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Abstract
The human retina and underlying choroid have multiple well-organized vascular layers. However, retinal and choroidal neovascularization can develop in diseases such as diabetic retinopathy or neovascular age-related macular degeneration, which can result in destructive vision loss. Ocular macrophages are implicated in the pathogenesis of such neovascular diseases. To identify a potential pro-angiogenic macrophage subtype, we integrated 13 single-cell RNA sequencing datasets from human and mouse eyes comprising 173 unique samples. From 99,184 mononuclear phagocytes, we identified a single macrophage subtype with high expression of SPP1, FABP5, and IL7R that was over-represented from neovascular disease samples. We fate mapped these cells and specifically depleted them, showing that they are monocyte-derived and necessary for laser-induced choroidal neovascularization in mice. Finally, we performed a high-resolution spatial RNA sequencing experiment on six human donors and found that the pro-angiogenic macrophage subtype localized to human choroidal neovascular membranes. These macrophages and other cells in neovascular membranes expressed MMP14, a potential therapeutic target, and MMP14 inhibition decreased choroidal neovascularization in mice. Collectively, we show that this Monocyte-derived Pro-angiogenic Ocular Macrophage (MoPOM) subtype is upregulated in multiple ocular neovascular diseases across species, necessary for choroidal neovascularization, and is a potential therapeutic target to reduce destructive angiogenesis in the eye.
Authors
Andrew P. Voigt, Joyce Gong, Steven Droho, Kelly Mulfaul, Amrita Rajesh, Greta E. Forbes, Jacob K. Sterling, Miles J. Flamme-Wiese, Robert F. Mullins, Jeremy A. Lavine
Abstract
Splenic myelopoiesis supplies immunosuppressive myeloid cells in cancer, yet its local regulation remains unclear. Here we identify a self-amplifying circuit between neutrophils and hematopoietic stem and progenitor cells (HSPCs) that sustains splenic myelopoiesis. Tumor-associated neutrophils produced reactive oxygen species (ROS) and created an oxidatively stressed milieu in the spleen, which activated FOXO1 in neighboring HSPCs and drove DRP1-mediated mitochondrial fission, reprogramming HSPCs toward myeloid-biased expansion and immunosuppressive output. Progeny neutrophils reinforced splenic oxidative stress and perpetuated this loop. Genetic ablation of Cybb or spleen-targeted ROS scavenging inhibited FOXO1 activation and preserved fused mitochondria morphology in HSPCs. Consistently, blockade of FOXO1 or DRP1 prevented mitochondrial fission and reduced ROS-producing myeloid cell generation. Such interventions reduced splenic myelopoiesis, curtailed the production of suppressive myeloid cells in the spleen and their infiltration into tumors, and enhanced cytotoxic T cell activity, thereby restoring antitumor immunity and restraining tumor progression. Analyses of spleen samples from cancer patients, together with cord-blood HSPC and neutrophil co-culture experiments, support the human relevance of this neutrophil–ROS–HSPC axis. These findings reveal a self-propagating neutrophil–HSPC feedback circuit that locally sustains tumor-promoting splenic myelopoiesis and provide a rationale for targeting neutrophil-to-progenitor signaling to normalize antitumor immunity.
