The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.
Keywords: CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; HGPS, Hutchinson-Gilford progeria syndrome; LA, lamin A; LMNAkd, LMNA knockdown; MEF, mouse embryonic fibroblasts; NHDF, normal human dermal fibroblasts; NT, non-targeting; OCR, oxygen consumtion rate; PDL, population doubling level; PLA, prelamin A; RD, restrictive dermopathy; ROS, reactive oxygen species; TBHP, tert-butyl hydrogen peroxide; TMRM, tetramethyl rhodamine methyl ester; ZMPSTE24; ZMPSTE24kd, ZMPSTE24 knockdown; apoptosis; hMSCs, human mesenchymal stem cells; high-content microscopy; lamin A/C; laminopathies; mitochondria; mitochondrial dysfunction; oxidative stress; prelamin A; senescence; Δψm, mitochondrial membrane potential.