助成研究報告

<div><p>As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) <i>Xpg^−/−</i> mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the <i>Xpg^−/−</i> mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.</p></div

Progeroid characteristics of <i>Xpg^−/−</i> mice.

<p>(A) Survival of <i>Xpg^−/−</i> mice in a C57Bl6 (red), FVB/N (green) or C57Bl6/FVB F1 hybrid (blue) background; n = 5 (C57Bl6), n = 10 (FVB/N), n = 14 (C57Bl6/FVB F1 hybrid). (B) Average body weight of embryonic 17.5-day old F1 hybrid <i>Xpg^−/−</i> and wild type (<i>wt</i>) littermates; n≥12 animals/group. (C) Average body weight of F1 hybrid <i>wt</i> males (black triangles), <i>wt</i> females (black circles), <i>Xpg^−/−</i> males (grey triangles), and <i>Xpg^−/−</i> females (grey circles); n≥4 animals/group. (D) Left: Photograph of a 7-day old F1 hybrid <i>Xpg^−/−</i> and <i>wt</i> littermate, showing no apparent differences except a slightly smaller size. Top right: Photograph of a 14-week old <i>Xpg^−/−</i> mouse. Bottom right: Side by side comparison of the same 14-week old <i>Xpg^−/−</i> and <i>wt</i> littermate showing a pronounced growth deficiency of the <i>Xpg^−/−</i> mouse. (E) Onset of hind limb clasping (orange), tremor (red) and kyphosis (green) with age and survival of F1 hybrid <i>Xpg^−/−</i> mice; n = 33 (clasping, tremor and kyphosis), n = 14 (survival). (F) CT-scan of a 16-week old F1 hybrid <i>wt</i> (left) and <i>Xpg^−/−</i> (right) mouse showing prominent curvature of the spine (kyphosis) in the <i>Xpg^−/−</i> mouse. (G) Bone strength of F1 hybrid <i>Xpg^−/−</i> and <i>wt</i> mice analyzed by a 3-point-bending assay of the femur at an average age of 15 weeks; n≥6 animals/group. (H) Cortical (left) and trabecular (right) thickness of the femora of F1 hybrid <i>Xpg^−/−</i> and <i>wt</i> mice at different ages; n = 4 animals/group. Error bars indicate standard error of the mean. *p<0.05, **p<0.01.</p

Age-related increase of neuronal stress in forebrain-specific <i>Xpg</i> knockout mice.

<p>(A) Average body weight of C57Bl6/FVB F1 hybrid wild type (<i>wt</i>) females (black circles) and forebrain-specific XPG-deficient (<i>Emx1-Xpg</i>) females (gray circles); n≥4 animals/group. (B) Onset of clasping of the hind limbs in <i>Emx1-Xpg</i> mice; n = 7 animals/group. (C) Representative images of GFAP immunostained sagittal neocortex sections of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice showing progressive astrocytosis in <i>Emx1-Xpg</i> mice. (D) Representative images of Mac2 immunostained sagittal brain sections of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice showing Mac2-positive microgliosis and a progressive decrease in size of the cerebral cortex and hippocampus of <i>Emx1-Xpg</i> mice. Arrows indicate microgliosis in corpus callosum and fimbria fornix. A thionin counterstaining was used. (E) Quantification of p53-positive cells in neocortex and cerebellum of 26- and 52-week old <i>Emx1-Xpg</i> and <i>wt</i> mice. Values are the average of four sections per genotype. Arrows indicate p53 positive cells. (F) Representative confocal images showing double labeled p53-NeuN cells in the neocortex (left) and p53-S100ß in the fimbria fornix (right) of 26-week old <i>Emx1-Xpg</i> mice. Arrows indicate p53 positive cells. NCx: neocortex, cc: corpus callosum, Str: striatum, ff: fimbria fornix, Hip: hippocampus. Scale bars: 50 µm (C), 500 µm (D), 200 µm (E) and 20 µm (F). Error bars indicate standard error of the mean. **p<0.01.</p

<i>Xpg^−/−</i> mice are born below Mendelian ratio in a C57BL6 background.

<p>Ratio of knockout mice born in different genetic backgrounds.</p><p>*p<0.05;</p><p>**p<0.01 deviation from Mendelian ratio (ChiSquaredTest).</p><p><i>Xpg^−/−</i> mice are born below Mendelian ratio in a C57BL6 background.</p

Comparison of the <i>Xpg^−/−</i> phenotype to that of XP/CS patients.

<p>y = present, n = not present, nd = not determined.</p><p>Comparison of the <i>Xpg^−/−</i> phenotype to that of XP/CS patients.</p

Aging features observed in the liver of liver-specific <i>Xpg</i> knockout mice.

