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1.
Neuron ; 111(21): 3435-3449.e8, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37918358

RESUMO

Interleukin-4 (IL-4) is a type 2 cytokine with pleiotropic functions in adaptive immunity, allergies, and cognitive processes. Here, we show that low levels of IL-4 in the early postnatal stage delineate a critical period in which microglia extensively prune cerebellar neurons. Elevating the levels of this cytokine via peripheral injection, or using a mouse model of allergic asthma, leads to defective pruning, permanent increase in cerebellar granule cells, and circuit alterations. These animals also show a hyperkinetic and impulsive-like phenotype, reminiscent of attention-deficit hyperactivity disorder (ADHD). These alterations are blocked in Il4rαfl/fl::Cx3cr1-CreER mice, which are deficient in IL-4 receptor signaling in microglia. These findings demonstrate a previously unknown role for IL-4 during a neuroimmune critical period of cerebellar maturation and provide a first putative mechanism for the comorbidity between allergic disease and ADHD observed in humans.


Assuntos
Interleucina-4 , Microglia , Animais , Humanos , Cerebelo , Encéfalo , Citocinas
2.
Pharmacol Ther ; 242: 108348, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36657567

RESUMO

Interleukin-4 (IL-4) is a pleiotropic cytokine mainly known for its role in type 2 immunity. Therapies antagonizing or blocking IL-4 activity have been developed to counteract diseases such as atopic dermatitis and asthma. In contrast, other disorders experimentally benefit from IL-4-related effects and IL-4 recently demonstrated beneficial activity in experimental stroke, spinal cord injury and the animal model of multiple sclerosis. To exploit IL-4-related activity for therapeutic concepts, current experimental efforts include modifying the pathway without inducing type 2 immune response and targeting of the cytokine to specific tissues. Here, we review different activities of IL-4 as well as therapeutic strategies.


Assuntos
Asma , Dermatite Atópica , Animais , Asma/tratamento farmacológico , Citocinas , Dermatite Atópica/tratamento farmacológico , Interleucina-33 , Interleucina-4/uso terapêutico , Humanos
3.
J Exp Med ; 219(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35587822

RESUMO

Evidence is emerging that immune responses not only play a part in the central nervous system (CNS) in diseases but may also be relevant for healthy conditions. We discovered a major role for the interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signaling pathway in synaptic processes, as indicated by transcriptome analysis in IL-4Rα-deficient mice and human neurons with/without IL-4 treatment. Moreover, IL-4Rα is expressed presynaptically, and locally available IL-4 regulates synaptic transmission. We found reduced synaptic vesicle pools, altered postsynaptic currents, and a higher excitatory drive in cortical networks of IL-4Rα-deficient neurons. Acute effects of IL-4 treatment on postsynaptic currents in wild-type neurons were mediated via PKCγ signaling release and led to increased inhibitory activity supporting the findings in IL-4Rα-deficient neurons. In fact, the deficiency of IL-4Rα resulted in increased network activity in vivo, accompanied by altered exploration and anxiety-related learning behavior; general learning and memory was unchanged. In conclusion, neuronal IL-4Rα and its presynaptic prevalence appear relevant for maintaining homeostasis of CNS synaptic function.


Assuntos
Interleucina-4 , Receptores de Interleucina-4 , Animais , Interleucina-4/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Receptores de Interleucina-4/metabolismo , Transdução de Sinais
4.
Cell Mol Life Sci ; 78(3): 1029-1050, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32468095

