RESUMEN
After central nervous system injury, a rapid cellular and molecular response is induced. This response can be both beneficial and detrimental to neuronal survival in the first few days and increases the risk for neurodegeneration if persistent. Semaphorin4B (Sema4B), a transmembrane protein primarily expressed by cortical astrocytes, has been shown to play a role in neuronal cell death following injury. Our study shows that after cortical stab wound injury, cytokine expression is attenuated in Sema4B-/- mice, and microglia/macrophage reactivity is altered. In vitro, Sema4B enhances the reactivity of microglia following injury, suggesting astrocytic Sema4B functions as a ligand. Moreover, injury-induced microglia reactivity is attenuated in the presence of Sema4B-/- astrocytes compared to Sema4B+/- astrocytes. In vitro experiments indicate that Plexin-B2 is the Sema4B receptor on microglia. Consistent with this, in microglia/macrophage-specific Plexin-B2-/- mice, similar to Sema4B-/- mice, microglial/macrophage reactivity and neuronal cell death are attenuated after cortical injury. Finally, in Sema4B/Plexin-B2 double heterozygous mice, microglial/macrophage reactivity is also reduced after injury, supporting the idea that both Sema4B and Plexin-B2 are part of the same signaling pathway. Taken together, we propose a model in which following injury, astrocytic Sema4B enhances the response of microglia/macrophages via Plexin-B2, leading to increased reactivity.
Asunto(s)
Astrocitos , Ratones Noqueados , Microglía , Proteínas del Tejido Nervioso , Semaforinas , Animales , Ratones , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Lesiones Encefálicas/genética , Comunicación Celular , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Semaforinas/metabolismo , Semaforinas/genéticaRESUMEN
The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodeling and returns to normal levels in recovering hearts. We wondered whether IGF2BP2 might play an adaptive role during cardiac stress and recovery. Enhanced expression of an IGF2BP2 transgene in a conditional, inducible mouse line leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks in newborn or adult hearts. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. IGF2BP2 is also upregulated in DCM or myocardial infarction patients. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in cardiomyopathies.
Asunto(s)
Cardiomiopatías , Cardiomiopatía Dilatada , Adulto , Animales , Humanos , Ratones , Cardiomiopatías/metabolismo , Cardiomiopatía Dilatada/genética , Miocitos Cardíacos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Background: High-grade gliomas (HGG) in children have a devastating prognosis and occur in a remarkable spatiotemporal pattern. Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), typically occur in mid-childhood, while cortical HGGs are more frequent in older children and adults. The mechanisms behind this pattern are not clear. Methods: We used mouse organotypic slice cultures and glial cell cultures to test the impact of the microenvironment on human DIPG cells. Comparing the expression between brainstem and cortical microglia identified differentially expressed secreted proteins. The impact of some of these proteins on DIPGs was tested. Results: DIPGs, pediatric HGGs of brainstem origin, survive and divide more in organotypic slice cultures originating in the brainstem as compared to the cortex. Moreover, brainstem microglia are better able to support tumors of brainstem origin. A comparison between the two microglial populations revealed differentially expressed genes. One such gene, interleukin-33 (IL33), is highly expressed in the pons of young mice and its DIPG receptor is upregulated in this context. Consistent with this observation, the expression levels of IL33 and its receptor, IL1RL1, are higher in DIPG biopsies compared to low-grade cortical gliomas. Furthermore, IL33 can enhance proliferation and clonability of HGGs of brainstem origin, while blocking IL33 in brainstem organotypic slice cultures reduced the proliferation of these tumor cells. Conclusions: Crosstalk between DIPGs and the brainstem microenvironment, in particular microglia, through IL33 and other secreted factors, modulates spatiotemporal patterning of this HGG and could prove to be an important future therapeutic target.
RESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. METHODS: We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. FINDINGS: We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. INTERPRETATION: Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.
