Issue published March 1, 2023 Previous issue | Next issue
- Volume 133, Issue 5
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On the cover: Synchronized cortical circuit dynamics mediate spontaneous pain
Ding et al. report that robustly synchronized neural dynamics of the primary somatosensory cortex are critical for pain-like behavior, based on a clinically relevant mouse model of trigeminal neuralgia. The cover image shows neuronal calcium dynamics, with blue representing lower activities and yellow representing higher activities, in a small region within the cortex that displays marked activation in response to foramen lacerum impingement of the trigeminal nerve root. Image credit: Tewodros Mulugeta Dagnew and Weihua Ding.
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Commentaries
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Abstract
A majority of the human genome is transcribed into noncoding RNAs, of which long noncoding RNAs (lncRNAs) form a large and heterogeneous fraction. While lncRNAs are mostly noncoding, recent evidence suggests that cryptic translation within some lncRNAs may produce proteins with important regulatory functions. In this issue of the JCI, Zheng, Wei, and colleagues used an integrative functional genomic strategy to systematically identify cryptic lncRNA-encoded ORFs that play a role in estrogen receptor–positive (ER+) breast cancer (BC). They identified 758 cryptic lncRNA-encoded ORFs undergoing active translation, of which 28 had potential functional and clinical relevance in ER+ BC. The LINC00992-encoded polypeptide GT3-INCP was upregulated in ER+ BC and drove tumor growth. GT3-INCP was regulated by estrogen and the ER and acted via the transcription factor GATA3 to regulate BC susceptibility and risk genes. These findings discern a largely unexplored class of molecules and have implications for many pathologies, including cancer.
Authors
Anindya Dutta, Hui Li, Roger Abounader
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The sodium-glucose cotransporter-2 (SGLT2) is expressed on the luminal side of proximal tubule epithelial cells in the kidney. While pharmacological inhibition of SGLT2 provides kidney protection in diabetic kidney disease (DKD), the molecular mechanisms remain unclear. In this issue of the JCI, Schaub et al. report on the changes in single-cell transcriptional profiles of young participants with type 2 diabetes who received SGLT2 inhibitors. Treatment with SGLT2 inhibitors restored metabolic perturbations in proximal tubular cells and reduced expression of the inflammatory signaling molecule mTORC1. Notably, changes in transcripts and mTORC1 were also found in the kidney of a diabetes mouse model treated with an SGLT2 inhibitor, supporting use of this model for further studies. These findings reveal cellular mechanisms of SGLT2 inhibitors and are important for advancing therapeutic targets in the treatment of DKD.
Authors
Katherine R. Tuttle
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Only three classes of pain medications have made it into clinical use in the past 60 years despite intensive efforts and the need for nonaddictive pain treatments. One reason for the failure involves the use of animal models that lack mechanistic similarity to human pain conditions, with endpoint measurements that may not reflect the human pain experience. In this issue of the JCI, Ding, Fischer, and co-authors developed the foramen lacerum impingement of trigeminal nerve root (FLIT) model of human trigeminal neuralgia that has improved face, construct, and predictive validities over those of current models. They used the FLIT model to investigate the role that abnormal, hypersynchronous cortical activity contributed to a neuropathic pain state. Unrestrained, synchronous glutamatergic activity in the primary somatosensory cortex upper lip and jaw (S1ULp–S1J) region of the somatosensory cortex drove pain phenotypes. The model establishes a powerful tool to continue investigating the interaction between the peripheral and central nervous systems that leads to chronic pain.
Authors
Norman E. Taylor, Luiz Ferrari
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Research Articles
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Abstract
Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.
Authors
Saket Jain, Jonathan W. Rick, Rushikesh S. Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chih-Chieh Chang, Nikita Choudhary, Alan T. Nguyen, Sweta Sudhir, Eric J. Chalif, Jia-Shu Chen, Ankush Chandra, Alexander F. Haddad, Harsh Wadhwa, Sumedh S. Shah, Serah Choi, Josie L. Hayes, Lin Wang, Garima Yagnik, Joseph F. Costello, Aaron Diaz, Dieter Henrik Heiland, Manish K. Aghi
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Background Merkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.Methods Here, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.Results Strikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain–containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain–dependent (TEAD-dependent) transcriptional repression of MCPyV LT.Conclusion These findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.Funding US Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.
