Rapamycin and HGPS雷帕霉素治疗早衰症

DOI: 10.1126/scitranslmed.3002346 , 89ra58 (2011);3 Sci Transl Med , et al.Kan Cao Clearance in Hutchinson-Gilford Progeria Syndrome Cells Rapamycin Reverses Cellular Phenotypes and Enhances Mutant Protein Editor's Summary The Young and the Youthless new notions are continually ''revealed to us in whose light all our previous knowledge must be rearranged.''−−Waugh according to−−children with HGPS and may offer insights into normal aging as well. Thus, in science, as in youth accumulation. The new work has helped to form the basis for a clinical trial of the rapamycin analog everolimus in insoluble aggregates. Blocking the expression of an autophagy-related gene with small RNAs enhanced progerin clearance of soluble progerin by activating the autophagic-lysosomal pathway, which inhibited the formation of untreated cells. Mechanistic experiments in normal human fibroblasts revealed that rapamycin treatment increased towith rapamycin showed enhanced progerin degradation, slowed senescence, and reduced nuclear blebbing relative helps to rid HGPS cells of progerin build-up and to remedy the resulting cellular quirks. Indeed, HGPS cells treated process by which cells clear junk protein and trashed organelles. Thus, Cao et al. tested whether rapamycin also a−−evidence that rapamycin might be useful in the treatment of these disorders by stimulating macroautophagy the cytoplasm. Protein aggregation is also a hallmark of several neurodegenerative diseases, and there is some inspan, nuclear blebbing (bulging of the nuclear membrane), and the characteristic accumulation of insoluble progerin cells (fibroblasts) from HGPS patients display a variety of blemishes, including slowed growth, an abbreviated life indicate that normal human cells also express tiny amounts of progerin, which accumulates as a person ages. Skin that accumulates in HGPS cells and wreaks havoc on cellular form and function. Several studies−−called progerin−− nuclear structural protein lamin A. This genetic defect creates an improperly processed version of the lamin A protein disease and stroke often by the age of 12. The disease is caused by a point mutation in the gene that encodes the phenotypes we associate with aging: hair loss, bone deficits, hardening of the skin by 1 or 2 years of age, and heart that rapamycin might be repurposed for the treatment of this dire disease. Children with HGPS display many of the that the drug reverses disease-distinguishing defects in cells from HGPS patients. These findings raise the possibility process, the versatile drug rapamycin has been shown to extend longevity in animal models. Now, Cao et al. reveal disease characterized by premature aging and death in adolescence or the teen years. In studies of the normal aging swiftly or more dramatically than in children suffering from Hutchinson-Gilford progeria syndrome (HGPS), a genetic how irrecoverably lost'' is the bloom. Although every adult can relate to this cri de coeur, in no case is youth lost more In the novel Brideshead Revisited, Evelyn Waugh reminisces about ''the langor of youth'' and laments ''how quickly, http://stm.sciencemag.org/content/3/89/89ra58.full.html can be found at: and other services, including high-resolution figures,A complete electronic version of this article http://stm.sciencemag.org/content/suppl/2011/06/27/3.89.89ra58.DC1.html can be found in the online version of this article at: Supplementary Material http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: article permission to reproduce this of this article or about obtaining reprintsInformation about obtaining is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title NW, Washington, DC 20005. Copyright 2011 by the American Association for the Advancement of Science; all last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine o n J ul y 2, 2 01 1 st m .s ci en ce m ag .o rg D ow nl oa de d fro m PROGER IA Rapamycin Reverses Cellular Phen Enhances Mutant Protein Clearanc Hutchinson-Gilford Progeria Synd R rd n a he si h Tr t ga iti in erative diseases through the activation of macroautophagy, a process by which the cell rids itself of unnecessary proteins and damaged or- on cultured fibroblasts d on analyzing nuclear cells. Cultured fibro- 69, HGADFN155, and viduals (HGFDFN168, were used in this study. l medium (MEM) con- me of vehicle [dimethyl y for a minimal duration f rapamycin, treatment ce approached. Because HGPS or control cell om one HGPS cell line N168) unless otherwise morphology, we labeled antibody to a-tubulin, . 