Extended Data Figure 4. Expression profile of IFITM3 and other markers in LOAD and age-matched controls.
(a) Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analyzed in the cortex (n=158) and hippocampus (n=123) of normal human brains using the Genotype-Tissue Expression (GTEx) cohort.. (b) mRNA expression in non-demented subject control (n=10) and LOAD samples (n=18) of MAP2 (ns), GFAP (**p=0.0046), and AIF1 (ns) were measured, which were used in Fig. 4c–d. (c) Expression profiles of MAP2 (ns), GFAP (****p<0.0001), and AIF1 (ns) in the temporal cortex of human control (n=76) and LOAD samples (n=80) using the Mayo Clinic cohort data. Correlation analyses were carried out and p values were calculated. (d) The protein levels of Nct (****p<0.0001) and PS1-NTF (**p=0.0042) in human brain membranes (control and LOAD). The samples were analyzed by WB and quantified (n=10 and 18, respectively). Signal was normalized to HeLa cell membrane. (e-f) IFITM3 SNP Genotypes. (e) Allelic discrimination plot depicting rs34481144 genotype calls for control (n=9), LOAD-L (n=10), and LOAD-H (n=8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n=2). (f) Allele frequency of rs34481144 genotype in control (n=9) and LOAD (n=18). (g) mRNA level of IFITM3 gene in four types of EGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons)(n=4 per group, GAD2-PV: ***p=0.0003, CCK-PV: ****p<0.0001, CCK-Cort *p=0.0363, PV-Cort: **p=0.0059). (h) mRNA levels of IFITM3 in human iPSC-derived neurons (n=4) and human primary astrocytes (n=3) were measured by qPCR (****p<0.0001). (i-j) Human iPSC-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bar = 200 and 500 μm. (k) Induction of IFITM3 by IFN-α in primary neurons. E16 mouse primary neurons were treated with 100 ng/ml of IFN-α at DIV12 for 24 hours. The protein levels of γ-secretase and IFITM3 were analyzed by WB (n=4 per group). β-Tubulin III was used as a loading control. (l) Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*p=0.0116) and Aβ42 (*p=0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-ΔID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n=12, 10). (m) JC8 whole cell photolabeling. Neuronal membranes were photolabeled with JC8 in the absence or presence of L458 and analyzed by anti-PS1-NTF antibody. Photolabeled PS1-NTF protein level was quantified by Odyssey imaging (n=3, *p=0.0210). (n) Spearman’s correlation between the expression level of IFITM3 and viruses. In the Brodmann Area 22 (BA-22 region) in the Mount Sinai Brain Bank (MSBB) cohort, the expression level of IFITM3 is positively correlated with the expression level of the human herpesvirus-6B (HHV-6B) (rho=0.248, p=0.044, n=66). In the Brodmann Area 36 (BA-36 region), the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho=0.255, p=0.033, n=70). All WB images and graphs are representative of two independent experiments (except; b/e: 1 replicate, h: data pooled from 2 experiments, k: 3 replicates, l: data pooled from 4 experiments). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, two-sided Student’s t-test (except; g: One-Way ANOVA followed by Tukey).