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The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemms canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type-specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-{beta} signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1, suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.
Early childhood stunting (ECS) affects millions of children globally and conjectured to result in suboptimal brain and cognitive development later in life. Charting out the trajectory of brain network development and most importantly how the compensation of function can be achieved gives windows of intervention to clinicians, educators and policy makers. In this study, advanced network neuroscience tools, graph theoretical methods applied on diffusion weighted MR-imaging (DWI) revealed the effects of ECS on white matter (WM) organization in a community-based birth cohort of 170 children (mean age = 9.18 years, SD = 0.28) from Vellore, India. Based on stunting status at ages two, five, and nine years, children were categorised into four groups: always stunted (AS; n=21), stunted until five with catch-up at nine (S5C9; n=31), stunted until two with catch-up at five (S2C5; n=28), and never stunted or typically developing (TD; n=90). The catch-up groups showed strikingly similar anthropometric measures compared to TD. However, all stunted groups (AS, S5C9, S2C5) showed significantly lower performance and verbal IQ scores compared to TD children. DWI data revealed AS children exhibited shorter tract lengths across select cortico-cortical connections, an increased number and strength of short- and medium-range connections, and a corresponding reduction in long-range connections and strength. Network analyses further revealed that AS and S5C9 groups displayed higher local clustering and local efficiency relative to TD, reflecting greater local segregation of brain networks. Hub-like modules was broadly conserved across groups, although both catch-up groups (S2C5 and S5C9) showed additional hubs, suggesting compensatory network reorganization via a more modular architecture. Together, these findings provide novel evidence that ECS is linked to altered structural reorganization of brain networks and reduced cognitive performance in later childhood. While persistent stunting (AS) is associated with the most pronounced alterations, partial catch-up (S2C5 and S5C9) is accompanied by compensatory adaptations, such as increased short-range connectivity and recruitment of additional hubs. These results underscore the critical importance of early nutritional interventions to support optimal brain network development.
Cancer cell dissemination critically determines clinical prognosis, yet metabolic dependencies and corresponding therapeutic targets during spread of lymphoid malignancies remain poorly understood. Here we show that the pyruvate branch point operates as a metabolic checkpoint for lymphoid cancer cell migration and disease dissemination through mitochondrial ROS (mROS)/HIF-1a signaling. Isolation of highly migratory mROShi cells led us to identify selective metabolic requirements of malignant lymphocyte migration and disease dissemination. Highly migratory cells show a reprogrammed metabolic profile characterized by increased glucose uptake and reduced glucose-carbon entry into the TCA cycle. Reprogramming of the TCA cycle with downregulation of citrate synthase provide the mechanistic basis for decreased pyruvate oxidation leading to increased migration and disease dissemination through mROS/HIF-1a signaling. Our findings connect central carbon metabolism and migratory capacity of lymphoid cancer cells and identify the pyruvate branch point as a metabolic switch and potential therapeutic target in lymphoid cancer cell dissemination.
Dysregulation of Phospholipase D1 (PLD1) has been implicated in the progression of neurodegenerative diseases, including Alzheimers Disease (AD). This study investigated PLD1 signaling in synaptic integrity and cognition during aging and in a late-onset AD mouse model, hypothesizing differential effects of PLD1 modulation on synaptic vulnerability. AAV2-mediated gene transfer was employed to overexpress (PLD1 OXP) or attenuate (PLD1 ATT) PLD1 in aged wild-type (WT) and 3xTg-AD mice. To validate these constructs, differentiated PC12 cells were utilized. Within these cells, a model of post-mitotic neurons, PLD1 OXP exhibited a notable reduction in both the average neurite length and the percentage of neurite-bearing cells. This suggests that elevated PLD1 activity exerts a significant influence on neurite outgrowth. Conversely, PLD1 ATT did not inhibit neurite formation, indicating it is not detrimental at the cellular level. These cellular findings paralleled in vivo observations - electrophysiological studies revealed PLD1 OXP impaired long-term potentiation (LTP) and synaptic transmission, particularly in aged WT mice, whereas PLD1 ATT improved synaptic function in 3xTg-AD mice. Behaviorally, PLD1 ATT enhanced spatial working memory and reduced anxiety-like behavior, notably in 3xTg-AD mice. These results highlight that tight PLD1 regulation is vital for maintaining synaptic integrity and cognitive resilience. Thus, PLD1 attenuation may serve as an important complement to immunotherapeutic approaches by strengthening synaptic resilience in neurodysfunctional states, including AD and related dementia (ADRD).
