Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Microglial phospholipase D4 deficiency influences myelination during brain development

doi: 10.2183/pjab.92.237

Figure Lengend Snippet: Generation of PLD4-deficient mice. (A) Genome structure of the PLD4 gene generated by homologous recombination of the STOP-tetO cassette in PLD4 knock-in (PLD4-deficient) mice. (B) Expression of PLD4 mRNA by quantitative real time PCR (qRT-PCR) in brain, spleen, liver and thymus of adult wild type (WT) and PLD4-deficient (Ho) mice. Graphs show the relative ratio of the level of PLD4 and β-actin mRNA from samples run in triplicate from 3 independent experiments. In wild type, PLD4 mRNA was expressed in brain and various reticuloendothelial tissues, including the spleen, liver and thymus. In PLD4-deficient mice, PLD4 mRNA was completely eliminated in these tissues. (C) PLD4 protein levels in adult wild type (WT), homozygote (Ho), and heterozygote (He) mice. Spleen homogenates (10 µg) with or without deglycosylation were separated by 10.5% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis was performed using an anti-PLD4 antibody. Since PLD4 has multiple glycosylation sites, various PLD4-related bands (70–75 kDa; indicated by *) were shown in non-treated samples. The arrow indicates deglycosylated PLD4 (∼45 kDa) by PNGase F (peptide-N-glycosidase F) treatment. The arrowhead indicates an unrelated protein product detected by the anti-PLD4 antibody. Note that this protein was also detected in PLD4-deficient spleen samples while PLD4 bands with or without sugars were completely eliminated. (D–F) Expression patterns of PLD1 (D), PLD2 (E), and PLD3 (F) mRNA in adult mice were examined by qRT-PCR. The quantitative analyses shown in B to F were obtained from three independent experiments. All experiments were performed in triplicate. Graphs indicate the mean ± standard error of the mean (SEM). Two-way ANOVA with Bonferroni multiple comparison tests were used for statistical analyses. ****P < 0.0001, **P < 0.01.

Article Snippet: For Western blotting: rat monoclonal anti-MBP antibody (1:4000; Chemicon); rabbit polyclonal anti-β-actin antibody (1:2000; Wako); horseradish peroxidase-conjugated species-specific secondary antibodies (1:10,000; Jackson Immunoresearch, West Grove, PA).

Techniques: Generated, Homologous Recombination, Knock-In, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Microglial phospholipase D4 deficiency influences myelination during brain development

doi: 10.2183/pjab.92.237

Figure Lengend Snippet: Influence of PLD4 deficiency on cerebellar myelination. (A, B) Western blot for MBP in the cerebellum of P7 wild type (WT) and PLD4-deficient (Ho) mice. Cerebellar homogenates (10 µg) from individual mice (three mice from each group) were separated by 12% SDS-PAGE and Western blot was performed using an anti-MBP antibody. After stripping off the antibody, the same blot was restained with an anti-β-actin antibody for quantification. B. The total intensity of all MBP bands (14, 17, 18.5, and 21 kDa) was quantified for each animal and the ratio of MBP to β-actin for each genotype was calculated. Graph indicates mean ± SEM. Statistical analysis with a student t test shows no significant difference between the two groups. (C–F ) Comparison of MBP immunoreactivity in wild type (C, E) and PLD4-deficient (D, F) cerebella at P5 (C, D) and P7 (E, F). Cell nuclei are stained with DAPI (blue). Representative images of 5 mice from each genotype were selected. As shown in Fig. C and D, myelination in the deep white matter is in progress at P5 and progresses to the folial white matter by P7. Images in C, D or E, F show the deep white matter tracts, or folial white matter, respectively. C'–F' are higher magnification views of the white squares in C–F without DAPI. At P5, MBP-positive myelin membranes were found in the deep white matter in wild type mice, while premyelinating oligodendrocytes with MBP-positive cell bodies (white arrowheads) were present in PLD4-deficient mice. At P7, myelin membranes were present in wild type folia, whereas premyelinating oligodendrocytes (white arrowheads) were still found in PLD4-deficeint mice in the same region. * represents the border between the cerebellum (upper) and the earlier myelinating brainstem (lower). Scale bars indicate 100 µm (D, F) and 20 µm (D', F'). (G, H) Double immunostaining of P7 wild type (G) and PLD4-deficient (H) cerebellar deep white matter around the cerebellar nuclei using anti-MBP (green) and anti-Iba1 (red) antibodies. The DAPI staining pattern is overlaid to indicate the cerebellar region. G' and H' are the same images as in G and H without DAPI. In mice of both genotypes, clusters of rounded Iba1-positive activated microglia are present in the area where myelination is progressing. Scale bar in H' represents 100 µm.

Article Snippet: For Western blotting: rat monoclonal anti-MBP antibody (1:4000; Chemicon); rabbit polyclonal anti-β-actin antibody (1:2000; Wako); horseradish peroxidase-conjugated species-specific secondary antibodies (1:10,000; Jackson Immunoresearch, West Grove, PA).

Techniques: Western Blot, SDS Page, Stripping Membranes, Staining, Double Immunostaining