RESUMO

Recent studies suggest that synaptic lysophosphatidic acids (LPAs) augment glutamate-dependent cortical excitability and sensory information processing in mice and humans via presynaptic LPAR2 activation. Here, we studied the consequences of LPAR2 deletion or antagonism on various aspects of cognition using a set of behavioral and electrophysiological analyses. Hippocampal neuronal network activity was decreased in middle-aged LPAR2-/- mice, whereas hippocampal long-term potentiation (LTP) was increased suggesting cognitive advantages of LPAR2-/- mice. In line with the lower excitability, RNAseq studies revealed reduced transcription of neuronal activity markers in the dentate gyrus of the hippocampus in naïve LPAR2-/- mice, including ARC, FOS, FOSB, NR4A, NPAS4 and EGR2. LPAR2-/- mice behaved similarly to wild-type controls in maze tests of spatial or social learning and memory but showed faster and accurate responses in a 5-choice serial reaction touchscreen task requiring high attention and fast spatial discrimination. In IntelliCage learning experiments, LPAR2-/- were less active during daytime but normally active at night, and showed higher accuracy and attention to LED cues during active times. Overall, they maintained equal or superior licking success with fewer trials. Pharmacological block of the LPAR2 receptor recapitulated the LPAR2-/- phenotype, which was characterized by economic corner usage, stronger daytime resting behavior and higher proportions of correct trials. We conclude that LPAR2 stabilizes neuronal network excitability upon aging and allows for more efficient use of resting periods, better memory consolidation and better  performance in tasks requiring high selective attention. Therapeutic LPAR2 antagonism may alleviate aging-associated cognitive dysfunctions.


Assuntos
Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Neurônios/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Envelhecimento , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Cromatografia Líquida de Alta Pressão , Giro Denteado/metabolismo , Análise Discriminante , Família de Proteínas EGF/deficiência , Família de Proteínas EGF/genética , Feminino , Fígado/metabolismo , Potenciação de Longa Duração , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Componente Principal , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/deficiência , Receptores de Ácidos Lisofosfatídicos/genética , Espectrometria de Massas em Tandem
5.
Cell Mol Neurobiol ; 40(8): 1327-1338, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32172457

RESUMO

Neurons of the central nervous system (CNS) that project long axons into the spinal cord have a poor axon regenerative capacity compared to neurons of the peripheral nervous system. The corticospinal tract (CST) is particularly notorious for its poor regeneration. Because of this, traumatic spinal cord injury (SCI) is a devastating condition that remains as yet uncured. Based on our recent observations that direct neuronal interleukin-4 (IL-4) signaling leads to repair of axonal swellings and beneficial effects in neuroinflammation, we hypothesized that IL-4 acts directly on the CST. Here, we developed a tissue culture model for CST regeneration and found that IL-4 promoted new growth cone formation after axon transection. Most importantly, IL-4 directly increased the regenerative capacity of both murine and human CST axons, which corroborates its regenerative effects in CNS damage. Overall, these findings serve as proof-of-concept that our CST regeneration model is suitable for fast screening of new treatments for SCI.


Assuntos
Axônios/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Humanos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/farmacologia , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismo
6.
J Exp Med ; 217(6)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32219436

RESUMO

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.


Assuntos
Sistema Nervoso Central/patologia , Inflamação/imunologia , Células Mieloides/patologia , Linfócitos T/imunologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Comunicação Celular , Morte Celular , Sobrevivência Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Glucosamina/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Fagocitose , Fosfatidilserinas/metabolismo , Receptores de Superfície Celular/metabolismo , Índice de Gravidade de Doença , Células Th17/imunologia , Células Th17/metabolismo
7.
Eur J Pharmacol ; 871: 172923, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31962100

RESUMO

The humanized anti-CD52 antibody alemtuzumab is successfully used in the treatment of multiple sclerosis (MS) and is thought to exert most of its therapeutic action by depletion and repopulation of mainly B and T lymphocytes. Although neuroprotective effects of alemtuzumab have been suggested, direct effects of anti-CD52 treatment on glial cells and neurons within the CNS itself have not been investigated so far. Here, we show CD52 expression in murine neurons, astrocytes and microglia, both in vitro and in vivo. As expected, anti CD52-treatment caused profound lymphopenia and improved disease symptoms in mice subjected to experimental autoimmune encephalomyelitis (EAE). CD52 blockade also had a significant effect on microglial morphology in organotypic hippocampal slice cultures but did not affect microglial functions. Furthermore, anti-CD52 neither changed baseline neuronal calcium, nor did it act neuroprotective in excitotoxicity models. Altogether, our findings argue against a functionally significant role of CD52 blockade on CNS neurons and microglia. The beneficial effects of alemtuzumab in MS may be exclusively mediated by peripheral immune mechanisms.


