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.

AgRP neurons control feeding behaviour at cortical synapses via peripherally derived lysophospholipids.

Phospholipid levels are influenced by peripheral metabolism. Within the central nervous system, synaptic phospholipids regulate glutamatergic transmission and cortical excitability. Whether changes in peripheral metabolism affect brain lipid levels and cortical excitability remains unknown. Here, we show that levels of lysophosphatidic acid (LPA) species in the blood and cerebrospinal fluid are elevated after overnight fasting and lead to higher cortical excitability. LPA-related cortical excitability increases fasting-induced hyperphagia, and is decreased following inhibition of LPA synthesis. Mice expressing a human mutation (Prg-1R346T) leading to higher synaptic lipid-mediated cortical excitability display increased fasting-induced hyperphagia. Accordingly, human subjects with this mutation have higher body mass index and prevalence of type 2 diabetes. We further show that the effects of LPA following fasting are under the control of hypothalamic agouti-related peptide (AgRP) neurons. Depletion of AgRP-expressing cells in adult mice decreases fasting-induced elevation of circulating LPAs, as well as cortical excitability, while blunting hyperphagia. These findings reveal a direct influence of circulating LPAs under the control of hypothalamic AgRP neurons on cortical excitability, unmasking an alternative non-neuronal route by which the hypothalamus can exert a robust impact on the cortex and thereby affect food intake.

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.

Age-related calcium dysregulation linked with tau pathology and impaired cognition in non-human primates.

INTRODUCTION: The etiology of sporadic Alzheimer's disease (AD) requires non-genetically modified animal models. METHODS: The relationship of tau phosphorylation to calcium-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) dysregulation was analyzed in aging rhesus macaque dorsolateral prefrontal cortex (dlPFC) and rat primary cortical neurons using biochemistry and immuno-electron microscopy. The influence of calcium leak from ryanodine receptors (RyRs) on neuronal firing and cognitive performance was examined in aged macaques. RESULTS: Aged monkeys naturally develop hyperphosphorylated tau, including AD biomarkers (AT8 (pS202/pT205) and pT217) and early tau pathology markers (pS214 and pS356) that correlated with evidence of increased calcium leak (pS2808-RyR2). Calcium also regulated early tau phosphorylation in vitro. Age-related reductions in the calcium-binding protein, calbindin, and in phosphodiesterase PDE4D were seen within dlPFC pyramidal cell dendrites. Blocking RyRs with S107 improved neuronal firing and cognitive performance in aged macaques. DISCUSSION: Dysregulated calcium signaling confers risk for tau pathology and provides a potential therapeutic target.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Aromatase and estrogen receptor immunoreactivity in the coronary arteries of monkeys and human subjects.

OBJECTIVE: The objective of this study was to determine whether estrogen could be formed locally in the coronary arteries. DESIGN: Coronary arteries were examined from monkeys (Macaca fascicularis, one male and one female) and human subjects (one premenopausal woman, one postmenopausal woman, and one man) by immunocytochemistry, using purified antisera against human placental estrogen synthetase (aromatase) and ER α. The arteries were graded for the amount of atherosclerosis. RESULTS: There was clear immunopositivity for both aromatase and estrogen receptors in all arteries studied. Although all endothelial cells (CD31 positive) stained for both antigens, the staining in macrophages, fibroblasts, and smooth muscle cells was irregular. CONCLUSION: The present results provide the first evidence for the local formation of estrogen in the coronary arteries. In addition to complementing the evidence of a cardioprotective effect of estrogen on the coronary circulation, our results highlight the potential importance of local regulation of estrogen formation and the role of available precursor androgens in maintaining the cardiovascular system.

Loss of Nucleobindin-2 Causes Insulin Resistance in Obesity without Impacting Satiety or Adiposity.

