Research Article
Abstract
Deposits of hydroxyapatite called Randall’s plaques are found in the renal papilla of calcium oxalate kidney stone formers and likely serve as the nidus for stone formation, but their pathogenesis is unknown. Claudin-2 is a paracellular ion channel that mediates calcium reabsorption in the renal proximal tubule. To investigate the role of renal claudin-2, we generated kidney tubule–specific claudin-2 conditional KO mice (KS-Cldn2 KO). KS-Cldn2 KO mice exhibited transient hypercalciuria in early life. Normalization of urine calcium was accompanied by a compensatory increase in expression and function of renal tubule calcium transporters, including in the thick ascending limb. Despite normocalciuria, KS-Cldn2 KO mice developed papillary hydroxyapatite deposits, beginning at 6 months of age, that resembled Randall’s plaques and tubule plugs. Bulk chemical tissue analysis and laser ablation–inductively coupled plasma mass spectrometry revealed a gradient of intrarenal calcium concentration along the corticomedullary axis in normal mice that was accentuated in KS-Cldn2 KO mice. Our findings provide evidence for the “vas washdown” hypothesis for Randall’s plaque formation and identify the corticomedullary calcium gradient as a potential target for therapies to prevent kidney stone disease.
Authors
Christine V. Behm, Duuamene Nyimanu, Ony Araujo Galdino, Sadhana Kanoo, Young Chul Kim, Natalia Lopez, Helen Goodluck, Peter S. Rowe, Andrew P. Evan, André J. Sommer, Matthew N. Barr, Tracy Punshon, Volker Vallon, Brian P. Jackson, James C. Williams Jr., Alan S.L. Yu
Abstract
Immune cells are constantly exposed to microbiota-derived compounds that can engage innate recognition receptors. How this constitutive stimulation is downmodulated to avoid systemic inflammation and autoimmunity is poorly understood. Here, we show that aryl hydrocarbon receptor (AhR) deficiency in monocytes unleashed spontaneous cytokine responses in vivo, driven by stimulator of interferon genes (STING)-mediated tonic sensing of microbiota. This effect was specific to monocytes, as mice deficient for AhR specifically in macrophages did not show any dysregulation of tonic cytokine responses. AhR inhibition also increased tonic cytokine production in human monocytes. Finally, in patients with systemic juvenile idiopathic arthritis, low AhR activity in monocytes correlated with elevated cytokine responses. Our findings reveal an essential role for AhR in monocytes in restraining tonic microbiota sensing and in maintaining immune homeostasis.
Authors
Adeline Cros, Alessandra Rigamonti, Alba de Juan, Alice Coillard, Mathilde Rieux-Laucat, Darawan Tabtim-On, Emeline Papillon, Christel Goudot, Alma-Martina Cepika, Romain Banchereau, Virginia Pascual, Marianne Burbage, Burkhard Becher, Elodie Segura
Abstract
Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of hereditary neuropathies. Despite progress in genetic sequencing, for around a quarter of patients the disease has lacked a genetic explanation. Here, we identified 16 recessive variants in the RhoGTPase activating protein 19 gene (ARHGAP19) causing motor-predominant neuropathy in 25 individuals from 20 unrelated families. The ARHGAP19 protein acts as a negative regulator of the RhoA GTPase. In vitro biochemical and cellular assays revealed that patient variants impair the GTPase-activating protein (GAP) activity of ARHGAP19 and reduce ARHGAP19 protein levels. Through the use of patient lines, in vitro GAP assays and in silico molecular modeling, we provided evidence that CMT-associated ARHGAP19 variants act through a loss-of-function (LOF) mechanism. LOF in ARHGAP19 orthologues in Drosophila melanogaster and Danio rerio induced motor defects in axonal and synaptic morphology. Similar cellular phenotypes were observed in ARHGAP19 patient-derived motoneurons. Transcriptomic studies further demonstrated that ARHGAP19 regulates cellular pathways associated with motor proteins and the cell cycle. Taken together, our findings establish ARHGAP19 variants as a cause of inherited neuropathy acting through a LOF mechanism.
