RESUMO
Macrophage colony stimulating factor (M-CSF) and its receptor are upregulated in the brain in Alzheimer's disease. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. Amyloid beta (Abeta) immunization experiments suggest that microglia have the capacity to aggressively clear Abeta from the brain under certain circumstances. We examined the role of M-CSF in phagocytosis of fluorescent microspheres and Abeta by cultured microglia. M-CSF treatment increased microglial cell phagocytosis of both microspheres and of Abeta. Antibody neutralization of M-CSF inhibited Abeta uptake induced by overexpression of the M-CSF receptor on microglia. These results suggest that M-CSF could be important in promoting microglial clearance of abnormal protein aggregates such as Abeta.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Microglia/citologia , Fagocitose , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Citometria de Fluxo , Corantes Fluorescentes/química , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Microglia/metabolismo , Microesferas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Macrophage colony stimulating factor (M-CSF) and its receptor are up-regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M-CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase-3 activation in organotypic hippocampal cultures, whereas treatment with M-CSF protected hippocampal neurons from NMDA-induced apoptosis. Caspase-3 activation was inhibited by M-CSF treatment to the same degree as with the caspase inhibitor Z-VAD-FMK. These results suggest that M-CSF has neuroprotective properties through inhibition of caspase-3 that could promote neuronal survival after excitotoxic insult. The role of M-CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.
Assuntos
Hipocampo/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , N-Metilaspartato/antagonistas & inibidores , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cultura , Inibidores de Cisteína Proteinase/farmacologia , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Ativação Enzimática/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/enzimologia , Marcação In Situ das Extremidades Cortadas , Neurônios/citologia , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease.
Assuntos
Dissecação/métodos , Lasers , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Laranja de Acridina , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Desoxirribonucleases/metabolismo , Expressão Gênica , Genótipo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteínas de Neurofilamentos/genética , RNA Mensageiro/genética , Análise de Regressão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Coloração e Rotulagem/métodosRESUMO
Macrophage colony stimulating factor (M-CSF) is a microglial activator expressed at increased levels in the brain in Alzheimer's disease. In monotypic microglial cultures, M-CSF strongly augments amyloid beta (Abeta) induced microglial production of proinflammatory cytokines and nitric oxide. However, this augmentation could be due to strong autocrine and paracrine effects in monotypic cultures. We used hippocampal organotypic cultures to test M-CSF/Abeta augmentation in a system modeling intact brain. Combined M-CSF/Abeta treatment increased interleukin-1 (IL-1) and macrophage inflammatory protein 1-alpha expression by microglia, whereas inducible nitric oxide synthase (iNOS) expression was localized primarily to astroglia. Induction of cytokines and iNOS was also observed after lipopolysaccharide treatment of organotypic hippocampal cultures, but iNOS expression was localized mainly to microglia rather than astrocytes. Treatment with M-CSF/Abeta did not result in neuronal death. These results demonstrate that combined M-CSF/Abeta treatment results in a strong inflammatory response in the organotypic environment without inducing neurotoxicity.