Authors
Lan Zhu, Shufeng Luo, Qiaomin Hua, Chang-An Zhao, Huiling Lin, Mingyu Liu, Lingyan Zhu, Jiabin Zheng, Huolun Feng, Yong Li, Chong Wu, Limin Zheng
Abstract
BACKGROUND. The penile urethra is a primary site of HIV acquisition in males, although little is known about the urethral determinants of HIV susceptibility. Inflammation at other genital sites enhances HIV risk, and studies suggest that some inflammatory urethral bacteria may be vaginally acquired. Here, we characterize the impact of sexual debut on the microbiome and immune milieu of the penile urethra in adolescent males from Rakai, Uganda. METHODS. A cohort of 185 self-reported sexually naïve adolescent males from Rakai, Uganda were followed longitudinally for 3 years, with questionnaires administered every 3 months and urethral swabs collected annually. Urethral soluble immune factors were quantified by chemiluminescent multiplex immunoassay and bacterial 16S rRNA sequencing was performed. Unsupervised clustering and mixed-effects models were used to assess associations between sexual debut and the urethral microbiome and immune milieu. RESULTS. Unsupervised clustering at 36 months identified two distinct urethral Community State Types (CSTs): CST-1 was dominated by Streptococcus mitis and CST-2 by bacteria associated with bacterial vaginosis (BV), particularly Sneathia amnii and Gardnerella vaginalis. CST-2 was greatly enriched at 36 months compared to baseline and was associated strongly with sexual debut. Individual bacterial taxa enriched in CST-2 were associated with both sexual debut and elevated urethral inflammatory cytokines, independent of serum testosterone and penile circumcision status. CONCLUSION. The penile urethra is colonized by BV-associated bacteria after penile-vaginal sex debut. Their presence in the urethra induces local inflammation and may serve as a reservoir for subsequent reintroduction into the vagina of female sexual partners.
Authors
Rameen Jamil, Sanja Huibner, Ping Yang, James Nnamutete, Jodie L. White, Lori Sokoll, James Pollock, Margaret Nakalanzi, Simon Peter Bukenya, Stephen D. Kiboneka, Mathias Agaba, Mary J. Nalubowa, Aggrey Anok, Godfrey Kigozi, Ronald M. Galiwango, Bryan Coburn, Cindy M. Liu, Jessica L. Prodger, Aaron A.R. Tobian, Rupert Kaul
Abstract
Alcohol-associated liver disease (ALD) is associated with higher incidence of colorectal liver metastasis (CRLM); prohibitin 1 (PHB1) defends against CRLM yet its role in ALD and ALD-CRLM is poorly understood and was investigated here. In human, murine, and in vitro ALD models, cytosolic and nuclear PHB1 were depleted, whereas mitochondrial PHB1 was preserved despite a loss in mitochondrial mass. Nuclear PHB1 loss was driven by CRM1-mediated export, and blocking this interaction with a PHB1 nuclear export signal (NES) peptide retained nuclear PHB1, ameliorating both ALD and ALD-CRLM. PHB1 NES prevented depletion of nuclear methionine adenosyltransferase α1, oxidative stress, and induction of matrix metalloproteinase 7 (MMP-7) and multiple oncogenes. Single-nucleus RNA sequencing of NIAAA diet-fed mouse livers and spatial transcriptomics of ALD-CRLM mice livers revealed that PHB1 NES treatment mitigated alcohol-induced metabolic and oncogenic stress, attenuated pro-inflammation and pro-fibrogenic responses, remodeled immunosuppressive microenvironment and contributed to overall hepatoprotection. These findings identify nuclear PHB1 depletion as a driver and a promising therapeutic target in ALD and ALD-CRLM.
Authors
Jyoti Chhimwal, Sonal Sinha, Jina Kim, Mario Alba, Lucía Barbier-Torres, Jiaohong Wang, Heping Yang, Youngyi Lim, Ramachandran Murali, Maria Lauda Tomasi, Sungyong You, José M. Mato, Shelly C. Lu
Abstract
Type 2 (T2) inflammation is a defining feature of asthma and associated with epithelial dysfunction, including goblet cell metaplasia and ciliary loss. Here, we investigated whether T2 inflammation and associated 15 lipoxygenase-1 (15LO1) activity alters mitochondrial metabolism in airway epithelial cells (AECs) and contributes to disease pathophysiology. Freshly isolated AECs from T2-high asthmatic participants exhibited increased oxygen consumption rates (OCR), including elevated basal respiration and proton leak, alongside reduced coupling efficiency compared to T2-low participants. These functional changes were accompanied by increased expression of electron transport chain (ETC) proteins, worse lung function and epithelial phenotypic changes. In vitro, IL-13 stimulation of differentiated AECs recapitulated these findings, inducing higher OCR, increased ETC protein levels, and enhanced proton leak while paradoxically reducing intracellular ATP. These metabolic alterations are associated with increased uncoupling protein 2 (UCP2) expression and goblet cell differentiation. Mechanistically, 15LO1 emerged as a key regulator, as ALOX15 knockdown or pharmacologic inhibition reduced OCR parameters, ETC protein expression, UCP2, and goblet cell markers, without effect on ATP levels, suggesting additional regulatory mechanisms. Collectively, these findings identify a metabolic axis linking T2 inflammation, mitochondrial dysfunction, and airway epithelial metabolism, while highlighting 15LO1-driven mitochondrial uncoupling as a potential therapeutic target in asthma.
