Jordy Sepulveda ¹, Michel Fallah³, Charlie Furlong¹, Stefano Vicini¹,³, and G. William Rebeck²,³.

1 Department of Pharmacology & Physiology, Georgetown University

2 Department of Neuroscience, Georgetown University

3 Interdisciplinary Program in Neuroscience, Georgetown University

Background: Neuroinflammation exacerbates the progression of AD pathology. Neuroinflammation is modulated by APOE4, the strongest genetic risk factor for AD. In the brain, apoE is primarily produced by astrocytes and microglia and acts as an immunomodulator of brain cytokines, suggesting a direct role of apoE in microglial activation. Previously, we demonstrated that astrocytes and microglia secrete different glycosylated forms of apoE and that APOE4 astrocytes and microglia produce significantly less apoE compared to APOE2 and APOE3 resulting in altered immunomodulation. Although the effects of APOE on cytokine release and phagocytosis have been studied, it remains unclear how APOE genotype affects other microglial homeostatic functions, such as chemotaxis and motility. We hypothesized that APOE4 confers a proinflammatory microglia phenotype in vivo that includes a simplification of microglial ramified processes, altered spontaneous motility, and impaired reactivity to chemotactic cues.

Methods: We generated a novel human APOE knock-in mice expressing GFP under the CX3CR1 promoter to study the effects of APOE genotype on homeostatic microglial function. Microglia activation was assessed in 30mm slices from 5 to 6 month old mice (n = 6 APOE3; n = 4 APOE4), by measuring microglial cell density, and total number of processes and lengths. To determine whether APOE genotype alters responsive microglia motility to chemotactic ligands, the velocity of ATP-directed microglial processes motility was tracked from 5 to 6 month old mice (n = 3 to 5 APOE3; n = 3 to 5 APOE4), using confocal microscopy in acute slices for 30 minutes.

Results: Microglia density and morphology was not altered by APOE genotype. Hippocampal APOE4 microglia displays a significantly lower mean processes velocity in response to 1mM ATP (0.96 ± 0.08 μm/min, p<0.0001, n=18 cells from 5 animals) compared to APOE3 microglia (1.5 ± 0.16 μm/min, n=9 cells from 3 animals) but not in the entorhinal cortex. Slower processes velocity was also displayed in APOE4 microglia in response to 3mM ATP (0.84 ± 0.05 μm/min, p<0.0015, n=19 from 5 animals), and 10mM ATP (0.79± 0.07 μm/min, p<0.023, n=29 cells from 5 animals) relative to APOE3 microglia 3mM ATP (1.4 ± 0.13 μm/min, n=19 cells from 4 animals), and 10mM ATP (1.06 ± 0.07 μm/min, n=20 cells from 4 animals).

Conclusion: Together, our finding demonstrated that in APOE4 microglia have an altered response to ATP despite no morphological changes. Based on these preliminary data, we will further investigate whether APOE genotype affects microglia sensitivity to ATP, or the number of purinergic receptors that responds to ATP. Understanding the effect of APOE genotype on homeostatic microglial function can potentially explain how APOE4 brain are more susceptible to neuronal degeneration.

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