Shi Z, Cassaglia PA, Pelletier NE, Brooks VL.
PMID: PMID: 30628058 | DOI: DOI:10.1113/JP277517
KEY POINTS: ICV insulin increased SNA and baroreflex control of SNA and HR dramatically more in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced LSNA responses were associated with reduced tonic inhibition of LSNA by NPY in the PVN. Yet, PVN NPY injection decreased LSNA similarly in OP/OR/CON rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the ArcN became resistant to the inhibitory effects of insulin. A HFD did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced "selective sensitization" of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. ABSTRACT: Obesity increases sympathetic nerve activity (SNA) in men, but not women; however, the mechanisms are unknown. We tested if intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by Neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane-anesthesia. The LSNA response was magnified in OP males, but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats, but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males, due to a decrease in NPY inputs. In contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females, but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on insulin's sympathoexcitatory actions and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.
Zhang N, Zhang HY, Bi SA, Moran TH and Bi S
PMID: 30902570 | DOI: 10.1016/j.neulet.2019.03.030
Thyrotropin-releasing hormone (TRH) plays an important role in the regulation of energy balance. While the regulation of TRH in the paraventricular nucleus (PVN) in response to changes of energy balance has been well studied, how TRH is regulated in the dorsomedial hypothalamus (DMH) in maintaining energy homeostasis remains unclear. Here, we assessed the effects of food restriction and exercise on hypothalamic Trh expression using Otsuka Long-Evens Tokushima Fatty (OLETF) rats. Sedentary ad lib fed OLETF rats (OLETF-SED) became hyperphagic and obese. These alterations were prevented in OLETF rats with running wheel access (OLETF-RW) or food restriction in which their food was pair-fed (OLETF-PF) to the intake of lean control rats (LETO-SED). Evaluation of hypothalamic gene expression revealed that Trh mRNA expression was increased in the PVN of OLETF-SED rats and normalized in OLETF-RW and OLETF-PF rats compared to LETO-SED rats. In contrast, the expression of Trh in the DMH was decreased in OLETF-SED rats relative to LETO-SED rats. This alteration was reversed in OLETF-RW rats as seen in LETO-SED rats, but food restriction resulted in a significant increase in DMH Trh expression in OLETF-PF rats compared to LETO-SED rats. Strikingly, while Trh mRNA expression was decreased in the PVN of intact rats in response to acute food deprivation, food deprivation resulted in increased expression of Trh in the DMH. Together, these results demonstrate the differential regulation of Trh expression in the PVN and DMH in OLETF rats and suggest that DMH TRH also contributes to hypothalamic regulation of energy balance.
Wang L, Huang J, Moore DC, Zuo C, Wu Q, Xie L, von der Mark K, Yuan X, Chen D, Warman ML, Ehrlich MG, Yang W.
PMID: 28983104 | DOI: 10.1038/s41598-017-12767-9
Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.
Journal of developmental biology
Vonk, AC;Hasel-Kolossa, SC;Lopez, GA;Hudnall, ML;Gamble, DJ;Lozito, TP;
PMID: 35225965 | DOI: 10.3390/jdb10010012
(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.
Brenna Ø, Furnes MW, Munkvold B, Kidd M, Sandvik AK, Gustafsson BI.
PMID: 27044258 | DOI: -
Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Ambler, M;Hitrec, T;Wilson, A;Cerri, M;Pickering, A;
PMID: 35440490 | DOI: 10.1523/JNEUROSCI.2102-21.2022
Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular projections from the preoptic area of the hypothalamus (POA) to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the POA and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor.SIGNIFICANCEDaily heterotherms such as mice employ torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the central nervous system controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.
Cellular and molecular gastroenterology and hepatology
Schumacher, MA;Liu, CY;Katada, K;Thai, MH;Hsieh, JJ;Hansten, BJ;Waddell, A;Rosen, MJ;Frey, MR;
PMID: 36414210 | DOI: 10.1016/j.jcmgh.2022.11.004
Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal IL-33/IL-13 signaling loop. Here we tested the hypothesis that IL-13, induced by epithelial Sprouty2 downregulation, promotes DCS cell differentiation and function.Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL-13 and IL-13 receptor; bulk RNA sequencing and qPCR were used to quantify changes in expression of identified HDPs. Cytokine treated colonoids were assessed for DCS cells. A requirement for an IL-33/IL-13 signaling loop in the regulation of DCS cells was assessed in vivo using IL-13 null mice.Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single cell transcriptomics showed the HDP Retnlb/RELMβ is highly enriched in DCS cells. Retnlb/RELMβ expression was increased in Spry2ΔIE colons. IL-13, but not IL-33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL-33-mediated expansion of the DCS cell population in vivo was dependent on IL-13, which was predominantly expressed by type II innate lymphoid cells (ILC2s) in the colonic mucosa.Sprouty2 limits colonic DCS cell differentiation through suppression of IL-13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMβ. Loss of epithelial Sprouty2 activates ILC2s to release IL-13, promoting expansion of the DCS cell population and increased colonic RELMβ levels.
