Final Report Summary - OLIGPROCESSEXTENSION (Study of proteins involved in oligodendrocyte process extension that regulate axon-glia interactions) Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). During CNS development and myelination, oligodendrocyte precursor cells (OPC) differentiate into mature OL by extending several membrane processes that will contact and wrap axons. The formation and extension of such processes requires controlled reorganization of the cytoskeleton. The main objective of this project consisted in the identification and validation of potential candidates involved in OL process extension and that may regulate early axon-glia interactions. Therefore, we established a Boyden chamber system in order to physically separate OPC soma from their processes for mRNA sequencing. With these data, we were able to identify and validate mRNAs mainly present in the processes of OPC. Such molecules are the most likely to translate for proteins that participate in OL process extension, wrapping and, later, myelination of axons. Accordingly, our data showed the distribution of such transcripts to be asymmetric with a larger number of transcripts being more highly expressed in the OPC processes than in the OPC soma. We found 489 transcripts to be significantly enriched in OPC processes. This clearly suggests that OPCs, as polarized cells, need to re-localize their mRNAs is order to regulate locally their translation and quickly respond to external stimuli (in our experimental set up, OPC responded to chemotactic gradients of PDGF-a and Netrin-1 and a haptotactic gradient of the extracellular matrix protein laminin-2). Moreover, a bioinformatic analysis revealed that the majority of mRNAs enriched in OPC processes were related to cytoskeleton re-organization and ribosomes, reinforcing the idea that mRNAs are being locally translated in OPC processes. After narrowing down the candidate list, we selected 25 candidates based on literature review, described functional roles, interface with the cytoskeleton, availability of antibodies and transgenic mice: Fam174b, Fam101b, Diras1, Yap1, Cgnl1, Cdc42bpg, Dusp19, Rab13, Net1, Gab2, Kank2, Synm, Pink1, Ovol1, Nbeal, Cntn2, Mmp2, Jmy, Palld, Cntnap4, Calm, Arhgef37, Tenm2, S1pr5 and Dpysl3. We validated their expression in OPC soma and processes and during OL differentiation by real-time PCR and selected 3 candidates for functional analysis: Yap1, Kank2, Dusp19. The roles of these proteins are not well understood and nothing is known about their function in OL and myelination. We are now functionally characterizing these 3 proteins in oligodendrocyte cultures and myelinating axon-glia co-cultures using lentivirus mediated shRNA delivery. Although we were not able to finalize all the planned experiments during this 24-month period, we are currently doing all the crucial experiments that will lead to scientifically relevant and publishable data. Moreover, we were able to prepare sufficient material to do small RNA sequencing (deep sequencing) of OPC soma and processes. This will generate valuable amount of data that will help to understand how genes relevant for OPC process extension are regulated. Finally, in Task 2, we aimed to evaluate if relevant candidates for OPC process extension are modulated in neuroinflammatory conditions, and analyse if the selected candidates are also relevant in disease conditions. Because this task was dependent on the accomplishment of Task 1, we were not able to complete it. In any case, we believe this will be easily done as we already have the necessary tools and samples established. Moreover, we performed some unplanned experiments that we think were complementary to the main plan of the grant. To enable us to finish the planned work, Sofia Domingues was recently awarded a 36-month grant from the Portuguese Science Foundation.We strongly believe that the knowledge obtained from this work will be relevant to better understand and promote remyelination in debilitating demyelinating diseases such as Multiple Sclerosis, which affects approximately half million people in Europe.In parallel to this project, we collaborated in 2 other projects. One of which resulted in a recent publication and the other produced data that will be summarized in a manuscript form and submitted for publication in a peer-review journal. Finally, during this period Sofia Domingues had the opportunity to extend her scientific knowledge in a different topic, attend relevant courses and learn new techniques. She also has the opportunity to participate in the dissemination of science actions to the public in general and to more specialized audiences concerning this project in particular.
