Fig 1.
The loss of FTO leads increased levels of corticosterone and ACTH.
(A-B) Quantification of plasma levels indicated that corticosterone (A) and ACTH (B) are significantly elevated in Fto-/- mice, n = 16 Fto+/+ / 19 Fto-/- (A); n = 13 Fto+/+ / 5 Fto-/- (B). Data are presented as mean ± S.E.M., unpaired t-test, *P < 0.05; **P < 0.01.
Fig 2.
The loss of FTO leads to anxiety-like behavior and impairments in working memory.
(A-B) The escape latency of 8 weeks old (A) and 16 weeks old (B) Fto+/+ and Fto-/- mice in the cage emergence test indicated a higher anxiety level in Fto-/- mice, n = 24 Fto+/+ / 20 Fto-/- (A); n = 11 Fto+/+ / 18 Fto-/- (B). (C-D) Measurements of the open arm time in the elevated plus maze test for 8 weeks old (C) and 16 weeks old (D) Fto+/+ and Fto-/- mice revealed a higher anxiety level in Fto-/- mice, n = 6 (C); n = 12 Fto+/+ / 9 Fto-/- (D). (E) Fto-/- mice show an unaltered escape latency during 5 days’ navigation training in the Morris water maze, n = 9 Fto+/+ / 12 Fto-/-. (F) Fto-/- mice need more time to visit every single arm at least once in the radial eight-arm maze, n = 7 Fto+/+ / 5 Fto-/-. (G) The total number of memory errors in the radial eight-arm maze was higher for Fto-/- mice, n = 7 Fto+/+ / 5 Fto-/-. Data are presented as mean ± S.E.M., unpaired t-test, *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3.
The loss of FTO leads to impairment of neuronal differentiation in the dentate gyrus of the hippocampus.
(A-B) Representative overview images of whole dentate gyrus of the hippocampus of Fto+/+ and Fto-/- mice.(C-H) Detailed representative immunofluorescence staining images show DCX+ neurons of the dentate gyrus in the hippocampus of Fto+/+ and Fto-/- mice. (I) Quantitative analysis indicated that Fto-/- mice had a lower percentage of DCX+ neurons per total cell number in the dentate gyrus than Fto+/+ mice, n = 4 Fto+/+ / 5 Fto-/-. Data are presented as mean ± S.E.M., unpaired t-test, **P < 0.01.
Fig 4.
The loss of FTO leads to a misbalance of mBDNF to proBDNF in the hippocampus and a decreased downstream pathway activation.
(A) Representative western blot images showing the protein levels of proBDNF and mBDNF with actin as loading control on one gel. For better clarity, the distance between the bands was cut out. (B-C) Quantification of images indicated significant lower mBDNF amounts and equal proBDNF amounts in the hippocampus of Fto-/- mice, n = 6. (D) Representative western blot images of p-MAP2K1/2 with HPRT as loading control. (E) Quantification of images indicated a significant reduced MAPK pathway activity by revealing a decreased p-MAP2K1/2protein amount in the hippocampus of Fto-/- mice, n = 4. (F-G) Western blot analysis of the nonphosphorylated form of MAP2K1/2 showed no change in Fto-/- mice, n = 4. (H) Comparison of the nonphosphorylated and phosphorylated MAP2K1/2 amounts revealed a significant decreased of p-MAP2K1/2 to MAP2K1/2 ratio in the hippocampus of Fto-/- mice. (I) Relative quantification results from real-time PCR analysis for Bdnf mRNA transcripts revealed a slight downregulation in Fto-/- mice in the hippocampus, n = 8 Fto+/+ / 11 Fto+/+. Data are presented as mean ± S.E.M., unpaired t-test, *P < 0.05; ***P < 0.001.
Fig 5.
FTO seems to regulate BDNF processing through modulation of the MMP-9 amounts.
(A) Representative western blot images for proteins involved in the cleavage of BDNF showing the protein levels of PC1/3, Furin, Plasminogen, TIMP1, MMP-7 and MMP-9 in the hippocampus. Actin or GAPDH were used as loading controls. (B-G) Quantification of western blots indicating no differences in the protein amounts of PC1/3 (B), Furin (C), Plasminogen (D), TIMP1 (E) and MMP-7 (F) between genotypes (n = 3) but a decreased protein amount of MMP-9 (G) in Fto-/- mice (n = 7). (H-J) Quantification of Mmp-3 (H), Mmp-7 (I) and Mmp-9 (J) mRNA transcripts detected in the hippocampus by real-time PCR. The mRNA expression of Mmp-9 is reduced in the hippocampus of Fto-/- mice, n = 6 Fto+/+ / 7 Fto-/-. Data are presented as mean ± S.E.M., unpaired t-test, **P < 0.01 ***P < 0.001.