Single-Cell Mapping of GLP-1 and GIP Receptor Expression in the Dorsal Vagal Complex
Ludwig, MQ;Todorov, PV;Egerod, KL;Olson, DP;Pers, TH;
PMID: 34176785 | DOI: 10.2337/dbi21-0003
The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor-expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain.
Methods in molecular biology (Clifton, N.J.)
Moissoglu, K;Lockett, SJ;Mili, S;
PMID: 36653713 | DOI: 10.1007/978-1-0716-2887-4_16
Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.
Investigative Ophthalmology & Visual Science
Anderson, K;Venkatesh, A;McKenty, T;
PURPOSE : ADOA is the most common inherited optic neuropathy, starting in the first decade of life and resulting in severe and progressive visual decline due to loss of RGCs. Most patients harbor loss-of-function mutations in the _OPA1 _gene that lead to haploinsufficiency. Reduced OPA1 protein levels result in impaired mitochondrial function in RGCs leading to cell death. Currently, there is no treatment for patients with ADOA. Targeted Augmentation of Nuclear Gene Output (TANGO) ASOs, such as STK-002, reduce the inclusion of a non-productive, alternatively spliced exon in _OPA1, _and leverage the wild-type allele to increase productive _OPA1_ mRNA and protein. We previously demonstrated that TANGO ASOs can increase OPA1 protein levels in human cell lines, rabbit retina, and ADOA patient fibroblasts. In this study, we evaluated ASO localization and OPA1 protein levels in the retina following intravitreal administration of STK-002 to NHPs.
Mendez-David, I;Schofield, R;Tritschler, L;Colle, R;Guilloux, JP;Gardier, AM;Corruble, E;Hen, R;David, DJ;
PMID: 34649711 | DOI: 10.1016/j.encep.2021.09.001
Recent contradictory data has renewed discussion regarding the existence of adult hippocampal neurogenesis (AHN) in humans, i.e., the continued production of new neurons in the brain after birth. The present review revisits the debate of AHN in humans from a historical point of view in the face of contradictory evidence, analyzing the methods employed to investigate this phenomenon. Thus, to date, of the 57 studies performed in humans that we reviewed, 84% (48) concluded in favor of the presence of newborn neurons in the human adult hippocampus. Besides quality of the tissue (such as postmortem intervals below 26hours as well as tissue conservation and fixation), considerations for assessing and quantify AHN in the human brain require the use of stereology and toxicological analyses of clinical data of the patient.
Culture and maintenance of urine derived, 3-dimensional canine transitional cell carcinoma organoids
Thenuwara, S;
| DOI: 10.31274/etd-20210609-187
Bladder Cancer is the 9th most common malignancy in the world. Transitional cell carcinoma (TCC) is the most common of bladder cancers, occurring in 90% of cases. There has been no great model established to study TCC in vitro. In this study, we explore urine-derived, 3-dimensional, canine TCC organoids as a possible model to study TCC in vitro. After establishing the cell line, we subjected the 3-D cells to RNA in situ hybridization (RNAish) and cell viability assays. Overall, 3-D cell culture from urine samples of TCC diagnosed canines expressed RNA biomarkers in a similar manner to parent tumors via RNAish and showed more sensitivity to Cisplatin treatment when compared to 2-D human TCC cells. With further experimentation, canine TCC organoids could be an ideal model to study TCC in vitro.
Journal of Vascular Surgery
Kasashima S, Kawashima A, Zen Y, Ozaki S, Kasashima F, Endo M, Matsumoto Y, Kawakami K.
PMID: 28434701 | DOI: 10.1016/j.jvs.2016.12.140
Abstract
OBJECTIVE:
Immunoglobulin (Ig) G4-related aortic aneurysms (IgG4-AAs) are a special aortic aneurysm among IgG4-related diseases (IgG4-RDs), which are inflammatory and fibrous conditions characterized by tumorous swelling of affected organs and high serum IgG4 concentrations. Recently, IgG4-RD pathogenesis was shown to be associated with T-helper-2 (Th2) and regulatory T (Treg) dominant cytokine production, such as interleukin (IL)-4, IL-10, and IL-13. IL-6 is a key proinflammatory cytokine contributing to lymphocyte and plasmacyte maturation and to atherosclerosis and aneurysm development. We serologically and histopathologically evaluated the cytokine profile in IgG4-AA patients.