Authors
Xiuxia Zhou, Krithika S. Rao, Sruti Shiva, Catharina van Heusden, Qi Wei, Nobuhiko Fukuda, Mayoko Tsuji, Angelina Kendi, Laura R. Bradley, Jinming Zhao, John B. Trudeau, Anuradha Ray, Richard C. Boucher, Sally E. Wenzel
Abstract
Hypoxia-inducible factor 2α (HIF-2α) is a central oncogenic driver in clear cell renal cell carcinoma (ccRCC) and a therapeutic target of the small-molecule inhibitor belzutifan. Genetic studies in murine models have suggested that hypoxia signaling may support T cell effector programs, raising concern that HIF-2α inhibition could impair antitumor immunity. However, whether pharmacologic HIF-2α inhibition alters human T cell biology remains unknown. Here, we investigated the cell-intrinsic effects of EPAS1 (encoding HIF-2α) perturbation in primary human T cells. CRISPR/Cas9-mediated deletion of HIF-2α demonstrated no notable transcriptional effects. Similarly, pharmacologic treatment with belzutifan produced minimal transcriptional changes and did not impair proliferation, cytokine production, polyfunctionality, or cytotoxic activity in T cells derived from healthy donor peripheral blood, peripheral blood from patients with ccRCC, or tumor-infiltrating lymphocytes under hypoxic conditions. High- dimensional immunophenotyping revealed preserved T cell differentiation and activation states following pharmacologic HIF-2α inhibition in vitro and in peripheral blood from patients receiving HIF-2α inhibitor therapy. Together, these findings demonstrate that pharmacologic HIF-2α inhibition preserves key effector programs, providing a mechanistic basis for the immunologic safety of HIF-2α–targeted therapy in ccRCC.
Authors
Katrine N. Madsen, Soki Kashima, Hanna Soulati, Lena Wirth, Katsuhiro Ito, Zachary A. Yochum, Vivien Moritz, Julia Walker, Marc Machaalani, Fady Ghali, Patrick Kenney, Adebowale Adeniran, Michael Hurwitz, Toni K. Choueiri, David A. Braun
Abstract
During organogenesis, stem cells undergo cellular and metabolic remodelling, facilitated by autophagy-mediated turnover of organelles. Autophagy impairment has been linked to human diseases, including neurodevelopmental disorders associated with disrupted neural stem/precursor cell (NPC) homeostasis, but the underlying mechanisms and pathogenic processes governing these connections remain poorly understood. Here, we report three de novo variants of uncertain significance (p.Gly223Asp, p.Gly889Glu, p.Met978Val) in the deubiquitinase USP15 in human probands with diverse clinical features, including a spectrum of brain malformations and metabolic phenotypes. Proband variants differentially altered USP15 activity and nucleocytoplasmic localization. USP15 showed dynamic localization in NPCs of embryonic mouse cerebral cortex. Using a knock-in mouse model carrying the p.Met978Val variant, we showed that aberrant cytoplasmic accumulation of USP15, but not its loss-of-function, impaired NPC self-renewal and differentiation, leading to reduced neuronal output and enlarged lateral ventricles. Mechanistically, USP15 deubiquitinated autophagy regulator ATG16L1, impeded its normal turnover, and impaired autophagy. Concurrently, lipid droplet mobilization and mitochondrial dynamics were attenuated. Reestablishing the ubiquitination-deubiquitination balance restored autophagy activity and normal neurogenesis. Our findings suggest that nucleocytoplasmic shuttling of USP15 creates a switch-like autophagy signal controlling NPC homeostasis, and its disruption may contribute to the pathogenesis of complex neurodevelopmental conditions.