<p>(A) Average body weight of C57Bl6/FVB F1 hybrid wild type (<i>wt</i>) males (black triangles), <i>wt</i> females (black circles), liver specific XPG-deficient (<i>Alb-Xpg</i>) males (gray triangles) and <i>Alb-Xpg</i> females (grey circles); n = 4 males/group, n = 2 females/group. (B) Average nucleus size of hepatocytes in the liver of 26- and 52-week old <i>Alb-Xpg</i> and <i>wt</i> mice; n = 4 animals/group. Bottom right: magnification of a nuclear inclusion found regularly in liver sections of 26- and 52-week old <i>Alb-Xpg</i> mice. (C) Quantification of p53-positive cells per cm² in the liver of 26- and 52-week old <i>Alb-Xpg</i> and <i>wt</i> mice; n = 3 animals/group. (D) Quantification of TUNEL-positive cells per cm² in the liver of 26- and 52-week old <i>Alb-Xpg</i> and <i>wt</i> mice; n = 3 animals/group. (E) Quantification of Ki67-positive cells per mm² in the liver of 26- and 52-week old <i>Alb-Xpg</i> and <i>wt</i> mice; n = 3 animals/group. (F) Relative mRNA expression levels of several antioxidant genes and the DNA damage response gene <i>p21</i> in liver tissue of 26-week old <i>Alb-Xpg</i> and <i>wt</i> mice. All values are corrected for <i>TubG2, Hprt, and Rps9</i> expression and normalized to <i>wt</i> expression levels; n = 3 animals/group. (G) Relative expression levels of the somatotrophic genes <i>Ghr, Igf1r and Igf1</i> in liver tissue of 26-week old <i>Alb-Xpg</i> and <i>wt</i> mice. All values are corrected for <i>TubG2, Hprt, and Rps9</i> expression as internal standard and normalized to <i>wt</i> expression levels; n = 3 animals/group. Scale bars: 25 µm (B, C, D). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.</p

Increased cell death, degeneration and stress responses in post-mitotic tissues of <i>Xpg^−/−</i> mice.

<p>(A) Representative images of GFAP immunostained sagittal neocortex sections of 4- and 14-week old <i>Xpg^−/−</i> and wild type (<i>wt</i>) mice showing progressive astrocytosis in <i>Xpg^−/−</i> mice. cc: corpus callosum. (B) Quantification of p53-positive cells per mm² in neocortex (NCx) and cerebellum (Cb) sections of 4- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice; n = 3 (14 weeks) and the average of five sections of a 4-week old <i>Xpg^−/−</i> and <i>wt</i> animal. (C) Representative images of calbindin immunostained sagittal cerebellum sections of 4- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice. Right panel: Magnification of the areas marked with dotted black boxes. Arrows indicate cerebellar torpedoes. ml: molecular layer, gl: granular layer. (D) Quantification of TUNEL-positive cells per cm² in neocortex and cerebellum sections of 4- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice; n≥3 animals/group. Arrows indicate positive cells. (E) Relative mRNA expression levels of the antioxidant genes <i>Nqo1</i>, <i>Nrf2</i>, and <i>HO-1</i> and the DNA damage response gene <i>p21</i> in 14-week old <i>Xpg^−/−</i> and <i>wt</i> cerebellum tissue. All values are corrected for <i>TubG2</i> expression and normalized to <i>wt</i> expression levels; n = 4 animals/group. (F) Quantification of TUNEL-positive cells per mm² in retinal sections of 4- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice; n = 6 animals/group. Arrows indicate positive cells. Scale bars: 250 µm (A), 50 µm (B), 100 µm (C), 25 µm (D, F). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.</p

Intestine and liver phenotype of <i>Xpg^−/−</i> mice.

<p>(A) Representative images of HE and Ki67 stained small intestine (SI) of 14-week old <i>Xpg^−/−</i> and wild type (<i>wt</i>) mice showing no gross morphological differences. (B) Average nucleus size of hepatocytes in the liver of 4- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice; n≥3 animals/group. Bottom right: magnification of a nuclear inclusion found sporadically in liver sections of 14-week old <i>Xpg^−/−</i> mice. (C) Relative mRNA expression levels of several antioxidant genes and the DNA damage response gene <i>p21</i> in liver tissue of 7- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice. All values are corrected for <i>TubG2, Hprt, and Rps9</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004686#pgen.1004686.s008" target="_blank">Table S1</a>) expression as internal standard and normalized to the 7-week old <i>wt</i> expression levels; n = 4 animals/group. (D) Relative expression levels of the somatotrophic genes <i>Ghr</i>, <i>Igf1r</i>, <i>Igf1</i>, and <i>Igfbp3</i> in liver tissue of 7- and 14-week old <i>Xpg^−/−</i> and <i>wt</i> mice. All values are corrected for <i>TubG2, Hprt, and Rps9</i> expression and normalized to the 7-week old <i>wt</i> expression levels; n = 4 animals/group. (E) Average basal blood glucose levels in groups of 4–7 and 12–18 week old <i>Xpg^−/−</i> and <i>wt</i> mice; n≥15 animals/group. Scale bars: 50 µm (A), 10 µm (B). Error bars indicate standard error of the mean. *p<0.05, **p<0.01.</p

Generation of <i>Xpg^−/−</i> mice.

