Grunddal, KV;Jensen, EP;Ørskov, C;Andersen, DB;Windeløv, JA;Poulsen, SS;Rosenkilde, MM;Knudsen, LB;Pyke, C;Holst, JJ;
PMID: 34662392 | DOI: 10.1210/endocr/bqab216
Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all β-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.
Lu, B;Chen, J;Xu, G;Grayson, TB;Jing, G;Jo, S;Shalev, A;
PMID: 35957590 | DOI: 10.1210/endocr/bqac133
Thioredoxin-interacting protein (Txnip) has emerged as a key factor in pancreatic beta cell biology and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of Txnip protects against diabetes in different mouse models. However, while Txnip is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We therefore now generated an alpha cell Txnip knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus, together the current studies suggest that downregulation of alpha cell Txnip is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.
Lee, H;Lee, HY;Chae, JB;Park, CW;Kim, C;Ryu, JH;Jang, J;Kim, N;Chung, H;
PMID: 35859009 | DOI: 10.1038/s42003-022-03676-3
Cellular senescence of the retinal pigment epithelium (RPE) is thought to play an important role in vision-threatening retinal degenerative diseases, such as age-related macular degeneration (AMD). However, the single-cell RNA profiles of control RPE tissue and RPE tissue exhibiting cellular senescence are not well known. We have analyzed the single-cell transcriptomes of control mice and mice with low-dose doxorubicin (Dox)-induced RPE senescence (Dox-RPE). Our results have identified 4 main subpopulations in the control RPE that exhibit heterogeneous biological activities and play roles in ATP synthesis, cell mobility/differentiation, mRNA processing, and catalytic activity. In Dox-RPE mice, cellular senescence mainly occurs in the specific cluster, which has been characterized by catalytic activity in the control RPE. Furthermore, in the Dox-RPE mice, 6 genes that have not previously been associated with senescence also show altered expression in 4 clusters. Our results might serve as a useful reference for the study of control and senescent RPE.
J Mol Cell Cardiol. 2019 Jan 3.
Satoh M, Nomura S, Harada M, Yamaguchi T, Ko T, Sumida T, Toko H, Naito AT, Takeda N, Tobita T, Fujita T, Ito M, Fujita K, Ishizuka M, Kariya T, Akazawa H, Kobayashi Y, Morita H, Takimoto E, Aburatani H, Komuro I.
PMID: 30611794 | DOI: 10.1016/j.yjmcc.2018.12.018
Abstract BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
Xing, J;Chen, K;Gao, S;Pousse, M;Ying, Y;Wang, B;Chen, L;Wang, C;Wang, L;Hu, W;Lu, Y;Gilson, E;Ye, J;
PMID: 36644807 | DOI: 10.1111/acel.13780
The contribution of cellular senescence to the behavioral changes observed in the elderly remains elusive. Here, we observed that aging is associated with a decline in protein phosphatase 2A (PP2A) activity in the brains of zebrafish and mice. Moreover, drugs activating PP2A reversed age-related behavioral changes. We developed a transgenic zebrafish model to decrease PP2A activity in the brain through knockout of the ppp2r2c gene encoding a regulatory subunit of PP2A. Mutant fish exhibited the behavioral phenotype observed in old animals and premature accumulation of neural cells positive for markers of cellular senescence, including senescence-associated β-galactosidase, elevated levels cdkn2a/b, cdkn1a, senescence-associated secretory phenotype gene expression, and an increased level of DNA damage signaling. The behavioral and cell senescence phenotypes were reversed in mutant fish through treatment with the senolytic ABT263 or diverse PP2A activators as well as through cdkn1a or tp53 gene ablation. Senomorphic function of PP2A activators was demonstrated in mouse primary neural cells with downregulated Ppp2r2c. We conclude that PP2A reduction leads to neural cell senescence thereby contributing to age-related behavioral changes and that PP2A activators have senotherapeutic properties against deleterious behavioral effects of brain aging.
Transplantation Proceedings (2019)
Mohamed M, Kang L, Zhang C, Edenfield B, Sykes J, Brown T, Johnson JL, Rehman F, Nguyen JH.
| DOI: doi. 10.1016/j.transproceed.2018.12.028
Small-for-size liver grafts (SFSG) in adult transplant recipients have elevated risk of graft dysfunction and graft failure, limiting its application in clinical liver transplantation. Relevant preclinical model of SFSG for deceased-donor split liver transplant is lacking. In this study, we present our initial characterization of SFSG model using monosegments of a discarded deceased-donor human liver.
Development (Cambridge, England)
Singh, VP;Hassan, H;Deng, F;Tsuchiya, D;McKinney, S;Ferro, K;Gerton, JL;
PMID: 37158673 | DOI: 10.1242/dev.201581
The placenta is essential for reproductive success. The murine placenta includes polyploid giant cells that are crucial for its function. Polyploidy occurs broadly in nature but the regulators and significance in the placenta are unknown. We discovered that many murine placental cell types are polyploid. We identified factors that license polyploidy using single-cell RNA seq. c-Myc is a key regulator of polyploidy and placental development and is required for multiple rounds of DNA replication, likely via endocycles, in trophoblast giant cells. Furthermore, c-MYC supports the expression of DNA replication and nucleotide biosynthesis genes along with ribosomal RNA. Increased DNA damage and senescence occur in trophoblast giant cells without c-Myc, accompanied by senescence in the neighboring maternal decidua. These data reveal c-Myc is essential for polyploidy to support normal placental development, thereby preventing premature senescence. Our study combined with the literature suggests c-Myc is an evolutionarily conserved regulator of polyploidy.
