PD-1 regulates ILC3-driven intestinal immunity and homeostasis.

Interleukin-(IL) 22 production by intestinal group 3 innate lymphoid cells (ILC3) is critical to maintain gut homeostasis. However, IL-22 needs to be tightly controlled; reduced IL-22 expression is associated with intestinal epithelial barrier defect while its overexpression promotes tumor development. Here, using a single-cell ribonucleic acid sequencing approach, we identified a core set of genes associated with increased IL-22 production by ILC3. Among these genes, programmed cell death 1 (PD-1), extensively studied in the context of cancer and chronic infection, was constitutively expressed on a subset of ILC3. These cells, found in the crypt of the small intestine and colon, displayed superior capacity to produce IL-22. PD-1 expression on ILC3 was dependent on the microbiota and was induced during inflammation in response to IL-23 but, conversely, was reduced in the presence of Notch ligand. PD-1+ ILC3 exhibited distinct metabolic activity with increased glycolytic, lipid, and polyamine synthesis associated with augmented proliferation compared with their PD-1- counterparts. Further, PD-1+ ILC3 showed increased expression of mitochondrial antioxidant proteins which enable the cells to maintain their levels of reactive oxygen species. Loss of PD-1 signaling in ILC3 led to reduced IL-22 production in a cell-intrinsic manner. During inflammation, PD-1 expression was increased on natural cytotoxicity receptor (NCR)- ILC3 while deficiency in PD-1 expression resulted in increased susceptibility to experimental colitis and failure to maintain gut barrier integrity. Collectively, our findings uncover a new function of the PD-1 and highlight the role of PD-1 signaling in the maintenance of gut homeostasis mediated by ILC3 in mice.

A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

Identification of aberrant luminal progenitors and mTORC1 as a potential breast cancer prevention target in BRCA2 mutation carriers.

Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2mut/+ tissue, including expansion of aberrant ERBB3lo luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3lo progenitors in BRCA2mut/+ breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3lo progenitors could generate both ER+ and ER- cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

Spatiotemporal mapping of sensory and motor innervation of the embryonic and postnatal mouse urinary bladder.

The primary function of the urinary bladder is to store urine (continence) until a suitable time for voiding (micturition). These distinct processes are determined by the coordinated activation of sensory and motor components of the nervous system, which matures to enable voluntary control at the time of weaning. Our aim was to define the development and maturation of the nerve-organ interface of the mouse urinary bladder by mapping the organ and tissue distribution of major classes of autonomic (motor) and sensory axons. Innervation of the bladder was evident from E13 and progressed dorsoventrally. Increasing defasciculation of axon bundles to single axons within the muscle occurred through the prenatal period, and in several classes of axons underwent further maturation until P7. Urothelial innervation occurred more slowly than muscle innervation and showed a clear regional difference, from E18 the bladder neck having the highest density of urothelial nerves. These features of innervation were similar in males and females but varied in timing and tissue density between different axon classes. We also analysed the pelvic ganglion, the major source of motor axons that innervate the lower urinary tract and other pelvic organs. Cholinergic, nitrergic (subset of cholinergic) and noradrenergic neuronal cell bodies were present prior to visualization of these axon classes within the bladder. Examination of cholinergic structures within the pelvic ganglion indicated that connections from spinal preganglionic neurons to pelvic ganglion neurons were already present by E12, a time at which these autonomic ganglion neurons had not yet innervated the bladder. These putative preganglionic inputs increased in density prior to birth as axon terminal fields continued to expand within the bladder tissues. Our studies also revealed in numerous pelvic ganglion neurons an unexpected transient expression of calcitonin gene-related peptide, a peptide commonly used to visualise the peptidergic class of visceral sensory axons. Together, our outcomes enhance our understanding of neural regulatory elements in the lower urinary tract during development and provide a foundation for studies of plasticity and regenerative capacity in the adult system.

Transcriptomic Profiling of Human Pluripotent Stem Cell-derived Retinal Pigment Epithelium over Time.

Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of diseases associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate time points of retinal pigment epithelium (RPE) cultures that reflect native counterparts, we characterised the transcriptomic profiles of the hPSC-derived RPE cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single-cell RNA-sequencing of a total of 16,576 cells to assess the molecular changes of the RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial-mesenchymal transition, and with the maturing populations of the RPE observed with time in culture. Assessment of Gene Ontology pathways revealed that as the cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirms a maturing transcriptional profile of RPE cells in culture. These results suggest that long-term in vitro culture of RPE cells allows the modelling of specific phenotypes observed in native mature tissues. Our work highlights the transcriptional landscape of hPSC-derived RPE cells as they age in culture, which provides a reference for native and patient samples to be benchmarked against.

Membrane budding is a major mechanism of in vivo platelet biogenesis.

