Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.     

Proteomic profiling of adipose tissue from Zmpste24-/- mice, a model of lipodystrophy and premature aging, reveals major changes in mitochondrial function and vimentin processing

Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.     

Amelioration of age‐related brain function decline by Bruton's tyrosine kinase inhibition

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24^?/? progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24^?/? mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1

Cisplatin is the most potent and widespread used chemotherapy drug for lung cancer treatment. However, the development of resistance to cisplatin is a major obstacle in clinical therapy. The principal mechanism of cisplatin is the induction of DNA damage, thus the capability of DNA damage response (DDR) is a key factor that influences the cisplatin sensitivity of cancer cells. Recent advances have demonstrated that miRNAs (microRNAs) exerted critical roles in DNA damage response; nonetheless, the association between DNA damage responsive miRNAs and cisplatin resistance and its underlying molecular mechanism still require further investigation. The present study has attempted to identify differentially expressed miRNAs in cisplatin induced DNA damage response in lung cancer cells, and probe into the effects of the misexpressed miRNAs on cisplatin sensitivity. Deep sequencing showed that miR-33b-3p was dramatically down-regulated in cisplatin-induced DNA damage response in A549 cells; and ectopic expression of miR-33b-3p endowed the lung cancer cells with enhanced survival and decreased γH2A.X expression level under cisplatin treatment. Consistently, silencing of miR-33b-3p in the cisplatin-resistant A549/DDP cells evidently sensitized the cells to cisplatin. Furthermore, we identified CDKN1A (p21) as a functional target of miR-33b-3p, a critical regulator of G1/S checkpoint, which potentially mediated the protection effects of miR-33b-3p against cisplatin. In aggregate, our results suggested that miR-33b-3p modulated the cisplatin sensitivity of cancer cells might probably through impairing the DNA damage response. And the knowledge of the drug resistance conferred by miR-33b-3p has great clinical implications for improving the efficacy of chemotherapies for treating lung cancers.     

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24^−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24^−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24^−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24^−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24^−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.     

Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin,promotes cell senescence and enhances senolysis

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.     

miR-29b-3p protects cardiomyocytes against endotoxin-induced apoptosis and inflammatory response through targeting FOXO3A

Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.     

miRNA expression profiling uncovers a role of miR-302b-3p in regulating skin fibroblasts senescence

Numbers of emerging evidence suggest that variable microRNA (miRNA) expression facilitates the aging process. In this study, we distinguished aberrant miRNA expression in aged skin and explored the biological functions and potential mechanism of upregulated miR-302b-3p. At first, miRNA microarray analysis was examined to explore miRNA expression profiling in the skin of aging mice model by D -galactose (d -gal) injection. We identified 29 aberrant miRNAs in aged mice skin. Next, KEGG enrichment analysis was conducted with DIANA-miPath v3.0, which was revealed that enrichment pathways involved in such processes as extracellular matrix-receptor interaction, MAPK signaling pathway, and mammalian target of rapamycin (mTOR) signaling pathway. The target genes of deregulated miRNAs were predicted from four bioinformatic algorithms (miRDB, Targetscan, miRwalk, and Tarbase). The interaction network of miRNAs and their targets were visualized using Cytoscape software. As a result, we found that some hub genes (including JNK2, AKT1/2/3, PAK7, TRPS1, BCL2L11, and IKZF2) were targeted by 12 potential miRNAs (including miR-302b-3p, miR-291a-5p, miR-139-3p, miR-467c-3p, miR-186-3p, etc.). Subsequently, we identified five upregulated miRNA via quantitative polymerase chain reaction and all of them were confirmed increased significantly in aged skin tissues compared with young control tissues. Among them, high expression of miR-302b-3p was verified in both aged skin tissues and senescence fibroblasts. Furthermore, miR-302b-3p mimic accelerated skin fibroblast senescence and suppressed the longevity-associated gene Sirtuin 1(Sirt1) expression, whereas miR-302b-3p inhibitor could delay skin fibroblast senescence and contribute Sirt1 expression. In addition, we demonstrated that c-Jun N-terminal kinase 2(JNK2) is a direct target of miR-302b-3p by a luciferase reporter assay. An inverse correlation was verified in fibroblasts between miR-302b-3p and JNK2. Most importantly, siRNA JNK2 confirmed that low expression of JNK2 could accelerate fibroblasts senescence. In conclusion, our results indicated that overexpressed miR-302b-3p plays an important biological role in accelerating skin aging process via directly targeting JNK2 gene.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.     

PP2Cdelta (Ppm1d, WIP1), an endogenous inhibitor of p38 MAPK, is regulated along with Trp53 and Cdkn2a following p38 MAPK inhibition during mouse preimplantation development

Preimplantation embryos utilize mitogen-activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cdelta (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cdelta proteins are expressed throughout mouse preimplantation development. Treatment of 2-cell embryos with SB220025 (potent inhibitor of p38 MAPK alpha/beta/MAPK 14/11) significantly increased Trp53, Ppm1d and Cdkn2a and Mapk14 mRNA levels at 12 and 24 hr. Treatment of 8-cell embryos with SB220025 for 12 hr increased Trp53, Ppm1d, and Cdkn2a mRNA levels, but not Mapk14 mRNA levels. Treatment of 8-cell embryos for 24 hr increased Trp53, and Ppm1d mRNA levels, but decreased Cdkn2a and Mapk14 mRNA levels. Therefore, blockade of p38 MAPK activity is associated with embryo stage specific influences on Trp53, Ppm1d, Cdkn2a, and Mapk14 expression during mouse preimplantation development. These results define downstream targets of p38 MAPK during preimplantation development and indicate that the p38 MAPK pathway regulates Trp53, Ppm1d, and Cdkn2a expression. This study increases our understanding of the mechanisms controlling preimplantation development and of the interactions between preimplantation embryos and their culture environments.