ABSTRACT
We previously reported that the bone morphogenetic protein (BMP) antagonist, noggin, improved the repair process with an increase in the reactive microglia/macrophage population in the ischemic brain. Since BMP plays a role in intracellular iron homeostasis via the hepcidin/ferroportin axis, and iron is required for myelination, this study was aimed to determine whether noggin affected iron status and remyelination in the brain following ischemic stroke. We further examined the effect of blocking the BMP/hepcidin pathway on reactive microglia (BV2) and myelination of oligodendroglial cells (MO3.13) to define the link between microglial iron status and myelin formation. Following the noggin infusion into the ischemic brain of mice, the induction of hepcidin and ferritin protein levels decreased, and the number of myelinated axons and myelin thickness increased at 8 weeks after ischemic stroke. Noggin repressed the increase in hepcidin and ferritin levels in BV2 exposed to lipopolysaccharide (LPS) and oxygen/glucose deprivation and reperfusion (OGD/R). When MO3.13 were exposed to the conditioned media from noggin-treated BV2 (noggin CM) during reperfusion, OGD/R-induced MO3.13â¯cell death was reduced. Under normal conditions, noggin CM induced myelin production with an increase in ferritin levels in MO3.13, which was reversed by the iron chelator, deferoxamine. These results indicated that noggin altered the iron status in reactive microglia from the iron-storing to the iron-releasing phenotype, which contributed to myelin synthesis by providing iron. We suggest that the BMP/hepcidin pathway can be a target for the regulation of the iron status in microglia to enhance remyelination in the ischemic brain.
Subject(s)
Brain Ischemia/pathology , Carrier Proteins/therapeutic use , Iron/metabolism , Microglia/drug effects , Myelin Proteins/metabolism , Myelin Sheath/drug effects , Animals , Body Weight/drug effects , Brain Ischemia/drug therapy , Cell Line, Transformed , Disease Models, Animal , Drug Delivery Systems , Functional Laterality/drug effects , Glucose/deficiency , Humans , Hypoxia/drug therapy , L-Lactate Dehydrogenase/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Muscle Strength/drug effects , Myelin Sheath/pathology , Myelin Sheath/ultrastructure , Recovery of Function/drug effectsABSTRACT
Less disruption of the blood-brain barrier (BBB) after severe ischemic stroke is one of the beneficial outcomes of ischemic preconditioning (IP). However, the effect of IP on tight junctions (TJs), which regulate paracellular permeability of the BBB, is not well understood. In the present study, we examined IP-induced changes in TJs before and after middle cerebral artery occlusion (MCAO) in mice, and the association between changes in TJs and tolerance to a subsequent insult. After IP, we found decreased levels of transmembrane TJ proteins occludin and claudin-5, and widened gaps of TJs with perivascular swelling at the ultrastructural level in the brain. An inflammatory response was also observed. These changes were reversed by inhibition of extracellular signal-regulated kinase1/2 (ERK1/2) via the specific ERK1/2 inhibitor U0126. After MCAO, reduced brain edema and inflammatory responses were associated with altered levels of angiogenic factors and cytokines in preconditioned brains. Pretreatment with U0126 reversed the neuroprotective effects of IP against MCAO. These findings suggest that ERK1/2 activation has a pivotal role in IP-induced changes in TJs and inflammatory response, which serve to protect against BBB breakdown and inflammation after ischemic stroke.
