Studies on Down's syndrome in tissue culture. I. Growth rates and protein contents of fibroblast cultures

Skin fibroblast cultures from six patients with Down's syndrome (Trisomy 21) were compared with four in vitro age-matched normal fibroblast cultures. Growth rates were calculated from increases in cell number and total protein during exponential growth, early in culture lifetime (less than 20 doublings). The Down's syndrome (D.S.) cultures had an average population doubling time of 35.6 ± 1.1 hours and average mass doubling time of 38.6 ± 3.2 hours, significantly lower (p<0.005) than the corresponding normal culture values of 23.0 ± 0.7 hours, and 23.3 ± 1.9 hours. D. S. Cells also contained 4.46 ± 0.19 ± 10^?4 μg protein/cell as compared to 3.06 ± 0.13 × 10^?4 μg/cell (p<0.001) for normal fibroblasts. Similar in vitro observations of increased doubling time and protein content have been reported in normal fibroblasts from older donors, and from individuals with premature aging syndromes, as well as in normal fibroblasts near the end of their in vitro lifetime. The present results, obtained from cultures young in vitro, may therefore suggest that D.S. fibroblast cultures age prematurely. This hypothesis is consistent with clinical manifestations of premature aging in D.S. patients and points to a defect in growth regulation, both in vivo and in vitro, resulting from an extra copy of chromosome 21.     

Abnormal protein metabolism in skin fibroblasts in vitro from patients with Duchenne muscular dystrophy

Rates of protein turnover have been measured on a statistical basis in Duchenne muscular dystrophy and normal skin fibroblasts populations in vitro. At comparable numbers of cumulative population doublings, protein synthesis was significantly reduced by about 24% in DMD fibroblasts as compared to normal fibroblasts (p less than 0.01, N = 12). Degradation of short lived proteins was significantly enhanced by about 60% (p less than 0.05, N = 18), and the degradation of long lived proteins was significantly increased by about 28% (p less than 0.05, N = 18) in DMD fibroblast populations in vitro. The enhanced degradation of long lived proteins in DMD fibroblasts can be reduced to basal levels of degradation by the use of the protease inhibitors leupeptin and Ep475 (p less than 0.05, N = 9).     

Gingival fibroblasts “in vitro” and Down's Syndrome

Gingival fibroblast cultures from four patients with Down's Syndrome (DS) and periodontal disease were compared with four in vitro age-matched fibroblast cultures of handicapped subjects (ND) also affected by periodontitis. The extra copy of cromosome 21 could alter growth regulation and biochemical mechanisms, so we examined quantitatively some DS phenotypical aspects to detect possible differences from those of controls. The growth properties of gingival fibroblast cultures from DS patients were more elevated than their ND age-matched controls. There were no differences in plasma membrane polarization and in neutral endopeptidase activity. The succinate-cytochrome C reductase activity decreases in DS fibroblasts compared with ND. Our results outline the difficulties to inusing fibroblast cultures as an in vitro system to study premature ageing Down's Syndrome.     

Collagen expression in fibroblasts with a novel LMNA mutation

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.     

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.     

Interleukin‐6 neutralization ameliorates symptoms in prematurely aged mice

Hutchinson–Gilford progeria syndrome (HGPS) causes premature aging in children, with adipose tissue, skin and bone deterioration, and cardiovascular impairment. In HGPS cells and mouse models, high levels of interleukin‐6, an inflammatory cytokine linked to aging processes, have been detected. Here, we show that inhibition of interleukin‐6 activity by tocilizumab, a neutralizing antibody raised against interleukin‐6 receptors, counteracts progeroid features in both HGPS fibroblasts and Lmna^G609G/G609G progeroid mice. Tocilizumab treatment limits the accumulation of progerin, the toxic protein produced in HGPS cells, rescues nuclear envelope and chromatin abnormalities, and attenuates the hyperactivated DNA damage response. In vivo administration of tocilizumab reduces aortic lesions and adipose tissue dystrophy, delays the onset of lipodystrophy and kyphosis, avoids motor impairment, and preserves a good quality of life in progeroid mice. This work identifies tocilizumab as a valuable tool in HGPS therapy and, speculatively, in the treatment of a variety of aging‐related disorders.     

