Article Figures & Data
Figures
- Figure 1.
Altered expression of METTL3 and RBM15B transcripts in the AD brain. In silico analyses of METTL3 (A) and RBM15B (B) mRNA expression in various postmortem human brain tissues from AD patients (pink dots) and control subjects (blue dots) from the Allen Institute for Brain Science transcriptomic database. Raw data are shown on the upper axes. Estimation plots below display the mean differences between the AD and control groups. METTL3 (HIP, Mdiff = −0.37 [95%CI −0.75,−0.03], p = 0.049; PCX, Mdiff = −0.26 [95%CI −0.63,0.12], p = 0.204; TCX, Mdiff = −0.25 [95%CI −0.63,0.15], p = 0.220). RBM15B (HIP, Mdiff = 0.45 [95%CI 0.06,0.83], p = 0.040; PCX, Mdiff = 0.42 [95%CI 0.09,0.81], p = 0.032; TCX, Mdiff = 0.48 [95%CI 0.13,0.83], p = 0.012). HIP, hippocampus (control, n = 29; AD, n = 21); PCX, parietal cortex (control, n = 27; AD, n = 21); TCX, temporal cortex (control, n = 28; AD, n = 23).
- Figure 2.
Analyses of METTL3, RBM15B, and METTL14 protein levels in the soluble fractions of postmortem AD hippocampal tissues. A, Postmortem hippocampal tissue from AD patients and control subjects from the Alzheimer’s Disease Research Center of the University of California, Irvine, was lysed in T-PER buffer. Detergent-soluble fractions of the lysates were subjected to SDS-PAGE and Western blotting with specific antibodies against METTL3, RBM15B, METTL14, Tau, and β-actin. Representative blots are shown. Densitometry analyses of the blots for METTL3 (B), RBM15B (C), METTL14 (D), and Tau (E) after normalization with β-actin are presented as estimation plots. Raw data are plotted on the left (control, blue dots; AD, pink dots), and mean differences are shown on the right. METTL3 (Mdiff = −0.39 [95%CI −0.75,−0.05], p = 0.030), RBM15B (Mdiff = 0.46 [95%CI 0.11,0.85], p = 0.036), METTL14 (Mdiff = −0.37 [95%CI −0.74,0.02], p = 0.074) and Tau (Mdiff = −0.13 [95%CI −0.33,0.08], p = 0.219). AD (n = 21–23), control (n = 12–17).
- Figure 3.
Elevated level of METTL3 protein in the insoluble fraction of human postmortem AD hippocampal tissues. A, Representative Western blots of detergent-insoluble fractions prepared from postmortem hippocampal tissue from AD patients and control subjects, probed with specific antibodies against METTL3, RBM15B, METTL14, and Tau. Densitometry analyses of the blots for METTL3 (B) and Tau (C) are presented as estimation plots. Raw data are plotted on the left (control, blue dots; AD, pink dots), and mean differences are shown on the right. METTL3 (Mdiff = 2.87 [95%CI 1.27,5.31], p = 0.012) and Tau (Mdiff = 9.92 [95%CI 5.53,18.76], p = 0.003). AD (n = 22), control (n = 16). D, Accumulation of METTL3 in the insoluble fraction of human hippocampal tissues positively correlates with Tau aggregates (control, blue dots; AD, pink dots; Spearman’s correlation coefficient = 0.50, p = 0.0012).
- Figure 4.
Analysis of METTL3 distribution and localization in postmortem human AD hippocampal tissue. Representative immunohistochemical staining of METTL3 in the human postmortem AD (A) and control hippocampal tissues (B). Magnified images of the selected regions are shown below. CA, cornu ammonis. Scale bar = 500 μm (low-power images) or 40 μm (high-power images). C, Negative staining performed on AD hippocampal tissue using the same protocol without the primary antibody against METTL3. D, Quantification of METTL3 OD measurement and mean differences in CA1 (Mdiff = 1.90 [95%CI 0.48,5.15], p = 0.015), CA2 (Mdiff = 1.03 [95%CI 0.42,1.87], p = 0.014), CA3 (Mdiff = 0.84 [95%CI 0.25,2.11], p = 0.025) and the DG (Mdiff = 0.97 [95%CI 0.36,1.70], p = 0.007). Data are presented as estimation plots, AD (n = 6–7), control (n = 7–8). E, Mettl3 does not interact with Tau in cells. HEK293T cells were transfected with the indicated plasmids for 48 h, lysed and immunoprecipitated with anti-myc antibodies. Bound proteins were eluted and resolved by SDS-PAGE, and analyzed by Western blottings with specific antibodies against myc, V5 and METTL14. F, Immunofluorescence staining revealed that METTL3 (red) and Tau (green) do not colocalize in AD hippocampal tissue. MAP2 and DAPI were used to stain for neuronal dendrites (blue) and nuclei (gray), respectively. Scale bar = 50 μm
Tables
- Table 1
Demographic summary of donors associated with the Allen Institute’s Aging, Dementia and TBI Study
Hippocampus Parietal cortex Temporal cortex Group Non-Dementia Dementia Non-Dementia Dementia Non-Dementia Dementia Sample size 29 21 27 21 28 23 Age (years) 88.2 ± 6.4 91.0 ± 6.0 88.4 ± 6.6 90.7 ± 6.1 88.2 ± 6.5 90.1 ± 5.8 Female percentage 48.3% 38.1% 51.9% 28.6% 46.4% 30.4% APOE4 alleles 7.1% 38.9%** 11.5% 36.8%* 11.1% 38.1%* - Table 2
Demographic summary of donors from the Alzheimer’s Disease Research left at University of California, Irvine
Group Control AD p value Sample size 19 26 - Age (years) 90.3 ± 4.4 88.6 ± 6.1 0.31 Female percentage 52.6% 53.8% 0.94 APOE4 alleles 31.6% 46.2% 0.32 χ2 test for gender and APOE4 composition among groups. An unpaired t test was used for age analysis.