Derr, AG;Arowosegbe, A;Satish, B;Redick, SD;Qaisar, N;Guo, Z;Vanderleeden, E;Trombly, MI;Baer, CE;Harlan, DM;Greiner, DL;Garber, M;Wang, JP;
PMID: 36346618 | DOI: 10.2337/db22-0521
Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. Here, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the pre-diabetic period. Single-cell RNA-Seq and differential expression analyses of islets from pre-diabetic rats reveal subsets of β and α cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ β and α cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed β and α cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.
New insights into molecular changes in skeletal muscle aging and disease: Differential alternative splicing and senescence
Mechanisms of ageing and development
Solovyeva, E;Ibebunjo, C;Utzinger, S;Eash, JK;Dunbar, A;Naumann, U;Zhang, Y;Serluca, FC;Demirci, S;Oberhauser, B;Black, F;Rausch, M;Hoersch, S;Meyer, A;
PMID: 34019916 | DOI: 10.1016/j.mad.2021.111510
Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.
Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells
American journal of physiology. Cell physiology
Young, LV;Morrison, W;Campbell, C;Moore, EC;Arsenault, MG;Dial, AG;Ng, S;Bellissimo, CA;Perry, CGR;Ljubicic, V;Johnston, AP;
PMID: 33979211 | DOI: 10.1152/ajpcell.00397.2020
Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21 and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence associated secretory phenotype which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne Muscular Dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-week-old) and D2-mdx mice (4-week and 8-week-old), two mouse models of DMD, that cells displaying canonical markers of senescence are found within skeletal muscle. 8-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation which were mostly Cdkn1a-positive macrophages while in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wildtype (WT) muscle which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes (Cdkn1a (p21), Cdkn2a (p16INK4A), Trp53 (p53)) which correlated with the quantity of SA-b-Gal-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.
Lee, SH;Rezzonico, MG;Friedman, BA;Huntley, MH;Meilandt, WJ;Pandey, S;Chen, YJ;Easton, A;Modrusan, Z;Hansen, DV;Sheng, M;Bohlen, CJ;
PMID: 34965428 | DOI: 10.1016/j.celrep.2021.110158
Non-neuronal responses in neurodegenerative disease have received increasing attention as important contributors to disease pathogenesis and progression. Here we utilize single-cell RNA sequencing to broadly profile 13 cell types in three different mouse models of Alzheimer disease (AD), capturing the effects of tau-only, amyloid-only, or combined tau-amyloid pathology. We highlight microglia, oligodendrocyte, astrocyte, and T cell responses and compare them across these models. Notably, we identify two distinct transcriptional states for oligodendrocytes emerging differentially across disease models, and we determine their spatial distribution. Furthermore, we explore the impact of Trem2 deletion in the context of combined pathology. Trem2 knockout mice exhibit severely blunted microglial responses to combined tau and amyloid pathology, but responses from non-microglial cell types (oligodendrocytes, astrocytes, and T cells) are relatively unchanged. These results delineate core transcriptional states that are engaged in response to AD pathology, and how they are influenced by a key AD risk gene, Trem2.
Prabakaran PJ, Javaid AM, Swick AD, Werner LR, Nickel KP, Sampene E, Hu R, Ong IM, Bruce JY, Hartig GK, Wieland A, Canon J, Harari PM, Kimple RJ.
PMID: 28659312 | DOI: 10.1158/1078-0432.CCR-17-0969
Abstract
PURPOSE:
Adenoid cystic carcinoma (ACC) is a rare cancer arising from the major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents or known radiosensitizers for ACC. Unlike the more common head and neck squamous cell carcinomas that frequently harbor TP53 mutations, ACC contain TP53 mutations at a rate of <5%, rendering them an attractive target for MDM2 inhibition. <p>Experimental Design: We report the successful establishment and detailed characterization of a TP53-WT ACC patient derived xenograft (PDX) which retained the histologic features of the original patient tumor. We evaluated this model for response to the MDM2 inhibitor AMG 232 as monotherapy and in combination with radiation (RT).</p> <p>Results: AMG 232 monotherapy induced modest tumor growth inhibition and RT monotherapy induced a transient tumor growth delay in a dose dependent fashion. Strikingly, combination treatment of AMG 232 with RT (including low dose RT of 2 Gy/fraction) induced dramatic tumor response and high local tumor control rates three months following treatment. Post treatment analysis revealed that while both AMG 232 and RT alone induced TP53 tumor suppressive activities, combination therapy amplified this response with potent induction of apoptosis after combination treatment.</p> <p>Conclusions: These data identify that MDM2 inhibition can provide potent radiosensitization in TP53-WT ACC. In light of the absence of effective systemic agents for ACC, the powerful response profile observed here suggests that clinical trial evaluation of this drug/RT combination may be warranted to improve local control in this challenging malignancy.
