While microglia are well known for their role as the immune cells of the brain, more recent evidence demonstrates their role in sculpting the synaptic landscape of the developing brain, including phagocytosing dendritic spines during peak pruning periods (1). It is unknown if microglia play similar roles in synaptic sculpting in th healthy adult brain. Additionally, microglia migrate to sites of neuronal injury, and could also participate in synaptic remodeling to aid in recovery - a function that could go awry with chronic neuroinflammatory responses. However, despite our knowledge of microglia in development, it is unknown if microglia possess these abilities in the adult and injured brain, and the functional consequences of them. I have developed tools that will allow me to address these key issues for the first time. My group has discovered that microglia in the adult brain are physiologically dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival, and that we can take advantage of this dependency through the administration of CSF1R inhibitors. This results in the rapid elimination of >99% of all microglia, thus allowing us to study the role f these cells in a fashion not previously possible. Additionally, I have been working with a highly novel transgenic model of inducible neuronal loss, which also sparsely expresses EGFP in neurons, allowing for visualization, counting, and classification of dendritic spines. Thus, by using these technologies together, I am in a unique position to answer if and how microglia sculpt the adult synaptic landscape, and how this is altered with neuronal injury. Such information is critical to understand how microglia activation throughout a chronic neurodegenerative disease, or following a brain injury, shapes the synaptic environment. To answer this question, CaM-Tet mice that undergo forebrain neuronal injury/loss upon induction of diphtheria toxin expression were crossed to mice that express EGFP in neuronal subsets (2, 3). Half of the mice in which neuronal injury is induced and half of the mice in which it is not induced were treated with a CSF1R antagonist that eliminates microglia from the brain for 2 months, thus allowing me to look at the effects of chronic microglial elimination, as well as a chronic neuronal injury. I have discovered that dendritic spine number is greatly increased in uninjured mice with their microglia eliminated compared to uninjured mice with their microglia intact, showing for the first time that microglia are involved in modulating dendritic spine numbers in the healthy adult brain. Going forward, the impact of microglia on the synaptic landscape in the injured adult brain in other two groups of mice will be determined by analysis of the number and morphology of spines in the CA1 region of the hippocampus. It is also important to understand how microglia contribute uniquely to different types of synapses, so immunohistochemistry and biochemistry will be employed to determine how glutamatergic, GABAergic, and cholinergic signaling are differentially affected. The involvement of the complement cascade in microglia-mediated synaptic modeling will also be investigated, as recent evidence indicates that pruning during development by microglia is dependent on C3/C3R signaling (4). Immunohistochemical and biochemical techniques will be used to determine the involvement and expression of C1q and C3 in the four groups of mice previously described, and the correlation of these markers with microglial morphology and activation state. Indeed, we recently produced qPCR data from mice with their microglia eliminated demonstrating that C3 mRNA expression is attenuated to 17% of that in untreated mice, supporting the idea that microglia are the primary producers of C3. As a result, we will go forward by investigating the dependence of microglia on C3 signaling for dendritic spine pruning by treating both C3 knockout and wild type mice with either vehicle or CSF1R antagonist. Finally, chronically activated microglia contribute to long-term inflammation in many neurodegenerative disorders and brain injuries. Therefore, it is important to determine the consequences of chronic microglia activation, which extends beyond the initial insult period, on synapse structure and the resulting effects on cognition. To achieve this, four groups of mice comparable to those used in the initial experiments were subjected to expression of diphtheria toxin, resulting in neuronal loss. Half of unlesioned and half of lesioned mice were treated with the CSF1R antagonist following, but not during, the lesion period and cognition and motor abilities will be assessed via behavioral end points.
Neurodegenerative diseases, as well and traumatic brain injuries, result in chronic and dramatic activation of microglia. It is critical to understand how the acute and long-term upregulation in inflammatory signaling by microglia contributes to synaptic sculpting in the adult brain, as well as the functional consequences on cognitive abilities.
|Spangenberg, Elizabeth E; Lee, Rafael J; Najafi, Allison R et al. (2016) Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-Î² pathology. Brain 139:1265-81|
|Rice, Rachel A; Spangenberg, Elizabeth E; Yamate-Morgan, Hana et al. (2015) Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus. J Neurosci 35:9977-89|
|Elmore, Monica R P; Najafi, Allison R; Koike, Maya A et al. (2014) Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82:380-97|