Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

Synaptic Proteome Changes in a DNA Repair Deficient <i>Ercc1</i> Mouse Model of Accelerated Aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used <i>Ercc1</i> mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged <i>Ercc1</i> mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and <i>Ercc1</i> mutant hippocampal neurons displayed normal outgrowth and synapse formation <i>in vitro</i>. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline

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

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