RESUMO
INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.
Assuntos
Lesões Encefálicas Traumáticas , Encéfalo , Modelos Animais de Doenças , Doenças Neuroinflamatórias , Animais , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Camundongos , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/etiologia , Masculino , Encéfalo/metabolismo , Encéfalo/patologia , Astrócitos/metabolismo , Microglia/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Feminino , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Corpo Caloso/diagnóstico por imagem , Inflamação/metabolismo , Imagem de Tensor de Difusão/métodosRESUMO
OBJECTIVES: In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI. METHODS: Ex vivo microglial cultures and a mouse model of WMI induced by perinatal inflammation (interleukin-1-beta [IL-1ß] and postnatal days 1-5) were used to uncover and elucidate the role of microRNA-146b-5p in microglial activation and WMI. RESULTS: A specific reduction in vivo in microglia of Dicer, a protein required for microRNAs maturation, reduces pro-inflammatory activation of microglia and prevents hypomyelination in our model of WMI. Microglial miRNome analysis in the WMI model identified miRNA-146b-5p as a candidate modulator of microglial activation. Ex vivo microglial cell culture treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) led to overexpression of immunomodulatory miRNA-146b-5p but its drastic reduction in the microglial extracellular vesicles (EVs). To increase miRNA-146b-5p expression, we used a 3DNA nanocarrier to deliver synthetic miRNA-146b-5p specifically to microglia. Enhancing microglial miRNA-146b-5p overexpression significantly decreased LPS-induced activation, downregulated IRAK1, and restored miRNA-146b-5p levels in EVs. In our WMI model, 3DNA miRNA-146b-5p treatment significantly prevented microglial activation, hypomyelination, and cognitive defect induced by perinatal inflammation. INTERPRETATIONS: These findings support that miRNA-146b-5p is a major regulator of microglia phenotype and could be targeted to reduce the incidence and the severity of perinatal brain injuries and their long-term consequences. ANN NEUROL 2022;91:48-65.
Assuntos
Encéfalo/patologia , MicroRNAs/metabolismo , Microglia/patologia , Substância Branca/patologia , Animais , Camundongos , Neurogênese/fisiologiaRESUMO
Approximately 15 million babies are born prematurely every year and many will face lifetime motor and/or cognitive deficits. Children born prematurely are at higher risk of developing perinatal brain lesions, especially white matter injuries (WMI). Evidence in humans and rodents demonstrates that systemic inflammation-induced neuroinflammation, including microglial and astrocyte reactivity, is the prominent processes of WMI associated with preterm birth. Thus, a new challenge in the field of perinatal brain injuries is to develop new neuroprotective strategies to target neuroinflammation to prevent WMI. Serotonin (5-HT) and its receptors play an important role in inflammation, and emerging evidence indicates that 5-HT may regulate brain inflammation by the modulation of microglial reactivity and astrocyte functions. The present study is based on a mouse model of WMI induced by intraperitoneal (i.p.) injections of IL-1ß during the first 5 days of life. In this model, certain key lesions of preterm brain injuries can be summarized by (i) systemic inflammation, (ii) pro-inflammatory microglial and astrocyte activation, and (iii) inhibition of oligodendrocyte maturation, leading to hypomyelination. We demonstrate that Htr7 mRNA (coding for the HTR7/5-HT7 receptor) is significantly overexpressed in the anterior cortex of IL-1ß-exposed animals, suggesting it as a potential therapeutic target. LP-211 is a specific high-affinity HTR7 agonist that crosses the blood-brain barrier (BBB). When co-injected with IL-1ß, LP-211 treatment prevented glial reactivity, the down-regulation of myelin-associated proteins, and the apparition of anxiety-like phenotypes. Thus, HTR7 may represent an innovative therapeutic target to protect the developing brain from preterm brain injuries.
