RESUMO
Activating mutations in KRAS extensively reprogram cellular metabolism to support the continuous growth, proliferation, and survival of pancreatic tumors. Targeting these metabolic dependencies are promising approaches for the treatment of established tumors. However, metabolic reprogramming is required early during tumorigenesis to provide transformed cells selective advantage towards malignancy. Acinar cells can give rise to pancreatic tumors through acinar-to-ductal metaplasia (ADM). Dysregulation of pathways that maintain acinar homeostasis accelerate tumorigenesis. During ADM, acinar cells transdifferentiate to duct-like cells, a process driven by oncogenic KRAS. The metabolic reprogramming that is required for the transdifferentiation in ADM is unclear. We performed transcriptomic analysis on mouse acinar cells undergoing ADM and found metabolic programs are globally enhanced, consistent with the transition of a specialized cell to a less differentiated phenotype with proliferative potential. Indeed, we and others have demonstrated how inhibiting metabolic pathways necessary for ADM can prevent transdifferentiation and tumorigenesis. Here, we also find NRF2-target genes are differentially expressed during ADM. Among these, we focused on the increase in the gene coding for NADPH-producing enzyme, Glucose-6-phosphate dehydrogenase (G6PD). Using established mouse models of KrasG12D-driven pancreatic tumorigenesis and G6PD-deficiency, we find that mutant G6pd accelerates ADM and pancreatic intraepithelial neoplasia. Acceleration of cancer initiation with G6PD-deficiency is dependent on its NADPH-generating function in reactive oxygen species (ROS) management, as opposed to other outputs of the pentose phosphate pathway. Together, this work provides new insights into the function of metabolic pathways during early tumorigenesis.
RESUMO
For a long time, myelin was thought to be restricted to excitatory neurons, and studies on dysmyelination focused primarily on excitatory cells. Recent evidence showed that axons of inhibitory neurons in the neocortex are also myelinated, but the role of myelin on inhibitory circuits remains unknown. Here we studied the impact of mild hypomyelination on both excitatory and inhibitory connectivity in the primary auditory cortex (A1) with well-characterized mouse models of hypomyelination due to loss of oligodendrocyte ErbB receptor signaling. Using laser-scanning photostimulation, we found that mice with mild hypomyelination have reduced functional inhibitory connections to A1 L2/3 neurons without changes in excitatory connections, resulting in altered excitatory/inhibitory balance. These effects are not associated with altered expression of GABAergic and glutamatergic synaptic components, but with reduced density of parvalbumin-positive (PV+ ) neurons, axons, and synaptic terminals, which reflect reduced PV expression by interneurons rather than PV+ neuronal loss. While immunostaining shows that hypomyelination occurs in both PV+ and PV- axons, there is a strong correlation between MBP and PV expression, suggesting that myelination influences PV expression. Together, the results indicate that mild hypomyelination impacts A1 neuronal networks, reducing inhibitory activity, and shifting networks towards excitation.
Assuntos
Córtex Auditivo , Parvalbuminas , Camundongos , Animais , Parvalbuminas/metabolismo , Córtex Auditivo/metabolismo , Receptores ErbB/metabolismo , Interneurônios/metabolismo , Oligodendroglia/metabolismoRESUMO
Like all receptor tyrosine kinases (RTKs), ErbB4 signals through a canonical signaling involving phosphorylation cascades. However, ErbB4 can also signal through a non-canonical mechanism whereby the intracellular domain is released into the cytoplasm by regulated intramembrane proteolysis (RIP) and translocates to the nucleus where it regulates transcription. These different signaling mechanisms depend on the generation of alternative spliced isoforms, a RIP cleavable ErbB4-JMa and an uncleavable ErbB4-JMb. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of brain, heart, mammary gland, lung, and immune cell development. However, most studies on non-canonical ErbB4 signaling have been performed in vitro due to the lack of an adequate mouse model. We created an ErbB4-JMa specific knock out mouse and demonstrate that RIP-dependent, non-canonical signaling by ErbB4-JMa is required for the regulation of GFAP expression during cortical development. We also show that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia. Furthermore, we identify genes whose expression during cortical development is regulated by ErbB4, and show that the expression of three of them, CRYM and DBi, depend on ErbB4-JMa whereas WDFY1 relies on ErbB4-JMb. Thus, we provide the first animal model to directly study the roles of ErbB4-JMa and non-canonical ErbB4 signaling in vivo.
