During development, neural circuitry undergoes a remodeling process in which excess synapses are eliminated or pruned and the remaining synapses are strengthened. While it is clear that developmental synaptic remodeling is an activity-dependent process whereby weaker or less-active synapses are selectively eliminated, the precise molecular mechanisms have not been elucidated. Recently, our laboratory discovered that components of the classical complement cascade (C1q and C3) were necessary for synapse elimination in the developing and diseased central nervous system (CNS)(Stevens et al., 2007). In addition, C1q protein was specifically expressed in neurons and localized to synapses at ages and brain regions consistent with active synaptic pruning. One of the major questions arising from these findings is by what mechanism is complement facilitating synapse removal. The complement cascade is traditionally associated with the innate immune system in which complement components coat or opsonizes debris (e.g. pathogens, cell corpses, etc.) for removal. A canonical pathway for complement-opsonized debris removal is through phagocytosis by cells that express receptors for complement proteins Similar to the innate immune system, we suggest that complement is acting in the developing nervous system to tag weak synapses for removal by activated microglia, the primary phagocytic cell in the CNS. To characterize the role of microglia in synaptic pruning (Aim 1), we will test the capacity of microglia to phagocytose synaptic endings (subaim 1a) using various imaging techniques (e.g in vivo live imaging, etc.). In addition, we will deterimine if microglia-mediated synaptic removal is a complement-dependent process (subaim 1b) by assessing pruning deficits in complement receptor KO mice and microglia physiology in C3 and C1q KO mice. The final component of this proposal (Aim 2) is to test the pervasive nature of microglia-mediated synapse remodeling throughout the developing CNS. Previous and proposed work has been done in the developing retinogeniculate system;therefore, I will test whether microglia-mediated synaptic remodeling also occurs in another brain region, the cerebellum (subaim 2a). Furthermore, I will determine if this is a complement-dependent process (subaim 2b). In addition to normal development, deficits in this pruning process have been implicated in a broad range of developmental (e.g. autism) and psychiatric (e.g. schizophrenia) disorders (Innocenti et al., 2003;Pardo et al., 2005;Vargas et al., 2005;Woo and Crowell, 2005). Furthermore, there is evidence that synapse removal occurs during early stages of neurodegenerative disease (e.g. Alzheimer's)(Selkoe, 2002). Therefore, studying basic mechanisms of normal development provides both a basic understanding of a biological mechanism but also a framework for studying mechanisms of CNS disease and development of therapeutic strategies. PUBLIC HEALTH RELEVANCE: Following birth, in a process called synaptic pruning, many connections between neurons and their nervous system targets are removed leaving behind a highly refined circuitry. Abnormalities in this pruning process are associated with developmental (e.g. autism) and psychiatric (e.g. schizophrenia) disorders as well as early stages of neurodegenerative disease (e.g. Alzheimer's disease). Thus, in addition to elucidating basic mechanisms of nervous system development, understanding synaptic pruning has implications for development of therapeutic strategies targeted toward treatment and/or prevention of nervous system diseases. .
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