Probes for INS

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.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

Acetylcholine is an important modulator of striatal activity and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nicotinic acetylcholine receptors (nAChRs) expressed by striatal interneurons. In the present study, we used fluorescent in situ hybridization (FISH) to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons (CINs) were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes, that despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by CINs and play a significant role in behavior.SIGNIFICANCE STATEMENTA large variety of nicotinic acetylcholine receptors are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nicotinic acetylcholine receptors. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

Background: Inhaled antibiotics control chronic airway infection and maintain respiratory health in cystic fibrosis (CF). Given variation in patient responses to inhaled antibiotics, the ability to identify distinct responder phenotypes would facilitate delivery of personalized care. Previously a 10- gene panel was identified, measured directly from blood leukocytes, that predicted response to intravenous antibiotic treatment during pulmonary exacerbations. In the current study, we tested whether the same panel predicted clinical response in subjects receiving a month of inhaled antibiotic therapy with aztreonam lysine for inhalation (AZLI). Methods: A cohort of CF subjects infected with P. aeruginosa were enrolled before initiating 11 month’s treatment with AZLI using the Altera nebulizer system. Eighteen CF subjects underwent blood leukocyte gene panel measurements, sputum quantitative microbiology, spirometry, and CRP measurement before onset and at completion of 4 weeks of AZLI therapy. Results: ppFEV1 improvement was 3%. Significant reductions in sputum bacterial colony counts were detected with treatment. C-reactive protein (CRP) increased after treatment. Although no single gene within the panel changed significantly after treatment, multivariate analysis demonstrated that genes significantly predicted reduction in bacterial load (PLXND1 and HCA112) and improvement in FEV1 (HCA112). Hierarchical clustering based on gene expression yielded 2 distinctive molecular clusters before and after AZLI therapy. Based on overall expression patterns, subjects were identified as Pauci-inflammatory and inflammatory. In the analysis of pretreatment gene expression, the inflammatory group manifested greater systemic and airway inflammation, based on CRP and sputum neutrophil elastase. Consistent with sputum and systemic inflammatory variables, 4 genes (ADAM9, CSPG2, HCA112, HPSE) were more highly expressed in the inflammatory cluster at pre- and post-AZLI time points. In comparison, in the analysis of posttreatment molecular clusters, neutrophil elastase and CRP values were not significantly different between the 2 groups. Conclusion: Whole-blood gene leukocyte expression identifies distinct populations of CF subjects before inhaled antibiotic therapy with AZLI. Molecular quantification of systemic inflammation may indicate subgroups of CF subjects with baseline differences and with variable clinical responses to inhaled antibiotics. Application of a molecular panel may thus be valuable in sub-phenotyping patients before inhaled treatment. A goal of future, larger studies would be to validate whether inflammatory differences define phenotypes of responders to AZLI and to inhaled antibiotics in general, especially given challenges that will be present in future studies of antimicrobial agents in the setting of CFTR modulating agents. Clinical Trials.gov Identifier: NCT01736839.

The proinflammatory cytokine IFN-γ, which is chronically elevated in multiple sclerosis, induces pathological quiescence in human oligodendrocyte progenitor cells (OPCs) via upregulation of the transcription factor PRRX1. In this study using animals of both sexes, we investigated the role of heparan sulfate proteoglycans in the modulation of IFN-γ signaling following demyelination. We found that IFN-γ profoundly impaired OPC proliferation and recruitment following adult spinal cord demyelination. IFN-γ-induced quiescence was mediated by direct signaling in OPCs as conditional genetic ablation of IFNγR1 (Ifngr1) in adult NG2+ OPCs completely abrogated these inhibitory effects. Intriguingly, OPC-specific IFN-γ signaling contributed to failed oligodendrocyte differentiation, which was associated with hyperactive Wnt/Bmp target gene expression in OPCs. We found that PI-88, a heparan sulfate mimetic, directly antagonized IFN-γ to rescue human OPC proliferation and differentiation in vitro and blocked the IFN-γ mediated inhibitory effects on OPC recruitment in vivo Importantly, heparanase modulation by PI-88 or OGT2155 in demyelinated lesion rescued IFN-γ mediated axonal damage and demyelination. In addition to OPC-specific effects, IFN-γ augmented lesions were characterized by increased size, reactive astrogliosis and proinflammatory microglial/macrophage activation along with exacerbated axonal injury and cell death. Heparanase inhibitor treatment rescued many of the negative IFN-γ-induced sequalae suggesting a profound modulation of the lesion environment. Together, these results suggest that the modulation of the heparanome represents a rational approach to mitigate the negative effects of proinflammatory signaling and rescuing pathological quiescence in the inflamed and demyelinated human brain.SIGNIFICANCE STATEMENT: The failure of remyelination in multiple sclerosis contributes to neurological dysfunction and neurodegeneration. The activation and proliferation of oligodendrocyte progenitor cells (OPCs) is a necessary step in the recruitment phase of remyelination. Here, we show that the proinflammatory cytokine interferon-gamma directly acts on OPCs to induce pathological quiescence and thereby limit recruitment following demyelination. Heparan sulfate is a highly structured sulfated carbohydrate polymer that is present on the cell surface and regulates several aspects of the signaling microenvironment. We find that pathological interferon-gamma can be blocked by modulation of the heparanome following demyelination using either a heparan mimetic or by treatment with heparanase inhibitor. These studies establish the potential for modulation of heparanome as a regenerative approach in demyelinating disease.

Background Hyperglycaemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycaemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. However, diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Purpose (1) Characterise the expression of SGLT1 in the myocardium, (2) investigate if SGLT1 is involved in a glucotoxicity injury during IRI, and (3) whether inhibiting SGLT1 with an SGLT inhibitor may reduce infarct size. Methods RT-PCR and in-situ hybridization (RNAScope) combined with Immunoflurescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22 mmol) on myocardium at reperfusion and Canagliflozin (CANA) a non-selective SGLT inhibitor (1000 nmol/L to block both the SGLT1 receptor and SGLT 2 receptor and 5 nmol/L to block the SGLT2 receptor only) was introduced following ischaemia at two different concentrations, at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results RT-PCR found SGLT1 mRNA is expressed in whole myocardium and in individual cardiac chambers. RNAscope detected SGLT1 mRNA is expressed homogenously within intact myocardium, particularly evident within the vasculature. Importantly, hyperglycaemia (22 mmol) at reperfusion increased infarct size (51.80 ± 3.52% vs 40.80 ± 2.89%; p-value: 0.026) compared to normoglycaemia, low dose CANA (5 nmol/L) did not attenuate infarct size in low glucose or high glucose, whereas high CANA concentration (1μmoL/L) significantly reduced infarct size in high glucose (22 mM) when administered at reperfusion (P value = 0.0047). Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycaemia appears augment myocardial infarction and inhibition of SGLT1 attenuates this increase.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was up-regulated by heart and neural crest derivatives expressed 2 (HAND2) and down-regulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.