Defective Lamin A-Rb Signaling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disorder caused by a de novo heterozygous point mutation G608G (GGC>GGT) within exon 11 of LMNA gene encoding A-type nuclear lamins. This mutation elicits an internal deletion of 50 amino acids in the carboxyl-terminus of prelamin A. The truncated protein, progerin, retains a farnesylated cysteine at its carboxyl terminus, a modification involved in HGPS pathogenesis. Inhibition of protein farnesylation has been shown to improve abnormal nuclear morphology and phenotype in cellular and animal models of HGPS. We analyzed global gene expression changes in fibroblasts from human subjects with HGPS and found that a lamin A-Rb signaling network is a major defective regulatory axis. Treatment of fibroblasts with a protein farnesyltransferase inhibitor reversed the gene expression defects. Our study identifies Rb as a key factor in HGPS pathogenesis and suggests that its modulation could ameliorate premature aging and possibly complications of physiological aging

The Mutant Form of Lamin A that Causes Hutchinson-Gilford Progeria Is a Biomarker of Cellular Aging in Human Skin

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals

Human skin biopsies

<p>Classification of the skin biopsies originating from different body sites of unaffected individuals and collected at the Dermatology Department in accordance with the Columbia University Board protocols on human subject protection.</p><p>Total# 150 Samples</p

Progerin expression in human skin.

<p>A, RT-PCR analysis of HGPS cells and human skin biopsies of indicated age, primers amplifying wild-type and progerin transcripts. B, Direct sequencing of a short portion within exon 11 of wild type lamin A (LMNA), and progerin transcripts from HGPS and 93-year-old subjects. C, Western blot analysis of protein extracted from skin biopsies of indicated age with anti-progerin 972S9, anti-lamin A/C and anti-actin antibodies.</p

Progerin detection in a subset of terminally differentiated keratinocytes.

<p>Left panels correspond to anti-progerin monoclonal antibody staining of skin sections derived from individuals of indicated age. Right panels correspond to the merged signal of dapi and progerin signals. Square indicates the zoomed in region of the epidermis. ep denotes epidermis, and de the dermis.</p

In situ localization of progerin on human skin sections derived from a subject with HGPS and from unaffected individuals.

<p>A, HGPS skin sections immunostained with anti-progerin (prog), anti-lamin A (LMNA) antibody, or anti-α smooth muscle actin antibody (αSMA) and counterstained with a DNA stain (dapi). Morphologic entities are indicated: epidermis (ep), dermis (de), sweat glands (SG), capillary (c), arrector pili muscle (a), and hair follicle (h). B, Newborn foreskin sections immunolabelled with anti-progerin or anti-αSMA antibody. C, Breast skin sections from 22- and 46-year-old female subjects probed with anti-progerin and lamin A. The respective double or triple merged signals are indicated.</p

Progerin accumulation in human elderly skin biopsy sections.

<p>A, Forehead skin section from a 69-year-old individual probed with anti-progerin antibody and counterstained with dapi. Bars correspond to 100 and 50 µm, respectively. B, Forehead skin section from a 93-year-old donor. C, Skin sections from different body sites as indicated were probed with anti-progerin and anti- lamin A (LMNA) antibodies. Bar, 50 µm. Merged images are indicated.</p

Progerin expression in primary dermal fibroblast cultures.

<p>A, Immunofluorescence microscopy on primary dermal fibroblasts from an HGPS subject and unaffected individuals of indicated ages with rabbit monoclonal anti-progerin antibody. B, Immunofluoresence detection of progerin in HGADFN 127 (HGPS individual) and DR118 (86-year-old individual) at late PPDs. C, Western blot analysis of nuclear protein extracts derived from fibroblast cultures HGADFN 127 (HGPS) at PPD 20 and DR118 (86-year-old female) at PPD 15 and 30, respectively, were immunoprecipitated with anti-progerin mAb 972S9 and the corresponding Western blot was probed with the same antibody.</p