ABSTRACT
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
Neurodegenerative processes in Alzheimer's disease (AD) are thought to be driven, in part, by the deposition of amyloid beta (A beta), a 39-43-aminoacid peptide product resulting from an alternative cleavage of amyloid precursor protein (APP). In addition to its neurotoxic properties, A beta may influence neuropathology by stimulating glial cell cytokine and acute phase protein secretion in affected areas of the brain (e.g., cortex, hippocampus). Using an in vitro human astrocyte model (U-373 MG astrocytoma cells), the effects of A beta treatment on acute phase protein (APP and alpha-1-antichymotrypsin [alpha 1-ACT]) and interleukin-8 (IL-8) were examined. U-373 MG cells secreted increased levels of alpha 1-ACT and neurotrophic/neuroprotective alpha-cleaved APP (alpha APP) after exposure to interleukin-1 beta (IL-1 beta) for 24 hours. A beta treatment resulted in a similar, but modest increase in alpha 1-ACT secretion, a two- to threefold stimulation of IL-8 production, and, conversely, a profound reduction in the levels of secreted alpha APPs. A beta inhibited alpha APP secretion by U-373 MG cells in a concentration- and conformation-dependent manner. Moreover, the reduction in alpha APP secretion was accompanied by an increase in cell-associated APP. Another proinflammatory amyloidogenic peptide, human amylin, similarly affected APP processing in U-373 astrocytoma cells. These data suggest that A beta may contribute to Alzheimer's-associated neuropathology by lowering the production of neuroprotective/neurotrophic alpha APPs. Moreover, the concomitant increase in cell-associated APP may provide increased substrate for the generation of amyloidogenic peptides within astrocytes.
Subject(s)
Alzheimer Disease/immunology , Amyloid beta-Peptides/immunology , Amyloid beta-Protein Precursor/immunology , Cytokines/immunology , Alzheimer Disease/metabolism , Cell Line , Cytokines/metabolism , Humans , Inflammation , Protein Processing, Post-Translational/immunologyABSTRACT
Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta. Given this interaction with A beta, the goal of this study was to determine whether clusterin could modulate A beta neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against A beta (1-40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A beta (1-40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-beta plaque pathology.
Subject(s)
Amyloid beta-Peptides/toxicity , Complement Inactivator Proteins/physiology , Glycoproteins/physiology , Molecular Chaperones , Neurotoxins/toxicity , Animals , Cell Line/physiology , Clusterin , Complement Inactivator Proteins/pharmacology , Cricetinae , Gene Expression/physiology , Glycoproteins/pharmacology , Hippocampus/cytology , Humans , Nerve Tissue Proteins/pharmacology , Nerve Tissue Proteins/physiology , Neuroprotective Agents/pharmacology , Rats , TransfectionABSTRACT
Amylin, a 37-amino-acid amyloidogenic peptide, bears biophysical similarities to the amyloid-beta peptide (A beta) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with A beta(1-40). Treatment with human amylin(1-37) resulted in prominent toxicity as assessed by phase-contrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin-induced neurotoxicity was morphologically similar to that induced by A beta(1-40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full-length human amylin was toxic; various amylin peptide fragments including amino acid residues 20-29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and A beta can adopt a similar neurotoxic conformation in vitro. Similar conformation-dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of A beta in Alzheimer's disease.