Authors
Kaylan M.L. Burns, Mashiat Zaman, Daniel Young, Michelle Hua, Yu-Yun Gao, Yvonne Yan Yan Or, Farzaneh Nobakht, Bryn D. Webb, Julie A. Jurgens, Francis Ramond, Xing-Chang Wei, Caroline D. Robson, Alistair T. Pagnamenta, Marvel Megaly, Belal Tafech, Antoine Dufour, Timothy E. Shutt, Ping Yee Billie Au, Guang Yang
Abstract
Familial Episodic Pain Syndrome (FEPS) is a rare inherited disorder characterized by recurrent episodes of severe upper-body pain triggered by stimuli such as cold, stress, or fasting. A gain-of-function mutation (N855S) in the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel has been identified in affected individuals, yet the cellular mechanisms driving FEPS pain remain poorly defined. Although TRPA1 has been primarily studied in sensory neurons, emerging evidence suggests that non-neuronal cells may contribute to pain signaling. To define the cell-specific role of mutant TRPA1 (TRPA1mut), we generated mouse models selectively expressing TRPA1mut in Schwann cells or sensory neurons, using CRISPR-based and Cre-loxP strategies. Patch-clamp recordings confirmed enhanced agonist-evoked currents in TRPA1mut compared with wild-type channels. Behavioral analyses revealed that neuronal TRPA1mut mediates acute nociceptive responses, whereas Schwann cell TRPA1mut drives mechanical allodynia induced by subthreshold TRPA1 agonists and physiological triggers relevant to FEPS, including fasting, cold exposure, and restraint stress. These responses were associated with increased reactive oxygen species and accumulation of 4-hydroxynonenal in sciatic nerves and were attenuated by antioxidant treatment. These findings uncover a previously unrecognized Schwann cell-dependent mechanism of TRPA1-driven pain and identify non-neuronal TRPA1 signaling as a potential therapeutic target in FEPS and related pain channelopathies.
Authors
Matilde Marini, Martina Chieca, Elisabetta Coppi, Marco Montini, Lorenzo Bonacchi, Lorenzo Landini, Irene Scuffi, Kelvin Y. Kwan, Alice Papini, Gaetano De Siena, Elisa Bellantoni, Lucrezia Timotei, Elena Colzi, Valentina Albanese, Gabriele Ferroni, Evelynn D. N. Melo, Marie-Christine Birling, Romain Lorentz, Romina Nassini, Francesco De Logu
Abstract
Background. Myelodysplastic syndromes (MDS) are characterized by aberrant DNA methylation, and mutations in epigenetic modifiers are frequently found in these patients. Although DNA methyltransferase inhibitors (DNMTi) are used to treat MDS, response variability remains a challenge in the clinic, with limited predictive markers. Methods. We integrated genomic, epigenomic, and transcriptomic analyses of 98 MDS patients. Patients were classified into epigenetic subtypes via hierarchical clustering. Random forest classifiers were developed and validated using internal stratified testing and an independent external cohort to predict AZA response. Results. MDS is characterized by widespread DNA hypomethylation affecting distal regulatory elements. We identified seven epigenetic clusters correlated with distinct molecular drivers. Notably, Cluster VI exhibited low mutational burden but a high AZA response rate of 71% (P ≤ 0.01). While transcriptional profiles alone failed to distinguish responders, a DNAme-based classifier achieved an area under the curve (AUC) of 0.82. An integrative model combining DNAme, gene expression, mutations, and clinical parameters achieved an AUC of 0.93 in internal validation and 0.88 in the external cohort. Conclusion. Epigenetic signatures at distal genomic elements provide superior predictive power for AZA response compared to promoter-centric or transcriptional analyses. These findings establish a robust framework for personalized treatment strategies in MDS.