Autophagy and aging: new lessons from progeroid mice

It is widely-assumed that the autophagic activity of living cells decreases with age and probably contributes to the accumulation of damaged macromolecules and organelles during aging. Over the last few years, the study of segmental progeroid syndromes in which certain aspects of aging are manifested precociously or in exacerbated form, has increased our knowledge of the molecular basis of aging. We have recently reported the unexpected finding that distinct progeroid murine models exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. Further studies on Zmpste24-null progeroid mice, which are a reliable model of human Hutchinson-Gilford progeria, have revealed that the observed autophagic increase is associated with a series of metabolic alterations resembling those occurring under calorie restriction or in other situations reported to prolong lifespan. Here, we analyze these unexpected findings and discuss their possible implications for the development of premature aging.     

Donor age reflects the replicative lifespan of human fibroblasts in culture

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro . This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.     

(Na++K+)-ATPase of Duchenne muscular dystrophy erythrocyte ghosts.

Adenosine triphosphatase (ATPase) activity of erythrocyte ghosts from Duchenne muscular dystrophy (DMD) patients and carriers was stimulated by ouabain while nonmyopathic donor preparations were inhibited. Ethacrynic acid was an effective inhibitor in both DMD patients and carriers as well as in controls. However, responsiveness of the enzyme to suramin and harmaline generally differed in DMD groups from that of nonmyopathic donors. These data are consistent with the hypothesis that modified affinity of Na binding site(s) may account for some properties of the (Na⁺+K⁺)-ATPase activity of DMD erythrocytes.     

Autophagy and aging: New lessons from progeroid mice

It is widely-assumed that the autophagic activity of living cells decreases with age and probably contributes to the accumulation of damaged macromolecules and organelles during aging. Over the last few years, the study of segmental progeroid syndromes in which certain aspects of aging are manifested precociously or in exacerbated form, has increased our knowledge on the molecular basis of aging. We have recently reported the unexpected finding that distinct progeroid murine models exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. Further studies on Zmpste24-null progeroid mice, which are a reliable model of human Hutchinson-Gilford progeria, have revealed that the observed autophagic increase is associated with a series of metabolic alterations resembling those occurring under calorie restriction or in other situations reported to prolong lifespan. Here, we analyze these unexpected findings and discuss their possible implications for the development of premature aging.

Addendum to: Mariño G, Ugalde AP, Salvador-Montoliu N, Varela I, Quirós PM, Cadiñanos J, van der Pluijm I, Freije JM, López-Otín C. Premature aging in mice activates a systemic metabolic response involving autophagy induction. Hum Mol Genet 2008; 17:2196–211.     

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.     

Progressive muscular dystrophy (Duchenne): Biochemical studies by flow-cytometry

Summary The peanut lectin (PNL) receptor density of the cell membrane and several metabolic parameters of cultured fibroblasts of normal human individuals and of patients with muscular dystrophy were measured by simultaneous two and three parameter flow cytometry. The PNL-receptor density was significantly decreased on muscular dystrophy fibroblasts (between 20.7 and 33.6%) as compared to normal fibroblasts. The cell volume, the esterase activity, the intracellular pH, and the percentage of proliferating cells of both types of fibroblasts were not significantly altered. The mean cell volume of different fibroblast cultures varied between 2500 and 6000m³. The concentration of the intracellular esterase activity of fibroblasts was low (0.169 relative units) as compared to lymphocytes and granulocytes of the peripheral blood (1.56 and 2.17 relative units). The fibroblasts had an acidic intracellular pH of 6.52 while lymphocytes and granulocytes had basic pH values of 7.30 and 7.17. Some of the fibroblasts were in the S+G2/M phase of the cell cycle (20%). The study shows that the measurement of biochemical parameters of vital and fixed single fibroblasts by flow-cytometry is of great interest for the recognition of differences between normal individuals and muscular dystrophy patients.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

综述: 人类早衰症的发病机制及干预方法

衰老是一种在细胞和组织水平逐渐发生功能衰退的过程.早衰症是一类罕见的人类遗传性疾病,以加速衰老为特征.对早衰症的研究有助于理解人类衰老的生理过程,对衰老相关疾病的防治具有借鉴意义.成人早衰症和儿童早衰症是两种著名的人类早衰症,本文将综述这两种早衰症的发病机制及干预方法.     

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

Decreased total nitric oxide production in patients with duchenne muscular dystrophy

Plasma nitric oxide (NO) levels in Duchenne muscular dystrophy (DMD) patients were significantly lower than those observed in both healthy controls and in patients with other neuromuscular disorders. The correlation between NO level and ejection fraction was significant (r=–0.384, p=0.0391) in the DMD group. Disruption of NO systems may contribute to the development of muscular dystrophy and have implications for therapeutic strategies.