Modulation of LMNA splicing as a strategy to treat prelamin A diseases

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Abstract

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A–specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A–specific diseases.

Authors

John M. Lee, Chika Nobumori, Yiping Tu, Catherine Choi, Shao H. Yang, Hea-Jin Jung, Timothy A. Vickers, Frank Rigo, C. Frank Bennett, Stephen G. Young, Loren G. Fong

The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity

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Abstract

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

Authors

Michal Dudek, Nicole Gossan, Nan Yang, Hee-Jeong Im, Jayalath P.D. Ruckshanthi, Hikari Yoshitane, Xin Li, Ding Jin, Ping Wang, Maya Boudiffa, Ilaria Bellantuono, Yoshitaka Fukada, Ray P. Boot-Handford, Qing-Jun Meng

Immune activation caused by vascular oxidation promotes fibrosis and hypertension

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Abstract

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg^sm/p22phox mice, which overexpress the NADPH oxidase subunit p22^phox in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg^sm/p22phox mice produced high levels of IL-17A and IFN-γ. Crossing tg^sm/p22phox mice with lymphocyte-deficient Rag1^–/– mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg^sm/p22phox mice. Autologous pulsing with tg^sm/p22phox aortic homogenates promoted DCs of tg^sm/p22phox mice to stimulate T cell proliferation and production of IFN-γ, IL-17A, and TNF-α. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.

Authors

Jing Wu, Mohamed A. Saleh, Annet Kirabo, Hana A. Itani, Kim Ramil C. Montaniel, Liang Xiao, Wei Chen, Raymond L. Mernaugh, Hua Cai, Kenneth E. Bernstein, Jörg J. Goronzy, Cornelia M. Weyand, John A. Curci, Natalia R. Barbaro, Heitor Moreno, Sean S. Davies, L. Jackson Roberts II, Meena S. Madhur, David G. Harrison

Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors

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Abstract

The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS^V12, or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS^V12-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation — a common feature of aging — has the potential to limit aging-associated oncogenesis.

Authors

Curtis J. Henry, Matias Casás-Selves, Jihye Kim, Vadym Zaberezhnyy, Leila Aghili, Ashley E. Daniel, Linda Jimenez, Tania Azam, Eoin N. McNamee, Eric T. Clambey, Jelena Klawitter, Natalie J. Serkova, Aik Choon Tan, Charles A. Dinarello, James DeGregori

Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita

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Abstract

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Authors

Hemanth Tummala, Amanda Walne, Laura Collopy, Shirleny Cardoso, Josu de la Fuente, Sarah Lawson, James Powell, Nicola Cooper, Alison Foster, Shehla Mohammed, Vincent Plagnol, Thomas Vulliamy, Inderjeet Dokal

Lifespan of mice and primates correlates with immunoproteasome expression

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Abstract

There is large variation in lifespan among different species, and there is evidence that modulation of proteasome function may contribute to longevity determination. Comparative biology provides a powerful tool for identifying genes and pathways that control the rate of aging. Here, we evaluated skin-derived fibroblasts and demonstrate that among primate species, longevity correlated with an elevation in proteasomal activity as well as immunoproteasome expression at both the mRNA and protein levels. Immunoproteasome enhancement occurred with a concurrent increase in other elements involved in MHC class I antigen presentation, including β-2 microglobulin, (TAP1), and TAP2. Fibroblasts from long-lived primates also appeared more responsive to IFN-γ than cells from short-lived primate species, and this increase in IFN-γ responsiveness correlated with elevated expression of the IFN-γ receptor protein IFNGR2. Elevation of immunoproteasome and proteasome activity was also observed in the livers of long-lived Snell dwarf mice and in mice exposed to drugs that have been shown to extend lifespan, including rapamycin, 17-α-estradiol, and nordihydroguaiaretic acid. This work suggests that augmented immunoproteasome function may contribute to lifespan differences in mice and among primate species.

Authors

Andrew M. Pickering, Marcus Lehr, Richard A. Miller

Phosphatase WIP1 regulates adult neurogenesis and WNT signaling during aging

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Abstract

The number of newly formed neurons declines rapidly during aging, and this decrease in neurogenesis is associated with decreased function of neural stem/progenitor cells (NPCs). Here, we determined that a WIP1-dependent pathway regulates NPC differentiation and contributes to the age-associated decline of neurogenesis. Specifically, we found that WIP1 is expressed in NPCs of the mouse subventricular zone (SVZ) and aged animals with genetically enhanced WIP1 expression exhibited higher NPC numbers and neuronal differentiation compared with aged WT animals. Additionally, augmenting WIP1 expression in aged animals markedly improved neuron formation and rescued a functional defect in fine odor discrimination in aged mice. We identified the WNT signaling pathway inhibitor DKK3 as a key downstream target of WIP1 and found that expression of DKK3 in the SVZ is restricted to NPCs. Using murine reporter strains, we determined that DKK3 inhibits neuroblast formation by suppressing WNT signaling and Dkk3 deletion or pharmacological activation of the WNT pathway improved neuron formation and olfactory function in aged mice. We propose that WIP1 controls DKK3-dependent inhibition of neuronal differentiation during aging and suggest that regulating WIP1 levels could prevent certain aspects of functional decline of the aging brain.