Asunto(s)
Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Proteína C9orf72/genética , Células Madre Pluripotentes Inducidas/citología , Mutación , Estrés Oxidativo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Biomarcadores , Células Cultivadas , Reprogramación Celular , Senescencia Celular/genética , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Ratones , Neuronas Motoras/metabolismo , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Abnormal neuronal activity in sensory ganglia contributes to chronic pain. There is evidence that signals can spread between cells in these ganglia, which may contribute to this activity. Satellite glial cells (SGCs) in sensory ganglia undergo activation following peripheral injury and participate in cellular communication via gap junctions and chemical signaling. Nitric oxide (NO) is released from neurons in dorsal root ganglia (DRG) and induces cyclic GMP (cGMP) production in SCGs, but its role in SGC activation and neuronal excitability has not been explored. It was previously reported that induction of intestinal inflammation with dinitrobenzoate sulfonate (DNBS) increased gap junctional communications among SGCs, which contributed to neuronal excitability and pain. Here we show that DNBS induced SGC activation in mouse DRG, as assayed by glial fibrillary acidic protein upregulation. DNBS also upregulated cGMP level in SGCs, consistent with NO production. In vitro studies on intact ganglia from DNBS-treated mice showed that blocking NO synthesis inhibited both SGCs activation and cGMP upregulation, indicating an ongoing NO production. Application of NO donor in vitro induced SGC activation, augmented gap junctional communications, and raised neuronal excitability, as assessed by electrical recordings. The cGMP analog 8-Br-cGMP mimicked these actions, confirming the role of the NO-cGMP pathway in intraganglionic communications. NO also augmented Ca2+ waves propagation in DRG cultures. It is proposed that NO synthesis in DRG neurons increases after peripheral inflammation and that NO induces SGC activation, which in turn contributes to neuronal hyperexcitability. Thus, NO plays a major role in neuron-SGC communication.
Asunto(s)
Comunicación Celular/fisiología , Ganglios Espinales/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Células Satélites Perineuronales/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/farmacología , Femenino , Ganglios Espinales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Células Satélites Perineuronales/efectos de los fármacosRESUMEN
Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.
Asunto(s)
Uniones Comunicantes/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Ganglio del Trigémino/citología , Animales , Compuestos de Boro/farmacología , Carbenoxolona/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Ácido Flufenámico/farmacología , Uniones Comunicantes/efectos de los fármacos , Heptanol/farmacología , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Isoquinolinas/metabolismo , Lipopolisacáridos/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Probenecid/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
INTRODUCTION: Rat endovascular trophoblasts (EVasT) express smooth muscle (SM) proteins and contract ex vivo upon exposure to endothelin-1 (ET1) via receptors A and B (ETA, ETB). Presently, we investigated the EVasT response to NOS inhibition (N-Nitro-l-arginine methyl ester hydrochloride, l-NAME), and potentiation by NO donor [S-Nitroso-N-Acetyl-D,l-Penicillamine (SNAP)] following KCl precontraction. M&M: Luminal surface area (LSA) of remodeled spiral artery rings (SAR) devoid of SM was measured ex vivo upon exposure to l-NAME alone; l-NAME and ET1 representing the combined contractile effect of both ET1 receptors; l-NAME with ET1 and ETA antagonist, representing the isolated contractile effect via ETB. In another experiment we administered SNAP to KCl precontracted SAR. Statistical analysis was performed using 2-way mixed ANOVA and repeated measures. RESULTS: l-NAME reduced LSA by 2.22%, 95% CI [0.83%, 3.60%] compared with control. ET1 and l-NAME reduced LSA immediately, compared with a plateau at 60min by ET1 alone. The isolated ET1 constrictive effect via ETB, reduced LSA by 5.94%; 95% CI [3.47%, 8.41%], significantly more than that obtained via ETA following 36 min of the experiment by 0.88%; 95%CI [0.09%, 1.67%]. Addition of KCl reduced LSA by 11.9%, 95% CI [9.6%, 14.1%]. Addition of SNAP increased LSA by 3.0%, 95% CI [1.7%, 4.3%]. CONCLUSIONS: EVasT of the rat remodeled spiral artery react to ET1 and KCl similar to vascular SM: contract via both ET1 receptors and KCl and relax by ET1 via ETB and by SNAP. This phenomenon may play a role in rat models of gestational vasoactive systems dysregulation.
