Investigating the contribution of Wiskott-Aldrich syndrome protein (WASp) to brain-resident immune cell functions

The immune system (IS) plays a crucial role in the development and homeostasis of the central nervous system (CNS). Brain-resident immune cells, microglia and CNS-associated macrophages (CAMs), are essential for neurodevelopment. Microglia regulate neuronal survival, synaptic pruning, and myelination, while CAMs contribute to cerebrovascular formation and blood–brain barrier maintenance. Disruption of immune homeostasis during development can therefore result in neurological dysfunction. Primary immunodeficiencies (PIDs) highlight this link: mutations affecting immune cell functions can lead to neurodevelopmental and cognitive alterations, underlying the critical crosstalk between the immune and nervous systems. Wiskott-Aldrich syndrome is a rare X-linked genetic disease caused by mutations in the WAS gene, which encodes the Wiskott-Aldrich Syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells. Clinical manifestations appear early in life and include recurrent infections, autoimmune disorders, and an increased risk of malignancies. Neurological symptoms, like acute brain bleeding or a decrease in emotional and cognitive functioning, may occur, although the molecular and cellular mechanisms remain unclear. In brain, WASp is expressed by microglia and CAMs. To investigate whether WASp contributes to the functions of these cells, we generated microglia (iMicro) and CAMs (iMacs) from human induced pluripotent stem cells (hiPSCs). We characterized their identity by analyzing the expression of selective markers, like P2ry12 for iMicro, CD163 for iMacs, and assessed Was expression. We found that Was was highly expressed in iMicro, whereas iMac showed minimal expression under basal conditions. At variance Was expression was inducible upon LPS stimulation (1 µg/ml). LPS-activated iMac treated with WASp inhibitor Wiskostatin failed to acquire the characteristic elongated morphology typical of phagocytic cells, suggesting that WASp is also required for their activation and morphological differentiation. To evaluate WASp function in iMicro, we performed phagocytosis assay that showed active nanoparticle uptake (144±21.81 area under curve of pHrodoTM fluorescence), which was reduced after exposure to two WASp inhibitors, Wiskostatin and CK.666. Immunofluorescence revealed F-actin localization at membrane ruffles and filopodia, typical of rearranged cytoskeleton to support phagocytosis and chemotaxis, in iMicro, which was not observed in iMicro with WASp inhibitors. WASp showed an overlaid localization to F-actin and, consequently, WASp distribution was similarly altered by the treatment with the inhibitors. In co-cultures of microglia, neurons, and astrocytes, treatment with CK-666 led to a reduction in the number of synapses compared to control co-cultures, suggesting impaired neuronal wiring. To extend our findings in an in vivo model, we analyzed zebrafish embryos that were exposed to CK-666 to inhibit WASp. Zebrafish treated with CK-666 showed, at 3 days post fertilization, a delayed response to mechanical stimuli (>4 touches in 78.57% of embryos) when compared to vehicle-treated (<4 touches in 67%), indicating impaired sensorimotor deficits. At the same time point, these deficits were associated with increased accumulation of brain apoptotic bodies, suggesting a defect in their microglia-mediated elimination, which is a hallmark of normal neurodevelopment to favor myeloid cell brain homing. At 5 days post fertilization, CK-666-treated zebrafish had an altered body morphology (reduced area, perimeter, and aspect ratio) compared to vehicle-treated zebrafish, indicating a defective development. Our findings identify WASp as a critical regulator of microglial and CAMs’ functions involving cytoskeletal dynamics. Specifically, the inhibition of WASp affects microglia phagocytic behavior, with consequences for correct neurodevelopment.