Abstracts

Rationale: The mechanisms that drive post-insult epilepsy development are not fully known. Glia and neuro-inflammation are likely critical early aspects of acquired epileptogenesis. Microglia, brain immune cells, engage calcium signaling selectively after injury, inflammation, and hyperactivity. Given this pattern of calcium signaling, we hypothesized that it could be a critical secondary messenger system regulating post-injury microglial physiology.

Methods: To understand patterns of microglial calcium signaling, we performed awake mouse longitudinal two-photon imaging of microglia calcium activity (GCaMP6s) over time in a model of epileptogenesis (systemic kainate administration).

To identify potential mechanism(s) of microglial calcium signaling, we used pharmacology and brain slice preparations to screen key ligands for their ability to provoke calcium activity. 

A candidate molecule, the purine UDP, was further evaluated in vivo to determine its endogenous release patterns in the brain during epileptogenesis using two-photon imaging and a novel UDP biosensor (AAV-hSyn-UDP1.0).

To confirm UDP was activating its endogenous receptor, P2Y6, to evoke calcium signaling, we performed calcium imaging experiments in P2Y6 KO mice. 

Finally, to evaluate the effect of the P2Y6 pathway on multiple facets of epileptogenesis, we employed histology to evaluate microglial phagocytosis and neuronal survival, bulk RNA-seq to evaluate the transcriptome of WT and P2Y6 KO mice, and high-parameter cytometry to evaluate pro-inflammatory tissue states.

Results: Microglia have prolonged two week) enhancements in their spontaneous calcium signaling (in vivo, longitudinal 2P imaging).  Purine damage signals were the strongest candidate to drive microglial calcium signaling. We exogenously applied key purine compounds in brain slice (focal pipette application) and discovered that both ATP, ADP, and UDP can evoke microglial calcium signaling. However, UDP calcium responses were extremely pronounced and became even stronger (sensitized) in brain slices prepared during epileptogenesis. Using a real-time UDP biosensor developed by the Yulong Li lab, we discovered that UDP release can happen under multiple instances of hyperexcitability (0 Mg slice 'epileptiform burst' experiments and longitudinally following kainate status epilepticus). Similarly, KO of the high-affinity UDP receptor P2Y6 abolished calcium elevations in microglia. By restricting microglial calcium signaling through P2Y6 KO, we could attenuate pro-inflammatory cytokine production (TNFa and IL-1B), phagocytic transition in microglia (CD68), and recruitment of outside myeloid populations into the brain. These effects collectively reduced CA3 neuron loss and associated cognitive impairments.

Conclusions: Calcium signaling is a critical regulator of pro-inflammatory, pro-phagocytic microglial transition in a model of epileptogenesis, having early effects in promoting hippocampal neuron loss.

Funding:
This work was supported by grants from the NIH: K99 NS126417 to ADU; R01 NS103212, and RF1 NS122174 to AJJ; R01 NS088627 and R35 NS132326 to L-J.W.