Research Article
Abstract
Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are clinically heterogeneous malignancies whose biology and microenvironmental organization remain poorly understood. Here, we integrated single-nucleus multiomic and spatial transcriptomic profiling across 38 well-differentiated pancreatic (PanNET) and small-intestinal (siNET) tumors to define conserved malignant programs, their regulatory circuits, and spatial niches. We observed 2 conserved malignant cell programs spanning a continuous transcriptional spectrum: a neuronal-like program and a secretory neuroendocrine program. Matched chromatin accessibility profiles uncovered distinct, tissue-specific regulatory networks, including MAX:MYC and MITF transcription factor binding motifs in siNETs versus ISL1 and TFAP4 in PanNETs, indicating organ-specific epigenetic control. Spatial transcriptomic analyses revealed that neuronal-like–high regions localized to densely cellular tumor areas with relative depletion of stromal infiltration, whereas secretory neuroendocrine–high regions occupied fibrovascular and stromal niches enriched for endothelial, fibroblast, and myeloid populations and associated with TGFB1-ITGB1, VEGFA-FLT1, and LAMA2-ITGA1 signaling. Across both tumor types, the cNMF2 program was enriched in metastatic lesions and was enriched for profibrotic and proangiogenic gene signatures. Thus, GEP-NETs are organized along a conserved neuronal-to-secretory axis defined by distinct epigenetic programs and spatially coupled to specific microenvironmental niches. This framework unifies NET heterogeneity across organ sites and identifies pathway-specific, microenvironment-linked vulnerabilities for therapeutic targeting.
Authors
Julie Karam, Samantha E. Hoffman, Amanda Garza, Dan Gui, Hannah I. Hoffman, Breanna M. Titchen, Yutaro Tanaka, Erica Pimenta, Theodora Pappa, Laura Valderrabano, Kevin Bi, Riaz Gillani, Lauren Brais, Erin Shannon, Jason L. Hornick, Jihye Park, Jennifer Chan, Eliezer M. Van Allen
Abstract
Membranous nephropathy (MN) is an autoimmune kidney disease and a major cause of nephrotic syndrome in adults. Although autoantibodies against phospholipase A2 receptor 1 (PLA2R) and complement activation are central to disease pathogenesis, the mechanisms by which anti-PLA2R antibodies activate complement at the podocyte surface remain incompletely defined. Here, we cloned 14 patient-derived anti-PLA2R mAbs and found that they predominantly recognized the N-terminal cysteine-rich (CysR) and C-type lectin domain 1 (CTLD1) regions of PLA2R. Individual anti-PLA2R mAbs induced little or no complement-dependent cytotoxicity (CDC) of PLA2R-expressing podocytes in vitro. In contrast, paired mAbs targeting distinct epitopes, particularly CysR and CTLD1, markedly enhanced CDC. This effect was strongest for IgG1 and IgG3 antibodies, whereas IgG4 alone did not activate complement but modulated CDC in combination with IgG1. Purified IgG from patients with PLA2R-associated MN similarly induced CDC, which was augmented by addition of anti–PLA2R IgG1 and reduced by anti–PLA2R IgG4 or Fab fragments targeting CysR or CTLD1. In human PLA2R–expressing mice, paired anti-PLA2R antibodies increased glomerular complement deposition and induced albuminuria. These findings identify epitope pairing as a key determinant of complement activation in PLA2R-associated MN and support epitope-specific targeting strategies as a promising avenue for therapeutic intervention.
Authors
Tsai-Yi Wu, Kun-Hua Tu, Larissa Seifert, Kung-Wei Lin, Han-Po Shih, Yu-Fang Lo, Jhan-Jie Peng, Gunther Zahner, Oliver Kretz, You-Ning Lin, Chen-Xuan Kang, Jing-Ya Ding, Yi-Ran Tu, Li-Yi Ma, Ya-Ting Chuang, Chia-Chi Lo, Yu-Huan Tsai, Chih-Wei Yang, Nicola M. Tomas, Cheng-Lung Ku
Abstract
Glucagon-like peptide 1 (GLP-1) receptor agonists (GLP1RAs) effectively reduce feeding to treat obesity, although nausea and other aversive side effects of these drugs can limit their use. Brainstem circuits that promote satiation and mediate the physiological control of body weight can be distinguished from those that cause aversion. It remains unclear, however, whether brainstem Glp1r neurons contribute to the normal regulation of energy balance and whether GLP1RAs control appetite via circuits distinct from those that mediate aversive responses. Here, we silenced Glp1r neurons in the nucleus of the solitary tract (NTS) or area postrema (AP) (NTSGlp1r or APGlp1r neurons, respectively) or restored their GLP1R signaling on an otherwise GLP1R-deficient background to determine physiological and pharmacological roles for each neuron population. Although NTSGlp1r neurons contributed to the normal restraint of food intake and body weight, they failed to mediate GLP1RA-dependent weight loss. In contrast, while we detected no role for APGlp1r neurons in physiological feeding, they mediated both the weight-lowering and aversive effects of GLP1RAs. Therefore, while nonaversive NTSGlp1r neurons controlled physiologic satiation they did not contribute to weight loss during GLP1RA treatment. Rather, APGlp1r neurons mediated both the weight-lowering and aversive effects of GLP1RAs, preventing the separation of their nauseating and weight-loss effects at a circuit level.