Assuntos
Transdução de Sinais , Tirosina , Animais , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , Tirosina/metabolismoRESUMO
Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.
Assuntos
Hormônio Liberador de Gonadotropina , Maturidade Sexual , Astrócitos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/fisiologia , Neurônios/fisiologia , Maturidade Sexual/fisiologiaRESUMO
Neurotrophin-3 growth factor can improve cochlear neuron survival, and localized delivery of this protein to the round window membrane in the middle ear may be able to reverse sensorineural hearing loss. Thus, the goal of this work was to develop an injectable hydrogel delivery system that can allow localized release of neurotrophin-3 in a controlled and sustained manner. We identified a PEG hydrogel formulation that uses thiol-vinyl sulfone Michael addition for crosslinking. This injectable formulation provides elastic hydrogels with higher mechanical rigidity, better bio-adhesion and longer residence time than Poloxamer hydrogels currently being investigated clinically for hearing loss. In vivo, PEG hydrogels induce local immune responses comparable to biocompatible Poloxamer hydrogels, yet they released payloads at a ~5-fold slower rate in the subcutaneous area. Based on this injectable hydrogel formulation, we designed an affinity-based protein release system by modifying PEG hydrogels with affinity peptides specific to neurotrophin-3 proteins. We verified the sustained release of neurotrophin-3 from peptide-conjugated PEG hydrogels resulting from the reversible interaction between peptides and proteins. The rate of affinity-controlled release depends on the polymer concentrations, the affinity of peptides and the peptide-to-protein ratios. Collectively, we developed an injectable hydrogel formulation for localized delivery of neurotrophin-3, which provides affinity-controlled release and longer delivery time compared to Poloxamer hydrogels.
Assuntos
Hidrogéis , Peptídeos , Materiais Biocompatíveis , Poloxâmero , Polietilenoglicóis , ProteínasRESUMO
Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1-21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.
Assuntos
Cóclea/patologia , Dependovirus/metabolismo , Neurotrofina 3/metabolismo , Ruído , Sinapses/patologia , Animais , Limiar Auditivo , Cóclea/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neurotrofina 3/genética , Emissões Otoacústicas Espontâneas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/metabolismoRESUMO
Experience with procurement of research funding and grantsmanship is an essential skill and one that is rarely taught in a manner that adequately prepares trainees for the magnitude of this professional requirement. The aims of the program described in this article are (1) to provide a mentored experience in grantsmanship through designing and concisely outlining an individual research study and (2) to supplement extramural funding mechanisms for clinical trainees to produce meaningful and substantive clinical and/or basic science research. A total of $10,000 of departmental chair discretionary funds is allocated for resident research annually. The first 2 cycles have successfully allocated the allotted funding through a competitive, scored grant evaluation process. Awardees have already produced meaningful data that have been nationally presented, submitted for publication, and integrated into an National Institutes of Health grant submission. The feasibility of implementing an intramural competitive resident research grant may have broad application within varied training environments.
Assuntos
Pesquisa Biomédica/economia , Comportamento Competitivo , Otolaringologia/educação , Apoio à Pesquisa como Assunto , Educação de Pós-Graduação em Medicina , Humanos , Internato e Residência , National Institutes of Health (U.S.) , Competência Profissional , Estados UnidosRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1(G93A) mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Axônios/metabolismo , Neuregulina-1/metabolismo , Junção Neuromuscular/metabolismo , Quinazolinas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Lapatinib , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Denervação Muscular/métodos , Neurogênese/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.
