A hydrophobic groove in secretagogin allows for alternate interactions with SNAP-25 and syntaxin-4 in endocrine tissues.

Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.

Transient expression of the neuropeptide galanin modulates peripheral­to­central connectivity in the somatosensory thalamus during whisker development in mice.

The significance of transient neuropeptide expression during postnatal brain development is unknown. Here, we show that galanin expression in the ventrobasal thalamus of infant mice coincides with whisker map development and modulates subcortical circuit wiring. Time-resolved neuroanatomy and single-nucleus RNA-seq identified complementary galanin (Gal) and galanin receptor 1 (Galr1) expression in the ventrobasal thalamus and the principal sensory nucleus of the trigeminal nerve (Pr5), respectively. Somatodendritic galanin release from the ventrobasal thalamus was time-locked to the first postnatal week, when Gal1R+ Pr5 afferents form glutamatergic (Slc17a6+) synapses for the topographical whisker map to emerge. RNAi-mediated silencing of galanin expression disrupted glutamatergic synaptogenesis, which manifested as impaired whisker-dependent exploratory behaviors in infant mice, with behavioral abnormalities enduring into adulthood. Pharmacological probing of receptor selectivity in vivo corroborated that target recognition and synaptogenesis in the thalamus, at least in part, are reliant on agonist-induced Gal1R activation in inbound excitatory axons. Overall, we suggest a neuropeptide-dependent developmental mechanism to contribute to the topographical specification of a fundamental sensory neurocircuit in mice.

A brainstem-hypothalamus neuronal circuit reduces feeding upon heat exposure.

Empirical evidence suggests that heat exposure reduces food intake. However, the neurocircuit architecture and the signalling mechanisms that form an associative interface between sensory and metabolic modalities remain unknown, despite primary thermoceptive neurons in the pontine parabrachial nucleus becoming well characterized1. Tanycytes are a specialized cell type along the wall of the third ventricle2 that bidirectionally transport hormones and signalling molecules between the brain's parenchyma and ventricular system3-8. Here we show that tanycytes are activated upon acute thermal challenge and are necessary to reduce food intake afterwards. Virus-mediated gene manipulation and circuit mapping showed that thermosensing glutamatergic neurons of the parabrachial nucleus innervate tanycytes either directly or through second-order hypothalamic neurons. Heat-dependent Fos expression in tanycytes suggested their ability to produce signalling molecules, including vascular endothelial growth factor A (VEGFA). Instead of discharging VEGFA into the cerebrospinal fluid for a systemic effect, VEGFA was released along the parenchymal processes of tanycytes in the arcuate nucleus. VEGFA then increased the spike threshold of Flt1-expressing dopamine and agouti-related peptide (Agrp)-containing neurons, thus priming net anorexigenic output. Indeed, both acute heat and the chemogenetic activation of glutamatergic parabrachial neurons at thermoneutrality reduced food intake for hours, in a manner that is sensitive to both Vegfa loss-of-function and blockage of vesicle-associated membrane protein 2 (VAMP2)-dependent exocytosis from tanycytes. Overall, we define a multimodal neurocircuit in which tanycytes link parabrachial sensory relay to the long-term enforcement of a metabolic code.

Molecularly stratified hypothalamic astrocytes are cellular foci for obesity.