Authors
Qin Yang, Miguel Torres-Martin, Masataka Taguchi, Alice Brogi, Irene Casalin, Eleonora Ceneri, Stephanie Halene, Amy E. DeZern, Elizabeth A. Griffiths, Matilde Y. Follo, Carlo Finelli, Jerald P. Radich, Michael J. Rauh, Rafael Bejar, Mikkael A. Sekeres, Valeria Santini, Maria E. Figueroa
Abstract
Despite enormous advances in clinical genomics, idiopathic scoliosis remains an enigmatic condition with poorly understood genetic and pathophysiological underpinnings impeding molecular diagnosis and the development of targeted treatments. We performed linkage analysis, exome, genome and short- and long-read RNA sequencing in a multigenerational family affected by autosomal dominant early-onset scoliosis (EOS) with a unique pattern of spondylodysplastic elements and progressive endplate erosion and identified the LRR binding FLII interacting protein 1 gene (LRRFIP1) as the disease-causing gene. The underlying cause is a rare noncoding variant altering transcription factor binding of NR3C1 (glucocorticoid receptor) leading to changes in LRRFIP1-isoform expression. Transcriptomic changes in fibroblasts of affected individuals indicated a combination of disturbed Wnt-signaling during somitogenesis, planar cell polarity signaling and postnatal inflammatory dysregulation with clinical and molecular overlaps with Ankylosing Spondylitis and Scheuermann kyphosis. We conducted a rare variant enrichment analysis using genome data from 484,903 UK Biobank participants and found an enrichment of rare risk-increasing LRRFIP1-variants in individuals with scoliosis. Our analysis of an Lrrfip1tm1.1(KOMP)Wtsi KO mouse model showed increased prevalence of idiopathic kyphoscoliosis in Lrrfip1-deficient mice. Our work provides insights into the pathophysiology of rare and common spinal disorders and hints to potential future therapeutic approaches with selective NR3C1-inhibitors.
Authors
Tanja Frey, Elena M. Cabello, Gabriele Siegel, Carla Bello, Rike Schiller, Martina A. Trippel, Neguin Ranjbar, Paranchai Boonsawat, Stephanie E. van Gijn, Ivan Ivanovski, Michael Papik, Markus Zweier, Kan Min, Katharina Steindl, Anita Rauch
Abstract
Recent studies have revealed that, beyond their classical role in platelet production, megakaryocytes (MKs) express immune-related genes and exert important immunoregulatory functions. However, it remains unclear whether these functions arise from a single versatile population or from distinct specialized subtypes, and how such subtypes influence infection and inflammation. Here, we identified three specialized immune MK subtypes (imm-MKs)—macrophage-like MKs (Mac-MKs), neutrophil-like MKs (Neu-MKs), and antigen-presenting MKs (APC-MKs)—each defined by distinct transcriptional programs and regulatory networks, with comparable heterogeneity observed in human MKs. Developmental analyses showed that MK immune-related programs increased with maturation and that immune MK subtypes exhibited distinct tissue- and stage-dependent patterns. Functionally, MK subtypes exhibited phase-specific responses during bacterial pneumonia: early infection preferentially induced Mac-MKs and Neu-MKs, which contributed to pulmonary inflammation, whereas during the post-peak acute-to-early-recovery stage, APC-MKs supported a Treg-associated regulatory program that contributed to pulmonary inflammatory control. This MK–Treg axis uncovers a previously unrecognized mechanism of hematopoietic–immune crosstalk. Collectively, our study delineates organ- and stage-specific immune specialization of MKs and identifies immune MK subtypes as dynamic contributors to phase-specific inflammatory and Treg-linked regulatory programs during development and infection.
