Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DNA repair genes

Abstract

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development.

Figure 1

Motor dysfunction and abnormal behavior in XPA^−/−CSB^−/− mice. (A) Compared with control littermates of XPA^+/−CSB^+/− (Left), XPA^−/−CSB^−/− mice (Right) had to spread their hind limbs to maintain balance (P14). (B) When the mice were suspended by their tails, the XPA^−/−CSB^−/− mice (Right) often crossed or clasped their hind limbs, whereas XPA^+/−CSB^+/− mice (Left) usually extended and shook their hind limbs. XPA^−/−CSB^−/− mice frequently performed waltzing (C 1–4) or lost their balance and fell (D 1–4).

Figure 2

Comparison of the body and brain of each genotypic mouse. (A) XPA^−/−CSB^−/− mice show growth retardation from an early postnatal stage (P14). (B) The gross view of the brain of XPA^−/−CSB^−/− versus XPA^+/−CSB^+/− littermates. Comparison to XPA^+/−CSB^+/− control, XPA^−/−CSB^−/− cerebellum was remarkably smaller (red interrupted line), so the colliculi were more exposed (black arrowheads). (Bar = 10 mm.) (C) Averaged body and brain weight of each genotype at P14. Values are mean ± SD derived from mice of each genotype. *, P < 0.01, against XPA^+/−CSB^+/−.

Figure 3

Histological abnormalities of cerebellum in XPA^−/−CSB^−/− mice. Sagittal cerebellar sections of P14 (A–D), P8 (E and F), and P20 (G and H) XPA^+/−CSB^+/− (A, E, and G), XPA^−/−CSB^+/− (B), XPA^+/−CSB^−/− (C), and XPA^−/−CSB^−/− (D, F, and H) mice. XPA^−/−CSB^−/− mice (D) showed impaired cerebellar foliation pattern and some fissures (black arrowheads), and the tenth lobule (Flocculonodular lobe, white triangles) appeared to be small compared with other genotypes (A–C). (Bar = 1 mm.)

Figure 4

Immunohistochemical staining of cerebellar PCs with anticalbindin antibody. (A) PC morphologies of XPA^+/−CSB^+/− and XPA^−/−CSB^−/− mice at P8 (Left) and P14 (Right). EGL, external granule cell layer; PCL, PC layer; IGL, internal granule cell layer; ML, molecular layer. (Bar = 50 μm.) (B) Measures of the length of the dendritic tree of PCs in lobules I/II and III, and IX/X of each genotypic mice cerebellum at P8 and P14. Values are mean ± SD for six midsagittal sections derived from five to eight mice of each genotype. *, P < 0.001, and **, P < 0.0001, against XPA^+/−CSB^+/−.

Figure 5

Reduced neurogenesis in XPA^−/−CSB^−/− cerebellur EGL. Compared with the EGL of XPA^+/−CSB^+/− cerebellum (A), proliferating cells (black) were obviously decreased in number, and the row of cells disordered in XPA^−/−CSB^−/− cerebellum at P8 (B). (Bar = 50 μm.) (C) A comparison of proliferative ability of cells in the EGL of lobules I/II and III, and IX/X of each genotypes at P8 and P14. Values are mean ± SD for six sections derived from three to five mice of each genotype. *, P < 0.002, and **, P < 0.02, against XPA^+/−CSB^+/−.

Figure 6

Accelerated apoptosis in XPA^−/−CSB^−/− cerebellar EGL. (A–D) The cerebellum of each genotypic mouse at P8 was stained by TUNEL. The number of TUNEL-positive cells (arrowhead) was significantly higher in XPA^−/−CSB^−/− EGL (D) than XPA^+/−CSB^+/− EGL (A) but comparable between XPA^−/−CSB^+/− (B) and XPA^+/−CSB^−/− (C). At P14, only a few TUNEL-positive cells remained in the EGL of both XPA^+/−CSB^+/− (E) and XPA^−/−CSB^−/− (F). (Bar = 50 μm.) (G) A comparison of the number of TUNEL-positive cells in the EGL in lobules I/II and III, and IX/X of each genotypes at P8 and P14. Values are mean ± SD for six sections derived from three to five mice of each genotype. *, P < 0.004, and **, P < 0.03, against XPA^+/−CSB^+/−.