海马脑片和培养海马脑片中MAP 2的磷酸化状态 海马神经元 用32 P-代谢标记的海马细胞 正磷酸盐将用于确定机制，受体 兴奋性氨基酸诱导的特异性和时间特性 MAP 2的去磷酸化。 刺激MAP 2磷酸化 神经递质和生长因子依赖性蛋白激酶活性将 也要检查。 将研究两种形式的MAP 2：常规MAP 2 在成年神经元中发现，以及一种独特的选择性剪接变体， 称为MAP 2c的未成熟神经元。这种变体缺少一个大的中间部分 成人MAP 2，因此包含相对少量的 磷酸化位点。 MAP 2的不成熟形式将帮助我们定义 其磷酸化位点在NMDA受体的控制下。 一旦我们确定了MAP 2和MAP 2c的激酶和磷酸酶途径， 调节，我们将确定磷酸化的精确位置， 在较小的蛋白质MAP 2c上。 这些信息将是重要的 在最终确定MAP 2和MAP 2c磷酸化的作用中， 在调节神经元形态方面。 这些结果将有助于 了解细胞外信号如何调节MAP 2功能， vivo. 更一般地，这些实验将允许配制药物组合物。 神经元跨膜调节的分子模型 细胞骨架

This proposal addresses the molecular control of neuronal cytoskeletal proteins by extracellular signals. Understanding how the neuronal cytoskeleton is regulated is important because several disease processes, including Alzheimer's disease and schizophrenia are thought to affect the cytoskeleton and thereby alter neuronal function. During outgrowth, development, and synaptogenesis, the morphology of neurons is controlled by local environmental cues like growth factors and neurotransmitters. The microstructure of adult neurons is also thought to be modified in response to synaptic or neurohumoral activity. We propose to examine the cellular basis for morphological plasticity in neurons by focusing on the regulation of the phosphorylation state and function of the microtubule- associated protein MAP2. MAP2 is thought to play an important role in the development and maintenance of dendritic morphology via its interaction with microtubules and other cytoskeletal elements. Multiple protein kinases and protein phosphatases are known to modify MAP2 phosphorylation and MAP2 function in vitro, but little is known about the properties of MAP2 phosphorylation in vivo. Our previous studies revealed that one signal which alters MAP2 phosphorylation in intact neurons is activation of excitatory amino acid receptors. MAP2 becomes rapidly and selectively dephosphorylated as a result of N-methyl-D- aspartate receptor activation. This effect appears to involve a novel signal transduction pathway for NMDA receptors. In the proposed studies, we will define the signal transduction pathways which modulate the phosphorylation state of MAP2 in hippocampal slices and cultured hippocampal neurons. Hippocampal cells metabolically labeled with 32P- orthophosphate will be used to determine the mechanism, receptor specificity and temporal properties of excitatory amino acid-induced dephosphorylation of MAP2. The stimulation of MAP2 phosphorylation by neurotransmitter and growth factor-dependent protein kinase activity will also be examined. Two forms of MAP2 will be studied: conventional MAP2 found in adult neurons, and an alternatively spliced variant unique to immature neurons called MAP2c. This variant lacks a large middle portion of adult MAP2, and thus contains a relatively small number of phosphorylation sites. The immature form of MAP2 will help us to define which phosphorylation sites are under the control of NMDA receptors. Once we identify the kinase and phosphatase pathways for MAP2 and MAP2c regulation, we will determine the precise location of phosphorylation sites on the smaller protein MAP2c. Such information will be important in eventually determining the role of phosphorylation of MAP2 and MAP2c in regulating neuronal morphology. These results will contribute to an understanding of how extracellular signals regulate MAP2 function in vivo. More generally, these experiments will allow formulation of a molecular model for transmembrane regulation of the neuronal cytoskeleton.