Authors
Huizhen He, Yezi Ma, Yifei Cai, xiaoyuan chen, Tianran Cheng, Ziqi Huo, Sibei Guo, Meijuan Xia, Dan Feng, Minmin Li, Jingjing Zhao, Nananan Zhao, Pei Su, Wen Zhou, Fei Wang, Cuicui Liu, Hongtao Wang, Jiaxi Zhou
Receptor-tuned Interleukin-2 Mutein Decouples Treg Expansion from ILC2-driven Cutaneous Inflammation
Abstract
Low-dose interleukin-2 (IL-2) and IL-2 muteins are being developed to expand regulatory T cells (Tregs) for autoimmune disease therapy, but injection site reactions (ISRs) remain frequent and poorly understood. Here we show IL2Mut24, a murine surrogate of IL-2 receptor α (IL-2Rα, CD25)-biased IL-2 mutein, enhances Treg expansion yet paradoxically exacerbates cutaneous inflammation compared to wild-type IL-2. Using immunodeficient mice, antibody blockade and adoptive transfer, we identify group 2 innate lymphoid cells (ILC2s) as key drivers of IL-2-induced skin inflammation, defining a CD25-dependent innate activation axis that constrains immune tolerance. In cynomolgus monkeys, the CD25-biased IL-2 mutein efavaleukin alfa promotes greater peripheral Treg expansion than aldesleukin (recombinant human IL-2) but is accompanied by transient increases in IL-5 and dose-dependent ISRs, indicating conservation of this innate inflammatory program across species. To improve the therapeutic window, we engineer receptor clamps by linking IL2Mut24 to antibodies against CD25 to restrict IL-2 access to CD25. This receptor-tuned IL-2 preserves Treg selectivity, suppresses ISRs, and outperforms IL2Mut24 in experimental autoimmune encephalomyelitis by restraining Th17 responses. These findings reveal a conserved innate mechanism underlying IL-2–associated toxicity and establish receptor-tuning as a strategy to improve the safety and efficacy of IL-2–based immunotherapy.
Authors
Anupama Sahoo, Cody Moorman, Mina Tsenkova, Yi Jing, Alexis Valdovinos, Xin Luo, Shiping Lu, Songyu Wang, Renee R. Hukkanen, Madeline Fort, Helen S.H. Tang, Ronya Primack, Shweta Mandavalli, Weiwen Deng
Abstract
Atrial fibrillation (AF) is the most common sustained human cardiac arrhythmia and linked to a drastic increase in stroke and heart failure risk. While sequence variations in the PITX2 non-coding region are the strongest genetic signature of AF risk, the direct role of PITX2 in AF remains a topic of debate. Here, we generated a mouse model (Pitx2Pro41Ser) of a human PITX2 coding variant linked to increased AF risk in the Finnish population. The Pitx2Pro41Ser mice exhibit near-complete penetrance of pacing-induced AF, and transcriptional profiling indicates that Pitx2Pro41Ser is a loss-of-function mutation. In vivo cleavage under targets and tagmentation (CUT&Tag) reveals that PITX2 acts as a transcriptional repressor in developing left atrial cardiomyocytes independent of DNA methylation. Ectopic Pitx2 expression in postnatal right atrial cardiomyocytes via adeno-associated virus (AAV) delivery or genetic overexpression represses right atrial genes and induces a left atrial transcriptome, revealing unexpected plasticity of postnatal atrial cardiomyocytes. Strikingly, delivery of Pitx2 AAV into Pitx2Pro41Ser mice rescues AF inducibility uncovering a direct link between PITX2 activity and AF susceptibility.
Authors
Jeffrey D. Steimle, Yue Yuan, Shaohai Fang, Vaibhav Deshmukh, Christine Rodriguez, Fansen Meng, Taotao Tan, Md. Abul Hassan Samee, Yun Huang, Na Li, James F. Martin
Abstract
Chronic kidney disease (CKD) disrupts mineral homeostasis, leading to impaired skeletal mineralization and cardiovascular pathology, yet the mechanism linking these processes remains undefined. Here we identify glycerol-3-phosphate acyltransferase 2 (GPAT2) as a regulator that couples free fatty acid (FFA) partitioning in osteoblasts to systemic phosphate balance. In mouse and human CKD, elevated osteoblast GPAT2 routes FFA away from mitochondrial oxidation, limiting phosphate incorporation into bone, and toward lysophosphatidic acid synthesis, increasing production of the phosphaturic hormone fibroblast growth factor 23 (FGF23). By contrast, osteoblast-specific Gpat2 deletion restores osteoblast FFA oxidation and skeletal phosphate incorporation, lowers circulating phosphate and FGF23, and attenuates vascular calcification and cardiac hypertrophy in CKD. Further, a bone-targeted GPAT inhibitor recapitulates most of these beneficial effects. These findings establish a fundamental role for osteoblast lipid metabolism in mineral homeostasis and identify GPAT2 in bone as a promising therapeutic target for both skeletal and cardiovascular complications of CKD.
