Activity of dorsal raphe neurons is controlled by noradrenaline afferents. In this brain region, noradrenaline activates Gαq-coupled α1-adrenergic receptors (α1-AR), causing action potential firing and serotonin release. In vitro, electrical stimulation elicits vesicular noradrenaline release and subsequent activation of α1-AR to produce an excitatory postsynaptic current (α1-AR-EPSC). The duration of the α1-AR-EPSC (∼27 s) is much longer than that of most other synaptic currents, but the factors that govern the spatiotemporal dynamics of α1-AR are poorly understood. Using an acute brain slice preparation from adult male and female mice and electrophysiological recordings from dorsal raphe neurons, we found that the time-course of the α1-AR-EPSC was slow, but highly consistent within individual serotonin neurons. The amount of noradrenaline released influenced the amplitude of the α1-AR-EPSC without altering the time constant of decay suggesting that once released, extracellular noradrenaline was cleared efficiently. Reuptake of noradrenaline via noradrenaline transporters was a primary means of terminating the α1-AR-EPSC, with little evidence for extrasynaptic diffusion of noradrenaline unless transporter-dependent reuptake was impaired. Taken together, the results demonstrate that despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is temporally and spatially controlled and noradrenaline transporters are critical regulators of serotonin neuron excitability. Given the functionally distinct types of neurons intermingled in the dorsal raphe nucleus and the unique roles of these neural circuits in physiological responses, transporters may preserve independence of each synapse to encode a long-lasting but discrete signal.SIGNIFICANCE STATEMENTThe dorsal raphe nucleus is the predominant source of serotonin in the brain and is controlled by another monoamine, noradrenaline. In this brain region, noradrenaline activates G protein-coupled α1-adrenergic receptors causing action potential firing and serotonin release. In spite of high interest in pharmacotherapies to enhance serotonin signaling, the factors that govern noradrenaline α1-AR signaling have received little attention. Here we show using mouse brain slices that the time-course of α1-AR signaling is slow, persisting for tens of seconds. Despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is controlled temporally and spatially by efficient noradrenaline transporter-dependent clearance extracellular noradrenaline. Thus, noradrenaline transporters are critical regulators of serotonin neuron excitability.

It is well-established that Ang-(1-7) counteracts the effects of Ang II in the periphery, while stimulating vasopressin release and mimicking the activity of Ang II in the brain, through interactions with various receptors. The rapid metabolic inactivation of Ang-(1-7) has proven to be a limitation to therapeutic administration of the peptide. To circumvent this problem, Alves et al. (Clinical Science (2021) 135(18), https://doi.org/10.1042/CS20210599) developed a new transgenic rat model that overexpresses an Ang-(1-7)-producing fusion protein. In this commentary, we discuss potential concerns with this model while also highlighting advances that can ensue from this significant technical feat.

In mammals, the hypothalamic suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker, orchestrating behavioral and physiological rhythms in alignment to the environmental light/dark cycle. The neurons that comprise the SCN are anatomically and functionally heterogeneous, but despite their physiological importance, little is known about the pathways that guide their specification and differentiation. Here, we report that the stem/progenitor cell transcription factor, Sex determining region Y-box 2 (Sox2), is required in the embryonic SCN to control the expression of SCN-enriched neuropeptides and transcription factors. Ablation of Sox2 in the developing SCN leads to downregulation of circadian neuropeptides as early as embryonic day (E) 15.5, followed by a decrease in the expression of two transcription factors involved in SCN development, Lhx1 and Six6, in neonates. Thymidine analog-retention assays revealed that Sox2 deficiency contributed to reduced survival of SCN neurons during the postnatal period of cell clearance, but did not affect progenitor cell proliferation or SCN specification. Our results identify SOX2 as an essential transcription factor for the proper differentiation and survival of neurons within the developing SCN.