Assuntos
Regulação da Expressão Gênica , Oligodendroglia/citologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Corpo Caloso/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mutação , Bainha de Mielina/química , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Nervo Óptico/metabolismo , Transdução de Sinais , Tamoxifeno/química , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Small-fiber neuropathy (SFN) is a disorder of peripheral nerves commonly found in patients with diabetes mellitus, HIV infection, and those receiving chemotherapy. The complexity of disease etiology has led to a scarcity of effective treatments. Using two models of progressive SFN, we show that overexpression of glial cell line-derived neurotrophic factor (GDNF) in skin keratinocytes or topical application of XIB4035, a reported nonpeptidyl agonist of GDNF receptor α1 (GFRα1), are effective treatments for SFN. We also demonstrate that XIB4035 is not a GFRα1 agonist, but rather it enhances GFRα family receptor signaling in conjunction with ligand stimulation. Taken together, our results indicate that topical application of GFRα/RET receptor signaling modulators may be a unique therapy for SFN, and we have identified XIB4035 as a candidate therapeutic agent.
Assuntos
Eritromelalgia/tratamento farmacológico , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinolinas/uso terapêutico , Transdução de Sinais , Administração Tópica , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Quinolinas/administração & dosagem , Bibliotecas de Moléculas PequenasRESUMO
Early social isolation results in adult behavioral and cognitive dysfunction that correlates with white matter alterations. However, how social deprivation influences myelination and the significance of these myelin defects in the adult remained undefined. We show that mice isolated for 2 weeks immediately after weaning have alterations in prefrontal cortex function and myelination that do not recover with reintroduction into a social environment. These alterations, which occur only during this critical period, are phenocopied by loss of oligodendrocyte ErbB3 receptors, and social isolation leads to reduced expression of the ErbB3 ligand neuregulin-1. These findings indicate that social experience regulates prefrontal cortex myelination through neuregulin-1/ErbB3 signaling and that this is essential for normal cognitive function, thus providing a cellular and molecular context to understand the consequences of social isolation.
Assuntos
Comportamento Animal , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Córtex Pré-Frontal/metabolismo , Isolamento Social , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuregulina-1/genética , Neuregulina-1/metabolismo , Oligodendroglia/citologia , Córtex Pré-Frontal/citologia , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Transdução de SinaisRESUMO
SynCAM1 is an adhesion molecule involved in synaptic differentiation and organization. SynCAM1 is also expressed in astroglial cells where it mediates astrocyte-to astrocyte and glial-neuronal adhesive communication. In astrocytes, SynCAM1 is functionally linked to erbB4 receptors, which are involved in the control of both neuronal/glial development and mature neuronal and glial function. Here we report that mice carrying a dominant-negative form of SynCAM1 specifically targeted to astrocytes (termed GFAP-DNSynCAM1 mice) exhibit disrupted diurnal locomotor activity with enhanced and more frequent episodes of activity than control littermates during the day (when the animals are normally sleeping) accompanied by shorter periods of rest. GFAP-DNSynCAM1 mice also display high levels of basal activity in the dark period (the rodent's awake/active time) that are attenuated by the psychostimulant D,L-amphetamine, and reduced anxiety levels in response to both avoidable and unavoidable provoking stimuli. These results indicate that disruption of SynCAM1-dependent astroglial function results in behavioral abnormalities similar to those described in animals model of attention-deficit hyperactive disorder (ADHD), and suggest a hitherto unappreciated contribution of glial cells to the pathophysiology of this disorder.
Assuntos
Astrócitos/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/patologia , Comportamento Animal , Moléculas de Adesão Celular/metabolismo , Imunoglobulinas/metabolismo , Transdução de Sinais , Anfetamina/farmacologia , Animais , Ansiedade/complicações , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Transtorno do Deficit de Atenção com Hiperatividade/complicações , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Comunicação Celular/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Imunoglobulinas/genética , Comportamento Impulsivo/complicações , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Astrocytes in the hypothalamus release prostaglandin E(2) (PGE(2)) in response to cell-cell signaling initiated by neurons and glial cells. Upon release, PGE(2) stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patch-clamp recording we demonstrate that PGE(2) exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A (PKA) inhibitors. The acute blockade of prostaglandin synthesis by indomethacin (INDO) or the selective inhibition of astrocyte metabolism by fluoroacetate (FA) suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE(2) release due to defective erbB signaling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE(2) acts as a gliotransmitter within the GnRH neurosecretory system.
