Premature Aging Phenotype Research Articles

Abstract P2037: Bubr1 Is A Critical Regulator Of Cardiac Aging

BubR1 is a serine/threonine protein kinase with a well-established role in regulating the spindle assembly checkpoint during mitosis. Previous studies have shown that BubR1 insufficiency leads to premature aging phenotypes such as kyphosis, cataracts, loss of subcutaneous fat, and a shortened lifespan. Furthermore, BubR1 naturally declines with age a wide range of tissues due to enhanced proteosome-mediated degradation. BubR1 hypomorphic mice expressing approximately 10% of the protein die suddenly in a manner reminiscent of sudden cardiac death. Subsequent studies have further shown that these mice suffer from conduction abnormalities such as a prolonged QTc interval. In humans, the incidence of QTc interval prolongation increases with age, and is associated with sudden cardiac death. Given BubR1 protein levels decline with age, we reasoned that BubR1 is critical for the maintenance of adult cardiac structure and function and its loss with age may drive age-related pathophysiology in the heart. We generated a conditional BubR1 knockout mouse which we crossed to the cardiac-specific cre driver line, Nkx2.5-cre , to assess the consequence of loss of BubR1 specifically in the heart. Electrocardiography analysis of BubR1 heterozygous knockouts also have a prolonged QTc interval in vivo . Structural analysis of the hearts of these mice revealed that they suffer from cardiac hypertrophy as shown by an increase in both heart weight as well as ventricular cardiomyocyte size. Masson’s trichrome staining revealed that BubR1 heterozygous knockout hearts have increased fibrosis both in the ventricular space and the perivascular region. At the molecular level, these mice also show increased levels of p21 and phospho-γH2AX staining, which represent key hallmarks of cellular senescence. Transcriptomic analysis further revealed dysregulation of multiple pathways involved in hypertrophy and senescence. Therefore, reduced expression of BubR1 drives cardiac aging as observed by increases in cardiac hypertrophy, fibrosis, senescence, and DNA damage response. Our results indicate that BubR1 plays a vital role in regulating adult cardiac structure and function, and its loss with age may drive deleterious phenotypes during heart aging.

Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra)

In this study, the poplar doubled haploid (DH) plants were used as the experimental material to explore the huge phenotypic differences between homozygous DH plants and the paternal plants, and the molecular regulation mechanism of the differential phenotypes. In this experiment, through morphological and histological observation and statistics, we found that the double haploid plants had significantly reduced plant height and ground diameter, increased leaf aspect ratio, premature senescence phenotype of top bud, and significant changes in the shape and cell area of the shoot apical meristem. Significantly differentially expressed genes were obtained using RNA-seq transcriptome sequencing. They were subjected to GO enrichment and KEGG analysis. Transcription factors with key functions were screened out for qRT-PCR to verify gene expression changes to predict gene function. The results showed that after the IAA and ABA treatment, the expression levels of some hormone-responsive genes in wild type plants were significantly changed with different treatment time. In the dihaploid plants, the corresponding genes also changed to different degrees, which reflected the changes in the response of the dihaploid plants to hormones. Compared to in WT, the differential expressed genes in the double haploids were involved in multiple physiological process such as response to oxidative stress, response to salicylic acid, plant pathogen interaction, and plant hormone signal transduction. A TF–centered gene regulatory network for phytohormone synthesis and plant senescence was constructed with the expression patterns of differentially expressed transcription factors (TFs). This study increases researchers’ understanding of the regulation of poplar growth and development and provides new research ideas for the creation of new species of poplar.

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Histone variant HTB4 delays leaf senescence by epigenetic control of Ib bHLH transcription factor-mediated iron homeostasis.

Leaf senescence is an orderly process regulated by multiple internal factors and diverse environmental stresses including nutrient deficiency. Histone variants are involved in regulating plant growth and development. However, their functions and underlying regulatory mechanisms in leaf senescence remain largely unclear. Here, we found that H2B histone variant HTB4 functions as a negative regulator of leaf senescence. Loss of function of HTB4 led to early leaf senescence phenotypes that were rescued by functional complementation. RNA-seq analysis revealed that several Ib subgroup basic helix-loop-helix (bHLH) transcription factors (TFs) involved in iron (Fe) homeostasis, including bHLH038, bHLH039, bHLH100, and bHLH101, were suppressed in the htb4 mutant, thereby compromising the expressions of FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER (IRT1), two important components of the Fe uptake machinery. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that HTB4 could bind to the promoter regions of Ib bHLH TFs and enhance their expression by promoting the enrichment of the active mark H3K4me3 near their transcriptional start sites. Moreover, overexpression of Ib bHLH TFs or IRT1 suppressed the premature senescence phenotype of the htb4 mutant. Our work established a signaling pathway, HTB4-bHLH TFs-FRO2/IRT1-Fe homeostasis, which regulates the onset and progression of leaf senescence.

