The basis for memory loss in early Alzheimer's disease (AD) seems likely to involve synaptic damage caused by soluble A␤-derived oligomers (ADDLs). ADDLs have been shown to build up in the brain and CSF of AD patients and are known to interfere with mechanisms of synaptic plasticity, acting as gain-of-function ligands that attach to synapses. Because of the correlation between AD dementia and synaptic degeneration, we investigated here the ability of ADDLs to affect synapse composition, structure, and abundance. Using highly differentiated cultures of hippocampal neurons, a preferred model for studies of synapse cell biology, we found that ADDLs bound to neurons with specificity, attaching to presumed excitatory pyramidal neurons but not GABAergic neurons. Fractionation of ADDLs bound to forebrain synaptosomes showed association with postsynaptic density complexes containing NMDA receptors, consistent with observed attachment of ADDLs to dendritic spines. During binding to hippocampal neurons, ADDLs promoted a rapid decrease in membrane expression of memory-related receptors (NMDA and EphB2). Continued exposure resulted in abnormal spine morphology, with induction of long thin spines reminiscent of the morphology found in mental retardation, deafferentation, and prionoses. Ultimately, ADDLs caused a significant decrease in spine density. Synaptic deterioration, which was accompanied by decreased levels of the spine cytoskeletal protein drebrin, was blocked by the Alzheimer's therapeutic drug Namenda. The observed disruption of dendritic spines links ADDLs to a major facet of AD pathology, providing strong evidence that ADDLs in AD brain cause neuropil damage believed to underlie dementia.

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 ␤ (A␤). A newer hypothesis has emerged, however, in which early memory loss is considered a synapse failure caused by soluble A␤ 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 transgenicmouse AD models. The current work characterizes the manner in which A␤ 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 A␤ 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 Ͼ90% 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 A␤ oligomers may provide a molecular basis for the specific loss of memory function in early AD.

Oxidative stress is a major aspect of Alzheimer disease (AD) pathology. We have investigated the relationship between oxidative stress and neuronal binding of A␤ 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. Reactive oxygen species (ROS)4 are minor cytotoxic products of normal mitochondrial metabolism. Approximately 1-2% of the oxygen molecules consumed in electron transport generate species, such as superoxide and hydrogen peroxide (1, 2), mainly through side reactions catalyzed by respiratory complexes I (3) and III (1, 3-5). However, imbalance between mitochondrial ROS production and the intracellular levels of antioxidant defenses leads to oxidative stress, a condition that has been associated with apoptosis (6, 7), inflammation (8), ischemia-reperfusion injury (9, 10), and neurodegenerative diseases (11-15). Several cellular features of the brain suggest that it is highly sensitive to oxidative stress (reviewed in Ref. 16). The brain possesses the highest oxygen metabolic rate of any organ in the body (16), consuming approximately 20% of the total amount of oxygen in the body (17). This enhanced metabolic rate leads to an increased probability that excessive levels of ROS will be produced.In some circumstances, particularly in the brain, even small imbalances can be deleterious. Transient production of ROS plays a role in synaptic signaling, with ROS acting as messenger molecules in the process of long term potentiation (LTP), a well known model for synaptic plasticity and learning (18). On the other hand, abnormally elevated ROS ...

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.