Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon coculture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration. GLIA 2014;62:1982-1991 Key words: microglia, axon degeneration, regeneration, multiple sclerosis, phagocytosis Introduction M icroglia, the resident immune cells of the central nervous system (CNS), are rapidly activated in response to a variety of injuries, including trauma, stroke, and inflammation. One of the key functions of microglia in the setting of acute injury is the phagocytosis of dying cells and cellular debris, which can facilitate recovery . Indeed, microglial phagocytosis of both apoptic cells and myelin debris have been widely implicated in suppression of inflammation and ensuing CNS repair (Dubois-Dalcq et al., 2005; Fadok et al., 1998;Franklin and Kotter, 2008;Voll et al., 1997). However, the role of microglial phagocytosis in CNS injury has yet to be fully defined, as underscored by recent findings that the phagocytic capacity of microglia may also directly contribute to neuronal loss and neurodegeneration (Neher et al., 2011).Microglial phagocytosis is a highly co-ordinated process dependent upon extracellular signals interacting with cell surface receptors. Notably, mechanisms of microglial phagocytosis are remarkably diverse (Napoli and Neumann, 2009). While several Toll-like receptors (TLRs) have been wellestablished as playing a crucial role in the phagocytosis of microbes, other molecu...

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