Gamarra María, Blanco-Urrejola Maite, Batista Andreia F R, Imaz Josune, Baleriola Jimena
Achucarro Basque Center for Neuroscience, Leioa, Spain.
Department of Neurosciences, Faculty of Medicine and Nursing, University of the Basque Country, Bilbao, Spain.
Front Neurosci. 2020 Jun 3;14:547. doi: 10.3389/fnins.2020.00547. eCollection 2020.
Subcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. However, protein delivery can also rely on localization of mRNAs that are translated at target sites. Although once considered heretical, RNA localization has proven to be highly conserved in eukaryotes. RNA localization and localized translation are especially relevant in polarized cells like neurons where neurites extend dozens to hundreds of centimeters away from the soma. Local translation confers dendrites and axons the capacity to respond to their environment in an acute manner without fully relying on somatic signals. The relevance of local protein synthesis in neuron development, maintenance and disease has not been fully acknowledged until recent years, partly due to the limited amount of locally produced proteins. For instance, in hippocampal neurons levels of newly synthesized somatic proteins can be more than 20-30 times greater than translation levels of neuritic proteins. Thus local translation events can be easily overlooked under the microscope. Here we describe an object-based analysis used to visualize and quantify local RNA translation sites in neurites. Newly synthesized proteins are tagged with puromycin and endogenous RNAs labeled with SYTO. After imaging, signals corresponding to neuritic RNAs and proteins are filtered with a Laplacian operator to enhance the edges. Resulting pixels are converted into objects and selected by automatic masking followed by signal smoothing. Objects corresponding to RNA or protein and colocalized objects (RNA and protein) are quantified along individual neurites. Colocalization between RNA and protein in neurites correspond to newly synthesized proteins arising from localized RNAs and represent localized translation sites. To test the validity of our analyses we have compared control neurons to Aβ -treated neurons. Aβ is involved in the pathology of Alzheimer's disease and was previously reported to induce local translation in axons and dendrites which in turn contributes to the disease. We have observed that Aβ increases the synthesis of neuritic proteins as well as the fraction of translating RNAs in distal sites of the neurite, suggesting an induction of local protein synthesis. Our results thus confirm previous reports and validate our quantification method.
亚细胞蛋白质递送在信号转导和细胞行为中尤为重要，通常通过蛋白质内的定位信号来实现。然而，蛋白质递送也可依赖于在靶位点翻译的mRNA的定位。尽管RNA定位曾一度被视为异端，但现已证明在真核生物中高度保守。RNA定位和局部翻译在极化细胞（如神经元）中尤为相关，在神经元中，神经突从胞体延伸数十至数百厘米。局部翻译赋予树突和轴突以敏锐方式响应其环境的能力，而无需完全依赖体细胞信号。直到近年来，局部蛋白质合成在神经元发育、维持和疾病中的相关性才得到充分认识，部分原因是局部产生的蛋白质数量有限。例如，在海马神经元中，新合成的体细胞蛋白质水平可比神经突蛋白质的翻译水平高20 - 30倍以上。因此，局部翻译事件在显微镜下很容易被忽视。在这里，我们描述了一种基于对象的分析方法，用于可视化和量化神经突中的局部RNA翻译位点。新合成的蛋白质用嘌呤霉素标记，内源性RNA用SYTO标记。成像后，用拉普拉斯算子对与神经突RNA和蛋白质相对应的信号进行滤波以增强边缘。所得像素被转换为对象，并通过自动掩膜然后进行信号平滑来选择。沿着单个神经突对与RNA或蛋白质相对应的对象以及共定位对象（RNA和蛋白质）进行量化。神经突中RNA和蛋白质之间的共定位对应于由局部RNA产生的新合成蛋白质，并代表局部翻译位点。为了测试我们分析的有效性，我们将对照神经元与经Aβ处理的神经元进行了比较。Aβ参与阿尔茨海默病的病理过程，先前有报道称其可诱导轴突和树突中的局部翻译，进而导致疾病。我们观察到Aβ增加了神经突蛋白质的合成以及神经突远端位点翻译RNA的比例，表明局部蛋白质合成受到诱导。因此，我们的结果证实了先前的报道并验证了我们的量化方法。
