Probes for INS

Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.

In the gastro-intestinal tract, the complex network of multiple innate cell populations play critical roles not only as a first line of defense against invading pathogens and in driving adaptive immune responses but also in maintaining intestinal homeostasis. Here, we describe the roles of various innate immune cell populations in gut immunity and detail studies investigating the impact of acute and chronic HIV infection on these cell populations. Alterations in frequencies, phenotype and/or function of innate lymphoid cells, dendritic cells, macrophages, neutrophils, and innate-like T cells have been reported in people with HIV (PWH), with many of these features persisting despite anti-retroviral therapy and virological suppression. Dysregulated gut innate immunity in PWH is a feature of gut pathogenesis. A greater understanding of the mechanisms driving impairment in the multiple different gut innate immune cell populations and the downstream consequences of an altered innate immune response on host defense and gut homeostasis in PWH is needed to develop more effective HIV treatments and cure strategies.

Acetaminophen (N-acetyl-p-aminophenol, APAP) use in the neonatal intensive care unit is rapidly increasing. While APAP-related hepatotoxicity is rarely reported in the neonatal literature, other end-organ toxicity can occur with toxic exposures. APAP-induced lung injury has been reported with toxic exposures in adults, but whether this occurs in the developing lung is unknown. Therefore, we tested whether toxic APAP exposures would injure the developing lung. Neonatal C57BL/6 mice (PN7, early alveolar stage of lung development) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice (280 mg/kg, IP). This exposure induced significant lung injury in the absence of identifiable hepatic toxicity. This injury was associated with increased pulmonary expression of Cyp2e1, the xenobiotic enzyme responsible for the toxic conversion of APAP. Exposure was associated with increased pulmonary expression of antioxidant response genes and decreased pulmonary glutathione peroxidase activity level. Furthermore, we observed an increase in pulmonary expression of pro-inflammatory cytokines and chemokines. Lastly, we were able to demonstrate that this toxic APAP exposure was associated with a shift in pulmonary metabolism away from glycolysis with an increased oxidative phosphorylation, a finding consistent with increased mitochondrial workload, potentially leading to mitochondrial toxicity. This previously unrecognized injury and metabolic implications highlight the need to look beyond the liver and evaluate both the acute and long-term pulmonary implications of APAP exposure in the perinatal period.

Background Polymorphisms in GRM3, the gene encoding the mGlu3 metabotropic glutamate receptor, are associated with impaired cognition and neuropsychiatric disorders such as schizophrenia. Limited availability of selective genetic and molecular tools has hindered progress in developing a clear understanding of the mechanisms through which mGlu3 receptors regulate synaptic plasticity and cognition. Methods We examined associative learning in mice with trace fear conditioning, a hippocampal-dependent learning task disrupted in patients with schizophrenia. Underlying cellular mechanisms were assessed using ex vivo hippocampal slice preparations with selective pharmacological tools and selective genetic deletion of mGlu3 receptor expression in specific neuronal subpopulations. Results mGlu3 receptor activation enhanced trace fear conditioning and reversed deficits induced by subchronic phencyclidine. Mechanistic studies revealed that mGlu3 receptor activation induced metaplastic changes, biasing afferent stimulation to induce long-term potentiation through a mGlu5 receptor-dependent, endocannabinoid-mediated, disinhibitory mechanism. Selective genetic deletion of either mGlu3 or mGlu5 from hippocampal pyramidal cells eliminated effects of mGlu3 activation, revealing a novel mechanism by which mGlu3 and mGlu5 interact to enhance cognitive function. Conclusions These data demonstrate that activation of mGlu3 receptors in hippocampal pyramidal cells enhances hippocampal-dependent cognition in control and impaired mice by inducing a novel form of metaplasticity to regulate circuit function – providing a clear mechanism through which genetic variation in GRM3 can contribute to cognitive deficits. Developing approaches to positively modulate mGlu3 receptor function represents an encouraging new avenue for treating cognitive disruption in schizophrenia and other psychiatric diseases.

Abnormal angiogenesis and regression of the diseased retinal vasculature are key processes associated with ischemic retinopathies, but the underlying mechanisms that regulate vascular remodeling remain poorly understood. Here, we confirmed the specific expression of semaphorin 3G (Sema3G) in retinal endothelial cells (ECs), which was required for vascular remodeling and the amelioration of ischemic retinopathy. We found that Sema3G was elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and in the neovascularization regression phase of oxygen-induced retinopathy (OIR). Endothelial-specific Sema3G knockout mice exhibited decreased vessel density and excessive matrix deposition in the retinal vasculature. Moreover, loss of Sema3G aggravated pathological angiogenesis in mice with OIR. Mechanistically, we demonstrated that HIF-2α directly regulated Sema3G transcription in ECs under hypoxia. Sema3G coordinated the functional interaction between β-catenin and VE-cadherin by increasing β-catenin stability in the endothelium through the neuropilin-2 (Nrp2)/PlexinD1 receptor. Furthermore, Sema3G supplementation enhanced healthy vascular network formation and promoted diseased vasculature regression during blood vessel remodeling. Overall, we deciphered the endothelium-derived Sema3G-dependent events involved in modulating physiological vascular remodeling and regression of pathological blood vessels for reparative vascular regeneration. Our findings shed light on the protective effect of Sema3G in ischemic retinopathies.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc-/-Ihh-/- double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome.

