A␤ 1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid ␤ protein (A␤) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible A␤ oligomers (referred to as ADDLs, for A␤-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of A␤-derived diffusible ligands acting upon particular neural signal transduction pathways.

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.

A molecular basis for memory failure in Alzheimer's disease (AD) has been recently hypothesized, in which a significant role is attributed to small, soluble oligomers of amyloid ␤-peptide (A␤). A␤ oligomeric ligands (also known as ADDLs) are known to be potent inhibitors of hippocampal long-term potentiation, which is a paradigm for synaptic plasticity, and have been linked to synapse loss and reversible memory failure in transgenic mouse AD models. If such oligomers were to build up in human brain, their neurological impact could provide the missing link that accounts for the poor correlation between AD dementia and amyloid plaques. This article, using antibodies raised against synthetic A␤ oligomers, verifies the predicted accumulation of soluble oligomers in AD frontal cortex. Oligomers in AD reach levels up to 70-fold over control brains. Brain-derived and synthetic oligomers show structural equivalence with respect to mass, isoelectric point, and recognition by conformation-sensitive antibodies. Both oligomers, moreover, exhibit the same striking patterns of attachment to cultured hippocampal neurons, binding on dendrite surfaces in small clusters with ligand-like specificity. Binding assays using solubilized membranes show oligomers to be high-affinity ligands for a small number of nonabundant proteins. Current results confirm the prediction that soluble oligomeric A␤ ligands are intrinsic to AD pathology, and validate their use in new approaches to therapeutic AD drugs and vaccines.

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.

The amyloid-β oligomer (AβO) hypothesis was introduced in 1998. It proposed that the brain damage leading to Alzheimer’s disease (AD) was instigated by soluble, ligand-like AβOs. This hypothesis was based on the discovery that fibril-free synthetic preparations of AβOs were potent CNS neurotoxins that rapidly inhibited long-term potentiation and, with time, caused selective nerve cell death (Lambert et al., 1998). The mechanism was attributed to disrupted signaling involving the tyrosine-protein kinase Fyn, mediated by an unknown toxin receptor. Over 4,000 articles concerning AβOs have been published since then, including more than 400 reviews. AβOs have been shown to accumulate in an AD-dependent manner in human and animal model brain tissue and, experimentally, to impair learning and memory and instigate major facets of AD neuropathology, including tau pathology, synapse deterioration and loss, inflammation, and oxidative damage. As reviewed by Hayden and Teplow in 2013, the AβO hypothesis “has all but supplanted the amyloid cascade.” Despite the emerging understanding of the role played by AβOs in AD pathogenesis, AβOs have not yet received the clinical attention given to amyloid plaques, which have been at the core of major attempts at therapeutics and diagnostics but are no longer regarded as the most pathogenic form of Aβ. However, if the momentum of AβO research continues, particularly efforts to elucidate key aspects of structure, a clear path to a successful disease modifying therapy can be envisioned. Ensuring that lessons learned from recent, late-stage clinical failures are applied appropriately throughout therapeutic development will further enable the likelihood of a successful therapy in the near-term.

Synapse deterioration underlying severe memory loss in early Alzheimer's disease (AD) is thought to be caused by soluble amyloid beta (Aβ) oligomers. Mechanistically, soluble Aβ oligomers, also referred to as Aβ-derived diffusible ligands (ADDLs), act as highly specific pathogenic ligands, binding to sites localized at particular synapses. This binding triggers oxidative stress, loss of synaptic spines, and ectopic redistribution of receptors critical to plasticity and memory. We report here the existence of a protective mechanism that naturally shields synapses against ADDL-induced deterioration. Synapse pathology was investigated in mature cultures of hippocampal neurons. Before spine loss, ADDLs caused major downregulation of plasma membrane insulin receptors (IRs), via a mechanism sensitive to calcium calmodulin-dependent kinase II (CaMKII) and casein kinase II (CK2) inhibition. Most significantly, this loss of surface IRs, and ADDL-induced oxidative stress and synaptic spine deterioration, could be completely prevented by insulin. At submaximal insulin doses, protection was potentiated by rosiglitazone, an insulin-sensitizing drug used to treat type 2 diabetes. The mechanism of insulin protection entailed a marked reduction in pathogenic ADDL binding. Surprisingly, insulin failed to block ADDL binding when IR tyrosine kinase activity was inhibited; in fact, a significant increase in binding was caused by IR inhibition. The protective role of insulin thus derives from IR signaling-dependent downregulation of ADDL binding sites rather than ligand competition. The finding that synapse vulnerability to ADDLs can be mitigated by insulin suggests that bolstering brain insulin signaling, which can decline with aging and diabetes, could have significant potential to slow or deter AD pathogenesis.