Wnt3a induces the release of membrane-bound vesicles from primary microglia

Abstract

In healthy brain tissue microglia are present in a ramified quiescent state and comprise approximately 10-20% of the total population of glia. Classically, ramified microglia were considered to be inactive under physiological conditions, however, it is now known that microglia exhibit pinocytotic activity and localised motility. Microglial processes directly contact neuronal cell bodies, astrocytes and blood vessels, therefore it seems likely that microglia monitor the well-being of brain cells and also function to cleanse the extracellular fluid in order to maintain homeostasis. In this respect, it has been suggested that ramified microglia contribute to metabolite removal and to the clearance of toxic factors released from injured neurons. Ramified microglia also secrete a number of neuroprotective factors such as nerve growth factor, which promote neuronal survival and repair. Few studies have investigated the signalling properties of Wnt on microglia. Here we explore the effects of Wnt3a on protein secretion from primary cultured rat microglia. The canonical Wnt pathway negatively regulates GSK3 activity by physically displacing complexed GSK3 from its regulatory binding partners consequently precluding kinase activity on specific substrates. GSK3 plays a pivotal role in AD and is upregulated in AD. Wnt can also induce the activation of the planar cell polarity pathway (involving Rho and ROCK) and the calcium pathway; both of which play important roles during development. Primary cultured microglia were treated with recombinant Wnt3a for 8 hours and medium was collected and concentrated using centrifugal filters (3 kDa cut off). Secreted proteins were subsequently identified using SDS-PAGE coupled with mass spectrometry (LC-MS/MS). Proteomic profiling revealed that Wnt3a causes the release of small vesicular particles from primary microglia. Secretion was blocked by Y-27632, a ROCK inhibitor, implicating the planar-cell polarity branch of the Wnt signalling cascade. Wnt3a is a novel microglial modulator inducing the release of small membrane-bound vesicles. These findings have ramifications for inter-cellular communication between glia and neurons in health and in neurodegenerative diseases such as Alzheimer's disease.