Cellular senescence and premature aging in Down Syndrome

Down syndrome (DS) is a genetic disorder caused by an extra copy of chromosome 21, resulting in cognitive impairment, physical abnormalities, and an increased risk of age-related co-morbidities. Individuals with DS exhibit accelerated aging, which has been attributed to several cellular mechanisms, including cellular senescence, a state of irreversible cell cycle arrest that is associated with aging and age-related diseases. Emerging evidence suggests that cellular senescence may play a key role in the pathogenesis of DS and the development of age-related disorders in this population. Importantly, cellular senescence may be a potential therapeutic target in alleviating age-related DS pathology. Here, we discuss the importance of focusing on cellular senescence to understand accelerated aging in DS. We review the current state of knowledge regarding cellular senescence and other hallmarks of aging in DS, including its putative contribution to cognitive impairment, multi-organ dysfunction, and premature aging phenotypes.

Constitutive knock-in of MnSODK68R causes dilated cardiomyopathy and senescence in mice

The mitochondrial superoxide dismutase (SOD2 or MnSOD) is an essential enzyme for managing oxidative stress, particularly by removing the highly reactive superoxide generated during the normal functioning of the electron transport chain. The activity of MnSOD is regulated post-translationally by acetylation, specifically at lysine 68. The deacetylated form of MnSOD is catalytically active, and it has been shown that the deacetylation mimic, MnSOD K68R in which lysine 68 has been replaced with arginine, has upregulated dismutase activity in MCF7 and T47D human breast cancer cells. Overexpression of MnSOD has been shown to increase lifespan and protect against oxidative stress in the aging heart. We investigated a MnSOD K68R knock-in mouse with ubiquitous expression of the mutant enzyme and expected similar effects as the MnSOD overexpressing mice. However, we have found that MnSOD K68R homozygous knock-in mice develop dilated cardiomyopathy and have significantly decreased ejection fraction as evidenced by echocardiography at a young age. These mice also exhibit lower blood pressure compared to wild-type mice. Strikingly, these mice exhibit higher body weight, with increased lean mass and food consumption. Mitochondria isolated from MnSOD K68R knock-in mouse hearts showed higher respiratory capacity than WT counterparts. Biochemical analysis revealed increased lipid peroxidation, oxidized peroxiredoxin and senescence markers, possibly indicative of a premature aging phenotype. We conclude that MnSOD constitutive function upregulation may produce a sustained increase in hydrogen peroxide levels that could cause senescence and lead to dilated cardiomyopathy. This research was funded by NCI grant R01CA152601 and, R01CA214025, and the Cancer Prevention and Research Institute of Texas grant number RR20012. JS is supported by T32 AG 021890. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restoring bone marrow niche function rejuvenates aged hematopoietic stem cells by reactivating the DNA Damage Response

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.

