Elucidating the role of WDR47 in regulating neuronal migration, autophagy and tubulin dynamics

[摘要] ENGLISH ABSTRACT: Introduction Normal cerebral cortex development depends on extensive neuronal migration duringembryogenesis, permitting the formation of accurate synaptic circuits and a highly orderedlaminar neocortex. The motility of a migrating neuron is achieved by a dynamic microtubulecytoskeleton that alternates between states of stabilization/lengthening anddestabilization/shortening. This dynamic instability of the microtubule cytoskeleton iscontrolled by numerous microtubule-stabilizing and -destabilising proteins that bind directlyto microtubules.Autophagy ('self-eating), a major bulk intracellular degradation system, involves the fusionof autophagosomes with lysosomes, followed by proteolysis and recycling of cellularconstituents. Like neuronal migration, autophagy is a microtubule-dependent process. Thedynamic microtubule network serves as a track for autophagosomes to be transported tothe lysosomes.WDR47 is a protein that is expressed in the brain during development, but of which thefunction is largely unknown. Novel interactions have recently been identified betweenReelin and WDR47 and between the microtubule-destabilising protein superior cervicalganglion 10 (SCG10) and WDR47. These findings suggest that WDR47 may be regulatingmicrotubule-dependent processes such as neuronal migration and autophagy. Wehypothesize that WDR47 may play a role in regulating neuronal migration and/orautophagy, and that this regulation may be mediated by a tubulin stability-regulating role ofWDR47.Aims and Methods Our aims are to assess the cellular localization of WDR47 in GT1-7 cells and to determinewhether WDR47 is able to influence neuronal migration, filopodia extension, surfaceadhesion, ultra-structure, autophagy, tubulin stability, and tau or SCG10 protein levels.GT1-7 neuronal cells were cultured under normal conditions and transfected with WDR47siRNA for 24 hours, followed by western blot verification of the knock-down. A 36 hour neuronal in vitro cell migration assay was performed and images of the wound werecaptured every 6 hours; the migration distances and the wound areas for the different timepoints were measured and analysed. A 24 hour migration assay was performed, capturingimages every hour, and the direction of migration was determined. Scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM) were performed to analyseneuronal surface morphology and ultra-structure. Western blot analysis of SCG10,acetylated α-tubulin, Tau, LC3, and Sequestosome 1/p62 (SQTM1) protein levels wasperformed. Super-resolution structured Illumination microscopy (SR-SIM) three-dimensional(3-D) imaging of WDR47-YFP transfected cells, confocal microscopy of LC3 and acetylatedtubulin, co-localization analysis of WDR47 and acetylated tubulin, and fluorescence recoveryafter photo-bleaching (FRAP) analysis were performed.ResultsWDR47 siRNA treatment significantly reduced the average migration distance and themigration velocity, resulted in fewer filopodia-like extensions as well as perturbed surfaceadhesion of migrating neurons, and lead to an increased presence of endoplasmic reticulum(ER) structures as well as an expanded nuclear envelope. LC3-II protein levels weresignificantly lower with WDR47 siRNA treatment, but were significantly increased withWDR47 siRNA treatment in conjunction with Bafilomycin A1 treatment, indicating increasedautophagic flux. SCG10 protein levels were significantly decreased with WDR47 siRNAtreatment. SR-SIM and confocal microscopy of WDR47 siRNA treated cells revealed a robustpresence of highly convoluted acetylated tubulin in the perinuclear region as well asdecreased LC3 fluorescence signal. Confocal microscopy revealed co-localization of WDR47with acetylated tubulin. - Discussion and Conclusion: The results suggest that WDR47 is involved in regulating neuronal migration, neuronalsurface adhesion and filopodia formation, microtubule dynamics, and likely also autophagicflux. Taken together, we propose that WDR47 is regulating microtubule dynamics byfacilitating assembly of microtubule-regulating proteins such as SCG10, thereby affectingmicrotubule-dependent processes such as neuronal migration and autophagy.

[发布机构] Stellenbosch University