Professor University of California Irvine, United States
Introduction:: Microglia are the resident macrophages in the brain responsible for key functions such as synaptic pruning, turnover of amyloid aggregates, and resolution of inflammatory responses. Their role in amyloid-β maintenance is particularly important in Alzheimer’s disease, where the accumulation of aggregates leads to the formation of plaques that cause neuronal degeneration and death. Recently, microglia have been identified as key effector cells that accumulate in plaque sites and can aid progression of Alzheimer’s Disease through inflammatory potentiation. The accumulation of amyloid plaques additionally causes the brain, one of the softest organs, to contain regions of substantial stiffness. In our study, we investigate the role of the mechanoreceptor PIEZO1 on changes in microglia lipid metabolism, which is important to cellular immune function in neurodegeneration. To stimulate PIEZO1 we used pharmacological agonism with Yoda1 and engineered culture substrates of varying stiffness.
Materials and Methods:: BV2 mouse microglia were seeded either unprimed or primed to an inflammatory state using 0.1 µg/mL of LPS. Cells were then treated with increasing concentrations of Yoda1 ranging from 0 to 20 µM. For confocal visualization of lipid droplets, the cells were fixed, stained with BODIPY and mounted. BODIPY droplets were counted on imageJ software and quantified. The supernatant from the cells on the glass condition were saved to perform sandwich ELISA’s for the measurement of IL-6 and TNF-α. For qPCR analysis cells on the glass condition were collected with Tri reagent and RNA was collected, reverse transcribed to cDNA and qPCR was conducted for various lipid metabolism genes.
Results, Conclusions, and Discussions:: Increasing concentrations of Yoda1 led to a pronounced accumulation of neutral lipid droplets in the BV2 cells. This accumulation was more potent at lower Yoda1 doses and lower substrate stiffness in the LPS primed cells. Increase in lipid droplets is also observed and significant in unprimed cells as. Based on the strong affinity of BODIPY to neutral lipids, the lipid increase mediated by PIEZO1 activation with Yoda1 is characteristic of lipid biosynthesis or transport. In Alzheimer’s Disease several key risk factors are proteins involved in microglia lipid processing and homeostasis such as TREM2 and apoE. This gives early indication that stiffness in the brain in AD may be causing aberrant microglial activation and chronic inflammation through early PIEZO1 activity in microglia. Preliminary gene analysis showed differential regulation of several genes related to microglia activation. For cytokine expression on the other hand, both TNF-α and IL-6 showed a dose dependent increase with Yoda1 only in the LPS primed cells. Of the two cytokines, IL-6 is a prime mediator of microglia activation and proliferation. Because we see this increase only in LPS primed cells, it’s likely that the inflammatory microglia are more ‘mechano-competent’ to the effects of PIEZO1 stimulation which leads to robust lipid accumulation and steady expression of pro-inflammatory cytokines. We demonstrate a key role of PIEZO1 activation in both the biosynthesis of lipid droplets which occurred with increasing Yoda1 for both primed and unprimed cells, with the LPS primed subset more sensitive to this effect. In our current work we are examining how PIEZO1 activation differentially regulates key metabolic genes. We plan to validate these findings with the generation of a BV2 PIEZO1 knockout model, expand our lipid metabolism gene panel, and replicate these findings in human iPSC derived microglia where we also will observe calcium signaling dynamics. Additionally, since we’re seeing increased lipid biosynthesis, we plan to analyze lipidome wide changes in microglia and cholesterol dynamics upon PIEZO1 activation.
Acknowledgements (Optional): : This work was supported by the NIH/NIAD R21 AG069067 grant as well as support from NIH award number S10OD0250264 for the Olympus FV3000 Confocal Microscope.