The blood-brain barrier (BBB) consists of endothelial cells, astrocytic end feet, and pericytes to form a barrier that minimizes the entry of circulating proteins and cells into the brain. However, stroke is known to cause significant damage to the BBB, causing the barrier to become permeable, which allows immune cells and other substances to be extravasated into the brain parenchyma. Preservation of the BBB is associated with better ischemic stroke outcomes; therefore, this synopsis summarizes 3 new studies that aim to characterize specific mechanisms of BBB damage and identify potential therapeutic pathways to preserve barrier integrity. CD36 (cluster of differentiation 36) is a glycoprotein expressed by monocytes and macrophages, as well as by endothelial cells. Previous studies have shown that global knockout of CD36 prevents stroke-induced damage, and Kim et al in 2023 published a study in the Journal of Cerebral Blood Flow and Metabolism titled “Endothelial Cell CD36 Mediates Stroke-Induced Brain Injury via BBB Dysfunction and Monocyte Infiltration in Normal and Obese Conditions,” in which they explore the role of CD36 specifically in endothelial cells. Conditional deletion of CD36 in endothelial cells improved stroke outcomes, as indicated by reduced infarct size and hemispheric swelling. Moreover, this deletion improved survival and motor function. Additionally, CD36 deletion in endothelial cells reduced IgG expression in the brain, indicating improved vascular integrity. There was reduced monocyte infiltration into the brain and reduced MCP-1 (monocyte chemoattractant protein-1) and CCR2 (chemokine receptor type 2) expression in the mice with endothelial cell deletion of CD36. This reduced monocyte trafficking persisted even when normalized for infarct size, suggesting that vascular integrity is maintained independent of cell loss. Intriguingly, endothelial cell-specific deletion of CD36 also made mice resistant to developing an obesity phenotype, providing a potential molecular cause for obesity as a stroke risk factor. In the Proceedings of the National Academy for Science in a publication titled “Myeloid-Derived MIF Drives RIPK1-Mediated Cerebromicrovascular Endothelial Cell Death to Exacerbate Ischemic Brain Injury,” Li et al in 2023 describe how macrophage MIF (migration inhibitory factor) exacerbates endothelial cell death and increases BBB permeability after middle cerebral artery occlusion (MCAo). By treating endothelial cells with MIF and subjecting them to oxygen-glucose deprivation followed by reoxygenation, the authors demonstrated that MIF promotes endothelial cell death specifically by activating RIPK1 (receptor-interacting protein kinase 1). Surgical trauma in both mice and humans increases circulating MIF, and the authors use a perioperative ischemic stroke model to see how this surgically induced increase in MIF affects outcomes after distal MCAo. Two-photon imaging showed that perioperative ischemic stroke mice showed increased adhesion of myeloid cells to ischemic microvascular endothelial cells, and RNAscope analysis showed that MIF expression was increased in microglia surrounding endothelial cells in perioperative ischemic stroke mice. Perioperative ischemic stroke mice also exhibited larger infarct volumes and exacerbated BBB damage. The authors then used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) to delete MIF in myeloid cells, which resulted in reduced expression of necrosis markers phosphorylated RIPK1, phosphorylated RIPK3, and CC3 (cleaved caspase 3) in endothelial cells. Mice with myeloid-deleted MIF showed reduced zonulin-1 loss, a marker of endothelial tight junctions, and sensorimotor deficits after distal MCAo. Peripheral blood mononuclear cells from mice with myeloid-deleted MIF were given after MCAo and resulted in smaller infarct volume and reduced IgG extravasation as compared with mice who were given peripheral blood mononuclear cells from wild types. Endothelial cells were cocultured with peripheral blood mononuclear cells from mice with myeloid-deleted MIF and wild types, and zonulin-1 expression was preserved in endothelial cocultured with peripheral blood mononuclear cells from myeloid-deleted MIF mice. Administration of MIF inhibitor before stroke reduced infarct volume, prevented IgG extravasation, preserved tight junction integrity, and prevented endothelial cell death. Collectively, these data show that myeloid-derived MIF is detrimental to BBB integrity after stroke and deleting this source of MIF can improve outcomes through preserving BBB health. Finally, Li et al in 2023 show in ACS Nano in a publication titled “Inducible Pluripotent Stem Cell-Derived Small Extracellular Vesicles Rejuvenate Senescent Blood-Brain Barrier to Protect Against Ischemic Stroke in Aged Mice” that small extracellular vesicles (sEVs) from induced pluripotent stem cells (iPSCs) are able to restore BBB function in old mice by reversing cellular senescence. Mice treated with iPSC-sEVs showed reduced senescence-associated β-galactosidase, p16, p53, p21, and γ-H2AX (histone family member X), all of which are markers associated with cellular senescence. Pretreatment with iPSC-sEVs before MCAo reduced infarct volume in the aged mice, improved neurological score, and reduced sensorimotor deficits, indicating improved stroke outcomes. Moreover, these mice showed decreased leakage of Evans blue dye into the brain parenchyma and preservation of tight junction proteins, indicating that these sEVs preserve BBB integrity after stroke. Mice treated with sEVs showed decreased immune cell infiltration after stroke and attenuated expression of tumor necrosis factor-α, IL (interleukin)-17, IL-6, and IL-1β, Moreover, sEV treatment reduced ischemia-induced apoptosis of oligodendrocytes and neurons. Reversal of the senescent phenotype of the BBB was tested in vitro, by chemically inducing senescence using D-galactose in endothelial cell cultures and subsequent treatment with iPSC-sEVs. sEV treatment reduced senescence markers and prevented loss of tight junction proteins. Oxygen-glucose deprivation was then used to mimic stroke conditions in these cultures, and sEV treatment preserved angiogenic properties of endothelial cells and reduced dextran leakage in a transwell assay. These experiments affirm the in vivo findings that sEV treatment reverses BBB senescence. The 3 studies summarized in this synopsis show 3 different potential pathways that may serve as a target for preserving BBB function after stroke. Deleting endothelial cell CD36, deleting myeloid-derived MIF, or reversing BBB senescence using iPSC-sEVs resulted in improved vascular integrity and overall better stroke outcomes.

