ABSTRACT
The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.
Subject(s)
Blood-Brain Barrier/immunology , Cell Movement/immunology , Encephalitis, Viral/immunology , G-Protein-Coupled Receptor Kinase 1/immunology , HIV Infections/immunology , HIV-1/immunology , Monocytes/immunology , Tight Junctions/immunology , Blood-Brain Barrier/virology , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , Claudin-5 , Down-Regulation/drug effects , Down-Regulation/genetics , Down-Regulation/immunology , Encephalitis, Viral/genetics , Encephalitis, Viral/virology , Enzyme Activation/drug effects , Enzyme Activation/genetics , Enzyme Activation/immunology , G-Protein-Coupled Receptor Kinase 1/antagonists & inhibitors , G-Protein-Coupled Receptor Kinase 1/genetics , HIV Infections/genetics , HIV Infections/virology , Humans , Membrane Proteins/immunology , Monocytes/virology , Mutation/immunology , Occludin , Protein Kinase Inhibitors/pharmacology , Protein Transport/genetics , Protein Transport/immunology , Tight Junctions/genetics , TransfectionABSTRACT
Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between beta-amyloid precursor protein (APP)-expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co-cultured with neuronal cells expressing wild type APP or familial AD-linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro-inflammatory cytokines and were reversed by depletion of amyloid beta-peptide (Abeta) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine-glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Abeta from conditioned media prepared from APP-expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor-mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP-processing products can induce oxidative neurotoxicity through microglial activation.
Subject(s)
Amino Acid Transport System y+ , Amyloid beta-Peptides/toxicity , Amyloid beta-Protein Precursor/metabolism , Monocytes/drug effects , Neurons/drug effects , Phagocytes/drug effects , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/toxicity , Animals , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Cells, Cultured , Coculture Techniques , Free Radical Scavengers/pharmacology , Glutamic Acid/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/drug effects , Macrophages/physiology , Microglia/cytology , Microglia/drug effects , Microglia/physiology , Monocytes/cytology , Monocytes/physiology , Mutation , Neurons/cytology , Neurons/metabolism , Oocytes/metabolism , PC12 Cells , Patch-Clamp Techniques , Phagocytes/cytology , Phagocytes/physiology , Protein Processing, Post-Translational , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Transfection , Xenopus laevisABSTRACT
Alzheimer's disease (AD) remains one of the most challenging brain disorders facing modern medicine. Neuronal loss underlies the pathogenesis of AD and can occur, in part, by oxidative stress, by beta-amyloid peptide (Abeta), and by excitotoxins. The complement cascade, especially C1q, may affect reactive oxygen species (ROS) and mediate neuronal injury during AD. We demonstrate that incubation of neurons with purified C1q results in increased ROS, which can be partially blocked by low concentrations of Abeta. C1q-binding sites on neurons were demonstrated by 125I-C1q-binding and immunofluorescence tests performed on primary neurons. The blocking of neuronal calreticulin by its antibody abrogated ROS by C1q. We suggest that the C1q may be an important factor contributing to neuronal oxidative stress and neuronal demise during AD.
Subject(s)
Alzheimer Disease/etiology , Calreticulin/physiology , Complement C1q/toxicity , Neurons/drug effects , Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Calreticulin/analysis , Cells, Cultured , Complement C1q/metabolism , Female , Fluorescent Antibody Technique , Oxidative Stress , Pregnancy , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Superoxides/metabolismABSTRACT
Insertion of genetic material into bovine chromaffin cells employing various techniques have produced low to moderate transduction rates. Recent technology using adenoviral gene transfer has become one of the most powerful methods for introducing genes into mammalian cells. We examined whether a recombinant adenovirus could provide a convenient vector to transfer genes of interest for mechanistic studies on chromaffin cells. Our results show that 100% transduction of chromaffin cells was accomplished within 18 h with a recombinant adenovirus as revealed by the expression of green fluorescent protein (GFP) in chromaffin cells. Transduction was dependent on time and viral titer but independent of cell age and density in culture. There was no effect of the recombinant adenovirus on the secretory function [3H]-norepinephrine ([3H]-NE release) of the chromaffin cells. The results demonstrate that the recombinant adenovirus provides an effective process for the complete transfection of bovine chromaffin cells with a selected gene.