Characterization of Microglia and Phagocytic Signaling Molecules in Human and Experimental Epilepsy
Abstract
Microglia-mediated neuroinflammation is widely associated with seizures and epilepsy. Although microglial cells are professional phagocytes, less is known about the status or morphology of this phenotype in epilepsy. Recent evidence supports that phagocytosis-associated molecules from the classical complement (C1q-C3) play novel roles in microglia-mediated synaptic pruning. Interestingly, in human and experimental epilepsy, altered mRNA levels of complement molecules were reported. Also, more recently, microglia phagocytosis has been associated with the phospholipid, phosphatidylserine, and the receptors that it interacts with including MerTK, Pros1, Tim4, and Trem2. These receptors have been found to play a crucial role in the actin remodeling of microglia. These receptors and ligands have been termed as the “eat me” signals. There have also been more recent studies showing altered morphology of microglia, but the exact biochemical properties of these morphologies has yet to be determined. Therefore, to identify a potential role for “eat me” signals and the morphology of microglia/macrophages in the pathology of epilepsy, we determined the protein levels of the “eat me” signaling molecules in human epilepsy and in the pilocarpine model of status epilepticus (SE), as well as the inflammatory and phagocytic properties of the peak morphologies of microglia during the time course of SE. Cortical brain samples surgically resected from patients with refractory epilepsy (RE) and tumor adjacent non-epileptic lesions (NE) were examined as well as hippocampi from control rats and rats at 4hr-, 1day-, 3day-, 6day-, 14day-, 3.5wk-, and 5wk-post-SE. Western blotting was used to determine the levels of phagocytosis signaling proteins such as the complements C1q and C3, MerTK, Trem2, and Pros1 along with cleaved-caspase 3, for apoptosis. In addition, immunostaining was used to determine the distribution of C1q and co-localization to microglia/macrophages and dendrites in the human tissue. Immunostaining was also used to determine the distribution of the morphology of microglia/macrophages, IBA1, in the animal model at key timepoints including control, 3day-, and 14day-post-SE. This was followed by co-localization immunostaining with IBA1 and TNFα and CD68. We found that the RE samples had significantly increased protein levels of C1q (p = 0.034) along with those of its downstream activation product iC3b (p = 0.027), and decreased levels of Trem2 (p = 0.045) and Pros1 (p = 0.005) when compared to the NE group, but no significant changes were found for Trem2 and Pros1 during the time course in the animal model. We found C1q localization to microglia and dendrites in both NE and RE samples, and observed substantial microglia-dendritic interactions in the RE tissue. The distribution of microglia/macrophages in the 3day- and 14day-post-SE tissue also showed an increase in amoeboid morphology compared to the control tissue, while there was only an increase in TNF? at the 3day-post-SE amoeboid morphology. These data suggest that aberrant phagocytic signaling occurs in chronic epilepsy, but may not play an immediate role in the development of epilepsy. The altered morphology of microglia may be expressing multiple biochemical properties suggesting a more pronounced difference between inflammatory and phagocytic phenotypes. It is likely that alteration of phagocytic pathways may contribute to unwanted elimination of cells/synapses and/or impaired clearance of dead cells. Future studies will investigate whether altered “eat me” signals and microglia morphology contribute to the hyperexcitability that results in epilepsy.^
Degree
M.S.
Advisors
Amy L. Brewster, Purdue University.
Subject Area
Neurosciences|Physiology
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