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

Abstract

Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response in neurodegenerative diseases. Phagocytosis is very efficient in physiological conditions, as well as during apoptotic challenge induced by excitotoxicity or inflammation, but is impaired in mouse and human mesial temporal lobe epilepsy (MTLE). Here we extend our studies to a genetic model of progressive myoclonus epilepsy type 1 (EPM1) in mice lacking cystatin B (CSTB), an inhibitor of cysteine proteases involved in lysosomal proteolysis. We first demonstrated that microglial phagocytosis was impaired in the hippocampus in Cstb knock-out (KO) mice when seizures arise and hippocampal atrophy begins, at 1 month of age. To test if this blockage was related to the lack of Cstb in microglia, we used an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression but we found no significant effect in the phagocytosis of apoptotic cells. We then tested whether seizures were involved in the phagocytosis impairment, similar to MTLE, and analyzed Cstb KO mice before seizures begin, at postnatal day 14. Here, phagocytosis impairment was restricted to the granule neuron layer but not to the subgranular zone, where there are no active neurons. Furthermore, we observed apoptotic cells (both phagocytosed and not phagocytosed) in Cstb deficient mice at close proximity to active, cFos + neurons and used mathematical modeling to demonstrate that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. These results suggest a complex crosstalk between apoptosis, phagocytosis and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, this data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis. METHODS Mice. Tissues ofCstb KO mice 7 (129S2/SvHsd5-Cstb tm1Rm ; Jackson Laboratory stock no. #003486) were obtained from the Lehesjoki´s lab at Folkhälsan Research Center and University of Helsinki. Wild-type litter mates with the same genetic background were used as controls. For the FACS experiments, fms-EGFP mice (B6.Cg-Tg(Csf1r-EGFP)1Hume/J; Jackson Laboratory stock #018549), where microglia constitutively express the GFP protein 25,26 were used. All procedures followed the European Directive 2010/63/EU and NIH guidelines, and were approved by the Animal Ethics Committee of the State Provincial Office of Southern Finland (decisions ) and Ethics Committees of the University of the Basque Country EHU/ UPV (Leioa, Spain; CEBA/205/2011, CEBA/206/2011, CEIAB/82/2011, CEIAB/105/2012). BV2 cell line. BV2 cells (Interlab Cell Line Collection San Martino-Instituto ScientificoTumori-Instituto Nazionale per la Ricerca sul Cancro), a cell line derived from raf/mycimmortalized rat neonatal microglia was used to perform the in vitro knock-down model of Cstb. BV2 cells were grown...

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