The metabolic importance of brown adipose tissue (BAT) has been recognized, but the origins of BAT, particularly supraclavicular BAT (scBAT), are less clear. Here, we traced the origin of scBAT to Mef2c-anterior heart field (AHF)-marked cells. Mef2c-AHF-marked cells isolated from scBAT can spontaneously differentiate into brown adipocytes, express mesenchymal stem cell markers, and can be isolated from the stromal-vascular fraction (SVF) of wild-type scBAT as [CD31-CD45-SCA-1+CD29+CD34-CD24-] (CD34-) cells. Mef2c-AHF-marked cells substantially overlap with Prrx1-marked cells in scBAT, which also contribute to beige adipocytes in inguinal white adipose tissue (iWAT) during development. Similarly, CD34- cells isolated from the SVF of iWAT can spontaneously differentiate into beige adipocytes in vitro. Using intersectional tracing, we revealed that common progenitors of Mef2c-AHF- and Prrx1-marked cells in scBAT emerge from the developing heart. Thus, these studies reveal a common origin for scBAT and cardiac tissue and a close lineage relationship between scBAT and beige adipocytes of the iWAT.
Morphogenesis of the anterior segment (AS) is crucial for healthy ocular physiology and vision but is only partially understood. The Schlemms canal (SC) and trabecular meshwork (TM) are essential drainage tissues within the AS, and their proper development and function are critical for maintaining normal intraocular pressure; abnormalities in either tissue can result in elevated pressure and glaucoma. Here, we use single-cell transcriptomic profiling to provide high-resolution molecular detail of AS development with a particular focus on SC and TM. We report transcriptomes for [~]130,000 single cells at key developmental stages from postnatal day 2 (P2) to P60. We provide the first annotation of cell types across these developmental stages and crucial information about dynamic changes in pathways/gene expression. Further, we trace developmental trajectories for TM cell and SC endothelial cell (SEC) subtypes and determine genes and signaling networks driving their specific cell fates. We demonstrate dynamic changes in signaling interactions between SC and the TM cells during their synchronized development. Collectively, our data lay a deep molecular foundation for AS development that will direct understanding of normal ocular physiology, glaucoma, and other AS conditions.
A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve=0.579), they offered marginal prediction improvement in PRS-only-based models (P=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites--lactate (P=8.8E-12), pyruvate (P=1.9E-10), and citrate (P=0.02)--linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (Pinteraction=0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratiohighest vs. lowest total resilience metabolite quartile=0.71, 95% Confidence Interval=0.64-0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (P=0.002) and optic nerve damage (P<0.0003) in Lmx1bV265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.
Na+/Ca2+ exchangers (NCXs) transport Ca2+ across the plasma membrane in exchange for Na+ and play a vital role in maintaining cellular Ca2+ homeostasis. Our previous structural study of human cardiac NCX1 (HsNCX1) reveals the overall architecture of the eukaryotic exchanger and the formation of the inactivation assembly by the intracellular regulatory domain that underlies the cytosolic Na+-dependent inactivation and Ca2+ activation of NCX1. Here we present the cryo-EM structures of HsNCX1 in complex with a physiological activator phosphatidylinositol 4,5-bisphosphate (PIP2), or pharmacological inhibitor SEA0400 that enhances the inactivation of the exchanger. We demonstrate that PIP2 binding stimulates NCX1 activity by inducing a conformational change at the interface between the TM and cytosolic domains that destabilizes the inactivation assembly. In contrast, SEA0400 binding in the TM domain of NCX1 stabilizes the exchanger in an inward-facing conformation that facilitates the formation of the inactivation assembly, thereby promoting the Na+-dependent inactivation of NCX1. Thus, this study reveals the structural basis of PIP2 activation and SEA0400 inhibition of NCX1 and provides some mechanistic understandings of cellular regulation and pharmacology of NCX family proteins.
The NHGRI-EBI GWAS Catalog serves as a vital resource for the genetic research community, providing access to the most comprehensive database of human GWAS results. Currently, it contains close to 7,000 publications for more than 15,000 traits, from which more than 625,000 lead associations have been curated. Additionally, 85,000 full genome-wide summary statistics datasets - containing association data for all variants in the analysis - are available for downstream analyses such as meta-analysis, fine-mapping, Mendelian randomisation or development of polygenic risk scores. As a centralised repository for GWAS results, the GWAS Catalog sets and implements standards for data submission and harmonisation, and encourages the use of consistent descriptors for traits, samples and methodologies. We share processes and vocabulary with the PGS Catalog, improving interoperability for a growing user group. Here, we describe the latest changes in data content, improvements in our user interface, and the implementation of the GWAS-SSF standard format for summary statistics. We address the challenges of handling the rapid increase in large-scale molecular quantitative trait GWAS and the need for sensitivity in the use of population and cohort descriptors while maintaining data interoperability and reusability.