Authors
Thomas C. Frost, Ashley K. Gartin, Mofei Liu, Jingwei Cheng, Harita Dharaneeswaran, Derin B. Keskin, Catherine J. Wu, Anita Giobbie-Hurder, Manisha Thakuria, James A. DeCaprio
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Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α–positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the “hidden” lncRNA-encoded proteome might be a new space for therapeutic target discovery.
Authors
Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen
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Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain.
Authors
Xueming Hu, Lixia Du, Shenbin Liu, Zhou Lan, Kaikai Zang, Jing Feng, Yonghui Zhao, Xingliang Yang, Zili Xie, Peter L. Wang, Aaron M. Ver Heul, Lvyi Chen, Vijay K. Samineni, Yan-Qing Wang, Kory J. Lavine, Robert W. Gereau IV, Gregory F. Wu, Hongzhen Hu
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BACKGROUND The fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus–specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODS We used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTS We show that clonally expanded, high-avidity A. fumigatus–specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSION Our data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDING German Research Foundation (DFG), under Germany’s Excellence Strategy (EXC 2167-390884018 “Precision Medicine in Chronic Inflammation” and EXC 2051-390713860 “Balance of the Microverse”); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).
Authors
Carsten Schwarz, Patience Eschenhagen, Henrijette Schmidt, Thordis Hohnstein, Christina Iwert, Claudia Grehn, Jobst Roehmel, Eva Steinke, Mirjam Stahl, Laura Lozza, Ekaterina Tikhonova, Elisa Rosati, Ulrik Stervbo, Nina Babel, Jochen G. Mainz, Hilmar Wisplinghoff, Frank Ebel, Lei-Jie Jia, Matthew G. Blango, Peter Hortschansky, Sascha Brunke, Bernhard Hube, Axel A. Brakhage, Olaf Kniemeyer, Alexander Scheffold, Petra Bacher
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The transcription factor p63 guards genome integrity in the female germline, and its mutations have been reported in patients with premature ovarian insufficiency (POI). However, the precise contribution of the TP63 gene to the pathogenesis of POI needs to be further determined. Here, in 1,030 Chinese patients with POI, we identified 6 heterozygous mutations of the TP63 gene that impaired the C-terminal transactivation-inhibitory domain (TID) of the TAp63α protein and resulted in tetramer formation and constitutive activation of the mutant proteins. The mutant proteins induced cell apoptosis by increasing the expression of apoptosis-inducing factors in vitro. We next introduced a premature stop codon and selectively deleted the TID of TAp63α in mice and observed rapid depletion of the p63+/ΔTID mouse oocytes through apoptosis after birth. Finally, to further verify the pathogenicity of the mutation p.R647C in the TID that was present in 3 patients, we generated p63+/R647C mice and also found accelerated oocyte loss, but to a lesser degree than in the p63+/ΔTID mice. Together, these findings show that TID-related variants causing constitutive activation of TAp63α lead to POI by inducing oocyte apoptosis, which will facilitate the genetic diagnosis of POI in patients and provide a potential therapeutic target for extending female fertility.
Authors
Chengzi Huang, Simin Zhao, Yajuan Yang, Ting Guo, Hanni Ke, Xin Mi, Yingying Qin, Zi-Jiang Chen, Shidou Zhao
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BACKGROUND To date, only limited data are available on the mechanisms of protection against colonization with Bordetella pertussis in humans.METHODS In this study, the cellular responses to B. pertussis challenge were monitored longitudinally using high-dimensional EuroFlow-based flow cytometry, allowing quantitative detection of more than 250 different immune cell subsets in the blood of 15 healthy donors.RESULTS Participants who were protected against colonization showed different early cellular responses compared with colonized participants. Especially prominent for colonization-protected participants were the early expansion of CD36– nonclassical monocytes on day 1 (D1), natural killer cells (D3), follicular T helper cells (D1–D3), and plasma cells (D3). Plasma cell expansion on D3 correlated negatively with the CFU load on D7 and D9 after challenge. Increased plasma cell maturation on D11–D14 was found in participants with seroconversion.CONCLUSION These early cellular immune responses following experimental infection can now be further characterized and potentially linked to an efficient mucosal immune response, preventing colonization. Ultimately, their presence may be used to evaluate whether new B. pertussis vaccine candidates are protective against B. pertussis colonization, e.g., by bacterial challenge after vaccination.TRIAL REGISTRATION ClinicalTrials.gov NCT03751514.FUNDING Innovative Medicines Initiative 2 Joint Undertaking and the EuroFlow Consortium.