Assuntos
Alemtuzumab/imunologia , Antígeno CD52/imunologia , Microglia/patologia , Neurônios/patologia , Animais , Antígeno CD52/metabolismo , Cálcio/metabolismo , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/imunologia , Camundongos
8.
J Neurotrauma ; 37(12): 1481-1490, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830857

RESUMO

With increasing evidence for the existence of a cerebral thrombin system, coagulation factor IIa (thrombin) is suspected to influence the pathogenesis of secondary injury progression after intracerebral hemorrhage (ICH). We hypothesized that mechanisms associated with local volume expansion after ICH, rather than blood constituents, activate the cerebral thrombin system and are responsible for detrimental neurological outcome. To test this hypothesis, we examine the local thrombin expression after ICH in a C57BL/6N mouse model in the presence and absence of blood constituents. ICH was established using stereotaxic orthotopic injection of utologous blood (n = 10) or silicone oil as inert volume substance (n = 10) into the striatum. Intracranial pressure (ICP), cerebral blood flow (CBF), and mean arterial blood pressure (MAP) were monitored during and 30 min after the procedure. No significant differences between ICP, CBF, and MAP were found between both groups. Prothrombin messenger RNA expression was upregulated early after ICH. Immunohistochemistry showed an increase of perilesional thrombin in both groups (blood, 4.24-fold; silicone, 3.10-fold), whereas prothrombin fragment (F1.2) was elevated only in the absence of whole blood. Thrombin expression is colocalized with neuronal antigen expression. After 24 h, lesion size and neuronal loss were similar. Perihematomal thrombin correlated with increased neuronal loss and detrimental neurological outcome in vivo. In our study, we demonstrate, for the first time, that the local cerebral thrombin system is activated after ICH and that this activation is independent of the presence of whole-blood constituents. In our study, neuronal damage is driven by local thrombin expression and leads to an adverse clinical outcome.


Assuntos
Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patologia , Circulação Cerebrovascular/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Trombina/biossíntese , Animais , Coagulação Sanguínea/fisiologia , Células Cultivadas , Hemorragia Cerebral/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
Nat Neurosci ; 22(1): 144, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30405214

RESUMO

In the version of this article initially published, Inigo Ruiz de Azua's name was miscategorized. His given name is Inigo and his surname is Ruiz de Azua. This has been corrected in the HTML coding.

10.
Nat Neurosci ; 21(10): 1392-1403, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30258239

RESUMO

Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.


Assuntos
Córtex Cerebral/fisiopatologia , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/patologia , Hipercinese/etiologia , Recuperação de Função Fisiológica/fisiologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Carbazóis/uso terapêutico , Células Cultivadas , Córtex Cerebral/ultraestrutura , Cuprizona/toxicidade , Modelos Animais de Doenças , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacocinética , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Adjuvante de Freund/toxicidade , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Microglia/patologia , Proteína Proteolipídica de Mielina/toxicidade , Fragmentos de Peptídeos/toxicidade , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Quinoxalinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
11.
Sci Transl Med ; 10(430)2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491183

