Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray
Proceedings of the National Academy of Sciences of the United States of America
Yang, Y;Li, Y;Liu, B;Li, C;Liu, Z;Deng, J;Luo, H;Li, X;Wu, J;Li, H;Wang, CY;Zhao, M;Wu, H;Lallemend, F;Svenningsson, P;Hökfelt, TGM;Xu, ZD;
PMID: 34108238 | DOI: 10.1073/pnas.1922586118
Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.
Shimoda M, Yoshida H, Mizuno S, Hirozane T, Horiuchi K, Yoshino Y, Hara H, Kanai Y, Inoue S, Ishijima M, Okada Y.
PMID: 28284715 | DOI: 10.1016/j.ajpath.2017.01.005
Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (HYBID/KIAA1199; encoded by CEMIP) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/locus of crossover in P1 (loxP) system and analyzed their phenotypes. Hybid-deficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4 weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction in the Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated that Hybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes. High-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondral ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.
Invest Ophthalmol Vis Sci.
Sun M, Wadehra M, Casero D, Lin MC, Aguirre B, Parikh S, Matynia A, Gordon L, Chu A
PMID: 32031575 | DOI: 10.1167/iovs.61.2.3
PURPOSE:
Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR).
METHODS:
Eyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1? (Hif1?), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17.
RESULTS:
Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1? at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling.
CONCLUSIONS:
The protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression
Duan X, Bradbury SR, Olsen BR, Berendsen AD.
PMID: 26899202 | DOI: 10.1016/j.matbio.2016.02.005.
Deficiency of vascular endothelial growth factor A (VEGF) has been associated with severe craniofacial anomalies in both humans and mice. Cranial neural crest cell (NCC)-derived VEGF regulates proliferation, vascularization and ossification of cartilage and membranous bone. However, the function of VEGF derived from specific subpopulations of NCCs in controlling unique aspects of craniofacial morphogenesis is not clear. In this study a conditional knockdown strategy was used to genetically delete Vegfa expression in Osterix (Osx) and collagen II (Col2)-expressing NCC descendants. No major defects in calvaria and mandibular morphogenesis were observed upon knockdown of VEGF in the Col2+ cell population. In contrast, loss of VEGF in Osx+ osteoblast progenitor cells led to reduced ossification of calvarial and mandibular bones without affecting the formation of cartilage templates in newborn mice. The early stages of ossification in the developing jaw revealed decreased initial mineralization levels and a reduced thickness of the collagen I (Col1)-positive bone template upon loss of VEGF in Osx+ precursors. Increased numbers of proliferating cells were detected within the jaw mesenchyme of mutant embryos. Explant culture assays revealed that mandibular osteogenesis occurred independently of paracrine VEGF action and vascular development. Reduced VEGF expression in mandibles coincided with increased phospho-Smad1/5 (P-Smad1/5) levels and bone morphogenetic protein 2 (Bmp2) expression in the jaw mesenchyme. We conclude that VEGF derived from Osx+ osteoblast progenitor cells is required for optimal ossification of developing mandibular bones and modulates mechanisms controlling BMP-dependent specification and expansion of the jaw mesenchyme.
The Journal of physiology
Peltekian, L;Gasparini, S;Fazan, FS;Karthik, S;Iverson, G;Resch, JM;Geerling, JC;
PMID: 37291801 | DOI: 10.1113/JP283169
In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.
Zhang, L;Koller, J;Gopalasingam, G;Qi, Y;Herzog, H;
PMID: 35691527 | DOI: 10.1016/j.molmet.2022.101525
Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons.We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons.Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control.NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.
Aguilar, K;Comes, G;Canal, C;Quintana, A;Sanz, E;Hidalgo, J;
PMID: 35770802 | DOI: 10.1002/glia.24234
Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.
Kuchroo, M;DiStasio, M;Song, E;Calapkulu, E;Zhang, L;Ige, M;Sheth, AH;Majdoubi, A;Menon, M;Tong, A;Godavarthi, A;Xing, Y;Gigante, S;Steach, H;Huang, J;Huguet, G;Narain, J;You, K;Mourgkos, G;Dhodapkar, RM;Hirn, MJ;Rieck, B;Wolf, G;Krishnaswamy, S;Hafler, BP;
PMID: 37147305 | DOI: 10.1038/s41467-023-37025-7
Due to commonalities in pathophysiology, age-related macular degeneration (AMD) represents a uniquely accessible model to investigate therapies for neurodegenerative diseases, leading us to examine whether pathways of disease progression are shared across neurodegenerative conditions. Here we use single-nucleus RNA sequencing to profile lesions from 11 postmortem human retinas with age-related macular degeneration and 6 control retinas with no history of retinal disease. We create a machine-learning pipeline based on recent advances in data geometry and topology and identify activated glial populations enriched in the early phase of disease. Examining single-cell data from Alzheimer's disease and progressive multiple sclerosis with our pipeline, we find a similar glial activation profile enriched in the early phase of these neurodegenerative diseases. In late-stage age-related macular degeneration, we identify a microglia-to-astrocyte signaling axis mediated by interleukin-1β which drives angiogenesis characteristic of disease pathogenesis. We validated this mechanism using in vitro and in vivo assays in mouse, identifying a possible new therapeutic target for AMD and possibly other neurodegenerative conditions. Thus, due to shared glial states, the retina provides a potential system for investigating therapeutic approaches in neurodegenerative diseases.
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.
Tamma R, Annese T, Ruggieri S, Marzullo A, Nico B, Ribatti D.
PMID: 29761299 | DOI: 10.1007/s10735-018-9777-0
Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.
Caliò A, Brunelli M, Segala D, Pedron S, Doglioni C, Argani P, Martignoni G.
PMID: 30206412 | DOI: 10.1038/s41379-018-0128-1
Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.
Front Cell Neurosci. 2018 Oct 9;12:341.
Yoo T, Cho H, Lee J, Park H, Yoo YE, Yang E, Kim JY, Kim H, Kim E.
PMID: 30356810 | DOI: 10.3389/fncel.2018.00341
Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.
