RESUMO
BACKGROUND: The accumulation of soluble oligomeric amyloid-ß peptide (oAß) proceeding the formation of senile plaques contributes to synaptic and memory deficits in Alzheimer's disease. Our previous studies have indentified scavenger receptor A (SR-A), especially SR-A type I (SR-AI), as prominent scavenger receptors on mediating oAß clearance by microglia while glycan moiety and scavenger receptor cysteine-rich (SRCR) domain may play the critical role. Macrophage receptor with collagenous structure (MARCO), another member of class A superfamily with a highly conserved SRCR domain, may also play the similar role on oAß internalization. However, the role of N-glycosylation and SRCR domain of SR-AI and MARCO on oAß internalization remains unclear. RESULT: We found that oAß internalization was diminished in the cells expressing SR-AI harboring mutations of dual N-glycosylation sites (i.e. N120Q-N143Q and N143Q-N184Q) while they were normally surface targeted. Normal oAß internalization was observed in 10 SR-AI-SRCR and 4 MARCO-SRCR surface targeted mutants. Alternatively, the SRCR mutants at ß-sheet and α-helix and on disulfide bone formation obstructed receptor's N-glycosylation and surface targeting. CONCLUSION: Our study reveals that N-glycan moiety is more critical than SRCR domain for SR-A-mediated oAß internalization.
Assuntos
Proteínas de Transporte/metabolismo , Receptores Imunológicos/metabolismo , Substituição de Aminoácidos , Peptídeos beta-Amiloides , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Glicosilação , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Transporte Proteico/genética , Receptores Imunológicos/genética , Fatores de Processamento de Serina-ArgininaRESUMO
BACKGROUND: The roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection. RESULT: Neurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred. CONCLUSIONS: Our results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.
Assuntos
Caspase 3/genética , Epilepsia/fisiopatologia , Hipocampo/efeitos dos fármacos , Ácido Caínico/toxicidade , Neurogênese/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Caspase 3/metabolismo , Epilepsia/induzido quimicamente , Hipocampo/metabolismo , Imuno-Histoquímica , Infusões Intraventriculares , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Plasticidade Neuronal/efeitos dos fármacosRESUMO
BACKGROUND: Insufficient clearance of soluble oligomeric amyloid-ß peptide (oAß) in the central nervous system leads to the synaptic and memory deficits in Alzheimer's disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-ß peptide (oAß) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAß and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities. RESULT: Genetic ablation of SR-A attenuated the internalization of oAß and AcLDL by microglia. Half of oAß-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAß internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAß and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention. CONCLUSION: The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer's disease and atherosclerosis.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Estrutura Terciária de Proteína , Receptores Depuradores Classe A/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Células COS , Chlorocebus aethiops , Cisteína/genética , Citoplasma/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Dobramento de Proteína , Proteólise , Receptores Depuradores Classe A/química , Receptores Depuradores Classe A/genética , Propriedades de SuperfícieRESUMO
BACKGROUND: The specific role of microglia on Aß-mediated neurotoxicity is difficult to assign in vivo due to their complicated environment in the brain. Therefore, most of the current microglia-related studies employed the isolated microglia. However, the previous in vitro studies have suggested either beneficial or destructive function in microglia. Therefore, to investigate the phenotypes of the isolated microglia which exert activity of neuroprotective or destructive is required. RESULTS: The present study investigates the phenotypes of isolated microglia on protecting neuron against Aß-mediated neurotoxicity. Primary microglia were isolated from the mixed glia culture, and were further cultured to distinct phenotypes, designated as proliferating amoeboid microglia (PAM) and differentiated process-bearing microglia (DPM). Their inflammatory phenotypes, response to amyloid ß (Aß), and the beneficial or destructive effects on neurons were investigated. DPM may induce both direct neurotoxicity without exogenous stimulation and indirect neurotoxicity after Aß activation. On the other hand, PAM attenuates Aß-mediated neurotoxicity through Aß phagocytosis and/or Aß degradation. CONCLUSIONS: Our results suggest that the proliferating microglia, but not the differentiated microglia, protect neurons against Aß-mediated neurotoxicity. This discovery may be helpful on the therapeutic investigation of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Córtex Cerebral/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Camundongos , Microglia/citologia , Neurônios/citologia , Fenótipo , Ratos , Ratos Sprague-DawleyRESUMO