ObjectiveClinical studies suggest that compared to anti-TNF treatment, JAK inhibitors (JAKi) are superior in reducing pain in rheumatoid arthritis (RA). The underlying mechanisms for this observation are still unknown. Sensory neurons transmit noxious signals from inflamed joints to the central nervous system, where a pain percept is generated. We investigated whether JAKi exert direct effects on sensory neurons. MethodsIn-house and public RNA sequencing datasets of sensory neurons were analysed for relevant JAK/STAT and cytokine-receptor gene expression. Human induced pluripotent stem cell (IPSC)-derived sensory neurons were stimulated with serum and synovial fluid (SF) from individuals with RA, or with selected cytokines that were found in RA SF by Luminex. Phosphorylation of STAT3 (pSTAT3) was assessed by Western blot. Sensory neuron activation was examined by recording neuronal firing using multi-electrode array and measuring expression levels of pain-relevant genes with STAT3-binding sites. ResultsCell-free RA synovial fluid induced pSTAT3 in IPSC-derived sensory neurons, an effect which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA SF was enriched for the JAK/STAT cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present on sensory neurons. Stimulation of IPSC- derived sensory neurons with these recombinant cytokines recapitulated pSTAT3 induction in these cells. Furthermore, IL-6+sIL-6R or LIF upregulated expression of pain-relevant genes which was blocked by tofacitinib. Finally, we provided evidence that LIF can induce neuronal sensitisation. ConclusionOur data indicate that JAKi can act directly on sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. Further, co-infections with HIV and Mtb have severe effects in the host, with people infected with HIV being fifteen to twenty-one times more likely to develop active TB. The use of an appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a useful tool for conducting basic and translational research in HIV/Mtb infections. The present study was focused on developing a humanized mouse model for investigations on HIV-Mtb co-infection. Using NSG-SGM3 mice that can engraft human stem cells, our studies showed that they were able to engraft human CD34+ stem cells which then differentiate into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced the development of granulomatous lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Our results suggest that the humanized NSG-SGM3 mice are able to recapitulate the effects of HIV and Mtb infections and co-infection in the human host at pathological, immunological and metabolism levels, providing a dependable small animal model for studying HIV/Mtb co-infection.
With age, people tend to accumulate body fat and reduce energy expenditure1. Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles2,3. The activity of brown and beige depots inversely correlates with BMI and age4-11, suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease12-15. Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function16. Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis17. However, BCAAs contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux18. Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.
The exact sites and molecules that determine resistance to aqueous humor drainage and control intraocular pressure (IOP) need further elaboration. Proposed sites include the inner wall of Schlemmss canal and the juxtacanalicular trabecular meshwork ocular drainage tissues. The adherens junctions (AJs) of Schlemms canal endothelial cells (SECs) must both preserve the blood-aqueous humor (AQH) barrier and be conducive to AQH drainage. How homeostatic control of AJ permeability in SC occurs and how such control impacts IOP is unclear. We hypothesized that mechano-responsive phosphorylation of the junctional molecule VE-CADHERIN (VEC) by SRC family kinases (SFKs) regulates the permeability of SEC AJs. We tested this by clamping IOP at either 16 mmHg, 25 mmHg, or 45 mmHg in mice and then measuring AJ permeability and VEC phosphorylation. We found that with increasing IOP: 1) SEC AJ permeability increased, 2) VEC phosphorylation was increased at tyrosine-658, and 3) SFKs were activated at the AJ. Among the two SFKs known to phosphorylate VEC, FYN, but not SRC, localizes to the SC. Furthermore, FYN mutant mice had decreased phosphorylation of VEC at SEC AJs, dysregulated IOP, and reduced AQH outflow. Together, our data demonstrate that increased IOP activates FYN in the inner wall of SC, leading to increased phosphorylation of AJ VEC and, thus, decreased resistance to AQH outflow. These findings support a crucial role of mechanotransduction signaling in IOP homeostasis within SC in response to IOP. These data strongly suggest that the inner wall of SC partially contributes to outflow resistance.
Schlemms canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemms canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue ([~]4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics. Significance statementSchlemms canal is a critical ocular drainage structure that maintains intraocular pressure with important consequences for glaucoma. Despite this, Schlemms canal has been relatively understudied with the molecular process that mediate its function, maintain its health, or that are compromised in glaucoma needing much deeper characterization. This study is important as it provides the most comprehensive molecular characterization of Schlemms canal cells to date providing a wealth of molecular details. In addition to highlighting important biological pathways, this information will guide studies to develop new treatments against glaucoma.