1A). To quantitatively cored the percentage of t 200 randomly selected cells were scored by fluorescence microscopy for each cell line under R E S EARCH ART I C L E o n J ul y 2, 2 01 1 st m .s ci en ce m ag .o rg D ow nl oa de d fro m *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: francis.collins@nih.gov approved by the U.S. Food and Drug Administration (FDA) for a va- riety of clinical applications, including immunosuppression and as a potential anticancer treatment (15). In addition, rapamycin has been the results were quite consistent for all tested lines, we show only the representative data fr (HGADFN167) and one control line (HGFDF indicated. To examine rapamycin’s effects on nuclear cells with an antibody to lamin A/C and an which together reveal the overall cell shape (Fig judge the impact of rapamycin treatment, we s nuclear blebbing in a blinded fashion. At leas 1Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892–8004, USA. 2Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA. 3Department of Neurology, Massachusetts General Hospital, MassGeneral Institute for Neurodegenerative Disease, Harvard Medical School, Charlestown, MA 02129, USA. rapamycin reduces mTORC2 function as well (14). Rapamycin has been and life span, and broad epigenetic alterations in histone methylation that predate the changes in nuclear shape (5–7). Rapamycin is a macrolide antibiotic that has been shown to affect a number of biological pathways, including the ability to extend longev- ity in yeast, invertebrates, and mammals (8–11). Its major cellular tar- get, mammalian target of rapamycin (mTOR), is a central regulator of cell growth that integrates nutrient and hormonal signals and regu- lates diverse cellular processes (12). mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, both of which control cell growth (13). At low (nanomolar) concentrations, rapamycin inhibits mTORC1 by directly associating with its intracellular receptor FKBP12, but longer-term exposure of the cells to higher (millimolar) doses of Rapamycin rescues nuclear blebbing an senescence in HGPS fibroblasts We explored the potential effects of rapamycin derived from HGPS patients. First, we focuse blebbing, the phenotypic hallmark of HGPS blasts from three HGPS patients (HGADFN1 HGADFN167) and from four control indi HGFDFN320, HGFDFN319, and AG08470) The cells were fed with fresh minimal essentia taining 0.68 mM rapamycin or the same volu sulfoxide (DMSO), 0.025% (v/v)] every other da of 2 weeks. To evaluate the long-term effect o continued over 100 days until cellular senescen mulations of progerin in mitotic cytoplasm (4), a reduced growth rate d postpones Kan Cao,1,2* John J. Graziotto,3* Cecilia D. Blair,1 Joseph Dimitri Krainc,3† Francis S. Collins1† Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic diso is most commonly caused by a de novo single-nucleotide substitutio activates a cryptic splice donor site in exon 11, producing an abnorm lation of progerin in dividing cells adversely affects the integrity of t bing in cultured cells. Progerin is also produced in normal cells, increa Here, we report the effect of rapamycin, a macrolide antibiotic that organismal aging, on the cellular phenotypes of HGPS fibroblasts. blebbing, delayed the onset of cellular senescence, and enhanced Rapamycin also decreased the formation of insoluble progerin aggre agic mechanisms in normal fibroblasts. Our findings suggest an add rapamycin on longevity and encourage the hypothesis that rapamyc children with HGPS. INTRODUCTION Classic Hutchinson-Gilford progeria syndrome (HGPS) is an auto- somal dominant genetic disease that is observed in about 1 in 4 mil- lion live births (1, 2). Patients with HGPS appear normal at birth but begin to display alopecia, growth retardation, bone abnormalities, os- teoporosis, and sclerodermatous skin by 1 year of age (1, 3). HGPS patients typically die at a mean age of 12 years from myocardial in- farction or cerebrovascular accident (1). Primary fibroblasts from HGPS patients exhibit characteristic nuclear blebbing, punctate accu- (F.S.C.); krainc@helix.mgh.harvard.edu (D.K.) www.S ganelles via the lysosomal degradation pathway (16–18). Here, we test the effects of rapamycin on HGPS cells and characterize the drug’s ef- fect on progerin accumulation. RESULTS otypes and e in rome Cells . Mazzulli,3 Michael R. Erdos,1 er characterized by premature aging. HGPS in the lamin A/C gene (LMNA) that partially l lamin A protein termed progerin. Accumu- nuclear scaffold and leads to nuclear bleb- ng in abundance as senescence approaches. as been implicated in slowing cellular and eatment with rapamycin abolished nuclear he degradation of progerin in HGPS cells. tes and induced clearance through autoph- onal mechanism for the beneficial effects of treatment could provide clinical benefit for shown to be potentially beneficial in the treatment of neurodegen- each condition. Consistent with a previous report (19), with mock cienceTranslationalMedicine.org 29 June 2011 Vol 3 Issue 89 89ra58 1 mal bleb- proached -matched, exhibited rovement cin treat- e increase