Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers.

Alzheimer's disease (AD) is characterized by presence of extracellular fibrillar A beta in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble A beta oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing A beta oligomers from AD brains, but not by an extract from non-AD brains. A beta oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology.

Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine.

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in brain tissue of AD patients and are considered centrally related to pathogenesis. Using hippocampal neuronal cultures, we found that ADDLs stimulated excessive formation of reactive oxygen species (ROS) through a mechanism requiring N-methyl-d-aspartate receptor (NMDA-R) activation. ADDL binding to neurons was reduced and ROS formation was completely blocked by an antibody to the extracellular domain of the NR1 subunit of NMDA-Rs. In harmony with a steric inhibition of ADDL binding by NR1 antibodies, ADDLs that were bound to detergent-extracted synaptosomal membranes co-immunoprecipitated with NMDA-R subunits. The NR1 antibody did not affect ROS formation induced by NMDA, showing that NMDA-Rs themselves remained functional. Memantine, an open channel NMDA-R antagonist prescribed as a memory-preserving drug for AD patients, completely protected against ADDL-induced ROS formation, as did other NMDA-R antagonists. Memantine and the anti-NR1 antibody also attenuated a rapid ADDL-induced increase in intraneuronal calcium, which was essential for stimulated ROS formation. These results show that ADDLs bind to or in close proximity to NMDA-Rs, triggering neuronal damage through NMDA-R-dependent calcium flux. This response provides a pathologically specific mechanism for the therapeutic action of memantine, indicates a role for ROS dysregulation in ADDL-induced cognitive impairment, and supports the unifying hypothesis that ADDLs play a central role in AD pathogenesis.

Synaptic targeting by Alzheimer's-related amyloid beta oligomers.

The cognitive hallmark of early Alzheimer's disease (AD) is an extraordinary inability to form new memories. For many years, this dementia was attributed to nerve-cell death induced by deposits of fibrillar amyloid beta (Abeta). A newer hypothesis has emerged, however, in which early memory loss is considered a synapse failure caused by soluble Abeta oligomers. Such oligomers rapidly block long-term potentiation, a classic experimental paradigm for synaptic plasticity, and they are strikingly elevated in AD brain tissue and transgenic-mouse AD models. The current work characterizes the manner in which Abeta oligomers attack neurons. Antibodies raised against synthetic oligomers applied to AD brain sections were found to give diffuse stain around neuronal cell bodies, suggestive of a dendritic pattern, whereas soluble brain extracts showed robust AD-dependent reactivity in dot immunoblots. Antigens in unfractionated AD extracts attached with specificity to cultured rat hippocampal neurons, binding within dendritic arbors at discrete puncta. Crude fractionation showed ligand size to be between 10 and 100 kDa. Synthetic Abeta oligomers of the same size gave identical punctate binding, which was highly selective for particular neurons. Image analysis by confocal double-label immunofluorescence established that >90% of the punctate oligomer binding sites colocalized with the synaptic marker PSD-95 (postsynaptic density protein 95). Synaptic binding was accompanied by ectopic induction of Arc, a synaptic immediate-early gene, the overexpression of which has been linked to dysfunctional learning. Results suggest the hypothesis that targeting and functional disruption of particular synapses by Abeta oligomers may provide a molecular basis for the specific loss of memory function in early AD.

Self-assembly of Abeta(1-42) into globular neurotoxins.

Amyloid beta 1-42 (Abeta(1-42)) is a self-associating peptide that becomes neurotoxic upon aggregation. Toxicity originally was attributed to the presence of large, readily formed Abeta fibrils, but a variety of other toxic species are now known. The current study shows that Abeta(1-42) can self-assemble into small, stable globular assemblies free of fibrils and protofibrils. Absence of large molecules was verified by atomic force microscopy (AFM) and nondenaturing gel electrophoresis. Denaturing electrophoresis revealed that the globular assemblies comprised oligomers ranging from trimers to 24mers. Oligomers prepared at 4 degrees C stayed fibril-free for days and remained so when shifted to 37 degrees C, although the spectrum of sizes shifted toward larger oligomers at the higher temperature. The soluble, globular Abeta(1-42) oligomers were toxic to PC12 cells, impairing reduction of MTT and interfering with ERK and Rac signal transduction. Occasionally, oligomers were neither toxic nor recognized by toxicity-neutralizing antibodies, suggesting that oligomers could assume alternative conformations. Tests for oligomerization-blocking activity were carried out by dot-blot immunoassays and showed that neuroprotective extracts of Ginkgo biloba could inhibit oligomer formation at very low doses. The observed neurotoxicity, structure, and stability of synthetic Abeta(1-42) globular assemblies support the hypothesis that Abeta(1-42) oligomers play a role in triggering nerve cell dysfunction and death in Alzheimer's disease.