Assuntos
Lesões Encefálicas , Nascimento Prematuro , Substância Branca , Animais , Camundongos , Gravidez , Feminino , Criança , Recém-Nascido , Humanos , Substância Branca/patologia , Roedores , Doenças Neuroinflamatórias , Serotonina/metabolismo , Nascimento Prematuro/metabolismo , Nascimento Prematuro/patologia , Encéfalo/metabolismo , Lesões Encefálicas/etiologia , Lesões Encefálicas/prevenção & controle , Inflamação/patologia , Microglia/metabolismoRESUMO
Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Using a mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of pro-inflammatory IL-1ß, we sought to uncover causes of cerebellar damage. In this model, IL-1ß is administered between postnatal day (P) 1 to day 5, a timing equivalent to the last trimester for brain development in humans. Structural MRI analysis revealed that systemic IL-1ß treatment induced specific reductions in gray and white matter volumes of the mouse cerebellar lobules I and II (5% false discovery rate [FDR]) from P15 onwards. Preceding these MRI-detectable cerebellar volume changes, we observed damage to oligodendroglia, with reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) at P10 and P15. Increased density of IBA1+ cerebellar microglia were observed both at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebellums and cerebrums revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but cerebellar microglia displayed a unique type I interferon signaling dysregulation. Collectively, these data suggest that perinatal inflammation driven by systemic IL-1ß leads to specific cerebellar volume deficits, which likely reflect oligodendrocyte pathology downstream of microglial activation. Further studies are now required to confirm the potential of protective strategies aimed at preventing sustained type I interferon signaling driven by cerebellar microglia as an important therapeutic target.
Assuntos
Doenças Cerebelares , Doenças do Prematuro , Inflamação , Interferon Tipo I , Interleucina-1beta , Microglia , Animais , Encefalopatias/induzido quimicamente , Encefalopatias/imunologia , Encefalopatias/patologia , Doenças Cerebelares/induzido quimicamente , Doenças Cerebelares/imunologia , Doenças Cerebelares/patologia , Cerebelo/efeitos dos fármacos , Cerebelo/imunologia , Cerebelo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/induzido quimicamente , Doenças do Prematuro/imunologia , Doenças do Prematuro/patologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interferon Tipo I/imunologia , Interleucina-1beta/efeitos adversos , Interleucina-1beta/farmacologia , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/patologia , GravidezRESUMO
Preterm birth (PTB) represents 15 million births every year worldwide and is frequently associated with maternal/fetal infections and inflammation, inducing neuroinflammation. This neuroinflammation is mediated by microglial cells, which are brain-resident macrophages that release cytotoxic molecules that block oligodendrocyte differentiation, leading to hypomyelination. Some preterm survivors can face lifetime motor and/or cognitive disabilities linked to periventricular white matter injuries (PWMIs). There is currently no recommendation concerning the mode of delivery in the case of PTB and its impact on brain development. Many animal models of induced-PTB based on LPS injections exist, but with a low survival rate. There is a lack of information regarding clinically used pharmacological substances to induce PTB and their consequences on brain development. Mifepristone (RU-486) is a drug used clinically to induce preterm labor. This study aims to elaborate and characterize a new model of induced-PTB and PWMIs by the gestational injection of RU-486 and the perinatal injection of pups with IL-1beta. A RU-486 single subcutaneous (s.c.) injection at embryonic day (E)18.5 induced PTB at E19.5 in pregnant OF1 mice. All pups were born alive and were adopted directly after birth. IL-1beta was injected intraperitoneally from postnatal day (P)1 to P5. Animals exposed to both RU-486 and IL-1beta demonstrated microglial reactivity and subsequent PWMIs. In conclusion, the s.c. administration of RU-486 induced labor within 24 h with a high survival rate for pups. In the context of perinatal inflammation, RU-486 labor induction significantly decreases microglial reactivity in vivo but did not prevent subsequent PWMIs.
Assuntos
Microglia , Nascimento Prematuro , Animais , Animais Recém-Nascidos , Feminino , Humanos , Inflamação , Lipopolissacarídeos/toxicidade , Camundongos , Mifepristona/farmacologia , GravidezRESUMO
Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.
Assuntos
Receptores de Somatostatina/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Ligantes , Conformação Proteica , Transporte Proteico , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Receptores de Somatostatina/fisiologia , Transdução de Sinais , Terminologia como AssuntoRESUMO
Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/ß-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.