- Table 3
Statistical summary of the mRNA and protein changes associated with AD brain tissues
Figure Data Data structure (normality test) Type of test Power p value 1A METTL3 (HIP) Yes Permutation t test [95%CI –0.75,–0.03] 0.049 METTL3 (PCX) Yes Permutation t test [95%CI –0.63,0.12] 0.204 METTL3 (TCX) Yes Permutation t test [95%CI –0.63,0.15] 0.220 1B RBM15B (HIP) Yes Permutation t test [95%CI 0.06,0.83] 0.040 RBM15B (PCX) Yes Permutation t test [95%CI 0.09,0.81] 0.032 RBM15B (TCX) Yes Permutation t test [95%CI 0.14,0.83] 0.012 2B Soluble METTL3 No Permutation t test [95%CI –0.75,–0.05] 0.030 2C Soluble RBM15B No Permutation t test [95%CI 0.11,0.85] 0.036 2D Soluble METTL14 Yes Permutation t test [95%CI –0.74,0.02] 0.074 2E Soluble tau No Permutation t test [95%CI –0.33,0.08] 0.219 3B Insoluble METTL3 No Permutation t test [95%CI 1.27,5.31] 0.012 3C Insoluble tau No Permutation t test [95%CI 5.53,18.76] 0.003 4D IHC (CA1) Yes Permutation t test [95%CI 0.48,5.15] 0.015 IHC (CA2) No Permutation t test [95%CI 0.42,1.87] 0.014 IHC (CA3) Yes Permutation t test [95%CI 0.25,2.11] 0.025 IHC (DG) Yes Permutation t test [95%CI 0.36,1.70] 0.007 HIP = hippocampus; PCX = parietal cortex; TCX = temporal cortex; IHC = immunohistochemistry.
HIP PCX TCX p value p value p value Writer METTL3 ↓ 0.0487 * n.s. n.s. METTL14 n.s. n.s. n.s. MACOM CBLL1 n.s. n.s. n.s. RBM15 n.s. n.s. n.s. RBM15B ↑ 0.0388 * ↑ 0.0337 * ↑ 0.0141 * ZC3H13 n.s. n.s. n.s. VIRMA n.s. n.s. n.s. WTAP n.s. n.s. n.s. Eraser FTO n.s. n.s. n.s. ALKBH5 n.s. n.s. ↑ 0.0143 * Reader EIF3A n.s. n.s. n.s. ELAVL3 n.s. n.s. ↑ 0.0337 * ELAVL4 n.s. n.s. n.s. YTHDF1 n.s. n.s. n.s. YTHDF2 n.s. n.s. n.s. YTHDF3 n.s. n.s. n.s. HNRNPA2B1 n.s. ↑ 0.0142 * n.s. FMR1 n.s. n.s. n.s. IGF2BP1 n.s. n.s. n.s. IGF2BP2 n.s. n.s. n.s. IGF2BP3 n.s. n.s. n.s. YTHDC1 n.s. n.s. n.s. YTHDC2 n.s. n.s. n.s. HIP = hippocampus; PCX = parietal cortex; TCX = temporal cortex; MACOM = m6A-methyltransferase associated complex.
↑ and ↓ indicate an increase and decrease, respectively, in the gene expression pattern in AD compared with control subjects.
n.s. = not significant; *p < 0.05; unpaired t test.
Extended Data Table 2-1
Human brain samples from normal and Alzheimer’s disease patients analyzed in this study. Download Table 2-1, DOCX file.
Extended Data Figure 3-1
The levels of Aβ40 or Aβ42 do not correlate with METTL3 accumulation in the insoluble fractions. Relative levels of Aβ40 (A) or Aβ42 peptides (B) of control (n = 11–12, blue dots) and AD (n = 14–17, pink dots) as measured by ELISA. Data are presented as estimation plots. Accumulation of METTL3 in the insoluble fraction of human hippocampal tissues does not correlate with Aβ40 (C) or Aβ42 (D) levels. Spearman’s correlation coefficients and p values are shown on each graph. Download Figure 3-1, TIF file.
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