Mapping the mechanisms of protein synthesis-dependent synaptic plasticity

描述（由申请人提供）：我们建议改变目前理解记忆分子基础的方法。 我们的方法通过开发新技术来解决蛋白质合成依赖性突触可塑性：单突触CLIP和突触翻译谱来挑战目前对突触RNA定量鉴定的关注。 这将使我们能够从两个新的叠加视角重新定义问题： 需要确定特定突触中受调节的RNA-蛋白质复合物，以及需要确定它们在翻译调节中的作用。 将研究特定的突触： 小脑浦肯野神经元（运动学习部位）、海马体分子层中的CA 1锥体神经元（联想记忆部位）和视觉皮层的V层锥体神经元（活动依赖性可塑性部位）的顶树突。 在这些突触中待研究的关键RNA-蛋白质复合物将是Argonaute（Ago）-mRNA-miRNA三元复合物、经调节的FMRP-mRNA复合物和已知存在于树突中并结合3'UTR的神经元特异性RNA调节蛋白-mRNA复合物（nElavl（Hu蛋白质），Nova）。 将这些复合物与存在于相同树突中的所有核糖体-mRNA突触复合物的描绘进行比较，从而使我们能够通过鉴定突触调节的mRNA（突触翻译谱）来验证相互作用。 调节的树突状RNA将通过评估它们在两种充分研究的蛋白质合成依赖性突触可塑性范例中的翻译状态来进一步验证：在海马中的翻译状态，以及在黑暗和随后的光暴露后的视觉皮层中的翻译状态。 这些研究将彻底改变我们对记忆基础的局部突触mRNAs的性质和调节的理解，为对记忆的神经系统疾病（如阿尔茨海默氏症和其他神经退行性疾病）的新见解奠定基础。 公共卫生相关性：RNA是基因组DNA和蛋白质之间的关键分子，它的失调越来越被认为是人类神经系统疾病的根源。 更确切地说，记忆和复杂的认知功能一般被认为取决于神经元连接部位蛋白质合成的调节。 我们将通过一系列完全创新的实验来研究这种调节机制，这些实验是理解记忆和认知障碍的关键。

DESCRIPTION (provided by applicant): We propose to change the current approach to understanding the molecular basis of memory. Our approach challenges the current focus on quantitative identification of synaptic RNAs by developing new technologies to address protein synthesis-dependent synaptic plasticity: single- synapse-CLIP and synaptic translational profiling. These will allow us to redefine the problem from two new superimposed perspectives: the need to identify regulated RNA-protein complexes in specific synapses, and the need to define their role in translational regulation. Specific synapses will be studied: the apical dendrites of cerebellar Purkinje neurons (a site of motor learning), of CA1 pyramidal neurons in the stratum moleculare of the hippocampus (a site of associative memory), and of layer V pyramidal neurons of the visual cortex (a site of activity- dependent plasticity). Key RNA-protein complexes to be studied in these synapses will be Argonaute (Ago)-mRNA-miRNA ternary complexes, translationally regulated FMRP-mRNA complexes, and neuron-specific RNA regulatory protein-mRNA complexes known to be present in the dendrite and to bind 3' UTRs (nElavl (Hu proteins), Nova). These complexes will be compared with a delineation of all ribosome-mRNA synaptic complexes present in the same dendrites, allowing us to validate interactions by identifying translationally regulated synaptic mRNAs (synaptic translational profiling). Regulated dendritic RNAs will be further validated by assessing for their translational state in two well-studied paradigms of protein synthesis- dependent synaptic plasticity: that in the hippocampus, and in the visual cortex after dark rearng and subsequent light exposure. These studies will revolutionize our understanding of the nature and regulation of local synaptic mRNAs that underlie memory, setting the stage for new insight into neurologic diseases of memory such as Alzheimer's and other neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Dysregulation of RNA-the key molecule between genomic DNA and proteins-is increasingly recognized to lie at the root of human neurologic disease. More precisely, memory, and complex cognitive function in general, is thought to depend on the regulation of protein synthesis at the sites of neuronal connections. Understanding the mechanisms governing this regulation will be studied here in an entirely innovative set of experiments, and they hold the key to understanding disorders of memory and cognition.