Inducers of satiety are drug targets for weight loss to mitigate obesity-associated diseases. Nucleobindin-2 (Nucb2) is thought to be post-translationally processed into bioactive nesfatin-1 peptide, which reportedly induces satiety, causes weight loss, and thus improves insulin sensitivity. Here, we show that deletion of Nucb2 did not affect food intake or adiposity and, instead, caused insulin resistance in mice fed a high-fat diet. In addition, ablation of Nucb2 in orexigenic hypothalamic Agrp neurons did not affect food intake, and nesfatin-1 was detectable in serum, despite global deletion of Nucb2 protein. Upon high-fat diet feeding, the loss of Nucb2 exacerbated metabolic inflammation in adipose tissue macrophages in an NFκB-dependent manner without inducing classical M1 or alternative M2-like macrophage polarization. Furthermore, the loss of Nucb2 in myeloid cells but not in adipocytes mediated the insulin resistance in response to a high-fat diet. Our study reveals that Nucb2 links metabolic inflammation to insulin resistance without affecting weight gain and food intake.

Viral Vectors for Studying Brain Mechanisms that Control Energy Homeostasis.

Viral vectors have been shown to be potent and versatile tools for genome editing. In the present Minireview, we focus on lentiviruses and adeno-associated viruses as vectors and their use in the study of the hypothalamic circuits involved in the regulation of energy homeostasis.

Comparative Analysis of Zearalenone Effects on Thyroid Receptor Alpha (TRα) and Beta (TRß) Expression in Rat Primary Cerebellar Cell Cultures.

Thyroid receptors play an important role in postnatal brain development. Zearalenone (ZEN), a major mycotoxin of Fusarium fungi, is well known to cause serious health problems in animals and humans through various mechanisms, including the physiological pathways of thyroid hormone (TH). In the present study, we aimed to investigate the expression of thyroid receptors α (TRα) and ß (TRß) in primary cerebellar neurons in the presence or absence of glia and following ZEN treatment, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Primary cerebellar granule cells were treated with low doses of ZEN (0.1 nM) in combination with physiologically relevant concentrations of l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3) and 17ß-estradiol (E2). Expression levels of TRα and TRß at mRNA and protein levels were slightly modified by ZEN administered alone; however, along with thyroid and steroid hormones, modelling the physiological conditions, expression levels of TRs varied highly depending on the given treatment. Gene expression levels were also highly modulated by the presence or absence of glial cells, with mostly contrasting effects. Our results demonstrate divergent transcriptional and translational mechanisms involved in the expression of TRs implied by ZEN and hormonal milieu, as well as culturing conditions.

Myeloid sirtuin1 deficiency aggravates hippocampal inflammation in mice fed high-fat diets.

Chronic low-grade inflammation-induced insulin resistance is associated with neuroinflammation. Myeloid sirtuin1 (SIRT1) deficiency aggravates high-fat diet (HFD)-induced insulin resistance. However, the function of myeloid-specific SIRT1 in the hippocampus of obese mice is largely unknown. To address this question, we fed myeloid SIRT1 knockout (KO) mice a HFD for 40 weeks. We found that HFD-fed SIRT1 KO mice had increased insulin resistance and macrophage infiltration in adipose tissue than wild type (WT) mice. Levels of HFD-induced lipocalin-2 (LCN2) were lower in SIRT1 KO mice than in WT. HFD-induced hippocampal LCN2 expression was lower in HFD-fed SIRT1 KO mice than in WT. Hippocampal acetylation of nuclear factor-κB (NF-κB) and amyloid precursor protein levels were higher in HFD-fed SIRT1 KO mice than in HFD-fed WT mice. Taken together, our results suggest that targeted induction of the anti-inflammatory effects of SIRT1 and LCN2 may help prevent obesity-associated insulin resistance and neuroinflammation.

Thyroid hormone- and estrogen receptor interactions with natural ligands and endocrine disruptors in the cerebellum.