METHODS:
Patients with IgG4-AAs (n = 10), non-IgG4-related inflammatory abdominal aortic aneurysms (non-IgG4-AAAs; n = 5), atherosclerotic AAAs (aAAAs; n = 10), and normal aortas without dilatation (n = 10) were examined for serum IL-10, IL-13, and IL-6 levels. Resected aortic tissues were evaluated for cluster of differentiation (CD) 34 (in the endothelial cells and mesenchymal cells) and CD163 (by macrophages) expression using immunohistochemistry and in situ hybridization.
RESULTS:
Serum IL-10 levels were rather higher in IgG4-AA patients (median, 1.3 pg/mL) than in non-IgG4-AAA and aAAA patients and in patients with normal aortas. Elevated serum IL-13 levels relative to standard values were detected in two IgG4-AA patients but not in the other groups. Cells immunopositive for IL-10 and IL-13 were more frequent in IgG4-AAs and significantly correlated with serum IgG4 levels. Serum IL-6 levels (median, 78.5 pg/mL) were also significantly higher in IgG4-AA patients than in non-IgG4-AAA and aAAA patients and control patients with normal aortas (P = .01, P = .001, and P = .004, respectively). They positively correlated with serum IgG4 levels and adventitial thickness, but other cytokines did not. The number of IL-6-immunopositive cells in the adventitia was significantly higher in IgG4-AA patients (median, 17.8/high-power field) than in aAAA patients or patients with normal aortas (P =.001 and P = .002, respectively). In situ hybridization confirmed frequent IL-6 messenger (m)RNA expression in the endothelium, mesenchymal cells, and histiocytes in IgG4-AA adventitia. In the same cells of IgG4-AAs, coexpression of IL-6 and CD34 mRNA or CD163 mRNA was detected.
CONCLUSIONS:
The cytokine profiles of IgG4-AA patients had two characteristics: local IL-10 and IL-13 upregulation in IgG4-AAs was related to Th2 and Treg-predominant cytokine balance, similar to other IgG4-RDs, and IL-6 upregulation in the adventitia was characterized by activated immune reactions in IgG4-AA patients. IL-6 synthesis, through contributions of mesenchymal cells and macrophages in the adventitia, is strongly involved in IgG4-AA pathogenesis or progression, or both.
Su S, Dwyer DM, Miska KB, Fetterer RH, Jenkins MC, Wong EA.
PMID: 28521031 | DOI: 10.3382/ps/pew468
Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.
Marshall VA, Labo N, Hao XP, Holdridge B, Thompson M, Miley W, Brands C, Coalter V, Kiser R, Anver M, Golubeva Y, Warner A, Jaffe ES, Piatak M Jr, Wong SW, Ohlen C, MacAllister R, Smedley J, Deleage C, Del Prete GQ, Lifson JD, Estes JD, Whitby D.
PMID: 30001436 | DOI: 10.1371/journal.ppat.1007130
Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.
Haidar M, Tin K, Zhang C, Nategh M, Covita J, Wykes AD, Rogers J and Gundlach AL
PMID: 30906254 | DOI: 10.3389/fnana.2019.00030
Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.
Sheng, ZF;Zhang, H;Phaup, JG;Zheng, P;Kang, X;Liu, Z;Chang, HM;Yeh, ETH;Johnson, AK;Pan, HL;Li, DP;
PMID: 37041718 | DOI: 10.1093/cvr/cvad056
Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension.Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs.CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.We found that hyperactivity of CRH neurons in the CeA, likely due to diminished Kv7 channel activity, play a major role in the development of chronic stress-induced hypertension. Our study suggests that CRH neurons in the brain may be targeted for treating chronic stress-induced hypertension. Thus, increasing Kv7 channel activity or overexpressing Kv7 channels in the CeA may reduce stress-induced hypertension. Further studies are needed to delineate how chronic stress diminishes Kv7 channel activity in the brain.
