Reactive astrocytosis-induced perturbation of synaptic homeostasis is restored by nerve growth factor (original) (raw)
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Biotechnology Advances
Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methalloproteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.
Cellular and Molecular Neurobiology, 2015
Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/ inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.
PLOS ONE, 2016
Modulation of extracellular matrix (ECM) remodeling after peripheral nerve injury (PNI) could represent a valid therapeutic strategy to prevent maladaptive synaptic plasticity in central nervous system (CNS). Inhibition of matrix metalloproteinases (MMPs) and maintaining a neurotrophic support could represent two approaches to prevent or reduce the maladaptive plastic changes in the ventral horn of spinal cord following PNI. The purpose of our study was to analyze changes in the ventral horn produced by gliopathy determined by the suffering of motor neurons following spared nerve injury (SNI) of the sciatic nerve and how the intrathecal (i.t.) administration of GM6001 (a MMPs inhibitor) or the NGF mimetic peptide BB14 modulate these events. Immunohistochemical analysis of spinal cord sections revealed that motor neuron disease following SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) response in the ventral horn of the spinal cord, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters (glutamate GLT1 and glycine GlyT1), increased levels of the neuronal glutamate transporter EAAC1, and a net increase of the Glutamate/GABA ratio, as measured by HPLC analysis. These molecular changes correlated to a significant reduction of mature NGF levels in the ventral horn. Continuous i.t. infusion of both GM6001 and BB14 reduced reactive astrogliosis, recovered the expression of neuronal and glial transporters, lowering the Glutamate/GABA ratio. Inhibition of MMPs by GM6001 significantly increased mature NGF levels, but it was absolutely ineffective in modifying the reactivity of microglia cells. Therefore, MMPs inhibition, although supplies neurotrophic support to ECM components and restores neuro-glial transporters expression, differently modulates astrocytic and microglial response after PNI.
Neuron Glia Biology, 2007
Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain. p38 mitogenactivated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain.
Gliotoxicity and Glioprotection: the Dual Role of Glial Cells
Molecular Neurobiology
Glial cells (astrocytes, oligodendrocytes and microglia) are critical for the central nervous system (CNS) in both physiological and pathological conditions. With this in mind, several studies have indicated that glial cells play key roles in the development and progression of CNS diseases. In this sense, gliotoxicity can be referred as the cellular, molecular, and neurochemical changes that can mediate toxic effects or ultimately lead to impairment of the ability of glial cells to protect neurons and/ or other glial cells. On the other hand, glioprotection is associated with specific responses of glial cells, by which they can protect themselves as well as neurons, resulting in an overall improvement of the CNS functioning. In addition, gliotoxic events, including metabolic stresses, inflammation, excitotoxicity, and oxidative stress, as well as their related mechanisms, are strongly associated with the pathogenesis of neurological, psychiatric and infectious diseases. However, glioprotective molecules can prevent or improve these glial dysfunctions, representing glial cells-targeting therapies. Therefore, this review will provide a brief summary of types and functions of glial cells and point out cellular and molecular mechanisms associated with gliotoxicity and glioprotection, potential glioprotective molecules and their mechanisms, as well as gliotherapy. In summary, we expect to address the relevance of gliotoxicity and glioprotection in the CNS homeostasis and diseases.
Reactive Astrocyte Gliosis: Production of Inhibitory Molecules
Spinal Cord Injury Therapy [Working Title]
The astrocytic cell responses to injury have been extensively studied in a variety of experimental models, and the term "astrogliosis" is often used to describe the astrocyte reactions to injury. Cells responding in these ways to injury are often referred to as "reactive astrocytes." Glial scarring appears to be a critical feature of wound healing in the central nervous system (CNS), since elimination of the mitotically active contingent of reactive astrocytes leads to increase in the size of the wound. Reactive astrogliosis is a term coined for the morphological and functional events seen in astrocytes responding to CNS injury. The concept of reactive astrogliosis and its molecular and cellular definition in spinal cord injury (SCI) is still incomplete. Producing several inhibitory molecules discourages regeneration of axons in the injured spinal cord. This inhibition is compounded by the poor regenerative ability of most CNS axons. This is probably a more achievable therapeutic target than axon regeneration, and an effective treatment would be of assistance to the majority of patients with partial cord injuries. Of course, understanding about astrogliosis and producing mediators and inhibitory molecules such as signaling pathways help us to develop new treatment strategies for SCI.
Neuroscience Letters, 1994
After excitotoxic lesion of the rat striatum, the time courses of local nerve growth factor (NGF) and NGF mRNA contents were investigated using a sensitive immunoassay (ELISA) and reverse transcription coupled to polymerase chain reaction (RT/PCR). To investigate a possible correlation of increased NGF expression and excitotoxin-induced reactive gliosis, striata were also analysed by immunohistochemistry with glial markers. We found elevated striatal NGF protein after lesion over the whole observation period. NGF mRNA showed a biphasic increase 10 h and 10 days after lesion, the latter coinciding with an increased astrogliosis. These results indicate that NGF accumulation after excitotoxin-induced neurodegeneration is partly due to local reactive astrocytes.