Authors
Warren T. Yacawych, Yi Wang, Guoxiang Zhou, Shad Hassan, Cagri Bodur, Elisabeth Walters, John G. Santinga, Frederike Sass, Martin deVaux, Stace Kernodle, Iris Wu, Jenny M. Brown, Dylan Belmont-Rausch, Alan C. Rupp, Abigail J. Tomlinson, Zitian Lin, Emma VanTongeren, Anna Secher, Kirsten Raun, Tune H. Pers, Randy J. Seeley, Martin G. Myers Jr., Weiwei Qiu
Abstract
Oil-producing sebaceous glands (SGs), attached to the upper/middle portion of hair follicles, are indispensable for maintaining skin hydration, and their dysfunction leads to dry skin. However, the molecular mechanisms underlying regulation of SG stem cells remain largely unknown. We identified transcription factor KROX20 as a marker of SG stem cells that sustains their stemness throughout SG morphogenesis and homeostasis. We developed an inducible mouse model in which ablation of KROX20-positive cells causes SG loss and rapidly and robustly induces dry, flaky, alopecia symptoms following induction. This model, termed xeroflacia (xero = xerosis, fla = flaky, cia = alopecia), provides a tool for studying the biology of dry skin. Furthermore, we found that KROX20 directly regulates Notch1 transcription to orchestrate the balance between SG stem cell self-renewal and differentiation. Small molecule drug modulation of Notch1 signaling to activate or inhibit the pathway enabled us to regulate SG differentiation to maintain skin oil levels, providing a proof of principle that other signaling pathways downstream of Krox20 could be therapeutic targets for SG-related diseases.
Authors
Yumeng Zhang, Pernelle Pulh, Michelle F. Pan, Yi He, Juanzhu Yan, Annie Li, Renée M. McKay, Lu Q. Le
Abstract
STXBP1 variants are a frequent cause of early onset developmental and epileptic encephalopathies and related neurodevelopmental disorders, but the clinical interpretation of these variants remains a major challenge. Most reported STXBP1 missense variants are classified as variants of uncertain significance (VUS), complicating diagnosis, counseling, and patient eligibility for precision therapies. Here, we developed EpiPred, a gene-specific machine learning classifier that predicts the pathogenicity of STXBP1 missense variants and tests these predictions using empirical evidence from well-established cellular assays. Trained on a curated set of pathogenic and benign variants, EpiPred outperformed global prediction tools in accuracy, sensitivity, and specificity. We validated the model’s predictions using variant effect assays that measure protein abundance, solubility, stability, and interaction with the SNARE complex partner syntaxin 1. These biochemical readouts aligned closely with model outputs and enabled reclassification of several possibly misdiagnosed variants, which warrant further validation and clinical reevaluation. We deployed EpiPred in an interactive web application that allows clinicians, researchers, and patients to explore predictions for all possible STXBP1 missense variants. By identifying likely pathogenic STXBP1 variants, including those that may respond to emerging therapies, such as protein stabilizers. By coupling gene-calibrated machine learning with orthogonal variant-effect assays and public deployment, EpiPred provides a transferable framework for VUS resolution, trial enrichment, and precision diagnosis across clinically actionable Mendelian disease genes.
