Research
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
Advanced prostate cancer has increasingly developed a lethal neuroendocrine form, small cell/neuroendocrine prostate cancer (NEPC), as a consequence of the widespread use of highly potent androgen receptor signaling inhibitors in castration-resistant disease. The molecular mechanisms remain unclear and no effective therapies currently exist. We report that tryptophan hydroxylase 1 (TPH1), the enzyme responsible for peripheral serotonin biosynthesis — a neurotransmitter enriched in neuroendocrine tumors and a classical neuroendocrine biomarker — was upregulated in both de novo and therapy-induced human NEPC. TPH1 upregulation was necessary and sufficient for neuroendocrine differentiation and the NEPC phenotype through its enzymatic activity. Silencing TPH1 suppressed neuroendocrine plasticity and various aggressive behaviors of NEPC cells, including proliferation, invasion, sphere formation, and NEPC tumor xenograft growth. Mechanistically, TPH1 activated mTOR via intracellular serotonin-dependent serotonylation of mTOR at glutamine 2453, which triggered the induction of FOXM1 and E2F1 to drive NEPC differentiation and growth. Importantly, pharmacological inhibition of TPH1 using the clinically available inhibitor LX1606 effectively restricted growth and neuroendocrine marker expression in multiple NEPC cell lines and patient-derived xenografts. Collectively, these findings characterize TPH1’s contribution to NEPC and suggest TPH1 as a potential therapeutic target.
Authors
Jing Wei, Jing Wang, Jingrui Chen, Michelle Zhang, Chia-Hui Chen, Tianjie Pu, Alivia O'Brien, Sephtis Hargrove, Eva Corey, Tzu-Ping Lin, Allen C. Gao, Boyang Jason Wu
Abstract
Pharmacologic immunosuppression is essential for preventing organ rejection and controlling autoimmunity, but profoundly impairs humoral immunity, increasing the risk of vaccine failure and infection. The mechanisms by which immunosuppressive therapies disrupt human B cell responses remain poorly defined. Here, we identified dysregulated lipid metabolism as a central determinant of impaired vaccine response in solid organ transplant recipients (SOTRs). Using high-dimensional immune profiling, single-cell transcriptomics, and functional metabolic assays, we found that effective B cell responses required a homeostatic balance between lipid synthesis and fatty acid oxidation. The widely used immunosuppressive agent, mycophenolic acid (MPA) was strongly associated with vaccine non-response and induced excessive lipid synthesis, lipid accumulation, and mitochondrial stress in B cells. In contrast, CD11c+ B cells retained the capacity to differentiate into plasmablasts in the presence of MPA through elevated expression of CPT1A, a mitochondrial fatty acid transporter, and enhanced fatty acid oxidation. These cells were found to be a key feature of early effective vaccine responses in healthy individuals and SOTRs. Notably, pharmacologic inhibition of cholesterol synthesis partially restored plasmablast differentiation in the presence of MPA. These findings identify B cell lipid metabolism as a critical and targetable regulator of human humoral immunity during immunosuppression.
Authors
Elizabeth A. Thompson, Alexis Figueroa, Katerina Roznik, Nicole E. Skinner, Santosh Dhakal, Shuai Li, Luca Biavati, Laura A. Sena, Laila Stoddart, Karli Redinger, Samuel B. Warner, Sabra L. Klein, Nadine Rouphael, Joel N. Blankson, Yolanda Eby, Robert D. Leone, Peter S. Heeger, Mark A. Robien, Christian P. Larsen, Erika L. Pearce, Edward J. Pearce, Hongkai Ji, Andrew H. Karaba, Dorry L. Segev, Aaron A.R. Tobian, William A. Werbel, Andrea L. Cox, Justin R. Bailey
Abstract
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 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, however. Here, we silenced Glp1r neurons in the nucleus of the solitary tract or area postrema (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 non-aversive NTSGlp1r neurons control physiologic satiation they do not contribute to weight loss during GLP1RA treatment. Rather, APGlp1r neurons mediate 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 M. 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
Obesity-associated inflammation impairs pancreatic β-cell function, yet the mechanisms by which immune cells acutely regulate insulin secretion remain poorly defined. Here, we identify myeloid Gq signaling as an immunometabolic node linking macrophage lipid sensing to impaired insulin secretion. Using chemogenetic DREADD-mediated activation of myeloid Gq, we show that acute macrophage Gq activation impairs glucose-stimulated insulin secretion (GSIS) in vivo, whereas myeloid Gαq ablation enhances GSIS. Mechanistically, Gq activation rapidly induced AMPK phosphorylation and sphingolipid remodeling independently of canonical inflammatory cytokines. Macrophage-derived sphingolipids impaired β-cell insulin signaling and GSIS through CD36-PKCζ, while inhibition of CD36, AMPK, or sphingolipid metabolism restored β-cell function. We further identified GPR18, a Gq-coupled endocannabinoid-responsive GPCR, as an upstream regulator. GPR18 activation with N-arachidonoyl glycine (NAGly) recapitulated this phenotype, whereas myeloid Gαq deletion or Gpr18/AMPK silencing abolished it. GPR18 signaling predominantly engaged Gq rather than Gi pathways. In human tissues, GPR18 was enriched in islet macrophages, and NAGly suppressed GSIS in primary human islets. Thus, a conserved macrophage GPR18-Gαq-AMPK-sphingolipid axis dynamically regulates β-cell function and represents a potential therapeutic target in obesity and type 2 diabetes.