Astrocyte-derived GDNF is a potent inhibitor of microglial activation
Neurobiology of Disease, 2012
Professora Doutora Graça Baltazar, UBI Co-orientadora Dr. Ana Clara Cristóvão, UBI Título da Tese GDNF libertado pelos astrócitos é um potente inibidor da activação microglial Thesis Title Astrocyte-derived GDNF is a potent inhibitor of microglial activation 2010 i Acknowledgment À Prof. Doutora Graça Baltazar, orientadora deste trabalho, dirijo o meu agradecimento. Obrigada pelo apoio, empenho e espírito crítico, que se tornou fundamental na conclusão dos objectivos propostos. Obrigada também pela enorme disponibilidade manifestada no desenrolar deste trabalho. Um agradecimento especial à Ana Clara Cristóvão, pela sua exemplar orientação ao longo deste trabalho, o suporte entusiástico, disponibilidade constante nas discussões construtivas e pelo despertar em mim, curiosidade de querer saber mais. Os seus ensinamentos foram decisivos para compreender muitos conceitos. Um grande obrigado pela tua preciosa presença e ajuda nos momentos difíceis. À Filipa e à Rita, pela transmissão de conhecimento, disponibilidade e acompanhamento na realização deste trabalho. Aos meus colegas do mestrado, o meu muito obrigada por me proporcionarem um ano fabuloso. Para finalizar, gostaria de expressar um sentimento especial ao Cláudio e à minha família. Aos meus pais e irmãos, pela enorme dedicação, estímulo e apoio incondicional ao longo de toda a minha vida. Ao Cláudio pelo incentivo, conforto e motivação a todas as horas. A ti, obrigado pelo constante apoio. Abstract viii List of Abbreviations x Chapter I 2010 iii 3.3. ELISA technique 28 3.4. Transfection of siRNA in astrocytes 28 3.5. Western blot analysis 29 3.6. Total RNA extraction, cDNA synthesis and RT-PCR 29 3.7. Immunocytochemistry 31 3.8. Stimulation of microglia 31 3.9. Phagocytosis assay 31 3.10. Determination of cellular ROS levels 31 3.11. Data analysis and statistics 32 4. Results 32 4.1. Effect of ACM on microglial activation induced by Zymosan A 32 4.2. GDNF, CDNF and BDNF are expressed by astrocytes 33 4.3. Effect of neurotrophic factors in microglial activation 34 4.4. Conditioned media from astrocytes silenciated for GDNF was unable to prevent Zymosan A induced microglial activation 36 4.5. Protection from Zymosan A induced microglial activation by ACM: Effect of blocking GFRα1 receptor 39 5. Discussion 40 6. References 44 2010 iv Resumo A doença de Parkinson é caracterizada pela perda selectiva de neurónios dopaminérgicos na substantia nigra pars compacta. A origem desta doença não está completamente esclarecida, no entanto têm sido propostas diversas hipóteses em relação aos possíveis factores envolvidos na degeneraração dos neurónios nesta zona. Entre elas, encontra-se a neuroinflamação, que é cada vez mais reconhecida como o principal factor na patogénese da doença de Parkinson, e inúmeras evidências sugerem que as células microgliais são a v microglial induzida pelo Zymosan A, foram usados anticorpos de forma a bloquear a acção de alguns factores neurotróficos conhecidos pelas suas propriedades neuroprotectoras na substantia nigra. Assim, o meio condicionado pelos astrócitos foi tratado com anti-GDNF, anti-CDNF e anti-BDNF, separadamente, e adicionado às culturas de microglia posteriormente expostas a Zymosan A. O factor neurotrófico derivado de células da glia (GDNF) parece ser um mediador solúvel capaz de prevenir completamente a activação microglial induzida pelo Zymosan A, sendo que os restantes mediadores parecem não exercer qualquer efeito na prevenção da activação microglial. Para confirmar este facto, silenciou-se especificamente o GDNF em culturas de astrócitos, recolhendo-se posteriormente o meio condicionado por estas culturas a aplicando-os a culturas de microglia. Este meio condicionado pelos astrócitos silenciados para o GDNF, não foi capaz de prevenir a activação microglial induzida pelo Zymosan A. Por último, para esclarecer se este efeito era directo e se não haveria outras moléculas a auxiliar o efeito exercido pelo GDNF nas células microgliais, quantificaram-se os níveis de GDNF no meio condicionado pelos astrócitos, e com base nesta quantificação, três concentrações de GDNF, 100 pg/mL, 200 pg/mL e 400 pg/mL, diluído em meio de cultura, foram testadas para avaliar os seu efeitos de prevenção da actividade microglial. Observou-se que todas as concentrações de GDNF suprimem a activação microglial induzida pelo Zymosan A. No entanto, não se verificou um efeito dose -resposta como era de esperar. Os resultados obtidos neste trabalho demonstram que o GDNF é um factor neurotrófico derivado de astrócitos, com capacidade de modular as respostas inflamatórias microgliais. Este efeito do GDNF poderá contribuir para o desenvolvimento de uma possível terapia de prevenção contra a neuroinflamação, e indirectamente reduzir o desenvolvimento da doença de Parkinson. Palavras-chave: Microglia, Astrócitos, Neuroinflamação, Doença de Parkinson.
Cells
The maladaptive response of the central nervous system (CNS) following nerve injury is primarily linked to the activation of glial cells (reactive gliosis) that produce an inflammatory reaction and a wide cellular morpho-structural and functional/metabolic remodeling. Glial acidic fibrillary protein (GFAP), a major protein constituent of astrocyte intermediate filaments (IFs), is the hallmark of the reactive astrocytes, has pleiotropic functions and is significantly upregulated in the spinal cord after nerve injury. Here, we investigated the specific role of GFAP in glial reaction and maladaptive spinal cord plasticity following sciatic nerve spared nerve injury (SNI) in GFAP KO and wild-type (WT) animals. We evaluated the neuropathic behavior (thermal hyperalgesia, allodynia) and the expression of glial (vimentin, Iba1) and glutamate/GABA system markers (GLAST, GLT1, EAAC1, vGLUT, vGAT, GAD) in lumbar spinal cord sections of KO/WT animals. SNI induced neuropathic behavior in both G...