RESUMO

Ongoing axonal degeneration is thought to underlie disability in chronic neuroinflammation, such as multiple sclerosis (MS), especially during its progressive phase. Upon inflammatory attack, axons undergo pathological swelling, which can be reversible. Because we had evidence for beneficial effects of T helper 2 lymphocytes in experimental neurotrauma and discovered interleukin-4 receptor (IL-4R) expressed on axons in MS lesions, we aimed at unraveling the effects of IL-4 on neuroinflammatory axon injury. We demonstrate that intrathecal IL-4 treatment during the chronic phase of several experimental autoimmune encephalomyelitis models reversed disease progression without affecting inflammation. Amelioration of disability was abrogated upon neuronal deletion of IL-4R. We discovered direct neuronal signaling via the IRS1-PI3K-PKC pathway underlying cytoskeletal remodeling and axonal repair. Nasal IL-4 application, suitable for clinical translation, was equally effective in improving clinical outcome. Targeting neuronal IL-4 signaling may offer new therapeutic strategies to halt disability progression in MS and possibly also neurodegenerative conditions.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Receptores de Interleucina-4/metabolismo , Administração Intranasal , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-4/administração & dosagem , Interleucina-4/uso terapêutico , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Esclerose Múltipla , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
12.
Glia ; 66(8): 1591-1610, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29575063

RESUMO

Axonal mRNA transport and local protein synthesis are crucial for peripheral axon regeneration. To date, it remains unclear how ribosomes localize to axons. They may be co-transported with mRNAs or, as suggested by recent studies, transferred from Schwann cells (SC). Here, we generated transgenic "RiboTracker" mice expressing tdTomato-tagged ribosomal protein L4 in specific cell types when crossed with Cre lines. Two neuronal RiboTracker-Cre lines displayed extremely low levels of axonal L4-tdTomato-positive ribosomes. In contrast, two glial RiboTracker-Cre lines revealed tagged ribosomes in sciatic nerve (SN) axons with increasing amounts after injury. Furthermore, non-RiboTracker dorsal root ganglia co-cultured with L4-tdTomato-expressing SCs displayed tagged ribosomes in axons. These data provide unequivocal evidence that SN axons receive ribosomes from SCs upon injury and indicate that glial cells are the main source of axonal ribosomes.


Assuntos
Axônios/metabolismo , Gânglios Espinais/metabolismo , Neuroglia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Camundongos Transgênicos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/patologia , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo
14.
PLoS One ; 10(7): e0134371, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26222542

RESUMO

Lesion-induced scarring is a major impediment for regeneration of injured axons in the central nervous system (CNS). The collagen-rich glial-fibrous scar contains numerous axon growth inhibitory factors forming a regeneration-barrier for axons. We demonstrated previously that the combination of the iron chelator 2,2'-bipyridine-5,5'-decarboxylic acid (BPY-DCA) and 8-Br-cyclic AMP (cAMP) inhibits scar formation and collagen deposition, leading to enhanced axon regeneration and partial functional recovery after spinal cord injury. While BPY-DCA is not a clinical drug, the clinically approved iron chelator deferoxamine mesylate (DFO) may be a suitable alternative for anti-scarring treatment (AST). In order to prove the scar-suppressing efficacy of DFO we modified a recently published in vitro model for CNS scarring. The model comprises a co-culture system of cerebral astrocytes and meningeal fibroblasts, which form scar-like clusters when stimulated with transforming growth factor-ß (TGF-ß). We studied the mechanisms of TGF-ß-induced CNS scarring and compared the efficiency of different putative pharmacological scar-reducing treatments, including BPY-DCA, DFO and cAMP as well as combinations thereof. We observed modulation of TGF-ß-induced scarring at the level of fibroblast proliferation and contraction as well as specific changes in the expression of extracellular matrix molecules and axon growth inhibitory proteins. The individual and combinatorial pharmacological treatments had distinct effects on the cellular and molecular aspects of in vitro scarring. DFO could be identified as a putative anti-scarring treatment for CNS trauma. We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord. DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion. We conclude that the in vitro model for CNS scarring is suitable for efficient pre-screening and identification of putative scar-suppressing agents prior to in vivo application and validation, thus saving costs, time and laboratory animals.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Cicatriz/prevenção & controle , Desferroxamina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Sistema Nervoso Central/metabolismo , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno Tipo IV/genética , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Técnicas In Vitro , Quelantes de Ferro/farmacologia , Regeneração Nervosa/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fator de Crescimento Transformador beta/farmacologia
16.
J Clin Invest ; 125(2): 699-714, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25607842