blebbing phenotype was observed even when rapamycin treatment was started in later-passage HGPS cells (passage 15 and beyond), suggesting that the drug is capable of reversing the nuclear blebbing phenotype in HGPS cells. Rapamycin-treated normal control cells also exhibited a significant decrease in blebbing compared with their mock-treated counterparts (Fig. 1B). Previous analysis has demonstrated that progerin accumulation induces progressive alterations in the trimethylation of Lys27 in histone i, ck-treated HGPS and control R E S EARCH ART I C L E o n J ul y 2, 2 01 1 st m .s ci en ce m ag .o rg D ow nl oa de d fro m fibroblast cells to DNA-damaging agent mitomycin C at the indi- cated concentration. An HGPS cell line (HGADFN155, female) at passage 12 and a control cell line (HGFDFN320, female) at pas- sage 15were used. Each experiment was counted independently three times, but the error bars were too small to be seen on a log scale. (E) Percentage of 53BP1 foci–positive cells in rapamycin- andmock-treated HGPS cells. (F) Growth curves of a primary con- trol fibroblast cell line (HGFDFN168) and an HGPS fibroblast cell line (HGADFN167) treatedwith vehicle (Mock) or rapamycin once every 2 days for 100 days (n = 3 for each condition). www.S blebbing and postpones senes- cence inHutchinson-Gilfordproge- ria syndrome (HGPS) fibroblasts. (A) Immunofluorescence images of an HGPS cell line (HGADFN167) at passage 15 treated with vehi- cle (DMSO) or rapamycin (Rap) for 14 days. Red, anti–a-tubulin (Tub, DM1a); green, anti–lamin A/C (MAB3211). Scale bar, 20 mm. (B) Percentage of nuclear blebbing when HGPS (HGADFN167) and control (HGFDFN168) fibroblast cells were treated once every oth- er day for 70 days with rapamycin (*P = 0.015; **P = 0.0002). These experiments were performed in three independent HGPS cell lines and in four control lines. Repre- sentative data from one HGPS cell line (HGADFN167) and one con- trol line (HGFDFN168) are shown. Percentage of nuclear blebbing was scored by a blinded observer who viewed more than 200 ran- domly chosen cells. For statisti- cal analysis, we used a two-tailed Student’s t test to compare the mock- and rapamycin-treated cells at four timepoints (days 10, 21, 30, and 70). A P value of <0.05 was considered significant. (C) Repre- sentative immunofluorescence images of an HGPS fibroblast cell line (HGADFN155, female) at pas- sage 10 and a control fibroblast cell line (HGFDFN320, female) at passage 10 treated with vehicle (Mock) or rapamycin for 14 days. Red, anti-H3K27me3; green: goat anti–lamin A/C (N18); blue, DAPI staining of DNA. Scale bar, 20 mm. X (D) Sensitivity of rapamycin- andmo inactivated X chromosome. treatment, more than 30% of the HGPS nuclei exhibited abnor bing, and this percentage gradually increased as the cells ap senescence in culture (Fig. 1B). In comparison with the passage mock-treated HGPS cells, the rapamycin-treated HGPS cells a significant reduction in nuclear blebbing (Fig. 1B). The imp in nuclear blebbing appeared as early as 10 days after rapamy ment and was retained in the long-term experiments despite th in cellular age (Fig. 1B). A similar reduction of the nuclear Fig. 1. Rapamycin rescuesnuclear cienceTranslationalMedicine.org 29 June 2011 Vol 3 Issue 89 89ra58 2 R E S EARCH ART I C L E o n J ul y 2, 2 01 1 st m .s ci en ce m ag .o rg D ow nl oa de d fro m H3 (H3K27me3), a mark of facultative heterochromatin (a reversible form of tightly packed, transcriptionally silent DNA) (6). The changes in H3K27me3 may be induced by the disruption of the nuclear scaf- fold and occur before changes in nuclear shape. To evaluate the po- tential effect of rapamycin on the epigenome, we examined the nuclear staining of H3K27me3 in rapamycin- or mock-treated female fibro- blasts. In female cells, it is known that H3K27me3 normally coats the inactivated X chromosome (Xi); in HGPS cells, this H3K27me3 staining on Xi is partially lost (Fig. 1C). We found that rapamycin treatment restored the normal distribution of H3K37me3 in HGPS fi- broblasts (Fig. 1C and fig. S2). In addition, because an increase in genome instability has been reported in HGPS cells (20, 21), we examined wheth- er rapamycin treatment can improve this phenotype. Using mitomycin C, a chemical that induces double-strand DNA breaks, we found that rapamycin-treated HGPS cells showed reduced sensitivity to DNA dam- age (Fig. 1D). Moreover, using immunofluorescence staining, we observed in rapamycin-treated cells a significant reduction in tumor protein p53-binding protein 1 (TP53BP1)–positive nuclei with multiple foci relative to control cells. TP53BP1 binds to sites of DNA damage in the nucleus (21). Thus, these observations indicate that rapamycin treat- ment helps to prevent DNA damage caused by accumulation of progerin (Fig. 1E). We also determined the HGPS