Abstract 1274: Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression

Abstract Melanoma is the deadliest form of skin cancer, with its incidence rising disproportionally to other cancers. Chronological aging is a major risk factor in all types of cancer as it has been shown to drive changes in noncancerous cells (stromal and immune) of the tumor microenvironment (TME) to promote tumor initiation and progression. Ultraviolet radiation (UVR) exposure, which is a major risk factor for melanoma, also can cause a physical premature aging phenotype, known as photoaging. Photoaging of the skin often occurs in tandem with chronological aging, and contributes to the physical features of aging through similar alterations in normal skin structure (eg. collagen degradation). However, the molecular mechanisms of how photoaging compares to chronological aging in changing the TME and on tumor progression is still lacking.Preliminary data have indicated that photoaging of young dermal fibroblasts induce some similar signature changes to that of intrinsically aged. We have shown in-vitro that UVA exposure in young fibroblasts cause upregulation in MMP-1, a matrix remodeling protein, and downregulation in β-catenin, the cell adhesion transcription regulator. These same changes were also shown in chronologically aged fibroblasts. In addition, both photoaging and chronological aging decreases ECM structural integrity, with increased collagen fragmentation and a reduction in the renewal of cross-linking proteins. To investigate how photoaging contributes to the pro-tumorigenic microenvironment of the aged dermis, we are using biochemical and phenotypic assays to study how chronic UVA exposure induces an intrinsically aged-like fibroblast behavior and secretome. We also hypothesize that photoaged fibroblasts reprogram the TME to become more permissible for the growth of tumor cells, and will test changes in invasion and metastasis or melanoma both in-vitro and in-vivo.Studying the impact of photoaging on aging in the skin microenvironment will help identify therapeutic targets and have direct clinical applications for cancer patients. Citation Format: Vania Wang, Yash Chhabra, Laura Hueser, Sneha Pramod, Alexis Carey, Ashani Weeraratna. Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1274.

Additional polgD257A mutation (mutator) does not influence dopaminergic neurodegeneration in aged parkin-deficient mice

Background: Parkinson’s disease is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Among the first identified causes of autosomal recessive Parkinson’s disease were mutations in the parkin gene. Independently, we and other groups have developed various parkin knockout mice, and none displayed dopaminergic degeneration in the substantia nigra. Interestingly, dopaminergic degeneration in the substantia nigra has been reported in a parkin knockout line (exon 3 deletion) carrying an additional mutation (D257A) in the mitochondrial DNA polymerase (polg) gene (mutator). The mutator mice show accelerated mutation rates in mitochondrial DNA resulting in a premature-aging phenotype. Methods: To verify this finding, we crossed our parkin-deficient mice with the mutator mice, and characterized phenotypic changes of the parkin/mutator double mutant mice up to one year of age. We examined their locomotion and motor coordination behaviors by using the open field, the rotarod, and the pole test, subsequently investigating their nigrostriatal axis by counting TH-positive cells in every tenth section throughout the entire substantia nigra pas compacta and their termini in the striatum. Results: The double mutants did not display additional deficiencies in locomotion in our behavior tests. We could also not detect dopaminergic neurodegeneration in the substantia nigra pars compacta of aged double mutants measured by levels of tyrosine hydroxylase positive neurons in the substantia nigra pars compacta as well as in striatal terminals. Conclusion: Our results do not support the hypothesis that the polgD257A mutation contributes to the age-related vulnerability of dopaminergic neurons in parkin-deficient mice. Keywords: Parkin, neurodegeneration, polgD257A, mutator, substantia nigra

INDETERMINATE1 autonomously regulates phosphate homeostasis upstream of the miR399-ZmPHO2 signaling module in maize.

The macronutrient phosphorus is essential for plant growth and development. Plants have evolved multiple strategies to increase the efficiency of phosphate (Pi) acquisition to protect themselves from Pi starvation. However, the crosstalk between Pi homeostasis and plant development remains to be explored. Here, we report that overexpressing microRNA399 (miR399) in maize (Zea mays) is associated with premature senescence after pollination. Knockout of ZmPHO2 (Phosphate 2), a miR399 target, resulted in a similar premature senescence phenotype. Strikingly, we discovered that INDETERMINATE1 (ID1), a floral transition regulator, inhibits the transcription of ZmMIR399 genes by directly binding to their promoters, alleviating the repression of ZmPHO2 by miR399 and ultimately contributing to the maintenance of Pi homeostasis in maize. Unlike ZmMIR399 genes, whose expression is induced by Pi deficiency, ID1 expression was independent of the external inorganic orthophosphate status, indicating that ID1 is an autonomous regulator of Pi homeostasis. Furthermore, we show that ZmPHO2 was under selection during maize domestication and cultivation, resulting in a more sensitive response to Pi starvation in temperate maize than in tropical maize. Our study reveals a direct functional link between Pi-deprivation sensing by the miR399-ZmPHO2 regulatory module and plant developmental regulation by ID1.

Yeast Chromatin Mutants Reveal Altered mtDNA Copy Number and Impaired Mitochondrial Membrane Potential.