Assuntos
Encéfalo/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Biologia Computacional , Humanos , Camundongos , Peixe-ZebraRESUMO
Neonatal acute ischemic stroke is a cause of neonatal brain injury that occurs more frequently in males, resulting in associated neurobehavioral disorders. The bases for these sex differences are poorly understood but might include the number, morphology and activation of microglia in the developing brain when subjected to stroke. Interestingly, poly (ADP-ribose) polymerase (PARP) inhibition preferentially protects males against neonatal ischemia. This study aims to examine the effects of PJ34, a PARP inhibitor, on microglial phenotypes at 3 and 8â¯days and on neurobehavioral disorders in adulthood for both male and female P9 mice subjected to permanent middle cerebral artery occlusion (pMCAo). PJ34 significantly reduced the lesion size by 78% and reduced the density of CX3CR1gfp-labeled microglial cells by 46% when examined 3â¯days after pMCAo in male but not in female mice. Eight days after pMCAo, the number of Iba1+/Cox-2+ cells did not differ between male and female mice in the cortical peri-infarct region. In the amygdala, Iba1+/Cox-2+ (M1-like) cell numbers were significantly decreased in PJ34-treated males but not in females. Conversely, Iba1+/Arg-1+ (M2-like) and Arg-1+/Cox-2+ (Mtransitional) cell numbers were significantly increased in PJ34-treated females. Regarding neurobehavioral disorders during adulthood, pMCAo induced a motor coordination deficit and a spatial learning deficit in female mice only. PJ34 prevented MBP fibers, motor coordination and learning disorders during adulthood in female mice. Our data show significant sex differences in the effects of PARP inhibition on microglia phenotypes following neonatal ischemia, associated with improved behavior and myelination during adulthood in females only. Our findings suggest that modulating microglial phenotypes may play key roles in behavior disorders and white matter injury following neonatal stroke.
Assuntos
Isquemia Encefálica/patologia , Microglia/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Animais Recém-Nascidos , Lesões Encefálicas/complicações , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Feminino , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fenantrenos/metabolismo , Fenantrenos/farmacologia , Fenótipo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fatores Sexuais , Acidente Vascular Cerebral/patologiaRESUMO
Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation.
Assuntos
Nanismo/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Microcefalia/genética , Osteocondrodisplasias/congênito , Proteínas/genética , Animais , Pré-Escolar , Regulação para Baixo , Retículo Endoplasmático Rugoso/metabolismo , Feminino , Complexo de Golgi/metabolismo , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Mutantes , Mutação , Bainha de Mielina/genética , Bainha de Mielina/fisiologia , Osteocondrodisplasias/genética , Transporte Proteico/genética , Transporte Proteico/fisiologiaRESUMO
Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH) axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a complex neurological phenotype, with abnormal glucose metabolism and gonadotropic axis deficiency due to a loss of GnRH neurons. Our findings identify rabconectin-3α as a key controller of neuronal and endocrine homeostatic processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Diabetes Mellitus Tipo 1/genética , Hipoglicemia/genética , Hipotireoidismo/genética , Infertilidade Masculina/genética , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Polineuropatias/genética , Deleção de Sequência , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adolescente , Animais , Sequência de Bases , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Haploinsuficiência , Homozigoto , Humanos , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Hipotálamo/patologia , Hipotireoidismo/metabolismo , Hipotireoidismo/patologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Hormônio Luteinizante/genética , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Neurônios/patologia , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Hipófise/patologia , Polineuropatias/metabolismo , Polineuropatias/patologia , Maturidade Sexual , Síndrome , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testículo/patologia , Adulto JovemRESUMO
Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.
