The nucleus is contained in a cell body or soma, from where several neurites emerge. Our results thus confirm previous reports and validate our quantification method.Īmong all cell types, neurons are the most morphologically complex. 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. 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. To test the validity of our analyses we have compared control neurons to Aβ 1 – 42-treated neurons. Colocalization between RNA and protein in neurites correspond to newly synthesized proteins arising from localized RNAs and represent localized translation sites. Objects corresponding to RNA or protein and colocalized objects (RNA and protein) are quantified along individual neurites. Resulting pixels are converted into objects and selected by automatic masking followed by signal smoothing. After imaging, signals corresponding to neuritic RNAs and proteins are filtered with a Laplacian operator to enhance the edges. Newly synthesized proteins are tagged with puromycin and endogenous RNAs labeled with SYTO. Here we describe an object-based analysis used to visualize and quantify local RNA translation sites in neurites. Thus local translation events can be easily overlooked under the microscope. 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. 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. Local translation confers dendrites and axons the capacity to respond to their environment in an acute manner without fully relying on somatic signals. 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. Although once considered heretical, RNA localization has proven to be highly conserved in eukaryotes. However, protein delivery can also rely on localization of mRNAs that are translated at target sites. Subcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. 6IKERBASQUE Basque Foundation for Science, Bilbao, Spain.5ICVS/3B’s, PT Associate Laboratory, Universidade do Minho, Guimarães, Portugal.4Life and Health Sciences Research Institute, School of Medicine, University of Minho, Braga, Portugal.3Department of Cell Biology and Histology, Faculty of Medicine and Nursing, University of the Basque Country, Leioa, Spain.2Department of Neurosciences, Faculty of Medicine and Nursing, University of the Basque Country, Bilbao, Spain.1Achucarro Basque Center for Neuroscience, Leioa, Spain.Batista 1,4,5, Josune Imaz 2 and Jimena Baleriola 1,3,6* María Gamarra 1,2, Maite Blanco-Urrejola 1,2,3, Andreia F.
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