Authors

Yunhua Zhu, Oleg N. Demidov, Amanda M. Goh, David M. Virshup, David P. Lane, Dmitry V. Bulavin

p16^INK4a reporter mice reveal age-promoting effects of environmental toxicants

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Abstract

While murine-based systems to identify cancer-promoting agents (carcinogens) are established, models to identify compounds that promote aging (gerontogens) have not been described. For this purpose, we exploited the transcription of p16^INK4a, which rises dynamically with aging and correlates with age-associated disease. Activation of p16^INK4a was visualized in vivo using a murine strain that harbors a knockin of the luciferase gene into the Cdkn2a locus (p16^LUC mice). We exposed p16^LUC mice to candidate gerontogens, including arsenic, high-fat diet, UV light, and cigarette smoke and serially imaged animals to monitor senescence induction. We show that exposure to a high-fat diet did not accelerate p16^INK4a expression, whereas arsenic modestly augmented, and cigarette smoke and UV light potently augmented, activation of p16^INK4a-mediated senescence. This work provides a toxicological platform to study mammalian aging and suggests agents that directly damage DNA promote molecular aging.

Authors

Jessica A. Sorrentino, Janakiraman Krishnamurthy, Stephen Tilley, James G. Alb Jr., Christin E. Burd, Norman E. Sharpless

p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes

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Abstract

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53β are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8⁺ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28^–CD57⁺) with decreased Δ133p53 and increased p53β expression. Human lung tumor-associated CD8⁺ T lymphocytes also harbored senescent cells. Cultured CD8⁺ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8⁺CD28^– cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53β overexpression in CD8⁺CD28⁺ cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53β in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Authors

Abdul M. Mondal, Izumi Horikawa, Sharon R. Pine, Kaori Fujita, Katherine M. Morgan, Elsa Vera, Sharlyn J. Mazur, Ettore Appella, Borivoj Vojtesek, Maria A. Blasco, David P. Lane, Curtis C. Harris

Essential amino acid supplementation in patients following total knee arthroplasty

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Abstract

Background. By the year 2030, 3.48 million older U.S. adults are projected to undergo total knee arthroplasty (TKA). Following this surgery, considerable muscle atrophy occurs, resulting in decreased strength and impaired functional mobility. Essential amino acids (EAAs) have been shown to attenuate muscle loss during periods of reduced activity and may be beneficial for TKA patients.

Methods. We used a double-blind, placebo-controlled, randomized clinical trial with 28 older adults undergoing TKA. Patients were randomized to ingest either 20 g of EAAs (n = 16) or placebo (n = 12) twice daily between meals for 1 week before and 2 weeks after TKA. At baseline, 2 weeks, and 6 weeks after TKA, an MRI was performed to determine mid-thigh muscle and adipose tissue volume. Muscle strength and functional mobility were also measured at these times.

Results. TKA patients receiving placebo exhibited greater quadriceps muscle atrophy, with a –14.3 ± 3.6% change from baseline to 2 weeks after surgery compared with –3.4 ± 3.1% for the EAA group (F = 5.16, P = 0.036) and a –18.4 ± 2.3% change from baseline to 6 weeks after surgery for placebo versus –6.2 ± 2.2% for the EAA group (F = 14.14, P = 0.001). EAAs also attenuated atrophy in the nonoperated quadriceps and in the hamstring and adductor muscles of both extremities. The EAA group performed better at 2 and 6 weeks after surgery on functional mobility tests (all P < 0.05). Change in quadriceps muscle atrophy was significantly associated with change in functional mobility (F = 5.78, P = 0.021).

Conclusion. EAA treatment attenuated muscle atrophy and accelerated the return of functional mobility in older adults following TKA.

Trial registration. Clinicaltrials.gov NCT00760383.

Funding. Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Office of the Director (OD), and the National Institutes of Health Office of Dietary Supplements (ODS), NIH grant K01HD057332, and the Medical Research Foundation, Oregon Health and Science University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the funders.

Authors

Hans C. Dreyer, Lisa A. Strycker, Hilary A. Senesac, Austin D. Hocker, Keith Smolkowski, Steven N. Shah, Brian A. Jewett