Authors
Natalia Dominik, Stephanie Efthymiou, Christopher J. Record, Xinyu Miao, Renee Q. Lin, Jevin M. Parmar, Annarita Scardamaglia, Reza Maroofian, Simon A. Lowe, Gabriel N. Aughey, Abigail D. Wilson, Riccardo Curro, Ricardo P. Schnekenberg, Shahryar Alavi, Leif Leclaire, Yi He, Kristina Zhelcheska, Yohanns Bellaïche, Isabelle Gaugué, Mariola Skorupinska, Liedewei Van de Vondel, Sahar I. Da’as, Valentina Turchetti, Serdal Güngör, Gavin V. Monahan, Ehsan Ghayoor Karimiani, Yalda Jamshidi, Phillipa J. Lamont, Camila Armirola-Ricaurte, Haluk Topaloglu, Albena Jordanova, Mashaya Zaman, Selina H. Banu, Wilson Marques, Pedro J. Tomaselli, Busra Aynekin, Ali Cansu, Huseyin Per, Ayten Güleç, Javeria Raza Alvi, Tipu Sultan, Arif Khan, Giovanni Zifarelli, Shahnaz Ibrahim, Grazia M. S. Mancini, M.M. Motazacker, Esther Brusse, Vincenzo Lupo, Teresa Sevilla, A. Nazli Başak, Seyma Tekgul, Robin J. Palvadeau, Jonathan Baets, Yesim Parman, Arman Çakar, Rita Horvath, Tobias B. Haack, Jan-Hendrik Stahl, Kathrin Grundmann-Hauser, Joohyun Park, Stephan Zuchner, Nigel G. Laing, Lindsay A. Wilson, Alexander M. Rossor, James Polke, Fernanda Barbosa Figueiredo, André Pessoa, Fernando Kok, Antônio Rodrigues Coimbra-Neto, Marcondes C. Franca Jr, Gianina Ravenscroft, Sherifa A. Hamed, Wendy K. Chung, Alan M. Pittman, Daniel P. Osborn, Michael Hanna, Andrea Cortese, Mary M. Reilly, James E.C. Jepson, Nathalie Lamarche-Vane, Henry Houlden
Abstract
Metabolic dysfunction–associated steatohepatitis (MASH) is a progressive liver disease characterized by complex interactions between lipotoxicity, ER stress responses, and immune-mediated inflammation. We identified enrichment of the proinflammatory alarmin S100 calcium-binding protein A11 (S100A11) on extracellular vesicles stimulated by palmitate-induced lipotoxic ER stress with concomitant upregulation of hepatocellular S100A11 abundance in an IRE1A-XBP1s–dependent manner. We next investigated the epigenetic mechanisms that regulate this stress response. Publicly available human liver ChIP-Seq GEO datasets demonstrated a region of histone H3 lysine 27 (H3K27) acetylation upstream of the S100A11 promoter. H3K27 acetylation ChIP-qPCR demonstrated a positive correlation between lipotoxic ER stress and H3K27 acetylation of the region, which we termed the lipotoxicity-influenced enhancer (LIE) domain. CRISPR-mediated repression of the LIE domain reduced palmitate-induced H3K27 acetylation and corresponding S100A11 upregulation in Huh7 cells and immortalized mouse hepatocytes. Silencing of the murine LIE in 2 independent steatohepatitis models demonstrated reduced S100a11 upregulation and attenuated liver injury. We confirmed H3K27 acetylation and XBP1s occupancy at the LIE domain in human MASH liver samples and an increase in hepatocyte-derived S100A11-enriched extracellular vesicles in MASH patient plasma. Our studies demonstrate a LIE domain that mediates hepatic S100A11 upregulation. This pathway may be a potential therapeutic target in MASH.