Authors
Simran Singh, Ashish Kumar, Sudipta Paul, Mriganka Sarkar, Santhosh Duraisamy, Ganesh Timalsina, Raashidha Farhath, Harender Yadav, Seema Kuldeep, Soumita Bhaumik, Kunj Kumar Prajapati, Saahiba Thaleshwari, Anuj Gargya, Tamojit Santra, Sonal Amit, Rashmi Parihar, Santosh K. Misra, Hamim Zafar, Luiz F. Barella, Michael A. Kalwat, Dharmaraja Allimuthu, Sai Prasad Pydi
Abstract
Successful implantation requires precise coordination of uterine epithelial receptivity, stromal decidualization, and immune homeostasis during a narrow peri-implantation window. Although retinoic acid (RA) signaling has been implicated in female reproduction, the endogenous and isoform-specific roles of retinoic acid receptors (RARs) remain poorly defined. Here, we combined isoform-specific genetic mouse models, transcriptomic profiling, and functional studies in mouse and human stromal cells to determine how RAR signaling regulates early pregnancy. We found that RARG is the dominant RAR isoform required for female fertility in mice, as its deletion severely impaired implantation, whereas combined loss of all RAR isoforms caused complete reproductive failure. RAR deficiency disrupted multiple sequential reproductive processes, including sperm transport and fertilization, suppression of uterine estrogen receptor activity, acquisition of stromal decidualization competence, and maintenance of uterine immune homeostasis. Transcriptomic analyses identified conserved epithelial and mesenchymal programs altered across independent RAR-deficient mouse models and revealed significant overlap with endometrial gene signatures from women with recurrent implantation failure. In human endometrial stromal cells, suppression of the RARA isoform consistently disrupted decidualization across three independent primary cell lines and an immortalized cell model. Together, these findings identify RAR signaling as a critical regulator of early pregnancy and reveal conserved, isoform-specific functions required for early pregnancy.
Authors
Yan Yin, Emily Y. So, Eliana Wolf, Vivian Robles Pinos, Meade Haller, Renjie Shang, Sylvia C. Hewitt, Alex Tak, Brent M. Bany, David Y. Chen, Mengcheng Shen, Francesco J. DeMayo, Liang Ma
Abstract
Microglia play essential yet poorly understood roles in brain development, including axon guidance, regulation of neurogenesis, and pruning of neuronal projections. Congenital hydrocephalus (CH), characterized by enlarged cerebrospinal fluid (CSF)-filled ventricles, is a leading cause of pediatric brain surgery, but its molecular mechanisms remain unclear. We have identified what we believe to be novel, recurrent, damaging missense variants in the SH3-binding domain of the adaptor protein Growth Factor Receptor-Bound Protein 2 (GRB2) in unrelated patients with CH. GRB2 is significantly co-expressed with one of its known upstream receptor tyrosine kinase partners, CSF1R, in the developing human brain, particularly in a microglial subtype associated with regulation of neural stem cells. Immunoprecipitation validated GRB2-CSF1R binding in mouse microglial cells and human monocyte cell line. Cx3cr1-Grb2fl/fl mice engineered with conditional deletion of Grb2 in microglia exhibit congenital absence of microglia and early postnatal severe communicating (non-obstructive) hydrocephalus, mimicking GRB2-mutant patients. The severe ventriculomegaly of Cx3cr1-Grb2fl/fl mice is associated with both depletion of cerebral cortical neurons and impairment of glia-lymphatic-mediated CSF flow. Together, these findings implicate a role of GRB2 in microglia that could be essential for brain development and CSF homeostasis.
