Increased apoptosis, p53 up-regulation, and cerebellar neuronal degeneration in repair-deficient Cockayne syndrome mice

Abstract

Cockayne syndrome (CS) is a rare recessive childhood-onset neurodegenerative disease, characterized by a deficiency in the DNA repair pathway of transcription-coupled nucleotide excision repair. Mice with a targeted deletion of the CSB gene (Csb-/-) exhibit a much milder ataxic phenotype than human patients. Csb-/- mice that are also deficient in global genomic repair [Csb-/-/xeroderma pigmentosum C (Xpc)-/-] are more profoundly affected, exhibiting whole-body wasting, ataxia, and neural loss by postnatal day 21. Cerebellar granule cells demonstrated high TUNEL staining indicative of apoptosis. Purkinje cells, identified by the marker calbindin, were severely depleted and, although not TUNEL-positive, displayed strong immunoreactivity for p53, indicating cellular stress. A subset of animals heterozygous for Csb and Xpc deficiencies was more mildly affected, demonstrating ataxia and Purkinje cell loss at 3 months of age. Mouse, Csb-/-, and Xpc-/- embryonic fibroblasts each exhibited increased sensitivity to UV light, which generates bulky DNA damage that is a substrate for excision repair. Whereas Csb-/-/Xpc-/- fibroblasts were more UV-sensitive than either single knockout, double-heterozygote fibroblasts had normal UV sensitivity. Csb-/- mice crossed with a strain defective in base excision repair (Ogg1) demonstrated no enhanced neurodegenerative phenotype. Complete deficiency in nucleotide excision repair therefore renders the brain profoundly sensitive to neurodegeneration in specific cell types of the cerebellum, possibly because of unrepaired endogenous DNA damage that is a substrate for nucleotide but not base excision repair.

Fig. 1.

Overview of runting and cerebellar morphology in Csb^−/−/Xpc^−/− mice. (A) Relative size of Csb^−/−/Xpc^−/− mice and a littermate control at P21. (B) Mean weight of Csb^−/−/Xpc^−/− mice compared with littermate controls. (C) Frequency of Purkinje cells per unit area of cerebellum in Csb^−/−/Xpc^−/− mice compared with littermate controls. (D–I) Histological sections of cerebellum of control (D–F) and double-knockout Csb^−/−/Xpc^−/− (G–I) mice stained with H&E demonstrating a progressive loss of Purkinje cells in double-knockout Csb^−/− Xpc^−/− mice. Mice were examined at P8 (D and G), P14 (E and H), and P21 (F and I). [Scale bars: D, 50 μm (applies to D and G); E, 50 μm (applies to E, F, H, and I).]

Fig. 2.

Histological sections of mouse cerebella-stained with calbindin-D28k to identify Purkinje cells, showing lack of degeneration in wild type (A) and showing degeneration (indicated by arrows in B and C) in both Csb^−/− and compound heterozygous Csb^+/−/Xpc^+/− mice at 3 months of age.

Fig. 3.

Apoptosis in double-knockout Csb^−/− Xpc^−/− mice. Sections of cerebellum of control (A and B) and double-knockout Csb^−/−/Xpc^−/− (C and D) mice labeled with TUNEL at ages P14 (A and C) and P21 (B and D) showing apoptosis in the external granule cell layer (arrowheads, D) and occasionally in the Purkinje cell layer. [Scale bar: A, 50 μm (applies to A–D).] (E) Frequency of TUNEL-positive cells per unit area of cerebellar folia in the Csb^−/−/Xpc^−/− mice compared with controls.

Fig. 4.

BrdU staining indicating cell proliferation in the external granule cell layer of a control littermate (A) and Csb^−/−/Xpc^−/− (B) at age P14. [Scale bar: A, 50 μm (applies to A and B).]

Fig. 5.

p53 and calbindin immunohistochemistry in control littermate (A) and Csb^−/−/Xpc^−/− (B) mice at age P21. [Scale bar: A, 50 μm (applies to A and B).]

Fig. 6.

Immunohistochemical staining for the Xpc protein in cerebellar sections of wild-type (A) mice at age P21 and Xpc^−/− controls. (B) Demonstration of the presence of the Xpc protein in Purkinje cells (arrowheads, B). [Scale bar: A, 50 μm (applies to A and B).]

Fig. 7.

Survival of MEFs after UV irradiation. Data are shown for wild type (solid square), Csb^+/− (open circle), Csb^−/− (closed triangle), Xpc^−/− (solid diamond), compound heterozygous Csb^+/−/Xpc^+/− (open square), double-knockout Csb^−/−/Xpc^−/− (closed circle), and double-knockout Csb^−/−/Ogg^−/− (closed inverted triangle).