Authors
P. Vineeth Daniel, Hanna L. Erickson, Daheui Choi, Feda H. Hamdan, Yasuhiko Nakao, Gyanendra Puri, Takahito Nishihara, Yeriel Yoon, Amy S. Mauer, Debanjali Dasgupta, Jill Thompson, Alexander Revzin, Harmeet Malhi
Abstract
Genetic factors contributing to hearing loss (HL) are heterogeneous, and effective medical treatments remain limited. We identified 3 distinct missense variants in CPD, encoding carboxypeptidase D, in 5 individuals with congenital deafness from 3 unrelated families, affecting the catalytically active CP domain 2 of this protein. Subsequent analysis of a larger cohort from the 100,000 Genomes Project revealed an enrichment of rare protein-altering CPD variants in individuals with HL. We show that CPD localizes to sensory epithelium and nerve cells in the mouse cochlea, and the enzymatic activity of CPD, crucial for nitric oxide (NO) production through arginine processing, is impaired in affected individuals. The levels of arginine, NO, and cGMP in patient-derived fibroblasts are also decreased, leading to endoplasmic reticulum stress–mediated responses being triggered in the cells. Silencing of Cpd in organotypic mouse cochlea cultures leads to increased apoptosis. Finally, Drosophila models of CPD deficiency display defective Johnston’s organ, impaired auditory transduction, and sensory and movement abnormalities. Notably, these phenotypes are partially rescued by supplementation with arginine or sildenafil, a cGMP enhancer. Our findings establish CPD mutations as a cause of congenital HL, highlighting that the NO signaling pathway offers a promising therapeutic avenue.
Authors
Memoona Ramzan, Natalie Ortiz-Vega, Mohammad Faraz Zafeer, Amanda G. Lobato, Tahir Atik, Clemer Abad, Nirmal Vadgama, Duygu Duman, Nazım Bozan, Enise Avcı Durmuşalioǧlu, Sunny Greene, Shengru Guo, Suna Tokgöz-Yılmaz, Merve Koç Yekedüz, Fatma Tuba Eminoğlu, Mehmet Aydın, Serhat Seyhan, Ioannis Karakikes, Vladimir Camarena, Maria Camila Robayo, Tijana Canic, Güney Bademci, Gaofeng Wang, Amjad Farooq, Mei-ling Joiner, Katherina Walz, Daniel F. Eberl, Jamal Nasir, R. Grace Zhai, Mustafa Tekin
Abstract
The effectiveness of RAS/MAPK inhibitors in treating metastatic KRAS-mutant non–small cell lung cancer (NSCLC) is often hindered by the development of resistance driven by disrupted negative feedback mechanisms led by phosphatases like PP2A. PP2A is frequently suppressed in lung cancer to maintain elevated RAS/MAPK activity. Despite its established role in regulating oncogenic signaling, targeting PP2A with RAS/MAPK to prevent resistance has not been previously demonstrated. In this study, we aimed to establish a treatment paradigm by combining a PP2A molecular glue with a RAS/MAPK inhibitor to restore PP2A activity and counteract resistance. We demonstrated that KRASG12C and MEK1/2 inhibitors disrupted PP2A carboxymethylation and destabilized critical heterotrimeric complexes. Furthermore, genetic disruption of PP2A carboxymethylation enhanced intrinsic resistance to MEK1/2 inhibition both in vitro and in vivo. We developed RPT04402, a PP2A molecular glue that selectively stabilizes PP2A-B56α heterotrimers. In commercial cell lines and in a patient-derived model, combining RPT04402 with a RAS/MAPK inhibitor slowed proliferation and enhanced apoptosis. In mouse xenografts, this combination induced tumor regressions, extended median survival, and delayed the onset of treatment resistance. These findings highlight that promoting PP2A stabilization and RAS/MAPK inhibition presents a promising therapeutic strategy to improve treatment outcomes and overcome resistance in metastatic KRAS-mutant NSCLC.
Authors
Brynne Raines, Stephanie Tseng-Rogenski, Amanda C. Dowdican, Irene Peris, Matthew Hinderman, Kaitlin P. Zawacki, Kelsey Barrie, Gabrielle Hodges Onishi, Alexander M. Dymond, Tahra K. Luther, Sydney Musser, Behirda Karaj Majchrowski, J. Chad Brenner, Aqila Ahmed, Derek J. Taylor, Caitlin M. O’Connor, Goutham Narla
Abstract
Adams-Oliver syndrome (AOS) is a rare congenital disorder characterized by scalp, limb, and cardiovascular defects. Although variants in the NOTCH1 receptor, DLL4 ligand, and RBPJ transcription factor have been implicated in AOS, the driving tissue types and molecular mechanisms by which these variants cause pathogenesis are unknown. Here, we used quantitative binding assays to show that AOS-associated RBPJ missense variants compromise DNA binding but not cofactor binding. These findings suggest that AOS-associated RBPJ variants do not function as loss-of-function alleles but instead act as dominant-negative proteins that sequester cofactors from DNA. Consistent with this idea, mice carrying an AOS-associated Rbpj allele develop dominant phenotypes that include increased lethality and cardiovascular defects in a Notch1 heterozygous background, whereas Notch1 and Rbpj compound heterozygous null alleles are well tolerated. To facilitate studies into the tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vascular endothelium to the development of AOS-like phenotypes. Importantly, our studies show that expression of the Rbpj AOS allele in endothelial cells is both necessary and sufficient to cause lethality and cardiovascular defects. These data establish that reduced Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.