Subject(s)
Infarction, Middle Cerebral Artery/pathology , Ischemic Preconditioning, Myocardial , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Tight Junctions/enzymology , Animals , Brain Edema/etiology , Brain Infarction/etiology , Butadienes/pharmacology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/drug therapy , Male , Mice , Mice, Inbred C57BL , Muscle Strength/drug effects , Neurologic Examination , Nitriles/pharmacology , Tight Junctions/drug effects , Tight Junctions/ultrastructure , Time Factors , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Toll-like receptors (TLRs), which have been implicated in various neuroinflammatory responses, are thought to act in defense mechanisms by inhibiting neuronal cell death in Alzheimer's disease. In this study, we evaluated the effects of TLR2 on amyloid beta peptide 25-35 (Aß25-35)-induced neuronal cell death, synaptic dysfunction, and microglial activation in organotypic hippocampal slice cultures (OHSCs) from wild-type (WT) C57BL/6 mice and TLR2-knockout (KO) mice. In WT mice, Aß25-35 induced ß-amyloid aggregation and surrounding TLR2 expression. And, propidium iodide (PI) uptake, which is a measure of cell death, increased in a dose-dependent manner in slices with Aß25-35 treatment. In the Aß25-35-treated TLR2-KO OHSCs, the PI uptake was significantly attenuated to the control level, indicating that the cells were less susceptible to Aß25-35-induced neuronal toxicity. In the ultrastructural analysis, nuclear shrinkage, slightly swollen mitochondria, and degraded organelles were detected in the Aß25-35-treated slices from WT mice but not in the Aß25-35-treated slices from TLR2-KO, suggesting the resistance of TLR2-KO to Aß25-35-induced neurotoxicity. In Aß25-35-treated OHSCs of WT mice, the levels of phosphorylated tau were increased and the levels of synaptophysin were decreased in a dose-dependent manner, but they were not changed in OHSCs of TLR2-KO mice. In WT mice, Aß25-35 increased total protein level and immunoreactivity of Iba-1, which was colocalized with TLR2. However, there were no significant changes in the slices of Aß25-35-treated TLR2-KO mice. These results suggested that TLR2 may play a role in Aß25-35-induced neuronal cell loss and synaptic dysfunction through the activation of microglia in OHSCs.
Subject(s)
Amyloid beta-Peptides/toxicity , Hippocampus/metabolism , Peptide Fragments/toxicity , Toll-Like Receptor 2/physiology , Animals , Blotting, Western , Fluorescent Antibody Technique , Hippocampus/pathology , Hippocampus/ultrastructure , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Phosphorylation , Synaptophysin/metabolism , Toll-Like Receptor 2/genetics , tau Proteins/metabolismABSTRACT
Memory loss in Alzheimer's disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide A beta 25-35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, A beta 25-35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in A beta 25-35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed A beta 25-35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by A beta 25-35 in CA1, CA3 and DG area at concentrations >2.5 microM. In conclusion, A beta 25-35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 microM in organotypic hippocampal slice cultures. These suggest that A beta 25-35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.
Subject(s)
Amyloid beta-Peptides/pharmacology , Hippocampus/cytology , Hippocampus/drug effects , Neurons/cytology , Peptide Fragments/pharmacology , Presynaptic Terminals/drug effects , Analysis of Variance , Animals , Animals, Newborn , Annexin A5/metabolism , Caspase 3/metabolism , Cell Death/drug effects , Dose-Response Relationship, Drug , Microscopy, Electron, Transmission/methods , Neurons/drug effects , Organ Culture Techniques , Presynaptic Terminals/ultrastructure , Rats , Rats, Sprague-Dawley , Synaptophysin/metabolism , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Brain ischemia activates Ca(2+)-dependent synaptic vesicle exocytosis. The synaptosomal-associated protein 25 (SNAP-25) and syntaxin proteins, located on presynaptic terminals, are components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and play a key role in regulating exocytosis. Changes in the expression of SNAREs could affect SNARE complex formation, fusion of vesicles with the presynaptic membrane, and release of neurotransmitters through exocytosis. To investigate the relationship of glucose/oxygen deprivation (GOD)/reperfusion-induced neuronal damage and alteration of presynaptic function, we examined the expression of SNAREs and complexin during GOD and reperfusion using organotypic hippocampal slice cultures. Microtubule-associated protein 2 (MAP-2) staining and transmission electron microscopy showed that neuronal damage increased in a time-dependent manner and both types of neuronal death can occur at different times during GOD and reperfusion. The immunoreactivity of SNAREs such as SNAP-25, vesicle-associated membrane protein and syntaxin and complexin increased in pyramidal cell bodies in the CA1 and CA3 areas in a time-dependent manner following reperfusion. Our data suggest that alteration of presynaptic function may play a partial role in delayed neuronal death during GOD and reperfusion in organotypic hippocampal slice cultures.