Although the effects of phytoestrogens on brain function is widely unknown, they are often regarded as "natural" and thus as harmless. However, the effects of phytoestrogens or environmental pollutants on brain function is underestimated. Estrogen (17beta-estradiol, E2) and thyroid hormones (THs) play pivotal roles in brain development. In the mature brain, these hormones regulate metabolism on cellular and organismal levels. Thus, E2 and THs do not only regulate the energy metabolism of the entire organism, but simultaneously also regulate important homeostatic parameters of neurons and glia in the CNS. It is, therefore, obvious that the mechanisms through which these hormones exert their effects are pleiotropic and include both intra- and intercellular actions. These hormonal mechanisms are versatile, and the experimental investigation of simultaneous hormone-induced mechanisms is technically challenging. In addition, the normal physiological settings of metabolic parameters depend on a plethora of interactions of the steroid hormones. In this review, we discuss conceptual and experimental aspects of the gonadal and thyroid hormones as they relate to in vitro models of the cerebellum.

Cannabis in fat: high hopes to treat obesity.

Cannabinoid receptor type-1 (CB1) is known to have a substantial impact on the regulation of energy metabolism via central and peripheral mechanisms. In this issue of the JCI, Ruiz de Azua and colleagues provide important insights into the regulation of adipocyte physiology by CB1. Mice with adipocyte-specific deletion of the CB1-encoding gene had an overall improved metabolic profile in addition to reduced body weight and total adiposity. These changes were associated with an increase in sympathetic tone of the adipose tissue and expansion of activated macrophages, both of which occurred prior to changes in body weight, lending support to a causal relationship between loss of CB1 in adipocytes and systemic metabolic changes. This work identifies adipocyte CB1s as a potential novel peripheral target for affecting systemic metabolism with diminished CNS effects.

Regulation of body weight and energy homeostasis by neuronal cell adhesion molecule 1.

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Endothelial HIF-1α Enables Hypothalamic Glucose Uptake to Drive POMC Neurons.

Glucose is the primary driver of hypothalamic proopiomelanocortin (POMC) neurons. We show that endothelial hypoxia-inducible factor 1α (HIF-1α) controls glucose uptake in the hypothalamus and that it is upregulated in conditions of undernourishment, during which POMC neuronal activity is decreased. Endothelium-specific knockdown of HIF-1α impairs the ability of POMC neurons to adapt to the changing metabolic environment in vivo, resulting in overeating after food deprivation in mice. The impaired functioning of POMC neurons was reversed ex vivo or by parenchymal glucose administration. These observations indicate an active role for endothelial cells in the central control of metabolism and suggest that central vascular impairments may cause metabolic disorders.

Bisphenol A influences oestrogen- and thyroid hormone-regulated thyroid hormone receptor expression in rat cerebellar cell culture.

Thyroid hormones (THs) and oestrogens are crucial in the regulation of cerebellar development. TH receptors (TRs) mediate these hormone effects and are regulated by both hormone families. We reported earlier that THs and oestradiol (E2) determine TR levels in cerebellar cell culture. Here we demonstrate the effects of low concentrations (10-10 M) of the endocrine disruptor (ED) bisphenol A (BPA) on the hormonal (THs, E2) regulation of TRα,ß in rat cerebellar cell culture. Primary cerebellar cell cultures, glia-containing and glia-destroyed, were treated with BPA or a combination of BPA and E2 and/or THs. Oestrogen receptor and TH receptor mRNA and protein levels were determined by real-time qPCR and Western blot techniques. The results show that BPA alone decreases, while BPA in combination with THs and/or E2 increases TR mRNA expression. In contrast, BPA alone increased receptor protein expressions, but did not further increase them in combination with THs and/or E2. The modulatory effects of BPA were mediated by the glia; however, the degree of changes also depended on the specific hormone ligand used. The results signify the importance of the regulatory mechanisms interposed between transcription and translation and raise the possibility that BPA could act to influence nuclear hormone receptor levels independently of ligand-receptor interaction.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Comparison of Individual and Combined Effects of Four Endocrine Disruptors on Estrogen Receptor Beta Transcription in Cerebellar Cell Culture: The Modulatory Role of Estradiol and Triiodo-Thyronine.