Purpose : A subset of mice in our Cfh knockout (Cfh-/-) colony exhibited rapid retinal degeneration, suggesting a spontaneous mutation occurred on mouse chromosome 1 (Chr 1). The retinal phenotype was similar to that in AdipoR1 knockout (AdipoR1-/-) mice, whose gene is located near the Cfh locus on Chr 1. We attempted to determine if a mutation in AdipoR1 occurred on the Cfh-/- background. Methods : We performed an allele complementation test with a cross between a Cfh-/- mouse with retinal degeneration and an AdipoR1-/- mouse with retinal degeneration. RNA-seq, in situ hybridization, immunohistochemistry and protein analysis were used to profile Cfh-/- and AdipoR1-/- mice. Results : About 50% of Cfh-/- mice exhibited retinal degeneration. Cfh-/- mice, regardless of retinal phenotype, demonstrated elevated complement activation in the eye compared to littermate controls. All offspring from the complementation test exhibited retinal degeneration, implying that AdipoR1 mutant alleles were responsible for the retinal degeneration. AdipoR1 protein, normally present in the RPE apical microvilli, was notably absent in Cfh-/- mice with retinal degeneration. Cfh-/- mice with normal retinal anatomy expressed AdipoR1 protein and were comparable to littermates[QY1] . Adipor1 mRNA expression levels were comparable across the colony as measured by RNAscope and RNA-seq. Analysis of AdipoR1 mRNA sequence revealed a transversion at position c.841 C>T in the Adipor1 gene concordant with mice exhibiting retinal degeneration. Further breeding identified mutant AdipoR1 mRNA in two Cfh+/- mice and one Cfh+/+ mice with early onset retinal degeneration, implying a recent cross over on Chr 1. This missense mutation results in a proline to serine conversion (P281S) in the fifth transmembrane domain of AdipoR1 and is predicted to be detrimental to the AdipoR1 protein. Conclusions : The spontaneous mutation in the AdipoR1 gene results in early onset retinal degeneration in a subset of our Cfh-/- mouse colony. Elevated complement activity in the eye does not appear to affect the course of retinal degeneration, as gene signatures are comparable across all mice with retinal degeneration, regardless of Cfh genotype.