cell proliferation rate with and without rapamycin treatment by quantifying the total cell numbers during the course of treatment. Initially, we had predicted a decrease in growth rate because of rapamycin’s well-characterized antiprolifera- tive effect (22), which may cause the death of unhealthy cells with pronounced nuclear defects. Surprisingly, we only noticed a slight drop in cell number at the beginning of the treatment (less than 2 weeks), and no significant delay in proliferation rate was observed when rapamycin was used at the indicated concentration during the long-term treatment (Fig. 1F). Indeed, after 60 days of rapamycin treatment, when the pro- liferation rate in the mock-treated cells began to drop as cellular senes- cence approached, the cell count continued upward in both the control and the HGPS rapamycin-treated cell lines (Fig. 1F). Consistent with these findings, when senescence-associated b-galactosidase (SA-b-Gal) activity was measured at day 60 of treatment, we observed significantly fewer b-Gal–positive cells in rapamycin-treated versus mock-treated cells (fig. S2), indicating that rapamycin slowed the progression of cel- lular senescence. Accordingly, the maximum cellular life span was sig- nificantly extended by at least 30 days in both control and HGPS cell lines treated with rapamycin. In support of a rapamycin effect on se- nescence, fluorescence-activated cell sorting (FACS) analysis revealed a significant increase of S-phase cells in the context of rapamycin but not mock treatment, which suggests that a higher percentage of the rapamycin-treated cells were continuing through the cell cycle (fig. S3). We concluded that rapamycin treatment rescues nuclear phenotypes and postpones cellular senescence in HGPS and control cells. Rapamycin decreases progerin levels in HGPS fibroblasts Previous studies have shown that rapamycin may promote the degra- dation of unnecessary or toxic components inside the cell through ac- tivation of autophagy (23, 24). In cultured HGPS cells, the severity of nuclear phenotypes in later passages correlates with an apparent in- crease in progerin accumulation (5). Moreover, progerin is also produced in normal cells and increases in abundance as senescence approaches (4, 25). Therefore, we hypothesized that rapamycin induces the accelerated degradation of progerin in treated HGPS and control www.S cells. To test this hypothesis, we generated a custom antibody against progerin using a peptide located at the cryptic splicing junction as an antigen (anti-progerin). This antibody specifically recognized progerin, but not lamin A or C in Western (immuno) blotting and immuno- fluorescence analyses (fig. S4). When normalized with a b-actin control, quantification of protein by Western blot revealed a ~50% reduction in progerin amounts in rapamycin-treated HGPS cells on day 60 of treat- ment relative to mock-treated cells (Fig. 2, A and B). Quantification of the intensities of lamin A and C bands revealed that the amount of wild-type lamin A was reduced by about 25%, and the lamin C amount was not affected. To test rapamycin effectiveness, we also examined the phosphorylation state of ribosomal protein subunit S6 (rpS6, and p-rpS6 for the phosphorylated form), a target substrate in the mTOR signal- ing pathway (9), and detected the expected reduction in rpS6 phospho- rylation (Fig. 2A). To further evaluate the rapamycin-associated decrease in progerin, we used immunofluorescence microscopy to scrutinize individual HGPS cells that had been stained with anti-progerin. We consistently de- tected a weaker progerin staining signal in almost all of the rapamycin- treated HGPS cells (more than 1000 cells viewed from three independent HGPS cell lines), and their nuclear blebbing phenotype appeared to be substantially improved relative to mock-treated cells (Fig. 2C). Quan- tification confirmed the intensity reduction (Fig. 2D), consistent with the Western blotting results (Fig. 2B). We then performed quantitative reverse transcription–polymerase chain reaction (RT-PCR) assays using a pair of primers specific for progerin mRNA in rapamycin- and mock- treated HGPS and control cells. As shown in Fig. 2E, progerin mRNA expression was not down-regulated in rapamycin-treated cells, suggest- ing that the rapamycin effect on progerin occurs post-transcriptionally. Rapamycin treatment increases solubility of progerin Having shown that rapamycin decreases progerin concentrations in HGPS cells, we next examined whether the drug alters aggregation of the protein. To this end, we used sequential extractions to examine the effect of rapamycin on solubility of progerin. Analysis of mock- treated HGPS cells revealed a significant increase in the amount of both lamin A and progerin in the SDS- and formic acid–soluble extracts compared to control cel