Cell migration is impaired in XPA-deficient cells

Abstract

Xeroderma pigmentosum (XP) is a hereditary disorder characterized by photosensitivity, predisposition to skin cancers, and neurological abnormalities including microcephaly and progressive neurodegeneration. A lack of nucleotide excision repair (NER) in patients with XP can cause hypersensitivity to the sun, leading to skin cancer, whereas the etiology of the neuronal symptoms of XP remains ambiguous. There are various neurological disorders that perturb neuronal migration, causing mislocalization and disorganization of the cortical lamination. Here, we investigated the role of the XP group-A (Xpa) gene in directed cell migration. First, we adopted an in utero electroporation method to transduce shRNA vectors into the murine embryonic cerebral cortex for the in vivo knockdown of Xpa. Xpa-knockdown neurons in the embryonic cerebral cortex showed abnormal cell migration, cell cycle exit, and differentiation. The genotype-phenotype relationship between the lack of XPA and cell migration abnormalities was confirmed using both a scratch assay and time-lapse microscopy in XP-A patient-derived fibroblasts. Unlike healthy cells, these cells showed impairment in overall mobility and the direction of motility. Therefore, abnormal cell migration may explain neural tissue abnormalities in patients with XP-A.

FIGURE 1

Delayed migration of embryonic neurons in mice in which Xpa was knocked down. Using an in utero electroporation method, shRNAi (Xpa‐R1) targeting Xpa was introduced into the lateral ventricles of E14 C57BL/6 mice. Green fluorescence identifies the neurons into which EGFP was introduced. Gray signals indicate differential interference contrast images. EGFP‐induced cortices were removed from E17 embryos and cerebral slices were observed using a confocal microscope. (A) Control mice; (B) Xpa‐R1‐introduced mice; (C) Xpa‐R1+ hXPA‐introduced mice. After introducing Xpa‐R1 into the fetal mouse lateral ventricle at E14, embryonic cortices at E16 were removed, and the cerebral slices were observed with a confocal microscope (D, E: Control and Xpa‐R1, respectively). Embryonic cortices were removed from P0 (F, G: Control, Xpa‐R1). CP: cortical plate, IZ: intermediate zone, Dotted line: border between CP and IZ. Scale bar = 200 μm.

FIGURE 2

Delayed migration of Xpa knockdown neurons affects cell cycle termination and differentiation. Cell cycle and neural differentiation in Xpa knocked down neurons were analyzed by immunohistological analysis using Ki‐67 antibody (A‐H) or MAP2 antibody (I‐P). CP: cortical plate, IZ: intermediate zone, Dotted line: border between CP and IZ. Green indicates EGFP signals and red signals indicate Ki‐67 positive cells (A, B: Control E14‐E17 C, D: Xpa‐R1 E14‐E17, E, F: Control E14‐E18.5 G, H: Xpa‐R1 E14‐E18.5). Neurological differentiation was analyzed by immunohistological analysis using the MAP2 antibody. Green indicates EGFP signals and red signals indicate MAP‐2 positive cells (I, J: Control E14‐E17 low‐magnification, K, L: Control E14‐E17 high‐magnification, M, N Xpa‐R1 E14‐E17 low‐magnification, O, P: Xpa‐R1 E14‐E17 high‐magnification). Scale bar = 200 μm.

FIGURE 3

Migration of fibroblasts from the patient with XP‐A was found to be impaired in the modified migration assay (A: Healthy person's fibroblast, B: XP‐A patient's fibroblast). IVS3‐1G > C homozygous: homozygous G‐to‐C mutation in the last nucleotide of intron 3 of XPA. The dotted lines indicate the starting line of cells made by scratching. The bar graph (C) is a quantification by measuring the number of cells in (A) and (B).

FIGURE 4

The analysis of cell motility and tracking using a HoloMonitor®M4. (A) The cellular motility distance (upper panel) and the cellular motility speed (lower panel) are shown. (B) The indicated cells were tracked for 72 h (left panels). Spatial movement graphs for selected cells (the starting position was the center) are shown in the right panel.