Association of increased autophagic inclusions labeled for beta-galactosidase with fibroblastic aging.
Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p<0.0001). The increase of autophagic vacuoles in aging fibroblasts was confirmed by electron microscopy. We compared young versus senescent fibroblasts and showed that autophagic vacuoles, already present in young cells, became larger in senescent fibroblasts with a significant relative increase of inclusion area with respect to measured cell area (p=0.0041). However, autophagy-associated-gene expression remained stable in senescent compared to young fibroblasts, suggesting that the autophagy process per se is not enhanced. In parallel, transmission electron microscopy analysis showed that beta-galactosidase activity distribution was modified by aging: beta-galactosidase (an enzyme linked to lysosome) was scattered in young fibroblasts, but clustered at the level of autophagic vacuoles in senescent fibroblasts, suggesting a predominance of autolysosomes at this stage. These results support the hypothesis that, during fibroblast aging, the increase of autophagic vacuoles, as well as that of beta-galactosidase activity, may be associated to an increase of lysosomal mass and to an accumulation of degradative autolysosomes with lipofuscin. This phenomenon could be involved in the death of senescent fibroblasts.
Gerland
Luc-Marie
LM
INSERM U 453, Biologie des Gènes Suppresseurs de Tumeur, Faculté de Médecine, 8 avenue Rockefeller et Centre Léon Bérard, 69373 Lyon 08, France.
Peyrol
Simone
S
Lallemand
Christophe
C
Branche
Robert
R
Magaud
Jean-Pierre
JP
Ffrench
Martine
M
D001343:Autophagy / Q000502:physiology*; D015415:Biomarkers / Q000032:analysis; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000032:analysis; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D005347:Fibroblasts / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D002479:Inclusion Bodies / Q000648:ultrastructure*; D008062:Lipofuscin / Q000032:analysis; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D001616:beta-Galactosidase / Q000032:analysis*
D015415:Biomarkers; D008062:Lipofuscin; D001616:beta-Galactosidase; C008542:monodansylcadaverine; D002103:Cadaverine