Immunohistochemical analyses of VMAT2 in the striatum of iPLA₂β-KO mice at 100 weeks.

<p>(A, B): Abnormal structures strongly positive for VMAT2 were also seen in KO mice at 100 weeks (large arrows in A and B); these structures contact striatal neurons (N) (small arrows in insets in A and B). In serial sections, an abnormal structure positive for VMAT2 (an arrow in C) is also immunopositive for TH (arrow in D). Scale bar in (A) represents 25 μm in all panels (A-D).</p

Immunohistochemical analysis for TH in the striatum of WT control mice and iPLA₂β-KO mice.

<p>Representative photographs of the striatum immunostained with TH in WT mice at 15 weeks (A, B), 56 weeks (C, D), and 100 weeks (E, F) and iPLA₂β-KO mice at 15 weeks (G, H), 56 weeks (I, J), and 100 weeks (K, L). (A), (C), (E), (G), (I), and (K) are low power fields (LPF) and (B), (D), (F), (H), (J), (L) are high power fields (HPF). (A-F): In the low power fields, neuropils of the striatum are diffusely stained with TH in WT mice at 15 weeks (A), 56 weeks (C), and 100 weeks (E). In the high power fields, many nerve fibers strongly immunopositive for TH were observed in neuropils of WT mice at 15 weeks (B), 56 weeks (D), and 100 weeks (F). The insets in (B), (D), (F), and (H) are high magnifications of the dotted square from their respective panel. (G-L): In the low power fields, neuropils of the striatum are diffusely stained with TH in iPLA₂β-KO mice at 15 weeks (G), 56 weeks (I), and 100 weeks (K). In the high power fields, many fibers positive for TH were observed in neuropils of the striatum in iPLA₂β-KO mice at 15 weeks (H), which are almost equal in number to those of WT mice at 15 weeks (B). In iPLA₂β-KO mice at 56 weeks, focal loss of TH-positive fibers is seen in some areas (dotted circle in J-3), while the density of TH-positive fibers (arrows in J-3) is preserved in other areas (J-2, 3). In iPLA₂β-KO mice at 100 weeks, the density of TH-positive fibers (arrows in L-2) are lower than that of WT mice at 100 weeks (F) and iPLA₂β-KO mice at 56 weeks (J-2, 3). Panels (J-2), (J-3), and (L-2) are high magnifications of the dotted squares in (J) and (L), respectively. Scale bar in (A) represents 100 μm in (A), (C), (E), (G), (I), and (K), and 25 μm in (B), (D), (F), (H), (J), and (L).</p

Double immunohistochemistry to detect ubiquitin and TH in the striatum of iPLA₂β-KO mice.

<p>(A, B): Double immunostaining of TH (brown) and ubiquitin (blue) in the striatum of iPLA₂β-KO mice at 15 weeks (A) and 100 weeks (B). (A): At 15 weeks, a few round, TH-positive structures were seen (brown, an arrow), while blue staining was not found in this view (immunohistochemistry for ubiquitin is negative). (B): At 100 weeks, several ubiquitin-positive structures were found (blue staining, arrowheads), but these were distinct from the round, TH-positive structures (brown staining, arrows). Scale bar in (A) represents 25 μm in (A) and (B).</p

Immunohistochemical analyses of TH, SMI31, and MAP2 in the striatum iPLA₂β-KO mice at 100 weeks.

<p>(A), (C), and (E) are serial sections. (B), (D), and (F) are high magnifications of squares in (A), (C), and (E), respectively. Many TH-positive structures can be seen in the striatum of iPLA₂β-KO mice at 100 weeks (A, arrows in B), which were mostly negative for SMI31 (C, D) and MAP2 (E, F). Scale bar in (A) represents 200 μm in (A), (C), and (E), and 50 μm in (B), (D), and (F).</p

Quantitative analysis of optical densities of TH and DAT in the striatum.

<p>Histograms show quantitative analysis of optical densities of TH (A) and DAT (B) immunostaining in the striatum. WT mice, gray bars; KO mice, black bars. Data are presented as the mean ± standard deviation. The number (n) of animals examined is indicated in each histogram. Vertical axis in both (A) and (B) shows percent density relative to WT mice at 15 weeks. (A) Symbols indicate statistically significant differences; *<i>p</i> < 0.05 vs. age-matched WT mice and iPLA₂β-KO mice at 15 weeks (Wilcoxon’s rank sum test). (B) Symbols indicate statistically significant differences; *<i>p</i> < 0.05 vs. age-matched WT mice and iPLA₂β-KO mice at 15 weeks (Wilcoxon’s rank sum test) and **<i>p</i> < 0.05 vs. iPLA₂β-KO mice at 56 weeks (Wilcoxon’s rank sum test).</p

Immunohistochemical analysis of TH in the amygdala of WT control mice and iPLA₂β-KO mice.