Mitochondria are multifunctional, dynamic organelles important for stress response, cell longevity, ageing and death. Although the mitochondrion has its genome, nuclear-encoded proteins are essential in regulating mitochondria biogenesis, morphology, dynamics and function. Moreover, chromatin structure and epigenetic mechanisms govern the accessibility to DNA and control gene transcription, indirectly influencing nucleo-mitochondrial communications. Thus, they exert crucial functions in maintaining proper chromatin structure, cell morphology, gene expression, stress resistance and ageing. Here, we present our studies on the mtDNA copy number in Saccharomyces cerevisiae chromatin mutants and investigate the mitochondrial membrane potential throughout their lifespan. The mutants are arp4 (with a point mutation in the ARP4 gene, coding for actin-related protein 4-Arp4p), hho1Δ (lacking the HHO1 gene, coding for the linker histone H1), and the double mutant arp4 hho1Δ cells with the two mutations. Our findings showed that the three chromatin mutants acquired strain-specific changes in the mtDNA copy number. Furthermore, we detected the disrupted mitochondrial membrane potential in their chronological lifespan. In addition, the expression of nuclear genes responsible for regulating mitochondria biogenesis and turnover was changed. The most pronounced were the alterations found in the double mutant arp4 hho1Δ strain, which appeared as the only petite colony-forming mutant, unable to grow on respiratory substrates and with partial depletion of the mitochondrial genome. The results suggest that in the studied chromatin mutants, hho1Δ, arp4 and arp4 hho1Δ, the nucleus-mitochondria communication was disrupted, leading to impaired mitochondrial function and premature ageing phenotype in these mutants, especially in the double mutant.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

Missing checkpoints in premature aging.

Zhang and colleagues demonstrate how the premature aging phenotypes in progeria involve a mitotic spindle-assembly-checkpoint protein, BUBR1. BUBR1 is misanchored to the nuclear membrane by progerin and its mRNA is destabilized in progeria, preventing it from functioning properly.

DNA hypomethylation mediates flower opening and senescence in sweet osmanthus through auxin and ethylene responsive pathways

Recent studies have shown that DNA methylation is critical for fruit ripening and leaf senescence, but its effect on flowering is unclear. This study investigated the dynamic changes in DNA methylation and its regulatory role on flower opening and senescence of Osmanthus fragrans ‘Liuyejingui’ by whole-genome bisulfite sequencing and transcriptome sequencing at six developmental stages (unopened floral bud to late flower senescence, S1 to S6). In total 3222 differentially methylated regions (DMRs) were identified in senescent flowers, of which 89% were hypomethylated (hypo-DMRs), and the number increased form S1 to S6, suggesting that DNA demethylation is associated with the progression of flowering. Analysis of hypo-DMR-associated up-regulated differentially expressed genes revealed that small auxin up-regulated RNAs (OfSAURs) were the most highly significantly enriched from S3 to S5, and ethylene-responsive transcription factors (OfERFs) were strongly activated in senescent flowers. These results suggested that DNA hypomethylation was important for flower expansion and flower senescence through the auxin and ethylene response pathways. Spray application of a DNA methylation inhibitor to unopened floral buds triggered a premature opening and senescence phenotype by promoting endogenous ethylene production and carotenoid accumulation, and indicated that DNA demethylation mediated flower opening and senescence by activating the expression of OfSAURs and OfERFs. An epigenetic regulatory three-component is proposed, in which flower opening and senescence are regulated by DNA demethylation, mediating the expression of specific gene families, together with the plant hormones auxin and ethylene, to transition the flower into an opening- and senescence-competent state. Data availabilityThe genome and transcriptome sequencing data of S1-S6 in O. fragrans ‘Liuyejingui’ were under the public database of the National Center of Biotechnology Information (NCBI) BioProject under the accession number of PRJNA679852 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA679852). Whole Genome Bisulfate Sequencing and transcriptome sequencing data in DNA methylation inhibitor 5-azacytidine and ddH2O treatment have been deposited into the public database of NCBI BioProject under the accession number of PRJNA798828 and PRJNA798559.

