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 Aβ1–42-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.

中文翻译：

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在 FIJI/ImageJ 中进行基于对象的分析，以测量神经突中响应 Aβ1-42 寡聚体的局部 RNA 翻译位点

亚细胞蛋白质传递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实现。然而，蛋白质传递也可能依赖于在目标位点翻译的 mRNA 的定位。虽然曾经被认为是异端，但 RNA 定位已被证明在真核生物中是高度保守的。RNA 定位和定位翻译在极化细胞（如神经元）中尤其相关，其中神经突从胞体延伸数十至数百厘米。本地翻译赋予树突和轴突以敏锐的方式对其环境做出反应的能力，而无需完全依赖体细胞信号。直到最近几年，局部蛋白质合成与神经元发育、维持和疾病的相关性才得到充分承认，部分原因是本地生产的蛋白质数量有限。例如，在海马神经元中，新合成的体细胞蛋白的水平可能比神经炎蛋白的翻译水平高 20-30 倍以上。因此，在显微镜下很容易忽略本地翻译事件。在这里，我们描述了一种基于对象的分析，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，对应于神经炎 RNA 和蛋白质的信号用拉普拉斯算子过滤以增强边缘。生成的像素被转换为对象，并通过自动屏蔽和信号平滑进行选择。对应于 RNA 或蛋白质的对象和共定位对象（RNA 和蛋白质）沿着单个神经突量化。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 Aβ1-42 处理的神经元进行了比较。Aβ 参与阿尔茨海默病的病理学，之前有报道称其可诱导轴突和树突的局部翻译，进而导致该疾病。我们观察到 Aβ 增加了神经炎蛋白的合成以及神经突远端部位翻译 RNA 的比例，这表明诱导了局部蛋白质合成。因此，我们的结果证实了之前的报告并验证了我们的量化方法。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 Aβ1-42 处理的神经元进行了比较。Aβ 参与阿尔茨海默病的病理学，之前有报道称其可诱导轴突和树突的局部翻译，进而导致该疾病。我们观察到 Aβ 增加了神经炎蛋白的合成以及神经突远端部位翻译 RNA 的比例，这表明诱导了局部蛋白质合成。因此，我们的结果证实了之前的报告并验证了我们的量化方法。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 Aβ1-42 处理的神经元进行了比较。Aβ 参与阿尔茨海默病的病理学，之前有报道称其可诱导轴突和树突的局部翻译，进而导致该疾病。我们观察到 Aβ 增加了神经突蛋白的合成以及神经突远端部位翻译 RNA 的比例，这表明诱导了局部蛋白质合成。因此，我们的结果证实了之前的报告并验证了我们的量化方法。