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的技术来解决依赖蛋白质合成的突触可塑性：单突触剪辑和突触翻译图谱，挑战了目前对突触RNA定量识别的关注。这些将使我们能够从两个新的重叠角度重新定义这个问题：需要识别特定突触中受调控的RNA-蛋白质复合体，以及需要定义它们在翻译调控中的作用。我们将研究特定的突触： 小脑浦肯野神经元的顶端树突(运动学习的部位)，CA1锥体神经元在海马层分子层的树突(联想记忆的部位)，以及视皮层的V层锥体神经元的顶端树突(活动依赖的可塑性部位)。在这些突触中需要研究的关键RNA-蛋白质复合体将是ArgAerte(AGO)-mRNA-miRNA三元复合体，翻译调控的FMRP-mRNA复合体，以及已知存在于树突中并与3‘UTRs结合的神经元特异性RNA调节蛋白-mRNA复合体(nElevl(Hu Proteins)，Nova)。这些复合体将与同一树突中存在的所有核糖体-mRNA突触复合体的描绘进行比较，使我们能够通过识别翻译调节的突触mRNAs(突触翻译图谱)来验证相互作用。受调控的树突RNA将通过评估它们的翻译状态在两个研究得很好的依赖于蛋白质合成的突触可塑性范例中得到进一步验证：在海马区，以及在黑暗和随后的光照下在视觉皮质中。这些研究将彻底改变我们对构成记忆基础的局部突触mRNAs的性质和调控的理解，为对阿尔茨海默氏症和其他神经退行性疾病等神经记忆疾病的新见解奠定基础。

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.