Assuntos
Cistinil Aminopeptidase/metabolismo , Hipocampo/metabolismo , Receptores de Somatostatina/metabolismo , Convulsões/metabolismo , Convulsões/prevenção & controle , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Sistema Límbico/metabolismo , Masculino , Camundongos , Transporte Proteico/fisiologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Perinatal ischemic stroke is the most frequent form of cerebral infarction in neonates; however, evidence-based treatments are currently lacking. We have previously demonstrated a beneficial effect of sildenafil citrate, a PDE-5 inhibitor, on stroke lesion size in neonatal rat pups. The present study investigated the effects of sildenafil in a neonatal mouse stroke model on (1) hemodynamic changes and (2) regulation of astrocyte/microglia-mediated neuroinflammation. METHODS: Ischemia was induced in C57Bl/6 mice on postnatal (P) day 9 by permanent middle cerebral artery occlusion (pMCAo), and followed by either PBS or sildenafil intraperitoneal (i.p.) injections. Blood flow (BF) velocities were measured by ultrasound imaging with sequential Doppler recordings and laser speckle contrast imaging. Animals were euthanized, and brain tissues were obtained at 72 h or 8 days after pMCAo. Expression of M1- and M2-like microglia/macrophage markers were analyzed. RESULTS: Although sildenafil (10 mg/kg) treatment potently increased cGMP concentrations, it did not influence early collateral recruitment nor did it reduce mean infarct volumes 72 h after pMCAo. Nevertheless, it provided a significant dose-dependent reduction of mean lesion extent 8 days after pMCAo. Suggesting a mechanism involving modulation of the inflammatory response, sildenafil significantly decreased microglial density at 72 h and 8 days after pMCAo. Gene expression profiles indicated that sildenafil treatment also modulates M1- (ptgs2, CD32 and CD86) and M2-like (CD206, Arg-1 and Lgals3) microglia/macrophages in the late phase after pMCAo. Accordingly, the number of COX-2(+) microglia/macrophages significantly increased in the penumbra at 72 h after pMCAo but was significantly decreased 8 days after ischemia in sildenafil-treated animals. CONCLUSIONS: Our findings argue that anti-inflammatory effects of sildenafil may provide protection against lesion extension in the late phase after pMCAo in neonatal mice. We propose that sildenafil treatment could represent a potential strategy for neonatal ischemic stroke treatment/recovery.
Assuntos
Isquemia Encefálica/patologia , Microglia/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Citrato de Sildenafila/farmacologia , Animais , Animais Recém-Nascidos , Isquemia Encefálica/enzimologia , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Infectious encephalitides are most often associated with acute seizures during the infection period and are risk factors for the development of epilepsy at later times. Mechanisms of viral encephalitis-induced epileptogenesis are poorly understood. Here, we evaluated the contribution of viral encephalitis-associated inflammation to ictogenesis and epileptogenesis using a rapid kindling protocol in rats. In addition, we examined whether minocycline can improve outcomes of viral-like brain inflammation. METHODS: To produce viral-like inflammation, polyinosinic-polycytidylic acid (PIC), a toll-like receptor 3 (TLR3) agonist, was applied to microglial/macrophage cell cultures and to the hippocampus of postnatal day 13 (P13) and postnatal day 74 (P74) rats. Cell cultures permit the examination of the inflammation induced by PIC, while the in vivo setting better suits the analysis of cytokine production and the effects of inflammation on epileptogenesis. Minocycline (50 mg/kg) was injected intraperitoneally for 3 consecutive days prior to the kindling procedure to evaluate its effects on inflammation and epileptogenesis. RESULTS: PIC injection facilitated kindling epileptogenesis, which was evident as an increase in the number of full limbic seizures at both ages. Furthermore, in P14 rats, we observed a faster seizure onset and prolonged retention of the kindling state. PIC administration also led to an increase in interleukin 1ß (IL-1ß) levels in the hippocampus in P14 and P75 rats. Treatment with minocycline reversed neither the pro-epileptogenic effects of PIC nor the increase of IL-1ß in the hippocampus in both P14 and P75 rats. CONCLUSIONS: Hippocampal injection of PIC facilitates rapid kindling epileptogenesis at both P14 and P75, suggesting that viral-induced inflammation increases epileptogenesis irrespective of brain maturation. Minocycline, however, was unable to reverse the increase of epileptogenesis, which might be linked to its absence of effect on hippocampal IL-1ß levels at both ages.