RESUMO

A body of experimental evidence suggests that T cells mediate neuroprotection following CNS injury; however, the antigen specificity of these T cells and how they mediate neuroprotection are unknown. Here, we have provided evidence that T cell-mediated neuroprotection after CNS injury can occur independently of major histocompatibility class II (MHCII) signaling to T cell receptors (TCRs). Using two murine models of CNS injury, we determined that damage-associated molecular mediators that originate from injured CNS tissue induce a population of neuroprotective, IL-4-producing T cells in an antigen-independent fashion. Compared with wild-type mice, IL-4-deficient animals had decreased functional recovery following CNS injury; however, transfer of CD4+ T cells from wild-type mice, but not from IL-4-deficient mice, enhanced neuronal survival. Using a culture-based system, we determined that T cell-derived IL-4 protects and induces recovery of injured neurons by activation of neuronal IL-4 receptors, which potentiated neurotrophin signaling via the AKT and MAPK pathways. Together, these findings demonstrate that damage-associated molecules from the injured CNS induce a neuroprotective T cell response that is independent of MHCII/TCR interactions and is MyD88 dependent. Moreover, our results indicate that IL-4 mediates neuroprotection and recovery of the injured CNS and suggest that strategies to enhance IL-4-producing CD4+ T cells have potential to attenuate axonal damage in the course of CNS injury in trauma, inflammation, or neurodegeneration.


Assuntos
Axônios/imunologia , Lesões Encefálicas/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-4/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Doenças Neurodegenerativas/imunologia , Animais , Axônios/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Linfócitos T CD4-Positivos/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interleucina-4/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia
17.
Front Mol Neurosci ; 4: 26, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21994489

RESUMO

Traumatic spinal cord injury (SCI) results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005) that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons, and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST). In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1-60 days post-operation (dpo). We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection, and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons toward enhanced cell maintenance and axonal regeneration.

18.
Arterioscler Thromb Vasc Biol ; 31(3): 624-33, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21164077

RESUMO

OBJECTIVE: Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors. METHODS AND RESULTS: In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4-activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor α gene expression. This enhancement of thrombin-stimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown. PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-ß and -δ isoforms or nuclear factor (NF)κB. Nuclear translocation of NFκB in high glucose-stimulated SMCs led to PKC-dependent NFκB binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels. CONCLUSIONS: High glucose enhances SMC responsiveness to thrombin through transcriptional upregulation of PAR-4, mediated via PKC-ß, -δ, and NFκB. This may play an important role in the vascular complications of diabetes.


Assuntos
Sinalização do Cálcio , Angiopatias Diabéticas/metabolismo , Glucose/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Movimento Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Angiopatias Diabéticas/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/genética , Fatores de Tempo , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Regulação para Cima
19.
Mol Cell Neurosci ; 42(2): 102-115, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19520167

RESUMO

We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for beta-actin, beta-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of beta-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of beta-actin in axons, the rate being increased by axotomy. Knock down of beta-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.


Assuntos
Axônios/fisiologia , Gânglios Espinais , Cones de Crescimento/fisiologia , Regeneração Nervosa/fisiologia , RNA Mensageiro/metabolismo , Actinas/genética , Animais , Animais Recém-Nascidos , Axotomia , Citoesqueleto/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/genética , Técnicas de Cultura de Tecidos
20.
J Neurosci ; 29(17): 5546-57, 2009 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-19403822

RESUMO

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.


Assuntos
Axônios/metabolismo , Cadeias alfa de Integrinas/fisiologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Células Receptoras Sensoriais/metabolismo , Tenascina/metabolismo , Animais , Axônios/ultraestrutura , Células Cultivadas , Gânglios Espinais/fisiologia , Gânglios Espinais/ultraestrutura , Humanos , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Masculino , Microscopia Confocal , Neuritos/ultraestrutura , Neurogênese/fisiologia , Células PC12 , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/fisiologia , Células Receptoras Sensoriais/ultraestrutura
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