A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination

During central nervous system development, oligodendrocyte progenitors elaborate multiple branched processes to contact axons and initiate myelination. Using cultured primary rat oligodendrocytes (OLGs), we have recently demonstrated that a cell surface protein belonging to the immunoglobulin superfamily, cell adhesion molecule-related, down-regulated by oncogenes (Cdon), is important in initiating OLG differentiation and axon myelination by promoting the formation of branched cellular processes; however, the molecular mechanism by which Cdon regulates OLG differentiation is not known. Here, using Cdon immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry analysis, we identified serine/threonine kinase TANK-binding kinase 1 (TBK1) as a candidate novel target of Cdon. We confirmed this interaction using co-IP and immunofluorescence with TBK1 antibodies, showing that TBK1 partly co-localizes with Cdon along cellular processes in puncta-like structures. We show that TBK1 is expressed throughout OLG differentiation, and surprisingly, that levels of phosphorylated TBK1 (ser172) increase during OLG maturation, while total levels of TBK1 protein decrease. To investigate function, TBK1 expression was knocked down using siRNA in OLG primary cultures, reducing protein levels by 69%. Two myelin-specific proteins, myelin basic protein and myelin-associated glycoprotein, were similarly reduced when examined at day 2 and day 4 of OLG differentiation. Reduced Cdon or TBK1 expression also decreased Akt phosphorylation at Threonine 308 in OLG. Our findings provide evidence that a Cdon-TBK1 complex is associated with Akt phosphorylation and early OLG differentiation.