Using murine-derived and human-derived microglia-like cell lines (BV2 and HMC3) and primary cultured murine microglia upon exposure to different immune challenges (TLR4 pathway, E. coli Lipopolysaccharide LPS; TLR3 activation, via the double-stranded RNA synthetic analog polyI:C; RIG-I/MDA-5-MAVS-IFN axis, using Lipofectamine-encapsulated polyI:C; and a pro-inflammatory activation cocktail, LPS + IFN-), we characterized the activation profile detecting the levels of IL1 and nitrites. LPS and LPS + IFN- stimuli increased drastically the levels of both pro-inflammatory mediators while polyI:C showed slight upregulation. To complement these, the levels of IFN- expression were measured by qRT-PCR, and found that any of these stimulati triggered an increase.
Then, we tracked MAM activities by measuring 3H-serine into 3H-phosphatidylserine and 3H-phosphatidylethanolamine will be tracked by thin-layer chromatography. By doing so, we found an upregulation of MAM upon 24h of stimulation with LPS or LPS + IFN- while polyI:C showed a decrease. Similar results were found in primary microglia when assaying the MAM-located ACAT1 (acyl-coenzyme A:cholesterol acyltransferase 1) activity by tracking the incorporation of 3H-cholesterol into 3H-cholesteryl esters. However, ACAT1 activity was below the detection limit in BV-2 and HMC3 cell lines, probably due to low uptake of 3H-cholesterol.
Proteomics and lipidomics analysis were performed to address the molecular landscape of MAM upon microglia activation. These results – still under analysis – will pinpoint relevant lipid pathways as potential targets for intervention.
Once MAM changes triggered by inflammatory stimuli were established, we aimed to modulate MAM by exposure to exogenously added recombinant sphingomyelinase (SMase), which has been shown to induce the mobilization of cholesterol from plasma membrane towards the ER, triggering MAM upregulation. By the same token, inhibition of MAM formation was achieved with specific inhibitors of SMase activity (GW4869 and/or desipramine), which are known to prevent cholesterol delivery to ER and impede the formation of MAM domains. Our results indicated, as expected, that SMase treatment enhanced MAM activities and microglia activation profile upon inflammatory stimulation. However, SMase inhibition showed no consistent alteration on the activation profile. Analyzing the lipidomic profile of microglia upon MAM intervention will help address whether this differential effect is due to alterations in any specific lipid pathway.
