Fig 1.
Age-dependent iron accumulation in the SN of iPLA2β-KO mice shown by Perl’s DAB enhanced staining.
(A–L), Perl’s DAB enhanced staining of the SN of WT mice at 15 weeks (A and B), 56 weeks (C and D), and 100 weeks (E and F), and iPLA2β-KO mice at 15 weeks (G and H), 56 weeks (I and J), and 100 weeks (K and L). (B), (D), (F), (H), (J), and (L) are the high magnification views of (A), (C), (E), (G), (I), and (K), respectively (low power field, LPF; high power field, HPF). (A–F) In WT mice at 15 weeks, only a few iron depositions are observed mainly in oligodendrocytes (A, small arrows in B). The inset in (B) is a high magnification of an iron-containing oligodendrocyte. The number of iron deposition increases in WT at 56 weeks (C, small arrows in D), compared with that at 15 weeks (A and B). In WT mice at 100 weeks, iron depositions become more prominent mainly in SN pars reticulate and are observed in nerve fibers (asterisks in F) as well as oligodendrocytes (E and F), in comparison with those of WT mice at 56 weeks (C and D). (G–L) In iPLA2β-KO mice at 15 weeks, iron depositions are observed in a few oligodendrocytes (G, small arrows in H), which are almost equal with those of WT mice at 15 weeks (A and B). In iPLA2β-KO mice at 56 weeks, iron depositions significantly increase and are also observed in the nerve fibers (I, asterisks in J) compared with those of WT mice at 56 weeks (C and D). In KO mice at 100 weeks, marked iron depositions are observed in a large number of oligodendrocytes and nerve fibers (K and L), which are more prominent than iPLA2β-KO mice at 56 weeks (I and J) and WT mice at 100 weeks (E and F). Scale bar in (A) represents 100 μm in (A), (C), (E), (G), (I), and (K), and 50 μm in (B), (D), (F), (H), (J), and (L).
Fig 2.
Age-dependent iron accumulation in other brain regions of iPLA2β-KO mice.
(A‒N) Perl’s DAB enhanced staining of the ST of WT mice at 15 weeks (A and B) and 56 weeks (C and D); the Gp of WT mice at 56 weeks (E and F); the ST of iPLA2β-KO mice at 15 weeks (G and H), 56 weeks (I, J), and 100 weeks (M); the Gp of iPLA2β-KO mice at 56 weeks (K and L) and 100 weeks (N). (B), (D), (F), (H), (J), and (L) are high magnification views of (A), (C), (E), (G), (I), and (K), respectively (low power field, LPF; high power field, HPF). In the ST of WT mice at 15 weeks (A and B) and iPLA2β-KO mice at 15 weeks (G and H), only a few iron depositions are observed in oligodendrocytes (small arrows in B and H), which show no significant differences between them. In the ST of iPLA2β-KO mice at 56 weeks, iron depositions increased significantly and are also observed in the nerve fibers (I, asterisks in J), in comparison with those of WT mice at 56 weeks (C and D) and those of iPLA2β-KO mice at 15 weeks (G and H). In the Gp of iPLA2β-KO mice at 56 weeks, prominent iron depositions are seen in a lot of oligodendrocytes as well as nerve fibers (K and L), in comparison with those in the Gp of WT mice at 56 weeks (E and F). Iron accumulation becomes more prominent in the ST (M) and the Gp (N) of iPLA2β-KO mice at 100 weeks than those in the ST and Gp of iPLA2β-KO mice at 56 weeks (I, J, K, and L). Scale bar in (A) represents 100 μm in (A), (C), (E), (G), (I), (K), 50 μm in (B), (D), (F), (H), (J), (L), (M), (N).
Fig 3.
Western blotting analyses of DMT1 and TfR1 in iPLA2β-KO mice at 100 weeks.
(A) Western blotting was applied to detect the expressions of DMT1 + IRE and TfR1 in iPLA2β-KO mice and WT mice at 100 weeks. (B) Statistical analysis. Data are presented as the ratio of TfR1 or DMT1 to GAPDH (WT mice = 1.0). Each bar represents the mean ± SD. *p < 0.01, **p < 0.05, Wilcoxon's rank-sum test.
Fig 4.
Western blotting analyses of IRPs in iPLA2β-KO mice at 100 weeks.
(A) Western blotting was applied to detect the expressions of IRP1 and IRP2 in iPLA2β-KO mice and WT mice at 100 weeks. (B) Statistical analysis. Data are presented as the ratio of IRP1 or IRP2 to GAPDH (WT mice = 1.0). Each bar represents the mean ± SD. *p < 0.01, Wilcoxon's rank-sum test.