Asunto(s)
Endotelina-1/metabolismo , Óxido Nítrico/metabolismo , Receptor de Endotelina B/metabolismo , Trofoblastos/metabolismo , Vasodilatación , Animales , Femenino , NG-Nitroarginina Metil Éster , Ratas Wistar , Receptor de Endotelina A/metabolismo , S-Nitroso-N-AcetilpenicilaminaRESUMEN
Satellite glial cell (SGCs) in trigeminal and dorsal root ganglia are altered structurally and functionally under pathological conditions associated with chronic pain. These changes include reactive gliosis, augmented coupling by gap junctions, and increased responses to ATP via purinergic P2 receptors. Similar information for nodose ganglia (NG), which receive sensory inputs from internal organs via the vagus nerves, is missing. Here, we investigated changes in SGCs in mouse NG after the intraperitoneal administration of lipopolysaccharide (LPS), which induces systemic inflammation. Using calcium imaging we found that SGCs in intact, freshly isolated NG are sensitive to ATP, acting largely via purinergic P2 receptors (mixed P2X and P2Y), with threshold at 0.1 µM. A single systemic injection of LPS (2.5 mg/kg) induced a 6-fold increase in the responses to ATP, largely by augmenting the sensitivity of P2X receptors. Immunohistochemical analysis revealed that at 1-14 days post-LPS injection the expression of glial fibrillary acidic protein in SGCs was 2-3-fold greater than controls. The expression of pannexin 1 channels increased 2-fold at day 7 after LPS injection. Using intracellular labeling we examined dye coupling among SGCs around different neurons, and observed an over 2-fold higher incidence of dye coupling after the induction of inflammation. Incubating the ganglia with ATP increased dye coupling by acting on neuronal P2X receptors, suggesting a role for ATP in the LPS-induced changes. We conclude that inflammation induces prominent changes in SGCs of NG, which might have a role in vagal afferent functions, such as the inflammatory reflex. GLIA 2015;63:2121-2132.
RESUMEN
Intercellular coupling by gap junctions is one of the main features of glial cells, but very little is known about this aspect of satellite glial cells (SGCs) in sympathetic ganglia. We used the dye coupling method to address this question in both a prevertebral ganglion (superior mesenteric) and a paravertebral ganglion (superior cervical) of mice. We found that in control ganglia, the incidence of dye coupling among SGCs that form the envelope around a given neuron was 10-20%, and coupling between SGCs around different envelopes was rare (1.5-3%). The dye injections also provided novel information on the structure of SGCs. Following peripheral inflammation, both types of coupling were increased, but most striking was the augmentation of coupling between SGCs forming envelopes around different neurons, which rose by 8-14.6-fold. This effect appeared to be non-systemic, and was blocked by the gap junction blocker carbenoxolone. These changes in SGCs may affect signal transmission and processing in sympathetic ganglia.
Asunto(s)
Uniones Comunicantes/fisiología , Inflamación Neurogénica/patología , Neuroglía/fisiología , Ganglio Cervical Superior/patología , Animales , Dinitrofluorobenceno/efectos adversos , Dinitrofluorobenceno/análogos & derivados , Modelos Animales de Enfermedad , Femenino , Adyuvante de Freund/efectos adversos , Uniones Comunicantes/patología , Isoquinolinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Inflamación Neurogénica/etiologíaRESUMEN
Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.
Asunto(s)
Neuroglía/fisiología , Fenotipo , Ganglio del Trigémino/citología , Animales , Comunicación Celular , Células Cultivadas , Femenino , Glutamato-Amoníaco Ligasa/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Neuronas/fisiología , Receptores Purinérgicos P2X7/metabolismo , Factores de TiempoRESUMEN
Peripheral injuries can lead to sensitization of neurons in dorsal root ganglia (DRGs), which can contribute to chronic pain. The neurons are sensitized by a combination of physiological and biochemical changes, whose full details are still obscure. Another cellular element in DRGs are satellite glial cells (SGCs), which surround the neurons, but little is known about their role in nociception. We investigated the contribution of SGCs to neuronal sensitization in isolated S1 DRGs from a mouse model of colonic inflammation induced by local application of dinitrosulfonate benzoate (DNBS). Retrograde labeling was used to identify DRG neurons projecting to the colon. Cell-to-cell coupling was determined by intracellular dye injection, and the electrical properties of the neurons were studied with intracellular electrodes. Pain behavior was assessed with von-Frey hairs. The dye injections showed that 10-12 days after DNBS application there was a 6.6-fold increase in gap junction-mediated coupling between SGCs surrounding adjacent neurons, and this occurred preferentially (another 2-fold increase) near neurons that project to the colon. Neuron-neuron coupling incidence increased from 0.7% to 12.1% by colonic inflammation. Inflammation led to an augmented neuronal excitability, and to a reduced pain threshold. Gap junction blockers abolished the inflammation-induced changes in SGCs and neurons, and significantly reversed the pain behavior. We propose that inflammation induces augmented cell coupling in DRGs that contributes to neuronal hyperexcitability, which in turn leads to visceral pain. Gap junction blockers may have potential as analgesic drugs.