Maynard, JP;Godwin, TN;Lu, J;Vidal, I;Lotan, TL;De Marzo, AM;Joshu, CE;Sfanos, KS;
PMID: 35971807 | DOI: 10.1002/pros.24424
Black men are two to three times more likely to die from prostate cancer (PCa) than White men. This disparity is due in part to discrepancies in socioeconomic status and access to quality care. Studies also suggest that differences in the prevalence of innate immune cells and heightened function in the tumor microenvironment of Black men may promote PCa aggressiveness.We evaluated the spatial localization of and quantified CD66ce+ neutrophils by immunohistochemistry and CD68+ (pan), CD80+ (M1), and CD163+ (M2) macrophages by RNA in situ hybridization on formalin-fixed paraffin-embedded tissues from organ donor "normal" prostate (n = 9) and radical prostatectomy (n = 38) tissues from Black and White men. Neutrophils were quantified in PCa and matched benign tissues in tissue microarray (TMA) sets comprised of 560 White and 371 Black men. Likewise, macrophages were quantified in TMA sets comprised of tissues from 60 White and 120 Black men. The phosphatase and tensin homolog (PTEN) and ETS transcription factor ERG (ERG) expression status of each TMA PCa case was assessed via immunohistochemistry. Finally, neutrophils and macrophage subsets were assessed in a TMA set comprised of distant metastatic PCa tissues collected at autopsy (n = 6) sampled across multiple sites.CD66ce+ neutrophils were minimal in normal prostates, but were increased in PCa compared to benign tissues, in low grade compared to higher grade PCa, in PCa tissues from White compared to Black men, and in PCa with PTEN loss or ERG positivity. CD163+ macrophages were the predominant macrophage subset in normal organ donor prostate tissues from both Black and White men and were significantly more abundant in organ donor compared to prostatectomy PCa tissues. CD68,+ CD80,+ and CD163+ macrophages were significantly increased in cancer compared to benign tissues and in cancers with ERG positivity. CD68+ and CD163+ macrophages were increased in higher grade cancers compared to low grade cancer and CD80 expression was significantly higher in benign prostatectomy tissues from Black compared to White men.Innate immune cell infiltration is increased in the prostate tumor microenvironment of both Black and White men, however the composition of innate immune cell infiltration may vary between races.
X-linked serotonin 2C receptor is associated with a non-canonical pathway for sudden unexpected death in epilepsy
Massey, C;Thompson, S;Ostrom, R;Drabek, J;Sveinsson, O;Tomson, T;Haas, E;Mena, O;Goldman, A;Noebels, J;
| DOI: 10.1093/braincomms/fcab149
Sudden Unexpected Death in Epilepsy is a leading cause of epilepsy-related mortality, and the analysis of mouse Sudden Unexpected Death in Epilepsy models is steadily revealing a spectrum of inherited risk phenotypes based on distinct genetic mechanisms. Serotonin (5-HT) signalling enhances post-ictal cardiorespiratory drive and, when elevated in the brain, reduces death following evoked audiogenic brainstem seizures in inbred mouse models. However, no gene in this pathway has yet been linked to a spontaneous epilepsy phenotype, the defining criterion of Sudden Unexpected Death in Epilepsy. Most monogenic models of Sudden Unexpected Death in Epilepsy invoke a failure of inhibitory synaptic drive as a critical pathogenic step. Accordingly, the G protein-coupled, membrane serotonin receptor 5-HT2C inhibits forebrain and brainstem networks by exciting GABAergic interneurons, and deletion of this gene lowers the threshold for lethal evoked audiogenic seizures. Here, we characterize epileptogenesis throughout the lifespan of mice lacking X-linked, 5-HT2C receptors (loxTB Htr2c). We find that loss of Htr2c generates a complex, adult-onset spontaneous epileptic phenotype with a novel progressive hyperexcitability pattern of absences, non-convulsive, and convulsive behavioural seizures culminating in late onset sudden mortality predominantly in male mice. RNAscope localized Htr2c mRNA in subsets of Gad2+ GABAergic neurons in forebrain and brainstem regions. To evaluate the contribution of 5-HT2C receptor-mediated inhibitory drive, we selectively spared their deletion in GAD2+ GABAergic neurons of pan-deleted loxTB Htr2c mice, yet unexpectedly found no amelioration of survival or epileptic phenotype, indicating that expression of 5-HT2C receptors in GAD2+ inhibitory neurons was not sufficient to prevent hyperexcitability and lethal seizures. Analysis of human Sudden Unexpected Death in Epilepsy and epilepsy genetic databases identified an enrichment of HTR2C non-synonymous variants in Sudden Unexpected Death in Epilepsy cases. Interestingly, while early lethality is not reflected in the mouse model, we also identified variants mainly among male Sudden Infant Death Syndrome patients. Our findings validate HTR2C as a novel, sex-linked candidate gene modifying Sudden Unexpected Death in Epilepsy risk, and demonstrate that the complex epilepsy phenotype does not arise solely from 5-HT2C-mediated synaptic disinhibition. These results strengthen the evidence for the serotonin hypothesis of Sudden Unexpected Death in Epilepsy risk in humans, and advance current efforts to develop gene-guided interventions to mitigate premature mortality in epilepsy.
