Ho Victoria M, Dallalzadeh Liane O, Karathanasis Nestoras, Keles Mehmet F, Vangala Sitaram, Grogan Tristan, Poirazi Panayiota, Martin Kelsey C
Interdepartmental Program for Neuroscience, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Department of Biology, University of Crete, Heraklion, Crete, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece.
Mol Cell Neurosci. 2014 Jul;61:1-12. doi: 10.1016/j.mcn.2014.04.006. Epub 2014 Apr 28.
AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.
AMPA 型谷氨酸受体介导大脑中的快速兴奋性神经传递，其在突触处的浓度是突触强度的重要决定因素。我们通过检查其 mRNA 的亚细胞分布并评估其在培养的小鼠海马神经元中受 microRNA 的翻译调控，来研究钙不渗透的 AMPA 受体亚基 GluA2 的转录后调控。使用计算方法，我们在 GluA2 的 3'UTR 中鉴定出一个保守的 microRNA-124（miR-124）结合位点，并在荧光素酶测定中证明 miR-124 以序列特异性方式调节 GluA2 mRNA 报告基因的翻译。虽然我们假设这种调控可能发生在树突中，但我们的生化和荧光原位杂交（FISH）数据表明，GluA2 mRNA 并不定位于小鼠海马神经元的树突或突触。相反，我们在树突中检测到显著浓度的 miR-124。通过免疫印迹和定量免疫细胞化学测量，在解离的神经元中过表达 miR-124 导致 GluA2 蛋白敲低 30%，而 GluA2 mRNA 浓度没有任何变化。虽然总 GluA2 浓度降低，但我们未检测到突触 GluA2 浓度有任何变化。从这些结果我们得出结论，miR-124 在细胞体中与 GluA2 mRNA 相互作用以下调翻译。我们的数据支持一个模型，即 GluA2 在细胞体中翻译，随后转运到神经元树突和突触，并表明突触 GluA2 浓度主要通过调节蛋白运输而不是通过调节局部翻译来改变。