<p>(A) Genomic organization and disruption strategy for <i>Xpg</i> depicting the wild type allele (<i>+</i>), the targeting construct, the targeted allele (<i>fn</i>), the conditional allele after Flp-mediated recombination of <i>Frt</i> sites (<i>f</i>) and the targeted <i>Xpg</i> allele following subsequent Cre-mediated recombination of <i>LoxP</i> sites (<i>−</i>). Exons 2–5 are indicated by black boxes. PCR primers are shown as arrows. (B) Southern blot and PCR analysis of an ES clone showing the correct insertion of the targeting construct. ES cell genomic DNA was digested with EcoRI for Southern blot analysis and hybridized with a 0.9 kb DpnI probe. The wild type (<i>wt</i>) allele yields a 7.4-kb fragment whereas the targeted (<i>tg</i>) allele yields a 4.1-kb fragment. The NheI-digested PCR product shows the 2.3-kb and 2.2-kb bands corresponding with the <i>wt</i> and <i>tg</i> allele, respectively (see also panel A). (C) PCR detection of mouse genotypes using the primers F1, NeoF and R1 as indicated as in A. (D) Immunoblot analysis of extracts from <i>Xpg^−/−</i> and <i>wt</i> MDFs using a rabbit polyclonal antibody raised against a peptide conserved between human and mouse XPG. Tubulin is used as loading control. (E) Primary <i>Xpg^−/−</i> and <i>wt</i> MDFs, cultured at low (3%) O₂ levels were irradiated with the indicated doses of UV-C (left) or treated with the indicated doses of Illudin S for 1 h (right). After 48 h recovery, survival was assessed by cell count. (F) UV-induced UDS in primary <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe GG-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. UDS levels are expressed relative to the non-irradiated <i>wt</i> cells. (G) UV-induced RRS in primary <i>Xpg^−/−</i> and <i>wt</i> MDFs reveals a severe TC-NER defect in <i>Xpg^−/−</i> cells. MDFs were irradiated with 16 J/m² of UV-C. 16 h after UV irradiation the <i>wt</i> cells show recovery of RNA synthesis, while <i>Xpg^−/−</i> MDFs only show residual activity in nucleoli (rRNA transcription). Arrowheads indicate nuclei. Error bars indicate standard error of the mean. **p<0.01.</p

Image3_The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.JPEG

Despite efficient repair, DNA damage inevitably accumulates with time affecting proper cell function and viability, thereby driving systemic aging. Interventions that either prevent DNA damage or enhance DNA repair are thus likely to extend health- and lifespan across species. However, effective genome-protecting compounds are largely lacking. Here, we use Ercc1Δ/− and Xpg−/− DNA repair-deficient mutants as two bona fide accelerated aging mouse models to test propitious anti-aging pharmaceutical interventions. Ercc1Δ/− and Xpg−/− mice show shortened lifespan with accelerated aging across numerous organs and tissues. Previously, we demonstrated that a well-established anti-aging intervention, dietary restriction, reduced DNA damage, and dramatically improved healthspan, strongly extended lifespan, and delayed all aging pathology investigated. Here, we further utilize the short lifespan and early onset of signs of neurological degeneration in Ercc1Δ/− and Xpg−/− mice to test compounds that influence nutrient sensing (metformin, acarbose, resveratrol), inflammation (aspirin, ibuprofen), mitochondrial processes (idebenone, sodium nitrate, dichloroacetate), glucose homeostasis (trehalose, GlcNAc) and nicotinamide adenine dinucleotide (NAD+) metabolism. While some of the compounds have shown anti-aging features in WT animals, most of them failed to significantly alter lifespan or features of neurodegeneration of our mice. The two NAD+ precursors; nicotinamide riboside (NR) and nicotinic acid (NA), did however induce benefits, consistent with the role of NAD+ in facilitating DNA damage repair. Together, our results illustrate the applicability of short-lived repair mutants for systematic screening of anti-aging interventions capable of reducing DNA damage accumulation.</p