ABSTRACT
The loss of synapses is a strong histological correlate of the cognitive decline in Alzheimer's disease (AD). Amyloid ß-peptide (Aß), a cleavage product of the amyloid precursor protein (APP), exerts detrimental effects on synapses, a process thought to be causally related to the cognitive deficits in AD. Here, we used in vivo two-photon microscopy to characterize the dynamics of axonal boutons and dendritic spines in APP/Presenilin 1 (APP(swe)/PS1(L166P))-green fluorescent protein (GFP) transgenic mice. Time-lapse imaging over 4 weeks revealed a pronounced, concerted instability of pre- and postsynaptic structures within the vicinity of amyloid plaques. Treatment with a novel sulfonamide-type γ-secretase inhibitor (GSI) attenuated the formation and growth of new plaques and, most importantly, led to a normalization of the enhanced dynamics of synaptic structures close to plaques. GSI treatment did neither affect spines and boutons distant from plaques in amyloid precursor protein/presenilin 1-GFP (APPPS1-GFP) nor those in GFP-control mice, suggesting no obvious neuropathological side effects of the drug.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Dendritic Spines/pathology , Plaque, Amyloid/drug therapy , Presynaptic Terminals/pathology , Quinolines/pharmacology , Sulfonamides/pharmacology , Amyloid beta-Protein Precursor/genetics , Animals , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Plaque, Amyloid/pathology , Presenilin-1/genetics , Quinolines/therapeutic use , Sulfonamides/therapeutic useABSTRACT
Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Dipeptides/administration & dosage , Endopeptidases/metabolism , Administration, Oral , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases , Brain/cytology , Brain/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Endopeptidases/drug effects , Enzyme Inhibitors/administration & dosage , Female , Humans , Injections, Subcutaneous , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Male , Mice , Mice, Transgenic , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Peptide Fragments/metabolismABSTRACT
alpha 4 beta 1 integrin (VLA-4) appears to be unique among the leukocyte integrins in that it can initiate the adhesion of circulating lymphocytes without cellular activation. It is not known how lymphocytes or other cell types maintain constitutive levels of alpha 4 beta 1 integrin activity. The current report describes a monoclonal antibody, 15/7, that recognizes a high affinity or ligand-occupied conformation of beta 1 integrin. Studies with 15/7 revealed that alpha 4 beta 1 integrin-dependent adhesion of leukocytic cell lines is mediated by a population of low affinity receptors that is conformationally responsive to ligand; the 15/7 epitope could be induced by nanomolar concentrations of soluble VCAM-1 or by micromolar concentrations of a peptide derived from the type III connecting segment domain of fibronectin (as ligands for alpha 4 beta 1 integrin). The same receptors were also responsive to adhesion activating reagents, such as Mn2+, activating anti-beta 1 integrin antibodies, and phorbol myristate acetate, which induced the 15/7 epitope directly and/or decreased the concentration of ligand required for epitope induction. In addition to the responsive receptor pool, cells expressed a second population of alpha 4 beta 1 integrin that was conformationally restrained, failing to respond to ligand or to any of the activating reagents. The relative size of the responsive and inactive receptor pools, as well as the affinity of the responsive receptors, represented a stable phenotype of different cell types and played important roles in defining the cells' adhesive capacity and ligand specificity. Similar receptor populations were measured on lymphocyte subsets in whole blood. These studies provide insight into how cells maintain different constitutive levels of alpha 4 beta 1 integrin activity, and how the activity of beta 1 integrin can be modulated by activators of cell adhesion.
Subject(s)
Integrins/metabolism , Monocytes/cytology , Receptors, Lymphocyte Homing/metabolism , Receptors, Very Late Antigen/metabolism , T-Lymphocytes/cytology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Cell Adhesion/immunology , Cell Line , Epitopes/immunology , Humans , Integrin alpha4beta1 , Integrins/immunology , Intercellular Adhesion Molecule-1/metabolism , L Cells/immunology , Ligands , Manganese/pharmacology , Mice , Molecular Sequence Data , Monocytes/immunology , Receptors, Lymphocyte Homing/immunology , Receptors, Very Late Antigen/chemistry , Receptors, Very Late Antigen/drug effects , T-Lymphocytes/immunology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.