BACKGROUND: Humans and animals are continuously exposed to a number of environmental substances that act as endocrine disruptors (EDs). While a growing body of evidence is available to prove their adverse health effects, very little is known about the consequences of simultaneous exposure to a combination of such chemicals; METHODS: Here, we used an in vitro model to demonstrate how exposure to bisphenol A, zearalenone, arsenic, and 4-methylbenzylidene camphor, alone or in combination, affect estrogen receptor ß (ERß) mRNA expression in primary cerebellar cell cultures. Additionally, we also show the modulatory role of intrinsic biological factors, such as estradiol (E2), triiodo-thyronine (T3), and glial cells, as potential effect modulators; RESULTS: RESULTS show a wide diversity in ED effects on ERß mRNA expression, and that the magnitude of these ED effects highly depends on the presence or absence of E2, T3, and glial cells; CONCLUSION: The observed potency of the EDs to influence ERß mRNA expression, and the modulatory role of E2, T3, and the glia suggests that environmental ED effects may be masked as long as the hormonal milieu is physiological, but may tend to turn additive or superadditive in case of hormone deficiency.

Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution.

Age-related thymic degeneration is associated with loss of naïve T cells, restriction of peripheral T-cell diversity, and reduced healthspan due to lower immune competence. The mechanistic basis of age-related thymic demise is unclear, but prior evidence suggests that caloric restriction (CR) can slow thymic aging by maintaining thymic epithelial cell integrity and reducing the generation of intrathymic lipid. Here we show that the prolongevity ketogenic hormone fibroblast growth factor 21 (FGF21), a member of the endocrine FGF subfamily, is expressed in thymic stromal cells along with FGF receptors and its obligate coreceptor, ßKlotho. We found that FGF21 expression in thymus declines with age and is induced by CR. Genetic gain of FGF21 function in mice protects against age-related thymic involution with an increase in earliest thymocyte progenitors and cortical thymic epithelial cells. Importantly, FGF21 overexpression reduced intrathymic lipid, increased perithymic brown adipose tissue, and elevated thymic T-cell export and naïve T-cell frequencies in old mice. Conversely, loss of FGF21 function in middle-aged mice accelerated thymic aging, increased lethality, and delayed T-cell reconstitution postirradiation and hematopoietic stem cell transplantation (HSCT). Collectively, FGF21 integrates metabolic and immune systems to prevent thymic injury and may aid in the reestablishment of a diverse T-cell repertoire in cancer patients following HSCT.

Estrogen- and Satiety State-Dependent Metabolic Lateralization in the Hypothalamus of Female Rats.

Hypothalamus is the highest center and the main crossroad of numerous homeostatic regulatory pathways including reproduction and energy metabolism. Previous reports indicate that some of these functions may be driven by the synchronized but distinct functioning of the left and right hypothalamic sides. However, the nature of interplay between the hemispheres with regard to distinct hypothalamic functions is still unclear. Here we investigated the metabolic asymmetry between the left and right hypothalamic sides of ovariectomized female rats by measuring mitochondrial respiration rates, a parameter that reflects the intensity of cell and tissue metabolism. Ovariectomized (saline injected) and ovariectomized+estrogen injected animals were fed ad libitum or fasted to determine 1) the contribution of estrogen to metabolic asymmetry of hypothalamus; and 2) whether the hypothalamic asymmetry is modulated by the satiety state. Results show that estrogen-priming significantly increased both the proportion of animals with detected hypothalamic lateralization and the degree of metabolic difference between the hypothalamic sides causing a right-sided dominance during state 3 mitochondrial respiration (St3) in ad libitum fed animals. After 24 hours of fasting, lateralization in St3 values was clearly maintained; however, instead of the observed right-sided dominance that was detected in ad libitum fed animals here appeared in form of either right- or left-sidedness. In conclusion, our results revealed estrogen- and satiety state-dependent metabolic differences between the two hypothalamic hemispheres in female rats showing that the hypothalamic hemispheres drive the reproductive and satiety state related functions in an asymmetric manner.

The ketone metabolite ß-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease.

The ketone bodies ß-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1ß and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1ß secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome, familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.