Fig. 2

Inhibition of γ-secretase activity decreases TREM2-dependent phagocytosis in microglial BV-2 cells. a Analysis of endogenous γ-secretase components PS1 and PS2 in microglial BV-2 cells by Western immunoblotting. Characteristic N-terminal fragment (NTF) and C-terminal fragment (CTF) of PS1 and PS2 were detected, indicating endogenous expression of both PS proteins. P-PS1 CTF denotes the migration of a phosphorylated variant of the PS1 CTF. b, c BV-2 cells were seeded on μ-IBIDI dishes (IBIDI, Germany) and transfected with myc-TREM2-GFP. Cells were cultured in the absence or presence of 1 μM of DAPT for 24 h. Four microliters of latex beads (Sigma-Aldrich, USA) was added, and TREM2 was stimulated for 2 h by addition of anti-myc antibody. Uptake of latex beads was analyzed by light microscopy (*p < 0.05; **p < 0.01; n = 97–105 cells per condition). Arrow heads indicate internalized leads. d BV-2 cells with endogenous expression of PS1 and PS2 (see a) were preincubated in the absence or presence of DAPT (d) for 24 h. Cells were stimulated with anti-myc antibodies, and changes in [Ca²⁺]_i were monitored by fluorescence microscopy. Treatment with DAPT abrogated the cellular response in [Ca²⁺]_i. e Cells stably overexpressing PS1 wild type (PS1 WT) or a dominant-negative variant of PS1 (PS1 DN) were stimulated with anti-myc. Only cells expressing PS1 WT, but not PS1 DN, responded to TREM2 activation with an increase in [Ca²⁺]_i. As observed in cells with endogenous PS expression, pretreatment of PS1 WT-overexpressing cells with DAPT also inhibited the increase in [Ca²⁺]_i (****p < 0.0001; n = 3 to 11 cells per condition)