The Role of Wiskott - Aldrich syndrome protein in the function of Microglia during Neurodevelopment

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is an X-linked rare disease caused by a mutated WAS gene. The WAS protein (WASp) is involved in hematopoiesis and in phagocytosis by immune cells. WAS patients develop immunodeficiency, with 40% of them showing neurological manifestations, whose molecular basis is unknown. In the brain, WASp is expressed by microglia, the main resident immune population of the central nervous system. Microglia physiologically sculpt neuronal networks during brain development by phagocytosis-mediated synaptic pruning. Our hypothesis posits that, in the absence of WASp, microglia functions during brain development are reduced, hence contributing to WAS-associated neurological deficits. METHODS: Microglia (iMicro) were derived from human-induced pluripotent stem cells (hIPSCs). iMicro were characterized for the gene expression of WAS and selective microglia markers by RT real-time PCR. To investigate the contribution of WASp to phagocytosis and cell motility, we did a confocal time-lapse phagocytic assay on living iMicro with or without WASp inhibitors Wiskostatin and CK-666. Post-fixation immunofluorescence was done to visualize cytoskeletal modification after WASp inhibition. Zebrafish embryos with or without CK-666 were used to assess the effects of WASp lack of function on behavior and neurodevelopment. TUNEL assay was used to stain apoptotic bodies in the Zebrafish brain. RESULTS: iMicro overexpressed the mature microglia marker P2RY12 (4.23±0.64 fold-change±SD compared to progenitor cells), downregulated the early stage microglia marker Sall1 (-6.75±1.98) and overexpressed WAS (2.32±0.52). The time-lapse confocal microscopy phagocytic assay showed that iMicro did active phagocytosis, measured as fluorescent pHrodo nanoparticle incorporation (144±21.81 area under curve of pHrodo fluorescence intensity±SD), which was prevented by a 1-hour exposure to WASp inhibitors prior of pHrodo exposure (84.87±21.65 for Wiskostatin; 25.56±6.63 for CK-666). Post-fixation immunofluorescence showed a highly organized arrangement of actin filaments with the presence of small filopodia in iMicro. At variance, when exposed to WASp inhibitors, iMicro displayed disorganized microfilaments throughout the cytoplasm with no filopodia. Zebrafish embryos exposed to CK-666 had reduced response to mechanical stimuli (78.57% of 42 zebrafish embryos showed bad outcomes) compared to control embryos (28.57% of 42 zebrafish embryos), along with increased apoptotic bodies in their brain. Conclusion: We could demonstrate that WAS is expressed by microglia in basal conditions. WASp was implicated in iMicro phagocytic functions and the cell's correct cytoskeleton assembly. Supporting the importance of these WASp-related functions of microglia in neurodevelopment, WASp inhibition in Zebrafish embryos caused altered behavior and reduced clearance of apoptotic neurons. These observations link microglia dysfunctions to WAS-associated neurological manifestations, lending reasons to further elucidate the underpinning mechanisms and develop future treatment/diagnostic tools.