Regional cellular heterogeneity is a fundamental feature of the human neocortex; however, details of this heterogeneity are still undefined. We utilized single-nucleus RNA-sequencing (snRNA-seq) to examine cell-specific transcriptional features in the dorsolateral prefrontal cortex (DLPFC) and the subgenual anterior cingulate cortex (sgACC)-regions implicated in major psychiatric disorders. Droplet-based nuclei-capture and library preparation were performed on replicate samples from eight male donors without history of psychiatric or neurological disorder. Unsupervised clustering identified major neural cell classes. Subsequent iterative clustering of neurons further revealed 20 excitatory and 22 inhibitory subclasses. Inhibitory cells were consistently more abundant in the sgACC and excitatory neuron subclusters exhibited considerable variability across brain regions. Excitatory cell subclasses also exhibited greater within-class transcriptional differences between the two regions. We utilized these molecular definitions to determine which cell classes might be enriched in loci carrying a genetic signal in genome-wide association studies (GWAS) or for differentially expressed genes (DEGs) in mental illness. We found that the heritable signals of psychiatric disorders were enriched in neurons and that while the gene expression changes detected in bulk-RNA-sequencing studies was dominated by glial cells, some alterations could be identified in specific classes of excitatory and inhibitory neurons. Intriguingly, only two excitatory cell classes exhibited concomitant region-specific enrichment for both GWAS loci and transcriptional dysregulation. In sum, by detailing the molecular and cellular diversity of the DLPFC and sgACC, we were able generate hypotheses on regional and cell-specific dysfunctions that may contribute to the development of mental illness.SIGNIFICANCE STATEMENT:Dysfunction of the subgenual anterior cingulate cortex (sgACC) has been implicated in mood disorders, particularly major depressive disorder, and the dorsolateral prefrontal cortex (DLPFC)-a subsection of the Prefrontal cortex involved in executive functioning-has been implicated in schizophrenia. Understanding the cellular composition of these regions is critical to elucidating the neurobiology underlying psychiatric and neurological disorders. We studied cell type diversity of the sgACC and DLPFC of humans with no neuropsychiatric illness utilizing a clustering analysis of single-nuclei RNA-sequencing (snRNA-seq) data. Defining the transcriptomic profile of cellular subpopulations in these cortical regions is a first step to demystifying the cellular and molecular pathways involved in psychiatric disorders.

Diseases of the airway such as Tracheobronchomalacia (TBM) and Complete tracheal rings (CTR) are prevalent conditions associated with abnormal patterning of the trachealis muscle and cartilage. However, the underlying mechanisms of tracheal patterning are poorly understood. We have demonstrated that Wnt signaling via Wls plays an essential role in determining the mesenchymal patterning of the trachea. Notum, a direct target of Wnt signaling, encodes an enzyme that inactivates Wnt ligands, thus attenuating Wnt signaling's strength in developing trachea. In Notum deficient mice, chondroblasts and smooth muscle cells of the trachea were specified properly. Meanwhile, deletion of Notum impaired and delayed mesenchymal condensations of chondroblasts causing abnormal cartilage and stenosis. Further, tracheal muscle organization was disrupted. We hypothesize that chondrocyte condensation influences the trachealis muscle organization during tracheal tubulogenesis. Methods: We utilized genetically modified mice wherein Notum, Wnt5a, and Ror2 were deleted in the germline or conditionally ablated in tracheal mesenchyme. Tracheal smooth muscle cells were genetically labeled using γSMAeGFP mice. RNA scope, whole-mount stain, immunofluorescence, and fluorescent microscopy were utilized to visualize changes in trachealis muscle cell and cartilage organization induced by in vivo gene deletion and ex vivo treatments. Results: Analysis of muscle cells of control tracheas at E12, E14, and E16 showed progressive trachealis muscle organization with myocytes oriented perpendicular to the elongation axis of the trachea. In contrast, Notum deficient tracheas showed disordered orientation of the muscle cells with changes in the cytoskeleton. The anomalous muscle cell arrangement was increasingly observed after E14 after mesenchymal condensations were completed, and cartilage formed. Ex vivo studies with ABC99, a pharmacological inhibitor of Notum affected trachealis muscle organization, recapitulating the in vivo data. The Planar Cell Polarity (PCP) branch of the non-canonical Wnt signaling pathway mediates organization and functioning of the trachealis muscle. Mesenchymal deletion of the non-canonical Wnt5a and its receptor Ror2 impaired trachealis muscle cell orientation, causing changes in myocytes' cytoskeletal organization similar to changes observed in Notum deficient trachea. Different from Notum deletion, changes in myocyte organization preceded cartilaginous mesenchymal condensation. Despite the abnormal muscle organization in Wnt5a, cartilaginous mesenchymal condensations occurred, although reduced in number. We conclude that tracheal chondrogenesis affects trachealis muscle formation by constraining the expansion and altering the cytoskeletal organization of tracheal myocytes after mesenchymal condensation takes place. Thus, delayed tracheal chondrogenesis may partially underlie the pathology of tracheal stenosis. These studies were partially supported by NIH-NHLBI R01HL144774-01A1 to DS.