Astrocytes safeguard the homeostasis of the central nervous system1,2. Despite their prominent morphological plasticity under conditions that challenge the brain's adaptive capacity3-5, the classification of astrocytes, and relating their molecular make-up to spatially devolved neuronal operations that specify behavior or metabolism, remained mostly futile6,7. Although it seems unexpected in the era of single-cell biology, the lack of a major advance in stratifying astrocytes under physiological conditions rests on the incompatibility of 'neurocentric' algorithms that rely on stable developmental endpoints, lifelong transcriptional, neurotransmitter, and neuropeptide signatures for classification6-8 with the dynamic functional states, anatomic allocation, and allostatic plasticity of astrocytes1. Simplistically, therefore, astrocytes are still grouped as 'resting' vs. 'reactive', the latter referring to pathological states marked by various inducible genes3,9,10. Here, we introduced a machine learning-based feature recognition algorithm that benefits from the cumulative power of published single-cell RNA-seq data on astrocytes as a reference map to stepwise eliminate pleiotropic and inducible cellular features. For the healthy hypothalamus, this walk-back approach revealed gene regulatory networks (GRNs) that specified subsets of astrocytes, and could be used as landmarking tools for their anatomical assignment. The core molecular censuses retained by astrocyte subsets were sufficient to stratify them by allostatic competence, chiefly their signaling and metabolic interplay with neurons. Particularly, we found differentially expressed mitochondrial genes in insulin-sensing astrocytes and demonstrated their reciprocal signaling with neurons that work antagonistically within the food intake circuitry. As a proof-of-concept, we showed that disrupting Mfn2 expression in astrocytes reduced their ability to support dynamic circuit reorganization, a time-locked feature of satiety in the hypothalamus, thus leading to obesity in mice. Overall, our results suggest that astrocytes in the healthy brain are fundamentally more heterogeneous than previously thought and topologically mirror the specificity of local neurocircuits.

Grabbing neuropeptide signals in the brain.

Bioengineered sensors resolve the dynamics of neuropeptide action.

Neuronal diversity of neuropeptide signaling, including galanin, in the mouse locus coeruleus.

The locus coeruleus (LC) is a small nucleus in the pons from which ascending and descending projections innervate major parts of the central nervous system. Its major transmitter is norepinephrine (NE). This system is evolutionarily conserved, including in humans, and its functions are associated with wakefulness and related to disorders, such as depression. Here, we performed single-cell ribonucleic acid-sequencing (RNA-seq) to subdivide neurons in the LC (24 clusters in total) into 3 NE, 17 glutamate, and 5 γ-aminobutyric acid (GABA) subtypes, and to chart their neuropeptide, cotransmitter, and receptor profiles. We found that NE neurons expressed at least 19 neuropeptide transcripts, notably galanin (Gal) but not Npy, and >30 neuropeptide receptors. Among the galanin receptors, Galr1 was expressed in ~19% of NE neurons, as was also confirmed by in situ hybridization. Unexpectedly, Galr1 was highly expressed in GABA neurons surrounding the NE ensemble. Patch-clamp electrophysiology and cell-type-specific Ca2+-imaging using GCaMP6s revealed that a GalR1 agonist inhibits up to ~35% of NE neurons. This effect is direct and does not rely on feed-forward GABA inhibition. Our results define a role for the galanin system in NE functions, and a conceptual framework for the action of many other peptides and their receptors.

Adverse effects of gestational ω-3 and ω-6 polyunsaturated fatty acid imbalance on the programming of fetal brain development.

Obesity is a key medical challenge of our time. The increasing number of children born to overweight or obese women is alarming. During pregnancy, the circulation of the mother and her fetus interact to maintain the uninterrupted availability of essential nutrients for fetal organ development. In doing so, the mother's dietary preference determines the amount and composition of nutrients reaching the fetus. In particular, the availability of polyunsaturated fatty acids (PUFAs), chiefly their ω-3 and ω-6 subclasses, can change when pregnant women choose a specific diet. Here, we provide a succinct overview of PUFA biochemistry, including exchange routes between ω-3 and ω-6 PUFAs, the phenotypes, and probable neurodevelopmental disease associations of offspring born to mothers consuming specific PUFAs, and their mechanistic study in experimental models to typify signaling pathways, transcriptional, and epigenetic mechanisms by which PUFAs can imprint long-lasting modifications to brain structure and function. We emphasize that the ratio, rather than the amount of individual ω-3 or ω-6 PUFAs, might underpin physiologically correct cellular differentiation programs, be these for neurons or glia, during pregnancy. Thereupon, the PUFA-driven programming of the brain is contextualized for childhood obesity, metabolic, and endocrine illnesses.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Brain-wide mapping of efferent projections of glutamatergic (Onecut3+ ) neurons in the lateral mouse hypothalamus.