Journal of cellular physiology
Zhang, CL;Lin, YZ;Wu, Q;Yan, C;Wong, MW;Zeng, F;Zhu, P;Bowes, K;Lee, K;Zhang, X;Song, ZY;Lin, S;Shi, YC;
PMID: 35312067 | DOI: 10.1002/jcp.30719
Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt-induced hypertension. Here, we demonstrate that wild-type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY-GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain-derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV-Cre recombinase into the Arc only of the NPY-targeted mutant mice carrying a loxP-flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt-dependent blood pressure. In summary, our study uncovers an important Arc NPY-originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.
Harris NA, Isaac AT, Günther A, Merkel K, Melchior J, Xu M, Eguakun E, Perez R, Nabit BP, Flavin S, Gilsbach R, Shonesy B, Hein L, Abel T, Baumann A, Matthews R, Centanni SW, Winder DG.
PMID: 30150361 | DOI: 10.1523/JNEUROSCI.0963-18.2018
Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress-responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress yet minimally affects relapse, potentially due to competing actions in the brain. Here we show that heteroceptor α2A-ARs postsynaptically enhance dorsal BNST (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, as inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons, and its activation elicits anxiety-like behavior in the elevated plus maze. Together, this data provides a framework for elucidating cell-specific actions of GPCR signaling and provides a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENTStress impacts the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here we show that guanfacine increases dBNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation (HCN) channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest 1) that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons, and 2) these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting care must be taken regarding interpretation of data obtained with these tools.
Chen, HJ;Barske, L;Talbot, JC;Dinwoodie, OM;Roberts, RR;Farmer, DT;Jimenez, C;Merrill, AE;Tucker, AS;Crump, JG;
PMID: 36905926 | DOI: 10.1016/j.devcel.2023.02.011
Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.
Shi, Z;Stornetta, DS;Stornetta, RL;Brooks, VL;
PMID: 34937769 | DOI: 10.1523/ENEURO.0404-21.2021
The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aR): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrus cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory Neuropeptide Y inputs, and activation of pro-opiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e. GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.SIGNIFICANCEThe arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII). Here we show that ArcN AngII activates AT1aR to increase AP in male and female rats by slowly increasing sympathetic nerve activity. In females, ArcN AngII also evoked an initial pressor response mediated by vasopressin-induced vasoconstriction. Pregnant and estrus females responded more than males, in association with higher ArcN AT1aR expression. AT1aR were identified in ArcN interneurons that express tyrosine hydroxylase (TH) and GABA. Since brain AT1aR and TH mediate stress responses, ArcN AT1aR TH neurons are well situated to locally coordinate autonomic, hormonal, and behavioral responses to stress.
Miura Y, Ota S, Peterlin M, McDevitt G, Kanazawa S.
| DOI: 10.1002/jbm4.10132
Specific MHC class II genes result in a high susceptibility to rheumatoid arthritis (RA), with co‐stimulatory molecules working together with MHC class II during the progression of the disease. To elucidate the involvement of the B7.1 co‐stimulatory molecule in RA, we analyzed the phenotype of B7.1 transgenic (named D1BC) mice and the sequential differentiation of synovial fibroblasts (SFs) by studying the expression of chondrogenic and osteogenic lineage markers together with lineage tracing experiment using B7.1 transgene in vivo. The B7.1 transgene was driven by a collagen type II (CII) promoter and enhancer in the D1BC mouse. A low‐dose of bovine CII (bCII) was used to induce chronic articular inflammation with interstitial pneumonitis. Joint damage was analyzed by histopathological examination and computed tomography. B7.1 was expressed in articular cartilage and SFs of D1BC mice. Chronic inflammatory arthritis in bCII‐D1BC mouse shared common features with those found in patients with RA, such as pannus formation, bone destruction, osteoporosis, and joint ankylosis. A subpopulation of SFs (Runx2+, Sox9+, Col10a1+, Osx+ and CX‐) in the pannus was classified as osteochondrogenic lineage rather than mesenchymal stromal lineage. These cells underwent differentiation into osteogenic lineage via hypertrophic chondrocytes at the end of the chronic phase. The ectopic expression of B7.1 in chondrocytes and SFs leads to an increased susceptibility to chronic inflammatory arthritis and subsequent new bone formation, reminiscent of ankylosis. The regulation of cartilage remodeling in pannus tissue is an important consideration in the treatment of RA.