Assuntos
Encéfalo , Encefalite Viral/complicações , Encefalite/etiologia , Epilepsia/etiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Anticonvulsivantes/uso terapêutico , Antivirais/farmacologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Encéfalo/virologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Encefalite/induzido quimicamente , Encefalite/virologia , Epilepsia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Excitação Neurológica/efeitos dos fármacos , Excitação Neurológica/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Microglia/efeitos dos fármacos , Minociclina/uso terapêutico , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Estatísticas não ParamétricasRESUMO
Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Mitocôndrias , Oxirredutases , Espécies Reativas de Oxigênio , Animais , Ciona intestinalis/genética , Transporte de Elétrons/genética , Transporte de Elétrons/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Oxirredução , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease characterized by ataxia, variously associating heart disease, diabetes mellitus and/or glucose intolerance. It results from intronic expansion of GAA triplet repeats at the FXN locus. Homozygous expansions cause silencing of the FXN gene and subsequent decreased expression of the encoded mitochondrial frataxin. Detailed analyses in fibroblasts and neuronal tissues from FRDA patients have revealed profound cytoskeleton anomalies. So far, however, the molecular mechanism underlying these cytoskeleton defects remains unknown. We show here that gene silencing spreads in cis over the PIP5K1B gene in cells from FRDA patients (circulating lymphocytes and primary fibroblasts), correlating with expanded GAA repeat size. PIP5K1B encodes phosphatidylinositol 4-phosphate 5-kinase ß type I (pip5k1ß), an enzyme functionally linked to actin cytoskeleton dynamics that phosphorylates phosphatidylinositol 4-phosphate [PI(4)P] to generate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Accordingly, loss of pip5k1ß function in FRDA cells was accompanied by decreased PI(4,5)P2 levels and was shown instrumental for destabilization of the actin network and delayed cell spreading. Knockdown of PIP5K1B in control fibroblasts using shRNA reproduced abnormal actin cytoskeleton remodeling, whereas over-expression of PIP5K1B, but not FXN, suppressed this phenotype in FRDA cells. In addition to provide new insights into the consequences of the FXN gene expansion, these findings raise the question whether PIP5K1B silencing may contribute to the variable manifestation of this complex disease.
Assuntos
Citoesqueleto/metabolismo , Ataxia de Friedreich/enzimologia , Inativação Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Citoesqueleto/genética , Fibroblastos/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Linfócitos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
BACKGROUND AND PURPOSE: The best conceivable treatment for hypoxia-ischemia (HI) is the restoration of blood flow to the hypoxic-ischemic region(s). Our objective was to examine whether boosting NO-cGMP signaling using sildenafil citrate, a phosphodiesterase-type 5 inhibitor, could modify cerebral blood flow and reduce lesions in the developing brain. METHODS: HI was induced in P7 Sprague-Dawley rats by unilateral carotid artery occlusion and hypoxia, and followed by either PBS or sildenafil. Blood-flow velocities were measured by ultrasound imaging with sequential Doppler recordings to evaluate collateral recruitment. Cell death, blood-brain barrier integrity, and glial activation were analyzed by immunohistochemistry. Motor behavior was evaluated using an open-field device adapted to neonatal animals. RESULTS: Sildenafil citrate (10 mg/kg) induced collateral patency, reduced terminal dUTP nick-end labeling-positive cells, reactive astrogliosis, and macrophage/microglial activation at 72 hours and 7 days post-HI. Sildenafil also reduced the number of terminal dUTP nick-end labeling-positive endothelial cells within lesion site. Seven days after HI and sildenafil treatment, tissue loss was significantly reduced, and animals recovered motor coordination. CONCLUSIONS: Our findings strongly indicate that sildenafil citrate treatment, associated with a significant increase in cerebral blood flow, reduces HI damage and improves motor locomotion in neonatal rats. Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.
