Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype

Abstract

Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13(-/-) mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.

FIG. 1.

Targeted disruption of Pex13. (A) Schematic representation of the wild-type allele. The positions of exons 2 (E2) to 4 (E4) and the directions and positions of genotyping PCR primers P1 to P4 are indicated. (B) Structures of proteins expressed from the wild-type (WT) Pex13 and disrupted (Pex13Δ) alleles. The numbered boxes depict the sections of the protein encoded by the Pex13 exons and are shown to scale. The positions of the transmembrane domain (TMD) and SH3 domain are indicated by the labeled solid lines. The dotted line represents the portion of the protein used to raise the Pex13 antibody. Two predicted translation termination codons (**) are indicated. (C) Genotyping by PCR screening of tissue genomic DNA. The P1/P2 primer pair generates an amplicon (490 bp) from the wild-type (WT) Pex13 allele, but not from the Pex13Δ allele because of the loss of the P2 primer binding sequence following Cre/loxP excision of exon 2. The P3/P4 primer pair generates an amplicon (385 bp) from the Pex13Δ allele, but not from the wild-type allele because of amplicon size. L, liver; B, brain; K, kidney. (D) Northern blot analysis of 10 μg of total RNA from the liver, kidney, and brain. The blot was probed with a radioactively labeled murine Pex13 cDNA probe, stripped, and reprobed with a labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. (E) Western blot analysis of total liver homogenate (10 μg of protein) from newborn (0.5 dpp) mice using anti-Pex13 antibody. The mobility of protein mass markers is indicated. (F) RT-PCR of RNA from Pex13^−/− mouse liver. Total liver RNA was reverse transcribed and PCR amplified using primers flanking the Pex13 open reading frame. Lane M, DNA ladder with selected markers (in base pairs) indicated. (G) Sequence chromatogram of a portion of the RT-PCR product from Pex13^−/− mouse liver RNA. The physiological translation start ATG (arrow) is shown. The position where exon 2 was removed (triangle) and positions of flanking exons 1 and 3 (Ex1 and Ex3, respectively) sequences are indicated. The positions of two in-frame translation termination codons (double underlines) are shown. (H) Appearance of newborn pups. (Left) An entire litter; (Right) one Pex13^+/− and one Pex13^−/− animal from a second litter. Symbols: +/+, wild-type; +/−, heterozygous for the disrupted Pex13 gene; −/−, homozygous for the disrupted Pex13 gene.

FIG. 2.

Pex13 deficiency disrupts protein import into peroxisomes. (A) Peroxisomal protein import in cultured skin fibroblasts from wild-type (+/+) and Pex13-deficient (−/−) mice. PTS1 protein import assessed by indirect immunofluorescence using an anti-SKL antibody (SKL panels), PTS2 protein import assessed by GFP autofluorescence in cells transfected with a plasmid that expresses a PTS2-EGFP fusion protein (GFP panels), and peroxisomal membranes detected by indirect immunofluorescence using an antibody to the peroxisomal integral membrane protein PMP70 (PMP panels) are shown. Note that GFP fluorescence and PMP70 immunofluorescence are from the same cell in each case. Bars, 10 μm. (B) Transfection of Pex13^−/− cells with a plasmid that expresses wild-type, myc-tagged Pex13 rescues import of PTS1 and PTS2-EGFP protein import. myc and SKL immunofluorescence in the same cell (top) and GFP fluorescence and SKL immunofluorescence in the same cell (bottom) are shown. (C) Western blot analysis of liver homogenates to determine the levels of the peroxisomal proteins catalase, peroxisomal bifunctional protein (PBP), and PMP70. Actin levels indicate protein loading. Liver homogenates from wild-type (+/+) and heterozygous (+/−) and homozygous (−/−) knockout animals were used. (D) Electron microscopic analysis of DAB-stained liver for detection of peroxisomes (PO) in wild-type and Pex13^−/− mice. Bars, 1 μm.

FIG. 3.

Peroxisomal biochemical abnormalities in Pex13-deficient mice. VLCFA measurements in the liver, brain, and cultured skin fibroblasts expressed as a ratio of C_26:0/C_22:0 (A) or C_24:0/C_22:0 (B) fatty acids. Oxidation of [1-¹⁴C]phytanic acid (C) and [1-¹⁴C]pristanic acid (D) by cultured skin fibroblasts, expressed as the production of [1-¹⁴C]CO₂ and 1-¹⁴C-water-soluble (Water-sol.) products. Levels of C_16:0 and C_18:0 plasmalogen in liver (E) and brain (F). (G) Activities of peroxisomal enzymes required for plasmalogen synthesis in cultured fibroblasts. Values are the means of three or four measurements on Pex13^+/+ (white bars), Pex13^+/− (grey bars), and Pex13^−/− (black bars) animals. Each error bar represents one SD.

FIG. 4.

Pex13 deficiency leads to abnormal lamination of the cerebral cortex. (A) Matched coronal sections of wild-type (+/+) and Pex13-deficient (−/−) mice stained with hematoxylin and eosin. Corresponding cortical layers are delimited by arrowheads. CP, cortical plate; IZ, intermediate zone. Bars, 200 μm. (B) Higher-magnification views of the cortical plate zone from the sections shown in panels A.

FIG. 5.

Tissue ultrastructural changes in Pex13-deficient mice. (A) Large lipid droplet (L) in a hepatocyte. N, nucleus. Bar, 1 μm. (B and C) Hepatic mitochondria with abnormal cristae. Bars, 200 nm. (D) Section through kidney glomeruli of Pex13^+/+ and Pex13^−/− mice. P, podocyte foot processes. Bars, 1 μm.