Assuntos
Astrócitos/metabolismo , Dinoprostona/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Hormônio Liberador de Gonadotropina/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Indometacina/farmacologia , Isoquinolinas/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Inibidores de Proteínas Quinases/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: Previous studies in infant rats and case-control studies of human infants undergoing surgery have raised concerns about potential neurodevelopmental toxicities of general anesthesia. Spinal anesthesia is an alternative to general anesthesia for some infant surgeries. To test for potential toxicity, a spinal anesthesia model in infant rats was developed. METHODS: Rats of postnatal ages 7, 14, and 21 days were assigned to no treatment, 1% isoflurane for either 1 h or 6 h, or lumbar spinal injection of saline or bupivacaine at doses of 3.75 mg/kg (low dose) or 7.5 mg/kg (high dose). Subgroups of animals underwent neurobehavioral testing and blood gas analysis. Brain and lumbar spinal cord sections were examined for apoptosis using cleaved caspase-3 immunostaining. The lumbar spinal cord was examined histologically.Rats exposed to spinal or general anesthesia as infants underwent Rotarod testing of motor performance as adults. Data were analyzed using ANOVA with general linear models, Friedman tests, and Mann-Whitney U tests, as appropriate. RESULTS: Bupivacaine 3.75 mg/kg was effective for spinal anesthesia in all age groups. Impairments in sensory and motor function recovered in 40-60 min. Blood gases were similar among groups. Brain and spinal cord apoptosis increased in rats receiving 6 h of 1% isoflurane, but not among the other treatments. All groups showed intact motor performance at adulthood. CONCLUSIONS: Spinal anesthesia is technically feasible in infant rats and appears benign in terms of neuroapoptotic and neuromotor sequelae.
Assuntos
Raquianestesia/efeitos adversos , Bupivacaína/efeitos adversos , Modelos Animais de Doenças , Isoflurano/efeitos adversos , Doenças do Sistema Nervoso/induzido quimicamente , Desempenho Psicomotor/efeitos dos fármacos , Raquianestesia/métodos , Animais , Animais Recém-Nascidos , Gasometria/métodos , Bupivacaína/administração & dosagem , Feminino , Isoflurano/administração & dosagem , Masculino , Doenças do Sistema Nervoso/diagnóstico , Desempenho Psicomotor/fisiologia , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
We previously identified synaptic cell adhesion molecule 1 (SynCAM1) as a component of a genetic network involved in the hypothalamic control of female puberty. Although it is well established that SynCAM1 is a synaptic adhesion molecule, its contribution to hypothalamic function is unknown. Here we show that, in addition to the expected neuronal localization illustrated by its presence in GnRH neurons, SynCAM1 is expressed in hypothalamic astrocytes. Cell adhesion assays indicated that SynCAM is recognized by both GnRH neurons and astrocytes as an adhesive partner and promotes cell-cell adhesiveness via homophilic, extracellular domain-mediated interactions. Alternative splicing of the SynCAM1 primary mRNA transcript yields four mRNAs encoding membrane-spanning SynCAM1 isoforms. Variants 1 and 4 are predicted to be both N and O glycosylated. Hypothalamic astrocytes and GnRH-producing GT1-7 cells express mainly isoform 4 mRNA, and sequential N- and O-deglycosylation of proteins extracted from these cells yields progressively smaller SynCAM1 species, indicating that isoform 4 is the predominant SynCAM1 variant expressed in astrocytes and GT1-7 cells. Neither cell type expresses the products of two other SynCAM genes (SynCAM2 and SynCAM3), suggesting that SynCAM-mediated astrocyte-astrocyte and astrocyte-GnRH neuron adhesiveness is mostly mediated by SynCAM1 homophilic interactions. When erbB4 receptor function is disrupted in astrocytes, via transgenic expression of a dominant-negative erbB4 receptor form, SynCAM1-mediated adhesiveness is severely compromised. Conversely, SynCAM1 adhesive behavior is rapidly, but transiently, enhanced in astrocytes by ligand-dependent activation of erbB4 receptors, suggesting that erbB4-mediated events affecting SynCAM1 function contribute to regulate astrocyte adhesive communication.