<p>Representative photographs of the amygdala immunostained for TH in WT mice at 15 weeks (A), 56 weeks (B), and 100 weeks (C) and iPLA₂β-KO mice at 15 weeks (D), 56 weeks (E, G), and 100 weeks (F). A section immunostained with TH in the striatum of iPLA₂β-KO mice at 56 weeks is also shown (H). (B), (E), (G), and (H) are frozen sections. The inset in (E) is a high magnification of the dotted square. (A–C): Distal regions of the axons of dopaminergic neurons are positive for TH in the amygdala of WT mice. (D): In iPLA₂β-KO mice at 15 weeks, TH-positive nerve fibers similar to those of WT mice were observed (A-C). (E, G): In iPLA₂β-KO mice at 56 weeks, round structures positive for TH are seen. Some are adjacent to TH-positive nerve fibers (small arrows in E). The round, TH-positive structures lie in a row like a string of beads (G). (F): Many round, TH-positive structures were seen in iPLA₂β-KO mice at 100 weeks (arrows in F). (H): Beaded, round TH-positive structures were also seen in the striatum of iPLA₂β-KO mice at 56 weeks. Scale bar in (A) represents 25 μm in all panels (A-H).</p

Induction of osteoblast differentiation by FoxOs.

<p>(A–C) Induction of osteoblast differentiation by FoxO3aTM. Primary osteoblasts from calvariae of wild-type mice were infected with adenovirus expressing GFP or FoxO3aTM, and ALP staining at 2 days and von Kossa staining at 6 days after infection (A), quantification of mineralization (B), and osteoblast marker gene expression (C) are shown. The value in GFP-introduced cells was set as 1 and the relative level is shown in B. Similar results were obtained in three independent experiments and representative data are shown. (D–F) Inhibition of the mineralization of MC3T3-E1 cells by sh<i>FoxO1</i> and sh<i>FoxO3a</i>. MC3T3-E1 cells were infected with retrovirus expressing GFP, sh<i>FoxO1</i>, or sh<i>FoxO3a</i>, and cultured in the presence of BMP2 (100ng/ml). The expression of <i>FoxO1</i> and <i>FoxO3a</i> was examined by real-time RT-PCR (D) and mineralization was examined by von Kossa staining (E) and its quantification (F) after culture for 2 weeks. The value in shGFP-introduced cells was set as 1 and the relative levels are shown in F. Similar results were obtained in three independent experiments and representative data are shown.</p

Bone morphometric analysis, BrdU and TUNEL staining, and real-time RT-PCR analysis of apoptosis-related genes in Bcl2^−/− mice.

<p>(A) Bone histomorphometric analysis. The trabecular bone volume (bone volume/tissue volume, BV/TV), number of osteoblasts (N.Ob/B.Pm), and number of osteoclasts (N.Oc/B.Pm) were compared in femurs between 6 wild-type and 4 Bcl2^−/− mice at 2 weeks of age. B.Pm, bone perimeter. (B–H) BrdU labeling (B, C) and TUNEL staining (D, E) of sections of femurs from wild-type mice (B, D) and Bcl2^−/− mice (C, E). Bars  = 50 µm. BrdU-positive osteoblastic cells (F), TUNEL-positive osteoblastic cells (G), and TUNEL-positive osteocytes (H) were counted and shown as a percentage of the number of osteoblastic cells or osteocytes. wild-type mice, n = 7; Bcl2^−/− mice, n = 5 in F. wild-type mice, n = 8; Bcl2^−/− mice, n = 5 in G and H. (I) Real-time RT-PCR analysis of apoptosis-related genes. RNA was directly extracted from newborn calvariae of wild-type and Bcl2^−/− mice. wild-type mice, n = 6; Bcl2^−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01.</p

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

<div><p>Mutations in <i>PLA2G6</i> have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the <i>PLA2G6</i> gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in <i>PLA2G6</i>-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in <i>PLA2G6</i>-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in <i>PLA2G6</i>-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.</p></div

Dopamine cell counts in the SNpc in WT control mice and iPLA₂β-KO mice.

<p>(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 (<i>p > 0</i>.<i>05</i>, Wilcoxon's rank sum test).</p