How platelets are produced by megakaryocytes in vivo remains controversial despite more than a century of investigation. Megakaryocytes readily produce proplatelet structures in vitro; however, visualization of platelet release from proplatelets in vivo has remained elusive. We show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet replacement. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. In support of this model, we demonstrate that in mice deficient for NF-E2 (the thrombopoietic master regulator), the absence of membrane budding correlates with failure of in vivo platelet production. Accordingly, we propose that membrane budding, rather than proplatelet formation, supplies the majority of the platelet biomass.

Identification of endothelin-converting enzyme-2 as an autoantigen in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.

Reduced Vglut2/Slc17a6 Gene Expression Levels throughout the Mouse Subthalamic Nucleus Cause Cell Loss and Structural Disorganization Followed by Increased Motor Activity and Decreased Sugar Consumption.

The subthalamic nucleus (STN) plays a central role in motor, cognitive, and affective behavior. Deep brain stimulation (DBS) of the STN is the most common surgical intervention for advanced Parkinson's disease (PD), and STN has lately gained attention as target for DBS in neuropsychiatric disorders, including obsessive compulsive disorder, eating disorders, and addiction. Animal studies using STN-DBS, lesioning, or inactivation of STN neurons have been used extensively alongside clinical studies to unravel the structural organization, circuitry, and function of the STN. Recent studies in rodent STN models have exposed different roles for STN neurons in reward-related functions. We have previously shown that the majority of STN neurons express the vesicular glutamate transporter 2 gene (Vglut2/Slc17a6) and that reduction of Vglut2 mRNA levels within the STN of mice [conditional knockout (cKO)] causes reduced postsynaptic activity and behavioral hyperlocomotion. The cKO mice showed less interest in fatty rewards, which motivated analysis of reward-response. The current results demonstrate decreased sugar consumption and strong rearing behavior, whereas biochemical analyses show altered dopaminergic and peptidergic activity in the striatum. The behavioral alterations were in fact correlated with opposite effects in the dorsal versus the ventral striatum. Significant cell loss and disorganization of the STN structure was identified, which likely accounts for the observed alterations. Rare genetic variants of the human VGLUT2 gene exist, and this study shows that reduced Vglut2/Slc17a6 gene expression levels exclusively within the STN of mice is sufficient to cause strong modifications in both the STN and the mesostriatal dopamine system.

Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson's Disease.

The ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain are associated with Parkinson's disease (PD), schizophrenia, mood disorders and addiction. Based on the recently unraveled heterogeneity within the VTA and SNc, where glutamate, GABA and co-releasing neurons have been found to co-exist with the classical dopamine neurons, there is a compelling need for identification of gene expression patterns that represent this heterogeneity and that are of value for development of human therapies. Here, several unique gene expression patterns were identified in the mouse midbrain of which NeuroD6 and Grp were expressed within different dopaminergic subpopulations of the VTA, and TrpV1 within a small heterogeneous population. Optogenetics-coupled in vivo amperometry revealed a previously unknown glutamatergic mesoaccumbal pathway characterized by TrpV1-Cre-expression. Human GRP was strongly detected in non-melanized dopaminergic neurons within the SNc of both control and PD brains, suggesting GRP as a marker for neuroprotected neurons in PD. This study thus unravels markers for distinct subpopulations of neurons within the mouse and human midbrain, defines unique anatomical subregions within the VTA and exposes an entirely new glutamatergic pathway. Finally, both TRPV1 and GRP are implied in midbrain physiology of importance to neurological and neuropsychiatric disorders.

Developing a functional urinary bladder: a neuronal context.

The development of organs occurs in parallel with the formation of their nerve supply. The innervation of pelvic organs (lower urinary tract, hindgut, and sexual organs) is complex and we know remarkably little about the mechanisms that form these neural pathways. The goal of this short review is to use the urinary bladder as an example to stimulate interest in this question. The bladder requires a healthy mature nervous system to store urine and release it at behaviorally appropriate times. Understanding the mechanisms underlying the construction of these neural circuits is not only relevant to defining the basis of developmental problems but may also suggest strategies to restore connectivity and function following injury or disease in adults. The bladder nerve supply comprises multiple classes of sensory, and parasympathetic or sympathetic autonomic effector (motor) neurons. First, we define the developmental endpoint by describing this circuitry in adult rodents. Next we discuss the innervation of the developing bladder, identifying challenges posed by this area of research. Last we provide examples of genetically modified mice with bladder dysfunction and suggest potential neural contributors to this state.

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity.

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations ("TH-Vglut2 Class1") also expressed the dopamine transporter (DAT) gene while one did not ("TH-Vglut2 Class2"), and the remaining population did not express TH at all ("Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion.

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.