Assuntos
Animais Recém-Nascidos/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Gasometria , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Morte Celular/efeitos dos fármacos , GMP Cíclico/fisiologia , Lateralidade Funcional/fisiologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Hipóxia-Isquemia Encefálica/psicologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/patologia , Ativação de Macrófagos/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Óxido Nítrico/fisiologia , Desempenho Psicomotor/efeitos dos fármacos , Purinas/farmacologia , Ratos , Citrato de SildenafilaRESUMO
Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cerebelo/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Doxiciclina/farmacologia , Eletrofisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Imuno-Histoquímica , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neuroglia/citologia , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
It has been known for a long time that epileptic seizures provoke brain neuroinflammation involving the activation of microglial cells. However, the role of these cells in this disease context and the consequences of their inflammatory activation on subsequent neuron network activity remain poorly understood so far. To fill this gap of knowledge and gain a better understanding of the role of microglia in the pathophysiology of epilepsy, we used an established zebrafish Dravet syndrome epilepsy model based on Scn1Lab sodium channel loss-of-function, combined with live microglia and neuronal Ca2+ imaging, local field potential (LFP) recording, and genetic microglia ablation. Data showed that microglial cells in scn1Lab-deficient larvae experiencing epileptiform seizures displayed morphological and biochemical changes characteristic of M1-like pro-inflammatory activation; i.e., reduced branching, amoeboid-like morphology, and marked increase in the number of microglia expressing pro-inflammatory cytokine Il1ß. More importantly, LFP recording, Ca2+ imaging, and swimming behavior analysis showed that microglia-depleted scn1Lab-KD larvae displayed an increase in epileptiform seizure-like neuron activation when compared to that seen in scn1Lab-KD individuals with microglia. These findings strongly suggest that despite microglia activation and the synthesis of pro-inflammatory cytokines, these cells provide neuroprotective activities to epileptic neuronal networks, making these cells a promising therapeutic target in epilepsy.
Assuntos
Modelos Animais de Doenças , Epilepsias Mioclônicas , Microglia , Neurônios , Peixe-Zebra , Animais , Microglia/metabolismo , Microglia/patologia , Epilepsias Mioclônicas/patologia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Interleucina-1beta/metabolismo , Larva , Cálcio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
By means of double-label immunocytochemistry, authors studied the presence of estrogen receptor α (ER-α) protein in vesicular glutamate transporter 2 (VGluT2) protein-immunoreactive neurons in the female rat hypothalamus and amygdala. They examined colocalization of the 2 immunoreactive proteins in structures in which they found a significant overlap in the localization of the distribution of ER-α- and VGluT2-immunopositive nerve cells, namely in the medial preoptic area, the ventral subdivision of the ventromedial hypothalamic nucleus, and the medial amygdaloid nucleus. In the medial preoptic area, only 2.74 % of ER-α-immunoreactive neurons were VGluT2 positive, and conversely, 5 % of VGluT2-immunoreactive neurons contained ER-α immunofluorescent labeling. Highest degree of colocalization was detected in the ventral subdivision of the ventromedial hypothalamic nucleus, where 22.81 % of the ER-α-immunopositive neurons were VGluT2 immunoreactive and 37.14 % of the VGluT2-immunolabeled neurons contained ER-α-positive nucleus. In the medial amygdaloid nucleus, 15.38 % of the ER-α and 18.1 % of the VGluT2-immunoreactive neurons were double labeled. The colocalizations suggest that glutamatergic (VGluT2 protein immunoreactive) neurons are involved in the mediation of the action of estrogen on the rat brain.
Assuntos
Tonsila do Cerebelo/citologia , Receptor alfa de Estrogênio/metabolismo , Hipotálamo/citologia , Neurônios/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Tonsila do Cerebelo/metabolismo , Animais , Feminino , Hipotálamo/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Ratos , Ratos Sprague-DawleyRESUMO
Somatostatin (SRIF) is a neuropeptide that acts as an important regulator of both endocrine and exocrine secretion and modulates neurotransmission in the central nervous system (CNS). SRIF also regulates cell proliferation in normal tissues and tumors. The physiological actions of SRIF are mediated by a family of five G protein-coupled receptors, called somatostatin receptor (SST) SST1, SST2, SST3, SST4, SST5. These five receptors share similar molecular structure and signaling pathways but they display marked differences in their anatomical distribution, subcellular localization and intracellular trafficking. The SST subtypes are widely distributed in the CNS and peripheral nervous system, in many endocrine glands and tumors, particularly of neuroendocrine origin. In this review, we focus on the agonist-dependent internalization and recycling of the different SST subtypes in vivo in the CNS, peripheral organs and tumors. We also discuss the physiological, pathophysiological and potential therapeutic effects of the intracellular trafficking of SST subtypes.