Regulation of Local BDNF Synthesis in Dendrites

描述(申请人提供)：脆性X智力低下蛋白(FMRP)的缺失导致脆性X综合征，这是最常见的遗传性智力低下形式，与认知障碍和皮质神经元中更多更细的树突有关。FMRP是一种mRNA结合蛋白，已被证明可以稳定特定的mRNA物种，并抑制树突中局部蛋白质的合成。然而，尽管它与运输RNA颗粒有关，但它在树突状靶向mRNAs中的作用仍有待确定。FMRP基因Fmr1缺失的小鼠在海马区依赖的学习中存在缺陷。与此表型一致的是，在Fmr1基因敲除(Fmr1 KO)小鼠制备的海马片上，由theta猝发刺激的阈值水平引发的长时程增强(LTP)受到损害，这可以通过应用脑源性神经营养因子(BDNF)来挽救。BDNF基因产生两种BDNF基因，其3‘非翻译区或3’非翻译区，或长3‘非翻译区。长3‘非编码区已被证明是必要的和充分的靶向BDNF的mRNA到树突。有趣的是，BDNF mRNA的树突状靶向受损的小鼠突变体也显示出更多的树突棘和海马CA1突触的LTP缺陷，Fmr1基因敲除也是如此。本研究项目将确定FMRP在BDNF mRNA转运到树突中的作用。在目标1中，将使用免疫沉淀法在脑裂解物中以及在培养的神经元中使用原位杂交和免疫细胞化学来检测FMRP和BDNF mRNA的相关性。凝胶迁移率改变分析将被用来确定长BDNF3‘非编码区中的FMRP结合位点。在目标2中，将进行两项研究，以确定FMRP是否需要将BDNF mRNA运输到树突。一是用原位杂交法检测Fmr1 KO神经元树突中BDNF mRNA的表达水平，二是确定FMRP结合位点突变对培养神经元树突靶向BDNF mRNA的影响。在目标3中，将在培养的神经元和突触神经体标本中检测FMRP在局部合成BDNF蛋白中的作用。公共卫生相关性：脆性X综合征是最常见的遗传性智力低下形式，与一系列神经症状和体征有关。这项研究将对了解脆性X综合征做出重大贡献。

DESCRIPTION (provided by applicant): Loss of fragile X mental retardation protein (FMRP) leads to fragile X syndrome, the most common form of inherited mental retardation, which is associated with cognitive impairment and more numerous and thinner dendritic spines in cortical neurons. FMRP is an mRNA binding protein and has been shown to stabilize select mRNA species and to repress local protein synthesis in dendrites. However, its role in dendritic targeting of mRNAs remains to be determined, although it is associated with transport RNA granules. Mice with deletion of the gene for FMRP, Fmr1, have deficits in hippocampus-dependent learning. In agreement with this phenotype, long-term potentiation (LTP) elicited by threshold levels of theta burst stimulation is impaired in hippocampal slices prepared from Fmr1 knockout (Fmr1 KO) mice, which can be rescued by application of brain-derived neurotrophic factor (BDNF). The Bdnf gene produces two pools of BDNF mRNA species, with either a short 3' untranslated region (3'UTR) or a long 3'UTR. The long 3'UTR has been shown to be necessary and sufficient to target BDNF mRNA to dendrites. Interestingly, a mouse mutant in which dendritic targeting of BDNF mRNA is impaired also exhibits more numerous dendritic spines and deficits in LTP at hippocampal CA1 synapses, as does the Fmr1 knockout. This research project will determine the role of FMRP in trafficking of BDNF mRNA to dendrites. In Aim 1 the association of FMRP with BDNF mRNA will be examined in brain lysates using immunoprecipitation and in cultured neurons using in situ hybridization and immunocytochemistry. Electrophoretic mobility shift assays will be employed to identify FMRP binding sites in the long BDNF 3'UTR. In Aim 2 two studies will be carried out to determine whether FMRP is required for trafficking of BDNF mRNA to dendrites. One is to examine levels of BDNF mRNA in dendrites of Fmr1 KO neurons using in situ hybridization, and the other is to determine the effect of mutations in the FMRP binding sites on dendritic targeting of BDNF mRNA in cultured neurons. In Aim 3 the role of FMRP in the local synthesis of the BDNF protein will be examined in cultured neurons and synaptoneurosome preparations. PUBLIC HEALTH RELEVANCE: Fragile X syndrome is the most common form of inherited mental retardation, which is associated with a wide range of neurological symptoms and signs. This research will make a significant contribution to the understanding of fragile X syndrome.