Authors
Phan Q. Duy, Benjamin C. Reeves, Huanxing Sun, Xueyan Peng, Pazhanichamy Kalailingam, Garrett Allington, Evan Dennis, Le Thi Hao, Lei Wang, David Rufino-Ramos, Shujuan Zhao, Qiang Li, Neel H. Mehta, William C. Davalan, Mason Blacker, Anthony J. Piscopo, Shozeb Haider, Baojian Fan, Kedous Y. Mekbib, Shuai Shao, Carol Nelson-Williams, TuKiet T. Lam, Benjamin P. Kleinstiver, Patricia L. Musolino, Seth L. Alper, Sheng Chih Jin, Erica L. Herzog, Kristopher T. Kahle
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 monoclonal antibodies (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
Pain is a common and disabling feature of myotonic disorders, yet its biological basis remains poorly understood and no targeted analgesic therapies currently exist. Here, we demonstrate that skeletal muscle hyperexcitability is sufficient to initiate a persistent pain state independent of inflammation, nerve injury, or overt tissue damage. Using complementary pharmacological and genetic models of myotonia resulting from loss of the voltage-gated skeletal muscle chloride channel ClC-1 function, we show that transient and chronic myotonia produce robust mechanical, thermal, and cold hypersensitivity, as well as spontaneous pain-like behavior. Notably, pain-like behaviors induced by transient myotonia persist long after overt motor symptoms have resolved, suggesting that a transient episode of muscle hyperexcitability is sufficient to trigger prolonged alterations in nociceptive processing. Physiological recordings revealed altered excitability of dorsal root ganglion and superficial dorsal horn neurons and enhanced sensory-evoked activity in the parabrachial nucleus, indicating altered nociceptive processing across multiple levels of the pain neuraxis. Transient myotonia increased total sodium current density in sensory neurons, with a shift toward a greater tetrodotoxin-resistant current fraction. Pharmacological inhibition with the NaV1.8-directed analgesic Suzetrigine markedly attenuated pain-like behaviors in both models of myotonia. Together, these findings establish a link between myotonia and persistent alterations in nociceptive processing and identify NaV1.8-directed analgesia as a promising therapeutic strategy for myotonia-associated pain.
Authors
Tyler S. Nelson, Aida Calderon-Rivera, Heather N. Allen, Alaina L Waters, Emanuel Loeza-Alcocer, Jorge B. Pineda-Farias, Narges Pachenari, Santiago Loya-Lopez, Kimberly Gomez, Erick J. Rodriguez-Palma, Paz Duran, Michael S. Gold, Rajesh Khanna
Abstract
Transport of nucleoside chemotherapeutic drugs into tumor cells is primarily accomplished through Equilibrative Nucleoside Transporter 1 (ENT1), considered to be constitutively-active, redistributing drugs across lipid bilayers via facilitated diffusion. Here we discover that ENT1 is not constitutively-active but rather requires activation of acid sphingomyelinase (ASMase) by gemcitabine, generating ceramide-rich platforms (CRPs) on external plasma membranes of endothelial and tumor cells into which ENT1 inserts, dimerizing therein to functionalize transmembrane gemcitabine transport. Whereas sarcoma cells synthesize minimal ASMase, they take up gemcitabine poorly in vitro and in murine xenografts. A strategy designed to augment gemcitabine-induced ASMase secretion into the extravascular space by ASMase-rich neo-angiogenic cells, which then targets tumor cell plasma membranes, yields “bystander” CRPs on sarcoma cells and ENT1 insertion therein, conferring markedly-enhanced gemcitabine uptake and xenograft response. Engaging this biology in a prospective Phase II clinical trial in advanced sarcoma yielded robust volumetric changes in evaluated tumors that developed early and were often durable.
Authors
Aditya Ganju, Shyam Rao, Mark A. Dickson, Robert A. Lefkowitz, Chris Thompson, Jin Cheng, Katia Manova, Adriana Haimovitz-Friedman, Gary Schwartz, Zhigang Zhang, Zvi Fuks, William D. Tap, Richard Kolesnick
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Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)