Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome - PubMed (original) (raw)

Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome

Paola Scaffidi et al. Nat Med. 2005 Apr.

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.

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Figures

Fig. 1

Fig. 1. Wild type GFP-lamin A is insufficient for phenotypic rescue of HGPS cells

(a–c) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (AG08469; population doublings: 25–30) (a) and a HGPS patient (AG01972; population doublings: 25–30), untreated (b) or transfected with wild type lamin A and GFP as a transfection marker (c). Cells were stained with DAPI (blue) and antibodies (red) against the indicated proteins. Four cells (indicated by arrowheads) with normal staining respectively for lamin B, LAP2, HP1α and Tri-Me-K9 are shown in panel b to directly compare the cellular level of the proteins in unaffected and affected cells. The percentage of cells showing aberrant phenotype is indicated. Scale bar: 10 μm. (d) FRAP analysis of wild type GFP-lamin A in living control and HGPS cells. Nuclei were imaged before and during recovery after the bleach pulse. Scale bar: 5 μm. (e) Kinetics of recovery of the fluorescence signal in the whole bleached area. The statistical significance of the difference between the two recovery curves is indicated. (f) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (AG08469; population doublings: 25–30) transfected with GFP-Δ50 lamin A. Cells were stained with DAPI (blue) and antibodies (red) against lamin A/C, lamin B, LAP2 proteins. The green fluorescent signal from GFP-Δ50 lamin A is overlaid with the DAPI signal. Scale bar: 10 μm.

Fig. 2

Fig. 2. Correction of aberrant splicing in the endogenous lamin A transcript in HGPS cells

(a) Schematic representation of the region of lamin A pre-mRNA targeted by exo11 antisense oilgonucleotide. The predicted splicing events (dotted line for the normal splicing, dashed line for the aberrant splicing) and the position of exo11 oligonucleotide (black bar) are indicated. (b) RT PCR analysis of control fibroblasts and lymphocytes and HGPS fibroblasts and lymphocytes subjected to 2 sequential electroporations with oligonucleotide or mock treated. (c) Quantitation of exo 11 (black symbols) and scrambled (with symbols) oligonucleotides titration in different cell lines. All values represent averages from 5 independent experiments ±S.D. (d) Schematic representation of the different fragments of lamin A (black bars) and lamin C (gray bar) cDNAs amplified by RT-PCR. (e) RT-PCR analysis and (f) quantification of the indicated fragments after oligonucleotide titration. The intensity of each band was normalized to the intensity of the corresponding β-actin band. (g) Western blot analysis of control cells and HGPS fibroblasts subjected to 3 sequential electroporations with oligonucleotide or mock treated, analyzed 6 days after the first delivery.

Fig. 3

Fig. 3. Phenotypic rescue of HGPS cells by treatment with morpholino oligonucleotide

(a–d) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (a) and a HGPS patient (AG01972) untreated (b) or treated with oligonucleotide (c). Cells were stained with DAPI (blue) and antibodies (red) against the indicated proteins. The percentage of cells showing aberrant phenotype is indicated. Scale bar: 10 μm. (d) Low magnification images of untreated and treated HGPS cells. Scale bar: 35 μm. (e) FRAP analysis of wt GFP-lamin A in HGPS cells upon splicing correction. A nucleus was imaged before and during recovery after the bleach pulse. Scale bar: 5 μm. (f) Kinetics of recovery of the fluorescence signal in the whole bleached area compared to control and untreated HGPS cells. The statistical significance of the differences between the curves is indicated.

Fig. 4

Fig. 4. Restoration of normal gene activity in HGPS cells by treatment with oligonucleotide

(a) RNAse protection assay on control cells (CRL-1474) and HGPS cells (AG11498) subjected to 3 electroporations with oligonucleotide or mock treated. A longer exposure of the gel is shown for CCL8, MMP3, HAS III and MMP14 due to the low expression level of those genes compared to STAT3, TIMP3, L32 and GAPDH. (b) Quantitation of gene expression levels. Values represent fold differences of the indicated mRNAs levels between untreated and treated HGPS cells and control cells. Values are averages from at least 3 independent experiments ±S.D.

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