Authors
Petra Simic, Han Xie, Wen Zhou, Yu Fan, Renata C. Pereira, Fangcong Dong, Jason D. Roh, Isidro B. Salusky, Charandeep Singh, Russell P. Goodman, Ashok Khatri, Eugene P. Rhee
Abstract
Authors
Tony Yao, Rita E. Chen, Melissa Yamada, J. Russell Moore, Marissa Jimenez, Tammy Huang, Lynn Cornelius, George Ansstas, Naresha Saligrama, David Y. Chen
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
Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare condition causing nephrotic syndrome, neuropathy, and other manifestations. SPLIS is caused by mutations in SGPL1, which encodes sphingosine-1-phosphate lyase (SPL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme needed to degrade the bioactive sphingolipid sphingosine-1-phosphate (S1P). Supplementation with the PLP precursor pyridoxine benefits some individuals with PLP-dependent enzymopathies. We investigated whether pyridoxine has therapeutic activity in SPLIS. Neurological improvement, plasma S1P normalization, and increased SPL activity in patient-derived fibroblasts were observed after pyridoxine supplementation in a patient with R222Q-variant SPLIS. Additionally, PLP dose-dependently augmented recombinant R222Q-variant SPL activity. To further explore pyridoxine’s effects, gene editing was employed to create an R222Q-variant SPLIS mouse model. SPLR222Q mice fed pyridoxine-enriched chow lacked obvious phenotypes. However, SPL inactivation, S1P accumulation, proteinuria, and glomerulosclerosis developed in SPLR222Q but not WT mice fed chow with reduced pyridoxine. Ultrastructural analysis and super-resolution microscopy showed podocyte loss and foot process effacement. Transcriptional profiling revealed patterns of cytokine upregulation and extracellular matrix remodeling. Inhibiting S1P production or RhoA/ROCK signaling prevented nephrosis in SPLR222Q mice fed chow lacking pyridoxine. Our findings establish a SPLIS mouse model that recapitulates R222Q-variant SPLIS, demonstrates its responsiveness to pyridoxine, and implicates a S1P/RhoA/ROCK pathway in its pathophysiology. Running Title: Cofactor supplementation in R222Q-variant SPLIS
Authors
Ranjha Khan, Maria L. Allende, Ehtesham Khalid, Joanna Y. Lee, Everett Stone, Max Rodnick-Smith, Audrey Izuhara, Vadym Buncha, Georgina Gyarmati, Janos Peti-Peterdi, Ranya Al-Khaledy, Jeffrey B. Hodgin, Gizachew Tassew, Babak Oskouian, Alina Chen, Yu-Tang Chang, Rachel Zhang, Richard L. Proia, Julie D. Saba
Abstract
Metabolic dysfunction-associated steatohepatitis (MASH) is rising globally despite recent therapeutic advances, highlighting the need to identify new targetable pathways. While dysregulated lipid and amino acid metabolism are established features of MASH, the interplay between these two metabolic pathways remains unexplored. Here, metabolomics of livers from humans and mice with MASH uncovered depletion of lipidated amino acids, where these two distinctive pathways converge. Notably, hepatic levels of N-oleoyl-leucine (C18:1-Leu) were inversely correlated with the severity of MASH-fibrosis. The C18:1-Leu-regulating enzyme, peptidase M20 domain containing 1 (PM20D1), was suppressed in MASH due to attenuated de novo transcription, and stable-isotope tracing confirmed impaired hepatic biosynthesis of C18:1-Leu in MASH. Hepatocyte-specific PM20D1 ablation lowered hepatic C18:1-Leu and exacerbated MASH, whereas hepatocyte-specific PM20D1 overexpression restored C18:1-Leu and ameliorated both MASH progression and established disease. Exogenous administration of C18:1-Leu similarly ameliorated MASH-fibrosis. In silico modeling, transcriptomics, metabolic flux analyses and hepatocyte-specific in vivo manipulations revealed that C18:1-Leu binds and activates peroxisome proliferator-activated receptor alpha to suppress C-C motif chemokine ligand 2, concurrently enhancing fatty acid β-oxidation and attenuating monocyte recruitment to reduce MASH-fibrosis. These findings highlight C18:1-Leu deficiency as a driver and a therapeutic target in MASH-fibrosis, warranting further clinical evaluation.
