Research Article
Abstract
Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle–specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type–specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.
Authors
Thuy-Linh Le, Louise Galmiche, Jonathan Levy, Pim Suwannarat, Debby M.E.I. Hellebrekers, Khomgrit Morarach, Franck Boismoreau, Tom E.J. Theunissen, Mathilde Lefebvre, Anna Pelet, Jelena Martinovic, Antoinette Gelot, Fabien Guimiot, Amanda Calleroz, Cyril Gitiaux, Marie Hully, Olivier Goulet, Christophe Chardot, Severine Drunat, Yline Capri, Christine Bole-Feysot, Patrick Nitschké, Sandra Whalen, Linda Mouthon, Holly E. Babcock, Robert Hofstra, Irenaeus F.M. de Coo, Anne-Claude Tabet, Thierry J. Molina, Boris Keren, Alice Brooks, Hubert J.M. Smeets, Ulrika Marklund, Christopher T. Gordon, Stanislas Lyonnet, Jeanne Amiel, Nadège Bondurand
Abstract
Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1β, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.
Authors
Matthew DeBerge, Kristofor Glinton, Manikandan Subramanian, Lisa D. Wilsbacher, Carla V. Rothlin, Ira Tabas, Edward B. Thorp
Abstract
Chronic HIV-1 infection is generally characterized by progressive CD4+ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4+ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4+ T cells, which revealed that pyroptotic and apoptotic CD4+ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4+ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1–infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4+ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4+ T cell loss in HIV-1–infected patients and implicate pyroptosis signaling as a target for anti–HIV-1 treatment.
Authors
Chao Zhang, Jin-Wen Song, Hui-Huang Huang, Xing Fan, Lei Huang, Jian-Ning Deng, Bo Tu, Kun Wang, Jing Li, Ming-Ju Zhou, Cui-Xian Yang, Qi-Wen Zhao, Tao Yang, Li-Feng Wang, Ji-Yuan Zhang, Ruo-Nan Xu, Yan-Mei Jiao, Ming Shi, Feng Shao, Rafick-Pierre Sékaly, Fu-Sheng Wang
Abstract
Glioblastoma (GBM) is composed of heterogeneous tumor cell populations, including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance in GBM, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by a reduction in cell proliferation and an increase in cell-cell adhesion and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which suppressed Wnt/β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin, a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation.
Authors
Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir
Abstract
Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat–induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate–Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.
Authors
Kunio Kawanishi, Sudeshna Saha, Sandra Diaz, Michael Vaill, Aniruddha Sasmal, Shoib S. Siddiqui, Biswa Choudhury, Kumar Sharma, Xi Chen, Ian C. Schoenhofen, Chihiro Sato, Ken Kitajima, Hudson H. Freeze, Anja Münster-Kühnel, Ajit Varki
Abstract
Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1–positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.
Authors
George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wüthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler
Abstract
Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of short- and middle-chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.
Authors
Verónica Miguel, Jessica Tituaña, J. Ignacio Herrero, Laura Herrero, Dolors Serra, Paula Cuevas, Coral Barbas, Diego Rodríguez Puyol, Laura Márquez-Expósito, Marta Ruiz-Ortega, Carolina Castillo, Xin Sheng, Katalin Susztak, Miguel Ruiz-Canela, Jordi Salas-Salvadó, Miguel A. Martínez González, Sagrario Ortega, Ricardo Ramos, Santiago Lamas
Abstract
In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal, and differentiation. Ablation of the kinase ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor phosphatase and tensin homolog (PTEN), which also regulates HSC function. We generated and analyzed a knockin mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/–, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSCs.
Authors
Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak
Abstract
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Authors
Eriene-Heidi Sidhom, Choah Kim, Maria Kost-Alimova, May Theng Ting, Keith Keller, Julian Avila-Pacheco, Andrew J.B. Watts, Katherine A. Vernon, Jamie L. Marshall, Estefanía Reyes-Bricio, Matthew Racette, Nicolas Wieder, Giulio Kleiner, Elizabeth J. Grinkevich, Fei Chen, Astrid Weins, Clary B. Clish, Jillian L. Shaw, Catarina M. Quinzii, Anna Greka
Abstract
Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney disease (CKD), often results in diffuse kidney scarring and predisposes to end-stage renal disease. Currently, there is no effective therapy against renal fibrosis. Recently, our laboratory identified an ER-resident protein, thioredoxin domain containing 5 (TXNDC5), as a critical mediator of cardiac fibrosis. Transcriptome analyses of renal biopsy specimens from patients with CKD revealed marked TXNDC5 upregulation in fibrotic kidneys, suggesting a potential role of TXNDC5 in renal fibrosis. Employing multiple fluorescence reporter mouse lines, we showed that TXNDC5 was specifically upregulated in collagen-secreting fibroblasts in fibrotic mouse kidneys. In addition, we showed that TXNDC5 was required for TGF-β1–induced fibrogenic responses in human kidney fibroblasts (HKFs), whereas TXNDC5 overexpression was sufficient to promote HKF activation, proliferation, and collagen production. Mechanistically, we showed that TXNDC5, transcriptionally controlled by the ATF6-dependent ER stress pathway, mediated its profibrogenic effects by enforcing TGF-β signaling activity through posttranslational stabilization and upregulation of type I TGF-β receptor in kidney fibroblasts. Using a tamoxifen-inducible, fibroblast-specific Txndc5 knockout mouse line, we demonstrated that deletion of Txndc5 in kidney fibroblasts mitigated the progression of established kidney fibrosis, suggesting the therapeutic potential of TXNDC5 targeting for renal fibrosis and CKD.
Authors
Yen-Ting Chen, Pei-Yu Jhao, Chen-Ting Hung, Yueh-Feng Wu, Sung-Jan Lin, Wen-Chih Chiang, Shuei-Liong Lin, Kai-Chien Yang
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