Probes for INS

Background Aging is accompanied by altered thinking (cognition) and feeling (mood), functions that depend on information processing by brain cortical cell microcircuits. We hypothesized that age-associated long-term functional and biological changes are mediated by gene transcriptomic changes within neuronal cell-types forming cortical microcircuits, namely excitatory pyramidal cells (PYC) and inhibitory GABAergic neurons expressing vasoactive intestinal peptide (Vip), somatostatin (Sst) and parvalbumin (Pvalb). Methods To test this hypothesis, we assessed locomotor, anxiety-like and cognitive behavioral changes between young (2 months, n=9) and old (22 months, n=12) male C57BL/6 mice, and performed frontal cortex cell-type specific molecular profiling, using laser-capture microscopy and RNA sequencing. Results were analyzed by neuroinformatics and validated by fluorescent in situ hybridization. Results Old-mice displayed increased anxiety and reduced working memory. The four cell-types displayed distinct age-related transcriptomes and biological pathway profiles, affecting metabolic and cell signaling pathways, and selective markers of neuronal vulnerability (Ryr3), resilience (Oxr1), and mitochondrial dynamics (Opa1), suggesting high age-related vulnerability of PYCs, and variable degree of adaptation in GABAergic neurons. Correlations between gene expression and behaviors suggest that changes in cognition and anxiety associated with age are partly mediated by normal age-related cell changes, and that additional age-independent decreases in synaptic and signaling pathways, notably in PYC and SST-neurons further contribute to behavioral changes. Conclusions Our study demonstrates cell-dependent differential vulnerability and coordinated cell-specific cortical microcircuit molecular changes with age. Collectively, the results suggest intrinsic molecular links between aging, cognition and mood-related behaviors with SST-neurons contributing evenly to both behavioral conditions.

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

The experience of rewarding or aversive stimuli is encoded by distinct afferents to dopamine (DA) neurons of the ventral tegmental area (VTA). Several neuromodulatory systems including oxytocin regulate DA neuron excitability and synaptic transmission that process socially meaningful stimuli. We and others have recently characterized oxytocinergic modulation of activity in mouse VTA DA neurons, but the mechanisms underlying oxytocinergic modulation of synaptic transmission in DA neurons remain poorly understood. Here, we find that oxytocin application or optogenetic release decrease excitatory synaptic transmission, via long lasting, presynaptic, endocannabinoid-dependent mechanisms. Oxytocin modulation of excitatory transmission alters the magnitude of short and long-term depression. We find that only some glutamatergic projections to DA neurons express CB1 receptors. Optogenetic stimulation of three major VTA inputs demonstrates that oxytocin modulation is limited to projections that show evidence of CB1R transcripts. Thus, oxytocin gates information flow into reward circuits in a temporally selective and pathway-specific manner.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/β-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike β-catenin gain-of-function models, which induce high Wnt/β-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/β-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing β-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/β-catenin activation, which is regulated by ZNRF3.

Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems.