In-Press Preview
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease. Emerging evidence suggests manifestations beyond the neuromuscular system. Bone alterations are part of the ALS clinical picture; it remains unclear whether they are secondary to muscle denervation or due to an autonomous process. We investigated skeletal involvement in the SOD1(G93A) mouse model at presymptomatic (P45) and symptomatic (P110) stage through biomechanical and transcriptomic approaches. Three-point bending revealed significant reductions in femoral rigidity and maximum bending force in SOD1 mutants at P45, indicating early structural deficits. Micro-CT analysis demonstrated reduced trabecular bone mineral density and thickness at P45, with progressive trabecular loss and cortical thinning by P110. Histological examination revealed marked osteoblast loss at P45 suggesting impaired bone formation as the primary early mechanism. Transcriptomics of bulk bone and cultured osteoblasts from P45 mice identified dysregulation of bone differentiation, including downregulation of osteoblast differentiation genes and upregulation of negative regulators of ossification and increased cell senescence signatures. Unfolded protein response was upregulated in SOD1 osteoblasts. Immunohistochemistry confirmed the senescence phenotype with increased p16Ink4a level in SOD1 osteoblasts. These findings suggest that bone deterioration precede overt motor symptoms and are linked to osteoblast premature senescence.
Authors
Burak Özkan, Jan-Moritz Ramge, Diana Wiesner, Jelena Scekic-Zahirovic, Stefano Antonucci, Sandra Nungeß, Dorothea Gebauer, Anita Ignatius, Jochen H. Weishaupt, Melanie Haffner-Luntzer, Francesco Roselli
Abstract
Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel diseases (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosa growth and the function of the gut epithelium barrier. The levels of tissue GAS5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from IBD patients. Transient and tissue-specific knockdown of GAS5 in mice using CRISPR-Cas9 enhanced the renewal of the mucosa of the small intestine, increased the levels of tight junction (TJ) proteins ZO-1, ZO-2, claudin-1, and claudin-2, and improved gut barrier function. Conversely, ectopic overexpression of GAS5 in intestinal organoids and in cultured intestinal epithelium cells decreased the levels of these TJ proteins and caused epithelial barrier dysfunction. Mechanistic studies revealed that GAS5 acted as a transcriptional enhancer of the gene encoding small noncoding vault RNAs (vtRNAs) and that GAS5 repressed TJ expression by increasing the levels of vtRNAs. Together, our results indicate that GAS5 disrupts the integrity of the intestinal epithelium by impairing mucosal growth and epithelial barrier function and that it represses TJ expression at least in part via vtRNAs.
Authors
Ting-Xi Yu, Hee Kyoung Chung, Amy VanderStoep, Bridgette Warner, Hongxia Chen, Haonan Zhao, Ana S. G. Cunnigham, Rosemary Kozar, Myriam Gorospe, Lan Xiao, Jian-Ying Wang
Abstract
Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Co-inheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR-deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering new insights into the complex role of PKR deficiency in SCD pathology.
Authors
Xunde Wang, Meghann Smith, Sayuri Kamimura, Quan Li, Niharika Shah, Martha Quezado, Luis E.F. Almeida, Sebastian Vogel, Mickias B. Tegegn, Kevin Y. Sun, Rafael Villasmil, Chengyu Liu, William A. Eaton, Swee Lay Thein, Zenaide M.N. Quezado
Abstract
Anoikis resistance or evasion of cell death triggered by matrix detachment is a hallmark of cancer cell survival and metastasis. We show that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance is associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance is not genetic, persisting for a finite duration without detachment stress, but is sensitive to CDK8/19 Mediator kinase inhibition that can also reverse anoikis resistance. Transcriptomic analysis reveals that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.
Authors
Mehri Monavarian, Resha Rajkarnikar, Emily Faith Page, Asha Kumari, Liz Quintero Macias, Felipe Massicano, Nam Y. Lee, Sarthak Sahoo, Nadine Hempel, Mohit Kumar Jolly, Lara Ianov, Elizabeth Worthey, Abhyudai Singh, Igor B. Roninson, Eugenia V. Broude, Mengqian Chen, Karthikeyan Mythreye
Abstract
Commotio retinae (CR) resulting from retinal trauma can lead to focal photoreceptor degeneration and permanent vision loss. Currently no therapies exist for CR-induced retinal degeneration, in part due to a lacking large animal model that replicates human injury pathology and allows testing of therapeutics. Severe CR is clinically characterized by subretinal fluid and focal photoreceptor outer nuclear layer thinning. To develop a porcine CR model, we developed a laser-guided projectile apparatus and optimized projectile delivery procedure using porcine cadaveric eyes embedded in a 3D-printed porcine skull. Scleral and corneal impacts, resulted in retinal damage consistent with patient injury but corneal impacts also led to cornea damage and opacification, which precluded follow up imaging. In live porcine eyes, scleral impacts of 39.5 m/s induced transient blood retinal barrier breakdown evidenced by subretinal fluid on optical coherence tomography (OCT), leakage observed on fluorescein and indocyanine green angiography, and transient photoreceptor outer segment disruption seen by OCT and multifocal electroretinography. Impacts above 39.5 m/s induced longer-lasting photoreceptor degeneration, but only transient blood retinal barrier breakdown. This porcine model, combined with clinically relevant imaging and diagnostic modalities will be valuable for testing the safety and efficacy of therapies to restore vision after focal photoreceptor degeneration.