Authors
Jeffrey D. Calhoun, Chengbing Wang, Carina G. Biar, Jonathan R. Gunti, John S. Lee, Aaron M. Geller, Jung H. Hong, Santiago Schnell, Louis T. Dang, Yu Wang, Jack M. Parent, Lori L. Isom, Michael D. Uhler, Heather C. Mefford, M. Elizabeth Ross, Vanessa Aguiar-Pulido, Gemma L. Carvill
Abstract
Regulatory T cells (Tregs) hold great promise as next-generation therapeutics for autoimmune diseases. However, maintaining their functional persistence within inflamed tissues remains a major translational challenge. Using an in vitro system that recapitulates the inflammatory CNS milieu of multiple sclerosis (MS), together with a pooled shRNA screen, we identify necroptotic signaling as a key driver of Treg death, thereby compromising Treg functional persistence under inflammatory conditions. We further demonstrate that Tregs in both a mouse model of MS and patients with MS exhibited a preferential susceptibility to receptor-interacting serine/threonine kinase 1–dependent (RIPK1-dependent) necroptosis. Mechanistically, a FOXP3-driven, low-glucose metabolic program rendered Tregs intrinsically susceptible to necroptosis by limiting O-GlcNAcylation (O-GlcNAc) modification on RIPK1. This vulnerability was not shared by conventional T cells under comparable inflammatory conditions. Finally, in combination with adoptive Treg transfer, we show that selective inhibition of necroptosis in Tregs enhanced their survival and suppressive function at sites of active inflammation, thereby reducing autoimmune pathology in mouse models of MS and systemic lupus erythematosus. Together, these findings identify necroptotic cell death as a barrier to Treg persistence within inflamed tissues and highlight the therapeutic potential of necroptosis-resistant Tregs for the treatment of autoimmune diseases.
Authors
Qiaoyan Wu, Na Cui, Li Gao, Zhihui Lu, Xiang Ao, Rui Pang, Xingyan Li, Heling Pan, Daichao Xu, Peiying Li, Junying Yuan, Chengyu Zou
Abstract
Cyclin-dependent kinase like 5 (CDKL5) is a serine-threonine kinase enriched in the mammalian brain whose loss of function causes a severe developmental and epileptic encephalopathy named CDKL5 Deficiency Disorder. We previously showed that CDKL5 phosphorylates the microtubule-associated protein MAP1S, but how this regulates microtubule-dependent functions is not well understood. To address this question, we generated MAP1S phosphomutant mice in which the CDKL5 phosphorylation sites S786 and S812 were mutated to alanine (MAP1S S786/812A; MAP1S SA). Using a microtubule cosedimentation assay, we found that dynein binding to microtubules was reduced in MAP1S SA and CDKL5 knockout (KO) brain lysates, and time-lapse imaging showed impaired dynein motility in dendrites from both genotypes. MAP1S SA mice also exhibited reduced AMPA receptor transport, dendritic spine density, and excitatory synapses, accompanied by anxiety-like behavior and motor, social, and memory deficits relevant to CDD. Mechanistically, MAP1S SA and CDKL5 KO neurons showed increased microtubule stability and reduced tubulin tyrosination, consistent with excessive MAP1S-mediated stabilization. Restoring tubulin tyrosination by expressing tubulin-tyrosine ligase rescued dynein transport defects. Together, these findings identify MAP1S phosphorylation as a critical regulator of microtubule dynamics and dynein-dependent transport.
Authors
André T. Lopes, Ondine Janiv, Suzanne Claxton, Sila K. Ultanir
Abstract
Mutations in the survival of motor neuron 1 (SMN1) can reduce functional SMN protein levels, which causes spinal muscular atrophy (SMA), a disease affecting the nervous system and peripheral tissues, including the immune system. Yet, SMN expression across immune cell subsets and the effect of SMN-modulating therapies on the immune system remain underexplored. We found that in neonatal mouse spleens, SMN expression was highest in B cells, and was massively reduced in SMA mice. In human PBMCs from adults, DCs and monocytes expressed the highest SMN levels, whereas patients with SMA showed reduced DC frequencies and increased B cell frequencies. Patients receiving systemic versus CNS-restricted therapy showed similar differences in immune cell composition and SMN expression levels. Similarly, an exploratory cohort including untreated patients did not show a substantial treatment-specific effect relative to controls. To assess the effect of differentiation on SMN, we analyzed PMA-treated THP-1 cells and found enhanced aberrant SMN splicing and increased SMN+ Cajal bodies. SMN levels varied across immune cell types, and reduced SMN levels were associated with altered immune cell composition. Immune alterations and decreased SMN levels were observed in both treated and untreated patients with SMA and may have contributed to dysfunctions of the immune system.