Authors
Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein
Abstract
Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing β cells, along with dysregulated glucagon secretion from pancreatic α cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors. The few surviving β cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In α cells, we observed hyperresponsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization, and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to α cell dysfunction, including MHC class I, which were closely linked with α cell dysfunction. Our data provide what we believe are novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual β cell function and modulate α cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D.
Authors
Theodore dos Santos, Xiao-Qing Dai, Robert C. Jones, Aliya F. Spigelman, Hannah M. Mummey, Jessica D. Ewald, Cara E. Ellis, James G. Lyon, Nancy Smith, Austin Bautista, Jocelyn E. Manning Fox, Norma F. Neff, Angela M. Detweiler, Michelle Tan, Rafael Arrojo e Drigo, Jianguo Xia, Joan Camunas-Soler, Kyle J. Gaulton, Stephen R. Quake, Patrick E. MacDonald
Abstract
The intratumor microenvironment shapes the metastatic potential of cancer cells and their susceptibility to any immune response. Yet, the nature of the signals within the microenvironment that control anticancer immunity and how they are regulated is poorly understood. Here, using melanoma as a model, we investigate the involvement in metastatic dissemination and the immune-modulatory microenvironment of Protein S-Acyl Transferases as an underexplored class of potential therapeutic targets. We find that ZDHHC13 suppresses metastatic dissemination by palmitoylation of CTNND1, leading to stabilization of E-cadherin. Importantly, ZDHHC13 also reshapes the tumor immune microenvironment by suppressing lysophosphatidylcholine (LPC) synthesis in melanoma cells, leading to inhibition of M2-like tumor-associated macrophages that we show degrade E-cadherin via MMP12 expression. Consequently, ZDHHC13 activity suppresses tumor growth and metastasis in immunocompetent mice. Our study highlights the therapeutic potential of targeting the ZDHHC13–E-cadherin axis and its downstream metabolic and immune-modulatory mechanisms, offering additional strategies to inhibit melanoma progression and metastasis.
Authors
Hongjin Li, Jianke Lyu, Yu Sun, Chengqian Yin, Yuewen Li, Weiqiang Chen, Suan-Sin Foo, Xianfang Wu, Colin R. Goding, Shuyang Chen
Abstract
Deficits in the mitochondrial energy–generating machinery cause mitochondrial disease, a group of untreatable and usually fatal disorders. Refractory epileptic events are a common neurological presentation of mitochondrial disease, including Leigh syndrome, a severe form of mitochondrial disease associated with epilepsy. However, the neuronal substrates and circuits for mitochondrial disease–induced epilepsy remain unclear. Here, using mouse models of Leigh syndrome that lack mitochondrial complex I subunit NDUFS4 in a constitutive or conditional manner, we demonstrated that mitochondrial dysfunction leads to a reduction of GABAergic neurons in the rostral external globus pallidus (GPe) and identified a specific affectation of pallidal Lhx6–expressing inhibitory neurons contributing to altered GPe excitability. Our findings revealed that viral vector–mediated Ndufs4 reexpression in the GPe effectively prevented seizures and improved survival in the models. Additionally, we highlight the subthalamic nucleus (STN) as a critical structure in the neural circuit involved in mitochondrial epilepsy, as its inhibition effectively reduces epileptic events. Thus, we have identified a role for pallido-subthalamic projections in epilepsy development in the context of mitochondrial dysfunction. Our results suggest STN inhibition as a potential therapeutic intervention for refractory epilepsy in patients with mitochondrial disease, providing promising leads in the quest to identify effective treatments.
Authors
Laura Sánchez-Benito, Melania González-Torres, Irene Fernández-González, Laura Cutando, María Royo, Joan Compte, Miquel Vila, Sandra Jurado, Elisenda Sanz, Albert Quintana
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