Fig 5.
Immunohistochemistry and Western blotting analyses for 4-HNE in iPLA2β-KO mice.
(A‒D) Immunohistochemistry for 4-HNE in the ST of WT mice (A) and iPLA2β-KO mice at 100 weeks (B‒D). (A) Almost no staining is observed in the ST of WT mice at 100 weeks. (B) In the ST of iPLA2β-KO mice at 100 weeks, the increase of 4-HNE is observed mainly in the white matter (small arrows), and the neuropil is also slightly immunostained with 4-HNE, compared with age-matched WT mice (A). (C, D) In high-power fields, some granules or dots strongly positive for 4-HNE are frequently observed (arrowheads in C) in the neuropil of the ST of iPLA2β-KO mice at 100 weeks. A few spheroids are also faintly immunopositive for 4-HNE (arrow in D). Scale bar in (A) represents 100 μm in (A), (B), and 50 μm in (C), (D), respectively. (E) On Western blotting, expression levels of 4-HNE-protein compound are increased in both ST and SN in iPLA2β-KO mice at 100 weeks in comparison with those of age-matched WT mice, with statistical significance (*p < 0.01, Wilcoxon's rank sum test). Data are presented as the ratio of 4-HNE-protein compound to GAPDH (WT mice = 1.0). Each bar represents the mean ± SD.
Fig 6.
Dopamine cell counts in the SNpc in WT control mice and iPLA2β-KO mice.
(A, B) Representative sections of SNpc immunostained with TH in WT (A) and KO mice (B). Panels A-2 and B-2 show high magnifications of dotted squares in A-1 and B-1, respectively. Scale bar in A-1 represents 100 μm in A-1 and B-1, and 25 μm in A-2 and B-2. (C) Histogram of the number of TH- and Nissl-double-positive neurons in the SNpc in WT (n = 3) and KO mice (n = 3) is shown. There is no significant difference between the two groups of mice (p > 0.05, Wilcoxon's rank sum test).
Fig 7.
Examination of mitochondrial function and cell viability in PLA2G6-KD cells.
Each experiment was performed at least three times. (A) Generation of PLA2G6-KD gene SH-SY5Y cells. Optimal KD efficacy is confirmed by Reverse transcriptase polymerase chain reaction (RT-PCR). (B, C) Western blotting analyses (B) and immunocytochemistry (C) for Tom20 (the marker for the mitochondrial outer membrane) and CCO (the marker of the mitochondrial inner membrane) in PLA2G6-KD cells. (B) Expression levels of CCO are decreased in PLA2G6-KD cells compared with those of SH-SY5Y cells treated with negative control siRNA with statistical significance (*p < 0.01, Wilcoxon's rank sum test), whereas there is no significant difference in expression levels of Tom20 between PLA2G6-KD cells and the negative control. Data are presented as the ratio of CCO or Tom20 to GAPDH (negative control = 1.0), respectively. The experiment was performed three times. Each bar represents the mean ± SD. (C) Immunocytochemistry for CCO (red) and Tom20 (green). Expression levels of CCO are reduced in PLA2G6-KD cells, and only a few cells are positive for CCO (white arrow). Scale bar represents 10 μm for each panel. (D) The amount of ATP in each cell is measured by a Kinshiro ATP luminescence kit. The ATP generation per total proteins is significantly reduced in PLA2G6-KD cells in comparison with negative control siRNA-transfected SH-SY5Y cells (*p < 0.01, Wilcoxon's rank sum test). The experiment was performed three times. Each bar represents the mean ± SD. (E) Knockdown of PLA2G6 gene significantly reduces cell viability in both CTB and LDH assays (*p < 0.01, compared with negative control, Wilcoxon's rank sum test). The experiment was performed five times and the averages of the results are shown.
Fig 8.
Western blotting analyses of the molecules involved in cellular iron homeostasis in PLA2G6-KD cells.
Each experiment was performed three times and the averages of the results are shown. (A, B) Expression levels of DMT1 + IRE, TfR1, IRP1, and IRP2 are significantly increased in PLA2G6-KD cells compared with SH-SY5Y cells treated with negative control siRNA with statistical significance (*p < 0.01, **p < 0.05, Wilcoxon's rank sum test), respectively. (B) Data are presented as the ratio of DMT1, TfR1, IRP1 or IRP2 to GAPDH (negative control = 1.0), respectively. Each bar represented the mean ± SD of four independent experiments.