Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia are the resident immune cells of the brain, crucial for the maintenance of the homeostasis in the central nervous system. They are responsible for removing cellular debris, pathogens, and damaged cells, as well as modulating neuronal function. Microglia actively and continuously patrol their local environment by using highly motile processes and ramified morphology. In response to triggering stimuli, such as infection, altered neuronal activity, trauma or neurodegenerative diseases, microglia undergo a process of activation that profoundly change their gene expression and functions. Microglia activation is a salient feature of neuroinflammation, prominent in many neurodegenerative diseases. Interestingly, microglia reactivity also entails a large-scale remodeling of cellular geometry, but while the role of the actin cytoskeleton in driving these morphological changes and the specialized functions of activated microglia has been extensively studied, the behavior of the microtubule cytoskeleton during these changes remains unexplored. Microtubules are hollow, cylindrical polymers composed of protofilaments of α/β tubulin heterodimers that arrange in a head-to-tail fashion, making microtubule polarized structures with a fast growing plus end and a slow growing minus end. Microtubules form the backbone of the cell's internal transport system for macromolecules and organelles and are also involved in cell division and the maintenance of cell shape and integrity. We hypothesize that rearrangement of the microtubule cytoskeleton would play a major role in the morphological changes guiding microglia transition from homeostatic to activated states providing a novel target to modulate microglia activation. Through a detailed in vitro phenotyping approach and validation in mouse retinal tissue we report that: 1) in homeostatic conditions, ramified microglia possess stable microtubule arrays, while microglia activation increases microtubule dynamic behavior. 2) A non-centrosomal microtubule organization in arrays with mixed polarity is a hallmark of homeostatic microglia, similar to the architecture typical of highly specialized cells such as neurons and oligodendrocytes, while activation induces recruitment of the majority of microtubule minus end at the centrosome. 3) Homeostatic microglia nucleate non-centrosomal microtubules at Golgi outposts, similar to what occurs in neurons, while activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results unveil the remodeling of the microtubule cytoskeleton as a striking hallmark of microglia reactivity and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of activated microglia, inhibition of microtubule dynamics may provide a novel strategy to reduce microglia reactivity in inflammatory disease.