Assuntos
Astrócitos/citologia , Moléculas de Adesão Celular/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/citologia , Hipotálamo/metabolismo , Imunoglobulinas/metabolismo , Neurônios/citologia , Sequência de Aminoácidos , Animais , Astrócitos/metabolismo , Adesão Celular , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Comunicação Celular , Linhagem Celular , Feminino , Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
Female sexual maturation requires erythroblastosis B (erbB)4 signaling in hypothalamic astrocytes; however, the mechanisms by which erbB4 contributes to this process are incompletely understood. Here we show that SynCAM1, a synaptic adhesion molecule with signaling capabilities, is not only expressed highly in neurons, but also in hypothalamic astrocytes and is functionally associated with erbB4 receptor activity. Whereas SynCAM1 expression is diminished in astrocytes with impaired erbB4 signaling, ligand-dependent activation of astroglial erbB4 receptors results in rapid association of erbB4 with SynCAM1 and activation of SynCAM1 gene transcription. To determine whether astrocytic SynCAM1-dependent intracellular signaling is required for normal female reproductive function, we generated transgenic mice that express in an astrocyte-specific manner a dominant-negative form of SynCAM1 lacking the intracellular domain. The mutant protein was correctly targeted to the cell membrane and was functionally viable as shown by its ability to block intracellular calcium/calmodulin-dependent serine protein kinase redistribution, a major SynCAM1-mediated event. Dominant-negative-SynCAM1 female mice had a delayed onset of puberty, disrupted estrous cyclicity, and reduced fecundity. These deficits were associated with a reduced capacity of neuregulin-dependent erbB4 receptor activation to elicit prostaglandin E2 release from astrocytes and GnRH release from the hypothalamus. We conclude that one of the mechanisms underlying erbB4 receptor-mediated facilitation of glial-neuronal interactions in the neuroendocrine brain involves SynCAM1-dependent signaling and that this interaction is required for normal female reproductive function.
Assuntos
Astrócitos/metabolismo , Receptores ErbB/metabolismo , Camundongos/metabolismo , Desenvolvimento Sexual , Animais , Astrócitos/citologia , Encéfalo/citologia , Encéfalo/metabolismo , Dinoprostona/metabolismo , Receptores ErbB/genética , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Camundongos/genética , Camundongos/crescimento & desenvolvimento , Camundongos Transgênicos , Ligação Proteica , Receptor ErbB-4 , Transdução de SinaisRESUMO
Currently available local anesthetics have analgesic durations in humans generally less than 12 hours. Prolonged-duration local anesthetics will be useful for postoperative analgesia. Previous studies showed that in rats, combinations of tetrodotoxin (TTX) with bupivacaine had supra-additive effects on sciatic block durations. In those studies, epinephrine combined with TTX prolonged blocks more than 10-fold, while reducing systemic toxicity. TTX, formulated as Tectin, is in phase III clinical trials as an injectable systemic analgesic for chronic cancer pain. Here, we examine dose-duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine 0.25% (2.5 mg/mL) solutions, with or without epinephrine 5 µg/mL (1:200,000) in rats. Percutaneous sciatic blockade was performed in Sprague-Dawley rats, and intensity and duration of sensory blockade was tested blindly with different Tectin-bupivacaine-epinephrine combinations. Between-group comparisons were analyzed using ANOVA and post-hoc Sidak tests. Nerves were examined blindly for signs of injury. Blocks containing bupivacaine 0.25% with Tectin 10 µM and epinephrine 5 µg/mL were prolonged by roughly 3-fold compared to blocks with bupivacaine 0.25% plain (P < 0.001) or bupivacaine 0.25% with epinephrine 5 µg/mL (P < 0.001). Nerve histology was benign for all groups. Combinations of Tectin in bupivacaine 0.25% with epinephrine 5 µg/mL appear promising for prolonged duration of local anesthesia.