The accumulation of soluble oligomeric amyloid-ß peptide (oAß) proceeds the formation of senile plaques and contributes to synaptic and memory deficits in Alzheimer's disease (AD). The mechanism of mediating microglial oAß clearance remains unclear and thought to occur via scavenger receptors (SRs) in microglia. SRs respond to their ligands in a subtype-specific manner. Therefore, we sought to identify the specific subtypes of SRs that mediate oAß internalization and proteases that degrade oAß species in naïve primary microglia. The component of oAß species were characterized by western blot analysis, analytical ultracentrifugation analysis, and atomic force microscopy. The oAß species remained soluble in the medium and microglial lysates during incubation at 37 °C. SR-A, but not CD36, mediated oAß internalization in microglia as suggested by the use of subtype-specific neutralizing antibodies and small interfering RNAs (siRNAs). Immunoprecipitation analysis showed that oAß interacted with SR-A on the plasma membrane. After internalization, over 40% of oAß vesicles were trafficked toward lysosomes and degraded by cysteine proteases, including cathepsin B. The inhibitors of proteasome, neprilysin, matrix metalloproteinases, and insulin degrading enzyme failed to protect internalized oAß from degradation. Our study suggests that SR-A and lysosomal cathepsin B are critical in microglial oAß clearance, providing insight into how microglia are involved in the clearance of oAß and their roles in the early stages of AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Receptores Depuradores/metabolismo , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Imuno-Histoquímica , Insulisina/metabolismo , Lisossomos/metabolismo , Camundongos , Neprilisina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , RNA Interferente PequenoRESUMO
BACKGROUND: Overactivated microglia that cluster at neuritic plaques constantly release neurotoxins, which actively contribute to progressive neurodegeneration in Alzheimer's disease (AD). Therefore, attenuating microglial clustering can reduce focal neuroinflammation at neuritic plaques. Previously, we identified CCL5 and CCL2 as prominent chemokines that mediate the chemotaxis of microglia toward beta-amyloid (Abeta)aggregates. Although transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate the expression of chemokines in activated microglia, whether TGF-beta1 can reduce the chemotaxis of microglia toward neuritic plaques in AD remains unclear. METHODS: In the present study, we investigated the effects of TGF-beta1 on Abeta-induced chemotactic migration of BV-2 microglia using time-lapse recording, transwell assay, real-time PCR, ELISA, and western blotting. RESULTS: The cell tracing results suggest that the morphological characteristics and migratory patterns of BV-2 microglia resemble those of microglia in slice cultures. Using this model system, we discovered that TGF-beta1 reduces Abeta-induced BV-2 microglial clustering in a dose-dependent manner. Chemotactic migration of these microglial cells toward Abeta aggregates was significantly attenuated by TGF-beta1. However, these microglia remained actively moving without any reduction in migration speed. Pharmacological blockade of TGF-beta1 receptor I (ALK5) by SB431542 treatment reduced the inhibitory effects of TGF-beta1 on Abeta-induced BV-2 microglial clustering, while preventing TGF-beta1-mediated cellular events, including SMAD2 phosphorylation and CCL5 down-regulation. CONCLUSIONS: Our results suggest that TGF-beta1 reduces Abeta-induced microglial chemotaxis via the SMAD2 pathway. The down-regulation of CCL5 by TGF-beta1 at least partially contributes to the clustering of microglia at Abeta aggregates. The attenuating effects of SB431542 upon TGF-beta1-suppressed microglial clustering may be mediated by restoration of CCL5 to normal levels. TGF-beta1 may ameliorate microglia-mediated neuroinflammation in AD by preventing activated microglial clustering at neuritic plaques.