Type 2 diabetes (T2D) and its comorbidities are a major public health concern. In addition to peripheral tissues, T2D also impacts the central nervous system leading to neurocognitive impairments, including memory deficits, anxiety, and depression. The metabolic determinants of these neurocognitive impairments remain unidentified. Here, we used a novel proprietary high-fat diet, in which glucose intolerance precedes weight gain, to decipher the metabolic determinants of neurocognitive affects. We show that this model exhibits anxiety-like behaviors, without eliciting depression nor recognition memory deficits. Long-term feeding leads to weight gain, brain glucose hypometabolism and impaired recognition memory alongside the early onset anxiety-like behavior. Using an established genetic model of T2D (db/db) and of diet-induced obesity we show that additional insulin resistance and obesity are associated with depressive-like behaviors and recognition memory deficits. Our findings indicate that glucose intolerance alone can elicit anxiety-like behaviors. Through this study we also provide a novel nutritional model to characterize the discrete effects of glucose intolerance on cognition, behavior, and the physiology of metabolic disease.
Coevolution is defined as the evolutionary change in one antagonist (host) in response to changes in the other antagonist (pathogen). At the genetic level, these changes are determined by genotype x genotype (GxG) interactions. We build on a general theoretical model of a host-pathogen interaction to derive four indices to retrieve key features of GxG interactions. The four developed indices extract relevant information from polymorphism data of randomly sampled uninfected hosts as well as infected hosts and their respective pathogen strains. Using these indices as summary statistics in an Approximate Bayesian Computation method, we can show their power to discriminate between GxG interaction matrices. Second, we apply our ABC method to a SNP data set of 451 European humans and their infecting Hepatitis C Virus (HCV) strains supplemented by polymorphism data of 503 individuals from the 1,000 genomes project. As our indices encompass and extend previous natural co-GWAs we recover many of the associations previously reported for this dataset and infer their underlying interaction matrix. We reveal a new candidate gene for resistance to HCV in the human genome, and two groups of significant GxG associations exhibiting gene-for-gene interactions. We suggest that the inferred types of GxG interactions result from the recent expansion, adaptation and low prevalence of the HCV virus population in Europe. Significance statementWhy are some host individuals susceptible/resistant to infection by certain pathogen genotypes and others not? Understanding the genetic characteristics of genes driving host-pathogen interactions is crucial to predict epidemics. We develop four indices based on a mathematical model and build a Bayesian statistical method computing these indices on full genome data of infected hosts and their infecting pathogen strains and data of non-infected hosts. We can pinpoint the genes underlying host-pathogen interactions and infer their characteristics. Applying our framework to data from European humans and the Hepatitis C virus, we discover a new potential resistance gene in humans and reveal how the virus has adapted in the last 150 years to match the genetic diversity of the European human population.
The Kolliker-Fuse nucleus (KF), which is part of the parabrachial complex, participates in the generation of eupnea under resting conditions and the control of active abdominal expiration when increased ventilation is required. Moreover, dysfunctions in KF neuronal activity are believed to play a role in the emergence of respiratory abnormalities seen in Rett syndrome (RTT), a progressive neurodevelopmental disorder associated with an irregular breathing pattern and frequent apneas. Relatively little is known, however, about the intrinsic dynamics of neurons within the KF and how their synaptic connections affect breathing pattern control and contribute to breathing irregularities. In this study, we use a reduced computational model to consider several dynamical regimes of KF activity paired with different input sources to determine which combinations are compatible with known experimental observations. We further build on these findings to identify possible interactions between the KF and other components of the respiratory neural circuitry. Specifically, we present two models that both simulate eupneic as well as RTT-like breathing phenotypes. Using nullcline analysis, we identify the types of inhibitory inputs to the KF leading to RTT-like respiratory patterns and suggest possible KF local circuit organizations. When the identified properties are present, the two models also exhibit quantal acceleration of late-expiratory activity, a hallmark of active expiration featuring forced exhalation, with increasing inhibition to KF, as reported experimentally. Hence, these models instantiate plausible hypotheses about possible KF dynamics and forms of local network interactions, thus providing a general framework as well as specific predictions for future experimental testing. Key pointsThe Kolliker-Fuse nucleus (KF), a part of the parabrachial complex, is involved in regulating normal breathing and controlling active abdominal expiration during increased ventilation. Dysfunction in KF neuronal activity is thought to contribute to respiratory abnormalities seen in Rett syndrome (RTT). This study utilizes computational modeling to explore different dynamical regimes of KF activity and their compatibility with experimental observations. By analyzing different model configurations, the study identifies inhibitory inputs to the KF that lead to RTT-like respiratory patterns and proposes potential KF local circuit organizations. Two models are presented that simulate both normal breathing and RTT-like breathing patterns. These models provide plausible hypotheses and specific predictions for future experimental investigations, offering a general framework for understanding KF dynamics and potential network interactions.