Authors
Juan Amaral, Irina Bunea, Arvydas Maminishkis, Maria M. Campos, Francesca Barone, Rohan Gupta, Mitra Farnoodian, Jonathan Newport, M. Joseph Phillips, Ruchi Sharma, David M. Gamm, Kapil Bharti, Richard J. Blanch
Abstract
Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that primarily affects the mucosal layer. Previously, we identified autoantibodies against integrin αvβ6 in patients with UC. In this study, we established monoclonal antibodies (mAbs) from patients with UC to reveal the features and functions of these anti-integrin αvβ6 autoantibodies. We identified two shared heavy chain complementarity-determining region (CDR) 3 amino acid sequences among different patients with UC. Notably, several mAbs contained the RGD sequence in their heavy chain CDR3 that mimicked the key recognition sequence of integrin αvβ6 ligands such as fibronectin. Almost all mAbs selectively reacted with integrin αvβ6 in the presence of divalent cations (Ca²⁺ and Mg²⁺) and blocked fibronectin–integrin αvβ6 binding. MAbs that shared the same heavy chain CDR3 amino acid sequence showed differences in reactivity to integrin αvβ6, indicating that the reactivity of these mAbs is also affected by the light chain. Some of the mAbs showed varying degrees of cross-reactivity with integrin αvβ3. The identification of shared CDR3 amino acid sequences in anti-integrin αvβ6 antibodies from several patients with UC suggests a common mechanism underlying their production, which may help elucidate the pathogenesis of UC.
Authors
Masahiro Shiokawa, Yoshihiro Nishikawa, Ikuhisa Takimoto, Takeshi Kuwada, Sakiko Ota, Darryl Joy C. Juntila, Takafumi Yanaidani, Kenji Sawada, Ayako Hirata, Muneji Yasuda, Koki Chikugo, Risa Nakanishi, Masataka Yokode, Yuya Muramoto, Shimpei Matsumoto, Tomoaki Matsumori, Tsutomu Chiba, Hiroshi Seno
Abstract
Cardiomyocyte growth is tightly controlled by multiple signaling pathways. Identification of master kinases in this process is essential in exploring potential targets for the treatment of pathological cardiac hypertrophy and heart failure. Here we identified the mTOR-independent activation of ribosomal protein S6 kinase b1 (Rps6kb1/S6K1) during cardiomyocyte growth. By utilizing phosphoproteomics in primary neonatal rat ventricular myocytes (NRVMs), we revealed Rps6kb1 as one of most activated kinases under growth stimulation. We further demonstrated the role of Rps6kb1 phosphorylation in pathological cardiac hypertrophy and heart failure. We showed that the phosphorylation of multiple sites at Rps6kb1, including T367 in the kinase domain and S418/T421/S424 in the C-terminal domain, is not directly regulated by the activity of mTOR, rather coupled with the activation of the MEK1-ERK axis. In mice, cardiomyocyte-specific deletion of Rps6kb1 significantly inhibited both constitutively active ERK- and pressure overload-induced cardiac hypertrophy. In contrast, cardiomyocyte-specific overexpression of wild-type Rps6kb1, rather than the phosphorylation-defective mutant, elevated cardiac hypertrophy and augmented pressure overload-induced heart failure. In conclusion, our findings reveal that the MEK-ERK axis primes Rps6kb1 activation through phosphorylation of two separate domains of Rps6kb1, which may play an essential role in cardiac hypertrophy and heart failure under hemodynamic stress.
Authors
Chao Li, Pengfei Zhang, Kai Zhang, Jane A. Cook, Weidan Song, Megan Virostek, Lily A. Slotabec, Nadiyeh Rouhi, Mohammed Hazari, Michael I. Adenawoola, Xiaofei Liu, Hao Zhang, Guangyu Zhang, Erica L Niewold, Qinfeng Li, Yong Fang, Waleed M. Elhelaly, Xue-Nan Sun, Xuejiang Guo, Andrew Lemoff, Yingfeng Deng, Thomas G. Gillette, Ji Li, Philipp E. Scherer, Zhao V. Wang
Abstract
Radiotherapy is a critical modality in cancer treatment, not only to eradicate cancer cells but also to trigger anti-tumor immunity. Interleukin-21 (IL-21), an immunomodulatory cytokine with potential in cancer therapy, has unexplored synergy with radiotherapy. Our study, leveraging human cancer databases and tissue microarrays, identified a positive correlation between IL-21 and radiotherapy outcomes, particularly in tumor microenvironment (TME) activation. In mouse tumor models, IL-21 combined with radiation significantly enhances TME, boosting CD8+ T cell activation and function, reducing tumor burden, and extending survival. Single-cell transcriptome sequencing revealed that the combination of IL-21 and radiation increased the cytotoxicity of effector and memory CD8+ T cells and prevented their exhaustion. These effects were further validated in humanized mice, where IL-21 combined with radiation reduced A549 tumor growth and enhanced CD8+ T cell function. Post-neoadjuvant radiotherapy samples from patients with esophageal cancer showed a positive correlation between IL-21 levels and CD8+ T cell infiltration. Our findings suggest that IL-21 is a promising adjuvant to radiotherapy, potentially improving the treatment efficacy through TME enhancement. This study provides a foundation for future clinical exploration of IL-21 for enhancing radiotherapy.