Assuntos
Peptídeos beta-Amiloides/imunologia , Quimiocina CCL5/fisiologia , Quimiotaxia/efeitos dos fármacos , Microglia/efeitos dos fármacos , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Benzamidas/farmacologia , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiocina CCL5/genética , Dioxóis/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/fisiologia , Ensaio de Imunoadsorção Enzimática , Fosforilação , Ratos , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Smad/genéticaRESUMO
The number of microglia surrounding senile plaques is correlated with the size of plaques in Alzheimer's disease (AD). It is unclear whether more microglia are passively recruited toward larger senile plaques or, conversely, microglia recruited to senile plaques directly contribute to the growth of plaques. In this study, BV-2 microglia were used to delineate the role of microglia in the growth of plaques using time-lapse recording. Aggregated beta amyloid peptide (Abeta)-induced BV-2 microglia to form clusters. The recruitment of BV-2 microglia bearing membrane-adhered Abeta enlarged preexisting Abeta aggregates. The receptors involved in the microglial uptake of Abeta, including integrin, formyl peptide like receptor 1, and scavenger receptors, also mediated the microglial clustering. Neutralization antibodies against chemokines significantly attenuated Abeta-induced microglial clustering and the enlargement of Abeta aggregates. Our results reveal a novel role of microglia in directly increasing the size of Abeta aggregates and suggest the targeting of Abeta-mediated microglial chemotactic migration in developing therapeutic interventions for AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Quimiocinas/metabolismo , Microglia/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Peptídeos beta-Amiloides/ultraestrutura , Análise de Variância , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Quimiocinas/imunologia , Relação Dose-Resposta a Droga , Integrinas/antagonistas & inibidores , Camundongos , Microglia/efeitos dos fármacos , Microscopia de Força Atômica/métodos , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Placa Amiloide/metabolismo , Polissacarídeos/farmacologia , Conformação Proteica , Receptores Depuradores/antagonistas & inibidores , Fatores de TempoRESUMO
Diabesity-associated metabolic stresses modulate the development of Alzheimer's disease (AD). For further insights into the underlying mechanisms, we examine whether the genetic background of APPswe/PS1dE9 at the prodromal stage of AD affects peripheral metabolism in the context of diabesity. We characterized APPswe/PS1dE9 transgenic mice treated with a combination of high-fat diet with streptozotocin (HFSTZ) in the early stage of AD. HFSTZ-treated APPswe/PS1dE9 transgenic mice exhibited worse metabolic stresses related to diabesity, while serum ß-amyloid levels were elevated and hepatic steatosis became apparent. Importantly, two-way analysis of variance shows a significant interaction between HFSTZ and genetic background of AD, indicating that APPswe/PS1dE9 transgenic mice are more vulnerable to HFSTZ treatment. In addition, body weight gain, high hepatic triglyceride, and hyperglycemia were positively associated with serum ß-amyloid, as validated by Pearson's correlation analysis. Our data suggests that the interplay between genetic background of AD and HFSTZ-induced metabolic stresses contributes to the development of obesity and hepatic steatosis. Alleviating metabolic stresses including dysglycemia, obesity, and hepatic steatosis could be critical to prevent peripheral ß-amyloid accumulation at the early stage of AD.
Assuntos
Peptídeos beta-Amiloides/sangue , Diabetes Mellitus Experimental/sangue , Fígado Gorduroso/sangue , Obesidade/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análise de Variância , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/sangue , Humanos , Leptina/sangue , Lipídeos/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/sangue , Presenilina-1/genética , Presenilina-1/metabolismo , Estresse Fisiológico/genética , Triglicerídeos/metabolismo , Aumento de PesoRESUMO
Although multiple subtypes of sodium channels are expressed in most neurons, the specific contributions of the individual sodium channels remain to be studied. The role of zebrafish Na(v)1.6 sodium channels in the embryonic locomotor movements has been investigated by the antisense morpholino (MO) knockdown. MO1 and MO2 are targeted at the regions surrounding the translation start site of zebrafish Na(v)1.6 mRNA. MO3 is targeted at the RNA splicing donor site of exon 2. The correctly spliced Na(v)1.6 mRNA of MO3 morphants is 6% relative to that of the wild-type embryos. Na(v)1.6-targeted MO1, MO2 and MO3 attenuate the spontaneous contraction, tactile sensitivity, and swimming in comparison with a scrambled morpholino and mutated MO3 morpholino. No significant defect is observed in the development of slow muscles, the axonal projection of primary motoneurons, and neuromuscular junctions. The movement impairments caused by MO1, MO2, and MO3 suggest that the function of Na(v)1.6 sodium channels is essential on the normal early embryonic locomotor activities.
Assuntos
Canais de Sódio/fisiologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Sequência de Bases , Marcação de Genes , Hibridização In Situ , Locomoção/genética , Locomoção/fisiologia , Neurônios Motores/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.6 , Oligodesoxirribonucleotídeos Antissenso/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Colinérgicos/metabolismo , Canais de Sódio/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Zebrafish scn8aa sodium channels mediate the majority of sodium conductance, which is essential for the embryonic locomotor activities. Here, we investigated the transcriptional regulation of scn8aa in developing zebrafish embryos by constructing a GFP reporter driven by a 15-kb fragment of scn8aa gene designed as scn8aa:GFP. GFP expression patterns of scn8aa:GFP temporally and spatially recapitulated the expression of endogenous scn8aa mRNA during zebrafish embryonic development, with one exception in the inner nuclear layer of the retina. Three novel elements, along with an evolutionarily conserved element shared with mouse SCN8A, modulated neuronal-specific expression of scn8aa. The deletion of each positive element reduced the expression levels in neurons without inducing ectopic GFP expression in non-neuronal cells. Our results demonstrate that these four regulatory elements function cooperatively to enhance scn8aa expression in the zebrafish nervous system.
Assuntos
Embrião não Mamífero/metabolismo , Neurônios/metabolismo , Canais de Sódio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Biologia Computacional , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.6 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Canais de Sódio/genética , Canais de Sódio/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.