ABSTRACT
The human apolipoprotein E4 (ApoE4) isoform is associated with genetic risk for Alzheimer's disease. To assess the effects of different ApoE isoforms on amyloid plaque formation, human ApoE3 and ApoE4 were expressed in the brains of transgenic mice under the control of the human transferrin promoter. Mice were crossed with transgenic mice expressing human amyloid precursor protein containing the Swedish mutation (APPsw), which facilitates amyloid beta peptide (A beta) production. The following progeny were selected for characterization: APPsw+/- x ApoE3+/- and APPsw+/-, APPsw+/- x ApoE4+/- and APPsw+/- littermates. All mice analyzed were wild type for the endogenous mouse APP and ApoE genes. Mice expressing ApoE4 in combination with APPsw have accelerated A beta deposition in the brain as assessed by enzyme immunoassay for A beta40 and A beta42 extractable in 70% formic acid, by assessment of amyloid plaque formation using thioflavin-S staining, and by immunohistochemical staining with antibodies specific for A beta40 or A beta42 and the 4G8 monoclonal or 162 polyclonal antibody. No difference in the rate of A beta deposition in the brain was seen in mice expressing ApoE3 in combination with APPsw. Thus, our data are consistent with the observation in Alzheimer's disease that ApoE4 is associated with increased accumulation of A beta in the brain relative to ApoE3.
Subject(s)
Amyloid beta-Peptides/metabolism , Apolipoproteins E/genetics , Brain Chemistry/genetics , Peptide Fragments/metabolism , Age Factors , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/immunology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Antibodies, Monoclonal , Apolipoprotein E4 , Brain/metabolism , Brain/pathology , Gene Expression , Humans , Immunoenzyme Techniques , Low Density Lipoprotein Receptor-Related Protein-1 , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Peptide Fragments/analysis , Peptide Fragments/immunology , Plaque, Amyloid/chemistry , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Receptors, Immunologic/metabolismABSTRACT
Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Cytokines/genetics , Encephalitis/metabolism , Plaque, Amyloid/metabolism , RNA, Messenger/metabolism , Aging/genetics , Aging/immunology , Aging/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/immunology , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/immunology , Animals , Cerebral Cortex/drug effects , Cerebral Cortex/immunology , Cerebral Cortex/metabolism , Chemokine CCL2/genetics , Disease Models, Animal , Encephalitis/genetics , Encephalitis/immunology , Female , Hippocampus/drug effects , Hippocampus/immunology , Hippocampus/metabolism , Interleukin-1/genetics , Male , Mice , Plaque, Amyloid/genetics , Plaque, Amyloid/immunology , RNA, Messenger/immunology , Tumor Necrosis Factor-alpha/genetics , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , GABA Antagonists/pharmacology , Picrotoxin/pharmacology , Receptors, GABA-A/drug effects , gamma-Aminobutyric Acid/pharmacology , Animals , Binding, Competitive , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Cell Line , Chlorides/metabolism , Cloning, Molecular , Dose-Response Relationship, Drug , Drug Interactions , GABA Antagonists/metabolism , Humans , Ion Channels/drug effects , Ion Channels/metabolism , Kidney/cytology , Kidney/drug effects , Kidney/embryology , Kinetics , Patch-Clamp Techniques , Picrotoxin/metabolism , Rats , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.
Subject(s)
Carbolines/pharmacology , Convulsants/pharmacology , GABA-A Receptor Agonists , Barbiturates/pharmacology , Cell Line , Chloride Channels/drug effects , Flumazenil/pharmacology , GABA Antagonists/pharmacology , Humans , Kidney/metabolism , Patch-Clamp Techniques , Plasmids , Receptors, GABA-A/genetics , Steroids/pharmacologyABSTRACT
Two forms of cDNA coding for the human GABAA beta 2 subunit have been cloned and sequenced. The two sequences differ by a 114 base pair insertion. The insert contains a phosphorylation consensus sequence for calmodulin-dependent protein kinase II. Quantitative PCR studies show that h beta 2L cDNA represents 10-15% of total h beta 2 cDNA in the 10 brain substructures tested. Analysis of human genomic southern blots suggests that the two forms might arise by differential splicing.
