Journal: Life Science Alliance
Article Title: Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum
Figure Lengend Snippet: Impaired TrkB signaling in the cerebellum of Pex14 ΔC/ΔC BL/ICR mice. (A) Sagittal sections of the cerebellum of wild-type (+/+) and Pex14 ΔC/ΔC ( ΔC/ΔC ) BL/ICR mice at P7 were stained with anti-TrkB (green) and calbindin-D 28k (red) antibodies. TrkB-positive punctate structures that were observed at P3 ( Fig 6A ) were not detected in the wild-type mouse cerebellum at P7, suggesting that climbing fiber terminals shifted to the Purkinje cell dendritic compartment. Scale bar, 10 μm. (B) Sagittal sections of the cerebellum at P7 were stained with antibodies against vGlut2 (red) and calbindin-D 28k (white). The number of dots stained with anti-vGlut2 antibody was quantified (right panel, n = 4). Scale bar, 10 μm. (C, D) Primary cerebellar neurons from a wild-type mouse at P0.5 were cultured for five DIV and then treated with 50 ng/ml BDNF for two DIV. Levels of TrkB-TK+ (C) and TrkB-T1 (D) mRNAs were determined by real-time PCR (n = 5). (E) Relative mRNA levels of c-fos and c-jun in the cerebellum at P3 were analyzed by real-time PCR (n = 3). (F) Cerebellar lysates of wild-type and Pex14 ΔC/ΔC BL/ICR mice at P0.5, P3, P5, and P7 were analyzed as in Fig 6E . TrkB-TK+, full-length TrkB; TrkB-T1, truncated isoform of TrkB; Dot, a non-specific band. (G) Amounts of TrkB-TK+ and TrkB-T1 were normalized by α-tubulin level and presented relative to those of TrkB-TK+ in control mice at P0.5 (n = 3). (H) Cerebellum lysates from wild-type and Pex14 ΔC/ΔC BL/ICR mice at P5 and P7 were analyzed by SDS–PAGE and immunoblotting with antibodies against TrkB (lower panels) and phosphorylated Trk (p-TrkB-TK+, Y496, upper panels). (I) Total ERK1/2 (lower panels) and phosphorylated ERK1/2 (p-ERK1 and 2, T202, and Y204, respectively, upper panels) at P5 and P7 were analyzed as in Fig 7C . The amount of phosphorylated ERK1/2 relative to total ERK1/2 is represented (P5; n = 3, P7; n = 4). (J, K) Sagittal sections of the cerebellum from wild-type (upper panels) and Pex14 ΔC/ΔC (lower panels) BL/ICR mice at P3 (J) and P7 (K) were stained with anti-calbindin-D 28k antibody (green) and phalloidin-TRITC (red). Scale bar, 10 μm. Higher magnification images of the boxed regions are shown (inset). Scale bar, 5 μm. Actin-positive structures located on the dendrites (arrowheads) were decreased in Pex14 ΔC/ΔC BL/ICR mice. ns, not significant, * P
Article Snippet: Construction of a targeting vector and generation of Pex14 ΔC/ΔC mice Genomic DNA corresponding to the Pex14 locus was isolated from 129/Sv mouse genomic library (Agilent Technologies).
Techniques: Mouse Assay, Staining, Cell Culture, Real-time Polymerase Chain Reaction, SDS Page