Cellular basis of ClC-2 Cl− channel–related brain and testis pathologies

Figure 1. Sertoli cell–specific Clcn2 disruption causes testicular degeneration in mice.A, Western blot analysis of ClC-2 of membrane fractions isolated from complete testis of adult Clcn2lox/lox, GC-ΔC2 (germ cell–specific Clcn2 knockout), Clcn2-/- and SC-ΔC2 mice. Equal amounts of protein, as determined by BCA assay, were loaded. This blot is representative for at least two independent experiments. B, upper row, testis cryosections of adult Clcn2lox/lox, SC-ΔC2 (Sertoli cell–specific Clcn2 knockout), and Clcn2-/- mice immunolabeled for ClC-2 (red) and costained with peanut agglutinin (green), a marker for acrosomes of spermatozoa (82). DNA in nuclei was stained with DAPI (blue). Dashed lines indicate the outer limit of the seminiferous tubules. Lower row, pictures as in upper row, showing ClC-2 staining (white). Staining in Sertoli cells (examples) is marked by arrows. The scale bar represents 50 μm. C, macroscopic view of the male reproductive system of adult Clcn2lox/lox and SC-ΔC2 mice. The scale bar represents 1 cm. D, testis and epididymis weights relative to body weight of adult Clcn2lox/lox and SC-ΔC2 mice (n = 5–7). Error bars, mean ± S.D. Statistical significance: ∗∗ p < 0.01…

Figure 2. Retinal degeneration upon Clcn2 knockout in the retinal pigment epithelium. H&E-stained retinal sections of 1-, 2-, and 4-week-old and adult control, Clcn2-/-, and RPE-ΔC2 mice. n = 1 to 3 per age and genotype; ∗ indicates the photoreceptor layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. The scale bar represents 50 μm.

Figure 3. ClC-2 expression and localization in glial cell–specific Clcn2 knockout mice.A and B, Western blot analysis of ClC-2 of membrane fractions isolated from cerebellum, cerebral cortex, and remainder of brain of adult control (Clcn2lox/lox), oligodendrocyte-specific (Cnp-Cre;Clcn2lox/lox), astrocyte-specific (GFAP-Cre;Clcn2lox/lox, GLAST-CreERT2;Clcn2lox/lox induced with tamoxifen), oligodendrocyte-/astrocyte-specific (Cnp-Cre;GFAP-Cre;Clcn2lox/lox) Clcn2 knockout mice and Clcn2−/− mice. Equal amounts of protein, as determined by BCA assay, were loaded with β-actin serving as loading control. This blot is representative for 1 to 3 independent experiments. C, immunofluorescent staining of ClC-2 (green/white) in brain cryosections of adult control, conditional Clcn2 knockout as indicated, and Clcn2−/− mice. Costaining for adenomatous polyposis coli (red) visualizes the cell bodies of oligodendrocytes in the myelin layer of the cerebellum. Costaining for the astrocytic cytoskeletal protein GFAP (red) visualizes Bergmann glia on the molecular layer of the cerebellum and costaining for perlecan (red) visualizes astrocytic endfeet at blood vessels at the hippocampus region. DNA in the…

Figure 4. Myelin vacuolization in glial cell–specific Clcn2 knockout mouse models. H&E-stained sagittal brain sections of the myelin layer of the cerebellum of control, Clcn2−/−, oligodendrocyte-specific (Cnp-Cre;Clcn2lox/lox), astrocyte-specific (GFAP-Cre;Clcn2lox/lox), and oligodendrocyte-/astrocyte-specific (Cnp-Cre;GFAP-Cre;Clcn2lox/lox) Clcn2 knockout mice in the age of 16 or 52 weeks (A), of the brain stem (B), and the corpus callosum (C) of 52-week-old Clcn2−/− and oligodendrocyte-/astrocyte-specific Clcn2 KO mice. n = 2 to 5. The scale bars represent 500 μm.

Figure 5. Similar myelin vacuolization phenotype of Clcn2op/opGlialcam−/−compared with Glialcam−/−mice. H&E-stained paraformaldehyde-fixed sagittal brain sections (myelin part of the cerebellum) of wildtype (Clcn2+/+;Glialcam+/+), Clcn2op/op;Glialcam+/+, Clcn2+/+;Glialcam−/−, and Clcn2op/op;Glialcam−/− mice at the age of 16 and 52 weeks. n = 1 to 3. The scale bar represents 500 μm.