Assuntos
Anestesia Local , Anestésicos Locais/farmacologia , Bupivacaína/administração & dosagem , Epinefrina/administração & dosagem , Tetrodotoxina/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Nervo Isquiático/fisiologiaRESUMO
Recent studies indicate that molecules released by glia can induce synapse formation. However, what induces glia to produce such signals, their identity, and their in vivo relevance remain poorly understood. Here we demonstrate that supporting cells of the vestibular organ--cells that have many characteristics of glia--promote synapse formation only when induced by neuron-derived signals. Furthermore, we identify BDNF as the synaptogenic signal produced by these nonneuronal cells. Mice in which erbB signaling has been eliminated in supporting cells have vestibular dysfunction caused by failure of synapse formation between hair cells and sensory neurons. This phenotype correlates with reduced BDNF expression in supporting cells and is rescued by reexpression of BDNF in these cells. Furthermore, knockdown of BDNF expression in supporting cells postnatally phenocopies the loss of erbB signaling. These results indicate that vestibular supporting cells contribute in vivo to vestibular synapse formation and that this is mediated by reciprocal signals between sensory neurons and supporting cells involving erbB receptors and BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Epitélio/fisiologia , Receptores ErbB/metabolismo , Neuroglia/fisiologia , Sinapses/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Receptores ErbB/genética , Proteína Glial Fibrilar Ácida , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Receptor ErbB-4 , Vestíbulo do Labirinto/citologiaRESUMO
Recent studies indicate that supporting cells play important roles in inner ear development, function, and regeneration after injury, but the molecular mechanisms underlying these processes remain poorly understood. Inducible cell-specific gene recombination in supporting cells could be a powerful tool to study the roles of specific molecules in these cells. Here we tested the feasibility, effectiveness, and cell specificity of inducible Cre-mediated gene recombination in the postnatal inner ear using mice that express an inducible form of Cre (CreER(T)) under the transcriptional control of the proteolipid protein (PLP) promoter. We assessed the pattern of tamoxifen-induced gene recombination in the inner ear using the ROSA26-LacZ reporter line, in which the beta-galactosidase gene is expressed only after Cre-mediated excision of a loxP-flanked stop cassette. Recombination was detected in cochlear inner phalangeal cells, supporting cells surrounding hair cells in vestibular maculae and cristae. Recombination also occurred in Schwann cells. We also found that this CreER(T) line can be used to increase and decrease the levels of expression of a trophic factor, brain-derived neurotrophic factor, specifically in supporting cells. These results show that PLP/CreER(T) mice are a powerful tool to dissect gene function in inner ear supporting cells.
Assuntos
Orelha Interna , Regulação da Expressão Gênica no Desenvolvimento , Neuroglia/fisiologia , Recombinação Genética/fisiologia , Células de Schwann/fisiologia , Animais , Antineoplásicos Hormonais , Fator Neurotrófico Derivado do Encéfalo/genética , Orelha Interna/citologia , Orelha Interna/embriologia , Orelha Interna/fisiologia , Genes Reporter , Células Ciliadas Vestibulares/fisiologia , Integrases/genética , Óperon Lac , Camundongos , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/genética , Regiões Promotoras Genéticas/genética , Tamoxifeno , Transgenes/genéticaRESUMO
Neuregulin-1 (NRG1) signaling has multiple functions in neurons and glia. The data in this study show that NRG1 may also possess significant signaling and cytoprotective properties in human brain microvascular endothelial cells (BMECs). Neuregulin-1 mRNA and protein expression are present in these cells, and NRG1 receptors erbB2 and erbB3 are phosphorylated in response to NRG1. Neuregulin-1 triggers clear biologic responses in BMECs--elevated phospho-Akt levels, increased ring formation in a Matrigel assay, and decreased cell death after oxidative injury with H(2)O(2). These data suggest that NRG1 signaling is functional and cytoprotective in BMECs.