Microglial phagocytosis dysfunction in the dentate gyrus is related to local neuronal activity in a genetic model of epilepsy

Abstract

Objective: Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very effi-cient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB). Methods: We used confocal imaging and stereology-based quantification of apop-tosis and phagocytosis of the hippocampus of Cstb knockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apopto-sis, phagocytosis, and active hippocampal neurons. Results: Microglial phagocytosis was impaired in the hippocampus of Cstb KO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present in Cstb KO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) in Cstb-deficient mice were at close proximity to active cFos+ neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos+neurons was specific for Cstb KO mice. Significance: These results suggest a complex crosstalk between apoptosis, phago-cytosis, and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, these data suggest that phago-cytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.