Authors
Ines Tapken, Katharina Rahmel-Stein, Christine Ehlers, Federica Cieri, Nora T. Detering, Tobias Schüning, Bogdan Bjelica, Charlotte Mindermann, Svenja Neuhoff, Linda-Isabell Schmitt, Markus Leo, Tim Hagenacker, Sabine Illsinger, Elia Di Schiavi, Theresa Graalmann, Susanne Petri, Ulrich Kalinke, Peter Claus
Abstract
Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor critical for host defense against microbial infections. To prevent aberrant activation, nuclear cGAS is restrained by nucleosome tethering or by barrier-to-autointegration factor–mediated restriction of its access to DNA. However, its physiological roles beyond enzymatic suppression in the nucleus remain poorly understood. Here, we present a genome-wide map of cGAS chromatin localization in human cells and unexpectedly find its binding beyond nucleosome tethering, where cGAS is recruited to chromatin by AP-1 transcription factors via a potentially previously uncharacterized transactivation domain. Integrated CUT&RUN, RNA-seq, and metabolomics analyses reveal that cGAS transcriptionally regulates cholesterol homeostasis. Mechanistically, cGAS promotes LGALS3 transcription, which inhibits ACAA2 activity to suppress fatty acid oxidation and acetyl-CoA production, thereby lowering intracellular cholesterol levels. Reduced cholesterol lowers plasma membrane phosphatidylinositol [3,4,5] triphosphate [PI(3,4,5)P3] levels and decreases mTOR complex 2 (mTORC2) activation. In contrast, cGAS depletion results in cholesterol accumulation and elevated PI(3,4,5)P3, driving mTORC2/Akt signaling, promoting xenografted tumor growth, and conferring resistance to DNA-damaging agents. In vivo, the elevated hepatic cholesterol levels observed in cGas–/– mice were largely reversed by inhibition of Akt but not STING, whereas cGAS deficiency had minimal effects on lung physiology. Collectively, our findings uncover an unexpected role for nuclear cGAS in orchestrating a transcriptional program that links lipid metabolism to cell proliferation, stress response, and cholesterol homeostasis in liver.
Authors
Yu Deng, Matthew J. Niederhuber, Zhichuan Zhu, Austin J. Hepperla, Le Yu, Quentin Hahn, Joshua Hartsell, Tooba Rashid, Piotr A. Mieczkowski, Hemant Kelkar, Jeremy M. Simon, Yanqiong Zhang, Alessandra Livraghi-Butrico, Meredith J. Zeller, Joseph G.C. Kennedy, Jazmin F. DePriest, Jiong Li, Yan Zhang, Xing Qiu, H. Ümit Kaniskan, Jian Jin, Corbin D. Jones, Laura E. Herring, Douglas H. Phanstiel, Xiaojing Liu, Jesse R. Raab, Brian D. Strahl, Pengda Liu
Abstract
Connections between vascular and neurocognitive diseases are emerging but remain poorly defined. Cognitive impairment is increasingly recognized in pulmonary hypertension (PH), a disease traditionally viewed as limited to the heart and lungs. Given that inflammation is shared by PH and neurodegenerative diseases, we investigated its role in driving brain and cognitive deficits in PH. Using structural and functional MRI data from UK Biobank (N = 248 PH) and a prospective validation cohort, we identified reduced cerebellar white matter volume, thinner frontal gyri, and weakened cerebellar-frontal connectivity in patients with PH but not in other heart or lung diseases. These changes were accompanied by worsened executive function and processing speed. Olink proteomics and targeted genomics linked inflammatory plasma proteins and a PH-associated nuclear receptor coactivator 7 (NCOA7) single nucleotide polymorphism, rs11154337, to these deficits. PH cerebellum showed increased microglial activation and white matter derangement. In PH mice, Ncoa7 deficiency worsened cerebellar inflammation and atrophy. Microglial functional assays, cerebellar single-nucleus RNA sequencing, and spatial transcriptomic analyses further implicated Ncoa7-dependent lysosomal, inflammatory, and cell-specific mechanisms of cerebellar pathobiology. These findings identify NCOA7-linked inflammatory dysfunction in the cerebellar-frontal axis in PH, redefining this disease with neurologic consequences and thus offering neural targets for diagnostic, prognostic, and therapeutic development.
Authors
Satoshi Okawa, Yinan Shi, Somanshu Banerjee, Yi Yin Tai, Jamal Nabhanizadeh, Stefan Guenther, Anisha N. Shah, Siyi Jiang, Timothy Kim, Yassmin Al Aaraj, Caroline Pidro, Metin Aytekin, Omolara Fatukasi, Nishita Kalepalli, Joseph Mettenburg, Timothy D. Girard, James T. Becker, Kathiravan Kaliyappan, Shanshan Song, Sandra Breuils-Bonnet, Steeve Provencher, Sébastien Bonnet, Theodore Huppert, Lloyd D. Harvey, Seyed Mehdi Nouraie, Rajkumar Savai, Dandan Sun, Soban Umar, Soni S. Pullamsetti, Stephen Y. Chan
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ISSN: 0021-9738 (print), 1558-8238 (online)