MicroRNA biogenesis pathway alterations in aging

Aging is characterized by genomic instability and dysregulation of gene expression. MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in post-transcriptional gene regulation. This work explores the impact of dysregulated miRNA biogenesis on the aging process. During aging, alterations in the transcription of primary miRNAs (pri-miRNAs) occur due to genomic changes, DNA damage, and epigenetic modifications. The microprocessor complex, comprising DGCR8 and Drosha proteins, is vital for pri-miRNA processing. Age-related changes in this complex affect miRNA biogenesis and miRNA expression profiles, linking these alterations with age-related conditions. Conversely, interventions like caloric restriction and mTOR inhibition enhance microprocessor activity, suggesting a connection between microprocessor function, aging-related pathways, and lifespan extension. Exportin-5 mediates the transport of pre-miRNAs from the nucleus to the cytoplasm. Although the role of miRNA export in aging is not well understood, accelerated export of pre-miRNAs is observed in response to DNA damage, and nucleocytoplasmic transport has been linked to cellular senescence. Dicer is responsible for processing pre-miRNAs into mature miRNAs. Reduced Dicer expression during aging is reported in various organisms and tissues and is associated with premature aging phenotypes. Conversely, the upregulation of Dicer improves stress resistance and metabolic adaptations induced by caloric restriction and exercise training. Understanding the role of miRNA biogenesis disruption in aging provides insights into the molecular mechanisms of aging and age-related diseases. Targeting this pathway may hold promise for therapeutic strategies and contribute to healthy aging.

A Novel Dominant Allele from 93-11, ES(4), Represses Reactive Oxygen Species Scavenging and Leads to Early-Senescence in Rice

Senescence is the last developmental process in plant, which has an important impact on crop yield and quality. In this study, a stable hereditary early-senescence line BC64 was isolated from the high-generation recombinant inbred lines of 93-11 and Wuyunjing7 (W7). Genetic analysis showed that the premature aging phenotype was controlled by a dominant gene derived from 93-11. By linkage analysis, the gene was primarily mapped in the region between marker B4 and B5 near the centromere of chromosome 4, described as ES(4). Through multiple backcrossing with W7, the near-isogenic line of ES(4) (NIL-ES(4)) was obtained. Compared with wild-type W7, NIL-ES(4) plants showed more sever senescence phenotype in both nature and dark conditions. In NIL plants, leaves turned yellow at the fully tillering stage; photosynthetic rate, pollen fertility and seed setting rate were decreased. Moreover, the malondialdehyde, proline content and relative conductivity in NIL-ES(4) were significantly higher than those in W7; both transcript level and activities of reactive oxygen species scavenging enzymes were repressed; H2O2 and O2− were significantly accumulated. This study provides a basis for further cloning and function identification of ES(4).

MAVS Antagonizes Human Stem Cell Senescence as a Mitochondrial Stabilizer.

Mitochondrial dysfunction is a hallmark feature of cellular senescence and organ aging. Here, we asked whether the mitochondrial antiviral signaling protein (MAVS), which is essential for driving antiviral response, also regulates human stem cell senescence. To answer this question, we used CRISPR/Cas9-mediated gene editing and directed differentiation techniques to generate various MAVS-knockout human stem cell models. We found that human mesenchymal stem cells (hMSCs) were sensitive to MAVS deficiency, as manifested by accelerated senescence phenotypes. We uncovered that the role of MAVS in maintaining mitochondrial structural integrity and functional homeostasis depends on its interaction with the guanosine triphosphatase optic atrophy type 1 (OPA1). Depletion of MAVS or OPA1 led to the dysfunction of mitochondria and cellular senescence, whereas replenishment of MAVS or OPA1 in MAVS-knockout hMSCs alleviated mitochondrial defects and premature senescence phenotypes. Taken together, our data underscore an uncanonical role of MAVS in safeguarding mitochondrial homeostasis and antagonizing human stem cell senescence.

Mutation of Leaf Senescence 1 Encoding a C2H2 Zinc Finger Protein Induces ROS Accumulation and Accelerates Leaf Senescence in Rice.