AIM: This study mapped the spatiotemporal positions and connectivity of Onecut3+ neuronal populations in the developing and adult mouse brain. METHODS: We generated fluorescent reporter mice to chart Onecut3+ neurons for brain-wide analysis. Moreover, we crossed Onecut3-iCre and Mapt-mGFP (Tau-mGFP) mice to visualize axonal projections. A dual Cre/Flp-dependent AAV construct in Onecut3-iCre cross-bred with Slc17a6-FLPo mice was used in an intersectional strategy to map the connectivity of glutamatergic lateral hypothalamic neurons in the adult mouse. RESULTS: We first found that Onecut3 marks a hitherto undescribed Slc17a6+ /Vglut2+ neuronal cohort in the lateral hypothalamus, with the majority expressing thyrotropin-releasing hormone. In the adult, Onecut3+ /Vglut2+ neurons of the lateral hypothalamus had both intra- and extrahypothalamic efferents, particularly to the septal complex and habenula, where they targeted other cohorts of Onecut3+ neurons and additionally to the neocortex and hippocampus. This arrangement suggests that intrinsic reinforcement loops could exist for Onecut3+ neurons to coordinate their activity along the brain's midline axis. CONCLUSION: We present both a toolbox to manipulate novel subtypes of hypothalamic neurons and an anatomical arrangement by which extrahypothalamic targets can be simultaneously entrained.

Regional redistribution of CB1 cannabinoid receptors in human foetal brains with Down's syndrome and their functional modifications in Ts65Dn+/+ mice.

AIMS: The endocannabinoid system with its type 1 cannabinoid receptor (CB1 R) expressed in postmitotic neuroblasts is a critical chemotropic guidance module with its actions cascading across neurogenic commitment, neuronal polarisation and synaptogenesis in vertebrates. Here, we present the systematic analysis of regional CB1 R expression in the developing human brain from gestational week 14 until birth. In parallel, we diagrammed differences in CB1 R development in Down syndrome foetuses and identified altered CB1 R signalling. METHODS: Foetal brains with normal development or with Down's syndrome were analysed using standard immunohistochemistry, digitalised light microscopy and image analysis (NanoZoomer). CB1 R function was investigated by in vitro neuropharmacology from neonatal Ts65Dn transgenic mice brains carrying an additional copy of ~90 conserved protein-coding gene orthologues of the human chromosome 21. RESULTS: We detected a meshwork of fine-calibre, often varicose processes between the subventricular and intermediate zones of the cortical plate in the late first trimester, when telencephalic fibre tracts develop. The density of CB1 Rs gradually decreased during the second and third trimesters in the neocortex. In contrast, CB1 R density was maintained, or even increased, in the hippocampus. We found the onset of CB1 R expression being delayed by ≥1 month in age-matched foetal brains with Down's syndrome. In vitro, CB1 R excitation induced excess microtubule stabilisation and, consequently, reduced neurite outgrowth. CONCLUSIONS: We suggest that neuroarchitectural impairments in Down's syndrome brains involve the delayed development and errant functions of the endocannabinoid system, with a particular impact on endocannabinoids modulating axonal wiring.

Resident Astrocytes can Limit Injury to Developing Hippocampal Neurons upon THC Exposure.

Cannabis legalization prompted the dilemma if plant-derived recreational drugs can have therapeutic potential and, consequently, how to address their regulation and safe distribution. In parallel, the steady worldwide decriminalization of cannabis and the enhanced content of its main psychoactive compound Δ9-tetrahydrocannabinol (THC), exposes populations to increasing amounts of cannabis and THC across all ages. While adverse effects of cannabis during critical stages of fetal neurodevelopment are investigated, these studies center on neurons alone. Thus, a gap of knowledge exists on how intercellular interactions between neighboring cell types, particularly astrocytes and neurons, could modify THC action. Here, we combine transcriptome analysis, transgenic models, high resolution microscopy and live cell imaging to demonstrate that hippocampal astrocytes accumulate in the strata radiatum and lacunosum moleculare of the CA1 subfield, containing particularly sensitive neurons to stressors, upon long term postnatal THC exposure in vivo. As this altered distribution is not dependent on cell proliferation, we propose that resident astrocytes accumulate in select areas to protect pyramidal neurons and their neurite extensions from pathological damage. Indeed, we could recapitulate the neuroprotective effect of astrocytes in vitro, as their physical presence significantly reduced the death of primary hippocampal neurons upon THC exposure (> 5 µM). Even so, astrocytes are also affected by a reduced metabolic readiness to stressors, as reflected by a downregulation of mitochondrial proteins. Thus, we find that astrocytes exert protective functions on local neurons during THC exposure, even though their mitochondrial electron transport chain is disrupted.

