Fig. 1

Trem2 H157Y increases TREM2 shedding. A. Trem2 H157Y knock-in mice were generated by introducing a C > T mutation (bold orange) via CRIPR/Cas9. Protospacer region recognized by guide RNA (gRNA) is shown in orange. Protospacer adjacent region (PAM) is indicated in green. B-C. Cortical Trem2 mRNA levels were examined using primers targeting exon 2 (N-terminal, B) or exon 4–5 (C-terminal, C), and normalized to WT mice for each genotype. D. Cycle threshold ratios of C-terminal Trem2 to N-terminal Trem2 (C/N Trem2) were calculated and normalized to WT mice for each genotype. E–F. TREM2 levels were examined by ELISA and normalized to WT mice in cortical TBS (E) and TBSX (F) lysates for each genotype. G-H. TREM2 levels were examined by ELISA in conditioned medium (CM) (G) and RIPA lysates (H) of primary microglia (MG). TREM2 amount was normalized to the total protein level of cell lysates followed by another normalization to WT littermates. N = 8–11 pups per genotype. Unknown sex of each pup. I. Serum TREM2 were examined by ELISA in mice from each genotype. J. SYK, phosphorylated SYK (pSYK) and actin were detected in the RIPA lysates of isolated microglia from WT and Hom mice. K-L. pSYK (K) and SYK (L) were quantified and normalized to WT. M. Ratios of pSYK/SYK were calculated and normalized to the WT mice. B-F, I. N = 11–14 mice per genotype at 6 months of age, mixed sex. Kruskal–Wallis tests with uncorrected Dun’s multiple comparisons were used in B-I. J-M. N = 6 mice/genotype at 6 months of age, mixed sex. Unpaired t-tests were used. Data are presented as Mean ± SEM. N.S., not significant. * p < 0.05. **p < 0.01