Authors
Xinyang Li, Xueqi Xie, Baochao Wei, Xiaozheng Sun, Minxin Chen, Rufei Liu, Qingxu Tao, Yiheng Huang, Qian Wang, Shuangshuang Ma, Ling Wei, Rong Xiao, Zhaoyun Liu, Jinming Yu, Meng Wu, Dawei Chen
Abstract
Congenital long QT syndrome (LQTS) promotes risk for life-threatening cardiac arrhythmia and sudden death in children and young adults. Pathogenic variants in the voltage-gated potassium channel KCNQ1 are the most frequently discovered genetic cause. Most LQTS-associated KCNQ1 variants cause loss-of-function secondary to impaired trafficking of the channel to the plasma membrane. There are currently no therapeutic approaches that address this underlying molecular defect. Using a high-throughput screening paradigm, we identified VU0494372, a small molecule that increases total and cell surface levels and trafficking efficiency of WT KCNQ1 as well as three LQTS-associated variants. Additionally, 16-hour treatment of cells with VU0494372 increased IKs (KCNQ1-KCNE1 current) for WT KCNQ1 and the LQTS-associated variant V207M in cells co-expressing KCNE1. VU0494372 had no impact on KCNQ1 transcription, degradation, or thermal stability, and increased the rate of KCNQ1 reaching the cell surface. We identified a potential direct interaction site with KCNQ1 at or near the binding site of the KCNQ1 potentiator ML277. Together, these findings demonstrate that small molecules can increase the expression levels and cell surface trafficking efficiency of KCNQ1 and introduce a potential new pharmacological approach for treating LQTS.
Authors
Katherine R. Clowes Moster, Carlos G. Vanoye, Ana C. Chang-Gonzalez, Ian M. Romaine, Katherine M. Stefanski, Mason C. Wilkinson, Joshua A. Bauer, Thomas P. Hasaka, Emily L. Days, Reshma R. Desai, Kathryn R. Butcher, Gary A. Sulikowski, Alex G. Waterson, Jens Meiler, Kaitlyn V. Ledwitch, Alfred L. George, Jr., Charles R. Sanders
Abstract
Sustained injury to renal tubular epithelial cells (TECs), driven by excessive autophagy, is a critical mechanism underlying kidney fibrosis. Our previous work identified JLP—a TEC-expressed scaffolding protein—as an endogenous anti-fibrotic factor that counteracts TGF-β1–induced autophagy and fibrogenesis. However, the mechanism underlying JLP downregulation in renal fibrosis remains unclear. Here, we delineated a TGF-β1/LEF1/β-catenin/JLP axis that governed TEC autophagy through a dichotomous regulatory circuit. Under physiological conditions, low levels of β-catenin and LEF1 with minimal nuclear localization permit normal JLP expression, which in turn maintains autophagy in check. In contrast, during renal injury, TGF-β1 promoted the expression and nuclear translocation of β-catenin and LEF1, which together suppressed JLP transcription. This loss of JLP-mediated inhibition led to unchecked autophagy and exacerbated fibrotic damage. Analyses of kidney tissues from patients with CKD, murine fibrotic kidneys, and cultured HK-2 cells confirmed consistent JLP downregulation accompanied by upregulation and nuclear accumulation of LEF1 and β-catenin. Therapeutic intervention using the β-catenin/LEF1 inhibitor iCRT3 or LEF1-targeted silencing in murine fibrosis models restored JLP expression, attenuated TEC autophagy, and ameliorated renal fibrosis. These findings revealed an autoregulatory circuit controlling TEC autophagy and fibrogenesis, and supported LEF1 and β-catenin as potential therapeutic targets in CKD.
Authors
Chen Li, Meng Zhang, Maoqing Tian, Zeyu Tang, Yuying Hu, Yuyu Long, Xiaofei Wang, Liwen Qiao, Jiefei Zeng, Yujuan Wang, Xinghua Chen, Cheng Chen, Xiaoyan Li, Lu Zhang, Huiming Wang
No posts were found with this tag.