Premature senescence of leaves causes a reduced yield and quality of rice by affecting plant growth and development. The regulatory mechanisms underlying early leaf senescence are still unclear. The Leaf senescence 1 (LS1) gene encodes a C2H2-type zinc finger protein that is localized to both the nucleus and cytoplasm. In this study, we constructed a rice mutant named leaf senescence 1 (ls1) with a premature leaf senescence phenotype using CRISPR/Cas9-mediated editing of the LS1 gene. The ls1 mutants exhibited premature leaf senescence and reduced chlorophyll content. The expression levels of LS1 were higher in mature or senescent leaves than that in young leaves. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were significantly increased and catalase (CAT) activity was remarkably reduced in the ls1 plants. Furthermore, a faster decrease in pigment content was detected in mutants than that in WT upon induction of complete darkness. TUNEL and staining experiments indicated severe DNA degradation and programmed cell death in the ls1 mutants, which suggested that excessive ROS may lead to leaf senescence and cell death in ls1 plants. Additionally, an RT-qPCR analysis revealed that most senescence-associated and ROS-scavenging genes were upregulated in the ls1 mutants compared with the WT. Collectively, our findings revealed that LS1 might regulate leaf development and function, and that disruption of LS1 function promotes ROS accumulation and accelerates leaf senescence and cell death in rice.

A Possible Modulator of Vitiligo Metabolic Impairment: Rethinking a PPARγ Agonist.

Vitiligo is a complex disease wherein derangements in multiple pathways determine the loss of functional melanocytes. Since its pathogenesis is not yet completely understood, vitiligo lacks a definitive safe and efficacious treatment. At present, different therapies are available; however, each modality has its baggage of disadvantages and side effects. Recently we have described several metabolic abnormalities in cells from pigmented skin of vitiligo patients, including alterations of glucose metabolism. Therefore, we conducted a study to evaluate the effect of Pioglitazone (PGZ), a Peroxisome proliferator-activated receptor-γ (PPARγ) agonist, on cells from pigmented vitiligo skin. We treated vitiligo melanocytes and fibroblasts with low doses of PGZ and evaluated the effects on mitochondrial alterations, previously reported by our and other groups. Treatment with PGZ significantly increased mRNA and protein levels of several anaerobic glycolytic enzymes, without increasing glucose consumption. The PGZ administration fully restored the metabolic network, replacing mitochondrial membrane potential and mitochondrial DNA (mtDNA) copy number. These effects, together with a significant increase in ATP content and a decrease in reactive oxygen species (ROS) production, provide strong evidence of an overall improvement of mitochondria bioenergetics in vitiligo cells. Moreover, the expression of HMGB1, Hsp70, defined as a part of DAMPs, and PD-L1 were significantly reduced. In addition, PGZ likely reverts premature senescence phenotype. In summary, the results outline a novel mode of action of Pioglitazone, which may turn out to be relevant to the development of effective new vitiligo therapeutic strategies.

Abstract 15027: Senescence-Associated Pathway Activation in Cardiac Fibroblasts in Dilated Cardiomyopathy

Introduction: Cardiac fibrosis is a pathologic driver in heart failure (HF) and aging is a major risk factor for HF. Persistence of activated myofibroblasts is a key feature of pathological fibrosis. In an aging model of lung fibrosis, senescent myofibroblasts exhibit apoptosis-resistance, which contributes to the sustained fibrotic response. Therapeutic targeting of NADPH oxidase-4 (NOX4), a primary source of reactive oxygen species (ROS) in myofibroblasts, can inhibit fibrosis development and reverse established lung fibrosis. We sought to understand the role of cellular senescence and NOX4 activation and persistence of cardiac fibroblasts with progression to HF. Methods and Results: Mice carrying a mutation in the phospholamban gene (PLNR9C) develop progressive dilated cardiomyopathy (DCM), extensive cardiac fibrosis and HF. Using the Langendorff hanging heart method and flow cytometry, cardiac fibroblasts were isolated and studied for markers of cellular senescence. Cardiac fibroblasts from PLNR9C mice with DCM display a senescent phenotype, with significantly elevated levels of the senescence markers p16 (2.1-fold, p<0.01, n=3, Figure) and beta-galactosidase (1.7-fold, p<0.01, n=3), along with significantly increased ROS production (Amplex Red assay 2.5-fold, p<0.01, n=3), as compared to fibroblasts from age-matched wild type (WT) mice. Nox4 is upregulated in cardiac fibroblasts from PLNR9C mice (mRNA 8.5-fold, n=4, p<0.01; protein 5.0-fold, p<0.01, n=4, Figure) with HF but not in young PLNR9C mice prior to development of DCM. Conclusions: These data reveal a premature aging phenotype in cardiac fibroblasts from mice with genetic DCM and identify NOX4 as a putative mediator of fibroblast activation and persistence.