Authors
Sumit Kumar Anand, Sandeep Das, Fabio Arias, Koral S.E. Richard, Sumati Rohilla, Alia Ghrayeb, M. Peyton McKinney, Lu Wang, Lin Tan, Jibin Ding, Dhananjay Kumar, Nilesh Pandey, Jennifer Lee, Ying Zhao, Suman Mohajan, Gurranna Male, Kelley Nunez, Alexandra C. Finney, Brenna H. Pearson-Gallion, Reethika Gade, Jemiah Maxie, Yu Liu, Harpreet Kaur, Rajan Pandit, Cyrine Ben Dhaou, Joseph Eniafe, Xiaolu Zhang, Zhipeng Liu, Andrew D. Yurochko, Minerva T. Garcia-Barrio, Babak Razani, Fei Chang, Francisco J. Schopfer, Xin Huang, Bishuang Cai, Eyal Gottlieb, Bo Wen, Duxin Sun, Wanqing Liu, Paul T. Thevenot, Ari J. Cohen, Jifeng Zhang, Y. Eugene Chen, A. Wayne Orr, Nirav Dhanesha, Arif Yurdagul Jr., Oren Rom
Abstract
Receptor activator of NFκB ligand (RANKL) is important for bone metabolism, but also modulates immune processes. We showed that mast cells (MCs) are involved in RANKL regulation, but the importance of MC-derived RANKL in skin inflammation has not yet been investigated. In contact hypersensitivity (CHS), the absence of MC-derived RANKL led to reduced skin inflammation due to impaired leukocyte infiltration and blood lymphopenia. Surprisingly, we observed a massive hyperplasia of the distant inguinal lymph nodes in the absence of MC-RANKL. Using adoptive transfers, flow cytometry and whole-mount 3D imaging, we demonstrated that this was not caused by structural maladaptation, but rather by the inability of lymphocytes to exit in a timely manner. Importantly, RANKL deletion in skin MCs only replicated the effect of LN hyperplasia and blood lymphopenia. Moreover, MCs were involved in serum sphingosine-1-phosphate (S1P) regulation during sensitization and challenge. Intravascular administration of S1P restored timely lymphocyte egress, demonstrating a MC-induced organ-spanning RANKL-S1P axis. Consequently, peripheral skin MC-derived RANKL is essential for the timely lymphocyte egress from distant LNs, which may have important implications for the targeted treatment of inflammatory skin diseases.
Authors
Konstantinos Katsoulis-Dimitriou, Waqar Umer, Laura Knop, Tanja Schickschneit, Aaron Hoffmann, Ali El-Bizri, Lea M. Schmitter, Kathleen Baumgart, Nouria Jantz-Naeem, Vladyslava Dovhan, Charlotte Heidelbach, Lars Philipsen, Andreas J. Müller, Stephan Fricke, Sascha Kahlfuß, Thomas Schüler, Jan Dudeck, Anne Dudeck
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 potential therapeutic targets for sebaceous gland-related diseases.
Authors
Yumeng Zhang, Pernelle Pulh, Michelle F. Pan, Yi He, Juanzhu Yan, Annie Li, Renée M. McKay, Lu Q. Le
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)