Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria

Abstract

Several progeroid disorders, including Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (ZMPSTE24 deficiency), arise when a farnesylated and methylated form of prelamin A accumulates at the nuclear envelope. Here, we found that a hypomorphic allele of isoprenylcysteine carboxyl methyltransferase (ICMT) increased body weight, normalized grip strength, and prevented bone fractures and death in Zmpste24-deficient mice. The reduced ICMT activity caused prelamin A mislocalization within the nucleus and triggered prelamin A-dependent activation of AKT-mammalian target of rapamycin (mTOR) signaling, which abolished the premature senescence of Zmpste24-deficient fibroblasts. ICMT inhibition increased AKT-mTOR signaling and proliferation and delayed senescence in human HGPS fibroblasts but did not reduce the levels of misshapen nuclei in mouse and human cells. Thus, targeting ICMT might be useful for treating prelamin A-associated progeroid disorders.

Fig. 1. Targeting Icmt ameliorates disease phenotypes and prevents death in 30-week-old Zmpste24^−/− mice

(A) Photograph of 24-week-old littermate mice. (B) Body-weight curves of male Zmpste24^−/−Icmt^+/+ (n = 11) and Zmpste24^−/−Icmt^hm/hm (n = 5) mice. (C) Kaplan-Meier plot showing the percentage of Zmpste24^−/−Icmt^+/+ (n = 21) and Zmpste24^−/−Icmt^hm/hm (n = 9) mice with normal grip strength. (D) Kaplan-Meier plot showing survival of Zmpste24^−/−Icmt^+/+ (n = 21) and Zmpste24^−/−Icmt^hm/hm (n = 9) mice. (E) Ventral view of spinal columns from an 18-week-old Zmpste24^−/−Icmt^+/+ mouse and a 30-week-old Zmpste24^−/−Icmt^hm/hm mouse. Arrowheads indicate rib fractures at costovertebral joints. (F) Number of rib fractures in Zmpste24^−/−Icmt^+/+ (n = 21) and Zmpste24^−/−Icmt^hm/hm (n = 9) mice. (G and H) Cellular parameters of L2 vertebrae (n = 6 per genotype). **P < 0.01; ***P < 0.001. Data are presented as mean ± SEM.

Fig. 2. Icmt deficiency mislocalizes prelamin A but does not reduce the frequency of misshapen nuclei in Zmpste24^−/− fibroblasts

(A) Confocal images of nuclei in primary mouse embryonic fibroblasts stained with a LAP2b antibody. Scale bar, 10 μm. (B) Frequency of misshapen nuclei in primary fibroblasts (n = 3 per genotype). Cre-adenovirus was used to produce Zmpste24^−/−Icmt^Δ/Δ cells from the parental Zmpste24^−/−Icmt^hm/hm cells. βgal-adenovirus was used as control. (C) Super-resolution structured illumination microscopy (SR-SIM) immunofluorescence images of nuclei in liver sections stained with prelamin A– and LAP2β-specific antibodies and counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. ***P < 0.001.

Fig. 3. Icmt deficiency prevents premature senescence of Zmpste24^−/− fibroblasts by increasing AKT pathway signaling

(A) Growth curves of primary fibroblasts (n = 4 per genotype) before (top) and after (bottom) incubation with Cre-adenovirus. (B) Immunoblots of fibroblast extracts show increased phosphorylation of AKT, S6, and 4E-BP1. Increased phosphorylation and inactivation of 4E-BP1 is evident by an increased ratio of γ and β/α isoforms. eIF4E and β-tubulin were the loading controls. (C) Immunoblots of fibroblast extracts with antibodies against prelamin A and cell cycle regulatory proteins. (D and E) Growth curves of primary fibroblasts incubated with inhibitors of (D) AKT (GSK690693, 10 μM) and (E) PTEN (VO-OHpic, 150 nM) (n = 3/genotype). (F) Immunoprecipitation (IP) and immunoblot (IB) analyses showing a methylation-dependent association between AKT and prelamin A. The lysates were also used directly for immunoblots for AKT (input). *P < 0.05; ***P < 0.001.

Fig. 4. Icmt knockdown in human HGPS fibro-blasts prevents premature senescence and increases AKT-mTOR pathway signaling

(A) TaqMan analyses showing ICMT mRNA levels in skin fibro-blasts from HGPS patients incubated with shRNA lentiviruses. Data are means of three independent cell lines incubated with two lentiviral clones expressing ICMT shRNAs or with a control clone containing a scrambled (SCR) sequence. (B) Growth curves of HGPS cells incubated with shIcmt or shSCR lentiviruses (n = 3 cell lines per treatment). (C) Immunoblots of fibroblast extracts; β-tubulin was the loading control. (D) Confocal images of fibroblasts stained with an antibody against LAP2b. Scale bar, 10 μm. (E) Frequency of nuclear shape abnormalities in control and HGPS fibroblasts incubated with shICMT and shSCR lentiviruses (n = 3 cell lines per treatment).