An accessory prefrontal cortex-thalamus circuit sculpts maternal behavior in virgin female mice.

The ability to care for the young is innate and readily displayed by postpartum females after delivery to ensure offspring survival. Upon pup exposure, rodent virgin (nulliparous) females also develop parental behavior that over time becomes displayed at levels equivalent to parenting mothers. Although maternal behavior in postpartum females and the associated neurocircuits are well characterized, the neural mechanisms underlying the acquisition of maternal behavior without prior experience remain poorly understood. Here, we show that the development of maternal care behavior in response to first-time pup exposure in virgin females is initiated by the activation of the anterior cingulate cortex (ACC). ACC activity is dependent on feedback excitation by Vglut2+ /Galanin+ neurons of the centrolateral nucleus of the thalamus (CL), with their activity sufficient to display parenting behaviors. Accordingly, acute bidirectional chemogenetic manipulation of neuronal activity in the ACC facilitates or impairs the attainment of maternal behavior, exclusively in virgin females. These results reveal an ACC-CL neurocircuit as an accessory loop in virgin females for the initiation of maternal care upon first-time exposure to pups.

A hypothalamic dopamine locus for psychostimulant-induced hyperlocomotion in mice.

The lateral septum (LS) has been implicated in the regulation of locomotion. Nevertheless, the neurons synchronizing LS activity with the brain's clock in the suprachiasmatic nucleus (SCN) remain unknown. By interrogating the molecular, anatomical and physiological heterogeneity of dopamine neurons of the periventricular nucleus (PeVN; A14 catecholaminergic group), we find that Th+/Dat1+ cells from its anterior subdivision innervate the LS in mice. These dopamine neurons receive dense neuropeptidergic innervation from the SCN. Reciprocal viral tracing in combination with optogenetic stimulation ex vivo identified somatostatin-containing neurons in the LS as preferred synaptic targets of extrahypothalamic A14 efferents. In vivo chemogenetic manipulation of anterior A14 neurons impacted locomotion. Moreover, chemogenetic inhibition of dopamine output from the anterior PeVN normalized amphetamine-induced hyperlocomotion, particularly during sedentary periods. Cumulatively, our findings identify a hypothalamic locus for the diurnal control of locomotion and pinpoint a midbrain-independent cellular target of psychostimulants.

Ontogenetic rules for the molecular diversification of hypothalamic neurons.

The hypothalamus is an evolutionarily conserved endocrine interface that, among other roles, links central homeostatic control to adaptive bodily responses by releasing hormones and neuropeptides from its many neuronal subtypes. In its preoptic, anterior, tuberal and mammillary subdivisions, a kaleidoscope of magnocellular and parvocellular neuroendocrine command neurons, local-circuit neurons, and neurons that project to extrahypothalamic areas are intermingled in partially overlapping patches of nuclei. Molecular fingerprinting has produced data of unprecedented mass and depth to distinguish and even to predict the synaptic and endocrine competences, connectivity and stimulus selectivity of many neuronal modalities. These new insights support eminent studies from the past century but challenge others on the molecular rules that shape the developmental segregation of hypothalamic neuronal subtypes and their use of morphogenic cues for terminal differentiation. Here, we integrate single-cell RNA sequencing studies with those of mouse genetics and endocrinology to describe key stages of hypothalamus development, including local neurogenesis, the direct terminal differentiation of glutamatergic neurons, transition cascades for GABAergic and GABAergic cell-derived dopamine cells, waves of local neuronal migration, and sequential enrichment in neuropeptides and hormones. We particularly emphasize how transcription factors determine neuronal identity and, consequently, circuit architecture, and whether their deviations triggered by environmental factors and hormones provoke neuroendocrine illnesses.

