Delayed and Accelerated Aging Share Common Longevity Assurance Mechanisms

Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of “survival” responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension

Antifibrotic Effects of Roscovitine in Normal and Scleroderma Fibroblasts

<div><p>Heightened production of collagen and other matrix proteins underlies the fibrotic phenotype of systemic sclerosis (SSc). Roscovitine is an inhibitor of cyclin-dependent kinases that promote cell cycling (CDK1, 2), neuronal development (CDK5) and control transcription (CDK7,9). In an <em>in vivo</em> glomerulonephritis model, roscovitine treatment decreased mesangial cell proliferation and matrix proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048560#pone.0048560-Pippin1">[1]</a>. We investigated whether roscovitine could regulate fibrotic protein production directly rather than through cell cycling. Our investigations revealed that roscovitine coordinately inhibited the expression of collagen, fibronectin, and connective tissue growth factor (CTGF) in normal and SSc fibroblasts. This effect occurred on a transcriptional basis and did not result from roscovitine-mediated cell cycle inhibition. Roscovitine-mediated suppression of matrix proteins could not be reversed by the exogenous profibrotic cytokines TGF-β or IL-6. To our knowledge, we are the first to report that roscovitine modulates matrix protein transcription. Roscovitine may thus be a viable treatment option for SSc and other fibrosing diseases.</p> </div

Roscovitine decreases transcription of fibrogenic mediators.

<p><b>A</b>. Roscovitine decreases mRNA expression of <i>CTGF</i>, collagen and fibronectin. RNA from confluent primary fibroblasts incubated for 36 hours with DMSO vehicle (1), 6.25 µg/mL roscovitine (2), or 12.5 µg/mL roscovitine (3) was blotted and probed for <i>CTGF</i>, collagen, and fibronectin mRNA. Equivalent loading is shown by ethidium bromide staining for total RNA. Fibronectin protein level in these cells is shown below. <b>B</b>. Decreased transcription driven by the COL2A1 promoter in the absence or presence of 6.25 µg/mL roscovitine (Rosc). Values average three independent transfections of 3T3 cells; representative of experiments repeated 3 times. Luciferase activity is normalized to GFP protein encoded by co-transfected plasmid. *, <i>p</i> = 0.01.</p

Inhibition of collagen and fibronectin expression by roscovitine in SSc and in normal fibroblasts.

<p><b>A.</b> Passage 4 primary fibroblasts from a scleroderma patient (SSc) or her unaffected twin (NL) that had been confluent for 2 days were exposed for 36 hours to DMSO vehicle (D), roscovitine (R, 12.5 µg/mL), AG-490 (AG, 25 µM) or TAT-p27 (27, 150 nM) as shown. Equal amounts of protein are present in all lanes from the cell pellets as indicated by a non-specific cross-reactive band. The effects of these agents on matrix protein levels in the cell pellets and in cell supernatants are shown as indicated. Equal amounts of protein are present in all lanes of the supernatants as shown by a different non-specific cross-reactive band present only in the supernatant. N.A., Not applicable. <b>B.</b> Immunohistochemical staining for fibronectin production by normal primary fibroblasts cultured in the absence (−) or presence (+) of 12.5 µg/mL of roscovitine for 36 hours. <b>C.</b> Levels of collagen in fibroblasts growth arrested by culture for 9 days in 0.2% serum and subsequently exposed for 24 hours to DMSO vehicle (−) or to roscovitine (+) as indicated. Ponceau as a loading control is included. The cell cycle profiles of these fibroblasts stained with propidium iodide (PI) is also shown. <b>D.</b> Roscovitine decreases fibronectin in primary fibroblasts (NL57) exposed to exogenous TGF-β or IL-6. Roscovitine also blocked TGF-β or IL-6 induction of connective tissue growth factor (CTGF). Cells treated for 23 hours with DMSO vehicle (D), 6.25 µg/mL roscovitine (R), 10 ng/mL TGF-β (T), or 50 ng/mL IL-6 (6). PARP band at 116 kD is also shown; no 89 kD cleaved PARP band (a marker of apoptosis) was detected. Total protein loading detected by Ponceau staining was equivalent in all lanes. <b>E.</b> Primary fibroblasts from a scleroderma patient (SSc) or an unaffected twin (NL) treated as described in panel D. CTGF, run on a parallel gel had marker lane (cut out, replaced by spacer) separating NL and SSc fibroblasts.</p

Roscovitine enhances STAT3 activation and does not block TGF-β signaling.

<p><b>A.</b> MRC-5 and NL57 human fibroblasts, as well as primary fibroblasts from a scleroderma patient (SSc) or an unaffected twin (NL) were incubated overnight in serum-free medium and stimulated for 45 minutes with DMSO vehicle (D), roscovitine (R, 6.25 µg/mL), TGF-β (T, 10 ng/mL), or IL-6 (6, 50 ng/mL) alone or in combination as indicated. Cells were directly lysed, and proteins fractionated and blotted for phosphorylated ERK (p-ERK), STAT3 (p-STAT3), or SMAD3 (p-SMAD3) and in parallel blots for total STAT3 or SMAD3. <b>B.</b> SMAD3 activation in serum-starved MRC-5 cells exposed to TGF-β without (−) or with (+) roscovitine for up to 60 minutes as indicated in 2 parallel experiments.</p

ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair▿ †

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1−/− Ku86−/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent

First Reported Patient with Human ERCC1 Deficiency Has Cerebro-Oculo-Facio-Skeletal Syndrome with a Mild Defect in Nucleotide Excision Repair and Severe Developmental Failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio- skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1

Delayed and accelerated aging share common longevity assurance mechanisms

Abstract Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of &apos;&apos;survival&apos;&apos; responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro-and anti-aging effects of treatments aimed at health-span extension