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high dose intravenous (IV) administration. To characterize biodistribution, transduction and toxicity amongst common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron-1 (SMN-1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2x1013, 5x1013, or 1x1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5x1013 vg/kg was found dead on Day 4. Clinical pathology data on Days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization (ISH) demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while qPCR confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after one month in rats, and suggest adding the autonomic system in the watch-list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Phagocytic clearance of apoptotic germ cells (GCs), as well as residual bodies (RBs) released from developing spermatids, is critical for Sertoli cells (SCs) to maintain inner environment homeostasis within testis. However, the molecular mechanisms controlling the phagocytosis are ill defined. Here, we identify a new role for alpha-enolase (ENO1), a key enzyme during glycolysis, as a molecule that facilitates testicular phagocytosis via transactivation of the engulfment and cell motility 1 (Elmo1) gene. Using immunohistochesmitry and double-labeling immunofluorescence, ENO1 was observed to be expressed exclusively in the nuclei of SCs and its expression correlated with the completion of Sertoli cell differentiation. By incubating TM4 cells with different pharmacological inhibitors and establishing TM4Tnfr1-/- cells, we demonstrated that Sertoli cell-specific expression of ENO1 was under a delicate paracrine control from apoptotic GCs. In turn, persistent blockade of ENO1 expression by a validated siRNA protocol resulted in the disturbance of spermatogenesis and impairment of male fertility. Furthermore, using chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter assay, we showed that in the presence of apoptotic GCs, ENO1 binds to the distal region of the Elmo1 promoter and facilitates transactivation of the Elmo1 gene. In agreement, overexpression of ELMO1 ameliorated ENO1 deficiency-induced impairment of phagocytosis in TM4 cells. These data reveal a novel role for Sertoli cell-specific expression of ENO1 in regulating phagocytosis in testis, identify TNF-α and ELMO1 as critical upstream and downstream factors in mediating ENO1 action, and have important implications for understanding paracrine control of Sertoli cell function by adjacent GCs.This article is protected by

Tracheobronchomalacia and Complete Tracheal Rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNTsignaling: Notum, and Axin2. In vitro, rBMP4 rescued the expression of Notum in Bmp4 deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4 deficient tracheas identified genes essential for chondrogenesis and muscle development co-regulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

SARS-CoV-2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. A mutation in the catalytic domain of ACE2, MDR504, significantly increased binding to SARS-CoV-2 spike protein, as well as to a spike variant, in vitro with more potent viral neutralization in plaque assays. Parental administration of the protein showed stable serum concentrations with excellent bioavailability in the epithelial lining fluid of the lung, and ameliorated lung SARS-CoV-2 infection in vivo. These data support that the MDR504 hACE2-Fc is an excellent candidate for treatment or prophylaxis of COVID-19 and potentially emerging variants.

Stress reduces cognitive flexibility and dopamine D1 receptor-related activity in the prelimbic cortex (PL), effects hypothesized to depend on reduced corticotropic releasing factor receptor type 1 (CRFr1) regulation of dopamine neurons in the ventral tegmental area (VTA). We assessed this hypothesis in rats by examining the effect of chronic unpredictable restraint stress (CUS), mild acute stress, or their combination on cognitive flexibility, CRFr1 expression in the VTA and D1-related activity in PL. In Experiment 1, rats received either CUS or equivalent handling for 14 days before being trained to press two levers to earn distinct food outcomes. Initial learning was assessed using an outcome devaluation test after which cognitive flexibility was assessed by reversing the outcomes earned by the actions. Prior to each reversal training session, half the CUS and controls receiving acute stress with action-outcome updating assessed using a second devaluation test and CRFr1 expression in the VTA assessed using in-situ hybridisation. Although CUS did not itself affect action-outcome learning, its combination with acute stress blocked reversal learning and decreased VTA CRFr1 expression after acute shock. The relationship between these latter two effects was assessed in Experiment 2 by pharmacologically disconnecting the VTA and PL, unilaterally blocking neurons expressing CRFr1 in the VTA and D1 receptors in the contralateral PL during reversal learning after acute stress. Acute stress again blocked reversal learning but only in the group with VTA-PL disconnection, demonstrating that VTA CRFr1-induced facilitation of dopaminergic activity in the PL is necessary for maintaining cognitive flexibility after acute stress. [250].

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function.The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 -/- and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC.Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 -/- mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells.This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.