Ouwenga Rebecca, Lake Allison M, O'Brien David, Mogha Amit, Dani Adish, Dougherty Joseph D
Division of Biology and Biomedical Sciences.
Departments of Genetics.
J Neurosci. 2017 Sep 6;37(36):8688-8705. doi: 10.1523/JNEUROSCI.3044-16.2017. Epub 2017 Aug 8.
Localized translation in neurites helps regulate synaptic strength and development. Dysregulation of local translation is associated with many neurological disorders. However, due to technical limitations, study of this phenomenon has largely been limited to brain regions with laminar organization of dendrites such as the hippocampus or cerebellum. It has not been examined in the cortex, a region of importance for most neurological disorders, where dendrites of each neuronal population are densely intermingled with cell bodies of others. Therefore, we have developed a novel method, SynapTRAP, which combines synaptoneurosomal fractionation with translating ribosome affinity purification to identify ribosome-bound mRNA in processes of genetically defined cell types. We demonstrate SynapTRAP's efficacy and report local translation in the cortex of mice, where we identify a subset of mRNAs that are translated in dendrites by neuronal ribosomes. These mRNAs have disproportionately longer lengths, enrichment for FMRP binding and G-quartets, and their genes are under greater evolutionary constraint in humans. In addition, we show that alternative splicing likely regulates this phenomenon. Overall, SynapTRAP allows for rapid isolation of cell-type-specific localized translation and is applicable to classes of previously inaccessible neuronal and non-neuronal cells Instructions for making proteins are found in the genome, housed within the nucleus of each cell. These are then copied as RNA and exported to manufacture new proteins. However, in the brain, memory is thought to be encoded by strengthening individual connections (synapses) between neurons far from the nucleus. Thus, to efficiently make new proteins specifically where they are needed, neurons can transport RNAs to sites near synapses to locally produce proteins. Importantly, several mutations that cause autism disrupt this process. It has been assumed this process occurs in all brain regions, but has never been measured in the cortex. We applied a newly developed method measure to study, for the first time, local translation in cortical neurons.
神经突中的局部翻译有助于调节突触强度和发育。局部翻译失调与许多神经系统疾病有关。然而，由于技术限制，对这一现象的研究主要局限于具有层状树突组织的脑区，如海马体或小脑。在皮质中尚未进行过研究，而皮质是大多数神经系统疾病的重要区域，其中每个神经元群体的树突与其他细胞的胞体密集交织。因此，我们开发了一种新方法SynapTRAP，它将突触神经小体分级分离与翻译核糖体亲和纯化相结合，以识别基因定义的细胞类型过程中与核糖体结合的mRNA。我们证明了SynapTRAP的有效性，并报告了小鼠皮质中的局部翻译，在那里我们鉴定出了由神经元核糖体在树突中翻译的一组mRNA。这些mRNA的长度不成比例地更长，富含FMRP结合和G-四联体，并且它们的基因在人类中受到更大的进化限制。此外，我们表明可变剪接可能调节这一现象。总体而言，SynapTRAP允许快速分离细胞类型特异性的局部翻译，并且适用于以前无法进入的神经元和非神经元细胞类别。蛋白质合成的指令存在于基因组中，位于每个细胞的细胞核内。然后这些指令被复制为RNA并输出以制造新的蛋白质。然而，在大脑中，记忆被认为是通过加强远离细胞核的神经元之间的单个连接（突触）来编码的。因此，为了在需要的特定位置有效地制造新蛋白质，神经元可以将RNA运输到突触附近的位置以局部产生蛋白质。重要的是，一些导致自闭症的突变会破坏这一过程。人们一直认为这一过程发生在所有脑区，但从未在皮质中进行过测量。我们应用一种新开发的方法首次研究皮质神经元中的局部翻译。