Gestational immune activation disrupts hypothalamic neurocircuits of maternal care behavior.

Immune activation is one of the most common complications during pregnancy, predominantly evoked by viral infections. Nevertheless, how immune activation affects mother-offspring relationships postpartum remains unknown. Here, by using the polyinosinic-polycytidylic acid (Poly I:C) model of gestational infection we show that viral-like immune activation at mid-gestation persistently changes hypothalamic neurocircuit parameters in mouse dams and, consequently, is adverse to parenting behavior. Poly I:C-exposed dams favor non-pup-directed exploratory behavior at the expense of pup retrieval. These behavioral deficits are underlain by dendrite pruning and lesser immediate early gene activation in Galanin (Gal)+ neurons with dam-specific transcriptional signatures that reside in the medial preoptic area (mPOA). Reduced activation of an exclusively inhibitory contingent of these distal-projecting Gal+ neurons allows for increased feed-forward inhibition onto putative dopaminergic neurons in the ventral tegmental area (VTA) in Poly I:C-exposed dams. Notably, destabilized VTA output specifically accompanies post-pup retrieval epochs. We suggest that gestational immunogenic insults bias both threat processing and reward perception, manifesting as disfavored infant caregiving.

Preclinical Establishment of a Divalent Vaccine against SARS-CoV-2.

First-generation vaccines against SARS-CoV-2 do not provide adequate immune protection. Therefore, we engineered a divalent gene construct combining the receptor-binding domain (RBD) of the spike protein and the immunodominant region of the viral nucleocapsid. This fusion protein was produced in either E. coli or a recombinant baculovirus system. Subsequently, the fusion protein was mixed with adjuvant and administered to mice in a prime-booster mode. Mice (72%) produced an IgG response against both proteins (titer: 10-4-10-5) 14 days after the first booster injection, which was increased to 100% by a second booster. Comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed IgGs beyond 90 days. In addition, cytolytic effector cell molecules were increased in lymphocytes isolated from peripheral blood. Ex vivo stimulation of T cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 among the CD4+ and CD8+ T cells of vaccinated mice. No side effect was documented in the central nervous system. Cumulatively, these data represent a proof-of-principle approach alternative to existing mRNA vaccination strategies.

A hypothalamic pathway for Augmentor α-controlled body weight regulation.

Augmentor α and ß (Augα and Augß) are newly discovered ligands of the receptor tyrosine kinases Alk and Ltk. Augα functions as a dimeric ligand that binds with high affinity and specificity to Alk and Ltk. However, a monomeric Augα fragment and monomeric Augß also bind to Alk and potently stimulate cellular responses. While previous studies demonstrated that oncogenic Alk mutants function as important drivers of a variety of human cancers, the physiological roles of Augα and Augß are poorly understood. Here, we investigate the physiological roles of Augα and Augß by exploring mice deficient in each or both Aug ligands. Analysis of mutant mice showed that both Augα single knockout and double knockout of Augα and Augß exhibit a similar thinness phenotype and resistance to diet-induced obesity. In the Augα-knockout mice, the leanness phenotype is coupled to increased physical activity. By contrast, Augß-knockout mice showed similar weight curves as the littermate controls. Experiments are presented demonstrating that Augα is robustly expressed and metabolically regulated in agouti-related peptide (AgRP) neurons, cells that control whole-body energy homeostasis in part via their projections to the paraventricular nucleus (PVN). Moreover, both Alk and melanocortin receptor-4 are expressed in discrete neuronal populations in the PVN and are regulated by projections containing Augα and AgRP, respectively, demonstrating that two distinct mechanisms that regulate pigmentation operate in the hypothalamus to control body weight. These experiments show that Alk-driven cancers were co-opted from a neuronal pathway in control of body weight, offering therapeutic opportunities for metabolic diseases and cancer.