Authors
Annieck M. Diks, Hans de Graaf, Cristina Teodosio, Rick J. Groenland, Bas de Mooij, Muktar Ibrahim, Alison R. Hill, Robert C. Read, Jacques J.M. van Dongen, Magdalena A. Berkowska, on behalf of the IMI-2 PERISCOPE Consortium
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The molecular mechanisms of sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) remain incompletely understood. Single-cell RNA sequencing and morphometric data were collected from research kidney biopsies donated by young persons with type 2 diabetes (T2D), aged 12 to 21 years, and healthy controls (HCs). Participants with T2D were obese and had higher estimated glomerular filtration rates and mesangial and glomerular volumes than HCs. Ten T2D participants had been prescribed SGLT2i (T2Di[+]) and 6 not (T2Di[–]). Transcriptional profiles showed SGLT2 expression exclusively in the proximal tubular (PT) cluster with highest expression in T2Di(–) patients. However, transcriptional alterations with SGLT2i treatment were seen across nephron segments, particularly in the distal nephron. SGLT2i treatment was associated with suppression of transcripts in the glycolysis, gluconeogenesis, and tricarboxylic acid cycle pathways in PT, but had the opposite effect in thick ascending limb. Transcripts in the energy-sensitive mTORC1-signaling pathway returned toward HC levels in all tubular segments in T2Di(+), consistent with a diabetes mouse model treated with SGLT2i. Decreased levels of phosphorylated S6 protein in proximal and distal tubules in T2Di(+) patients confirmed changes in mTORC1 pathway activity. We propose that SGLT2i treatment benefits the kidneys by mitigating diabetes-induced metabolic perturbations via suppression of mTORC1 signaling in kidney tubules.
Authors
Jennifer A. Schaub, Fadhl M. AlAkwaa, Phillip J. McCown, Abhijit S. Naik, Viji Nair, Sean Eddy, Rajasree Menon, Edgar A. Otto, Dawit Demeke, John Hartman, Damian Fermin, Christopher L. O’Connor, Lalita Subramanian, Markus Bitzer, Roger Harned, Patricia Ladd, Laura Pyle, Subramaniam Pennathur, Ken Inoki, Jeffrey B. Hodgin, Frank C. Brosius III, Robert G. Nelson, Matthias Kretzler, Petter Bjornstad
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Cortical neural dynamics mediate information processing for the cerebral cortex, which is implicated in fundamental biological processes such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes the cortical network into an aberrant state and, if so, whether it can be brought back to a “normal” operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain–like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos–expressing neurons and selective activation of GABAergic interneurons significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More important, restoring a “normal” range of neural dynamics through attenuation of pain-induced S1 synchronization alleviated pain-like behavior. These results suggest that spontaneous pain pushed the S1 regional network into a synchronized state, whereas reversal of this synchronization alleviated pain.
Authors
Weihua Ding, Lukas Fischer, Qian Chen, Ziyi Li, Liuyue Yang, Zerong You, Kun Hu, Xinbo Wu, Xue Zhou, Wei Chao, Peter Hu, Tewodros Mulugeta Dagnew, Daniel M. Dubreuil, Shiyu Wang, Suyun Xia, Caroline Bao, Shengmei Zhu, Lucy Chen, Changning Wang, Brian Wainger, Peng Jin, Jianren Mao, Guoping Feng, Mark T. Harnett, Shiqian Shen
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The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete’s dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb’s enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.
Authors
André A. Grassmann, Rafal Tokarz, Caroline Golino, Melissa A. McLain, Ashley M. Groshong, Justin D. Radolf, Melissa J. Caimano
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)