Mary P. Lambert

Alzheimer's disease-affected brain: Presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss

A molecular basis for memory failure in Alzheimers disease (AD) has been recently hypothesized, in which a significant role is attributed to small, soluble oligomers of amyloid β-peptide (Aβ). Aβ 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 Aβ 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 Aβ ligands are intrinsic to AD pathology, and validate their use in new approaches to therapeutic AD drugs and vaccines.

Synaptic Targeting by Alzheimer's-Related Amyloid β Oligomers

The cognitive hallmark of early Alzheimers 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 β (Aβ). A newer hypothesis has emerged, however, in which early memory loss is considered a synapse failure caused by soluble Aβ 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 Aβ 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 Aβ 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 Aβ oligomers may provide a molecular basis for the specific loss of memory function in early AD.

Aβ 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 Aβ 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.

Soluble oligomers of β amyloid (1-42) inhibit long-term potentiation but not long-term depression in rat dentate gyrus

The dementia in Alzheimer disease (AD) is usually attributed to widespread neuronal loss in conjunction with the pathologic hallmarks of intracellular neurofibrillary tangles and extracellular plaques containing amyloid (Aβ) in fibrillar form. Recently it has been demonstrated that non-fibrillar assemblies of Aβ possess electrophysiologic activity, with the corollary that they may produce dementia by disrupting neuronal signaling prior to cell death. We therefore examined the effects of soluble oligomers of Aβ1-42 on long-term potentiation (LTP) and long-term depression (LTD), two cellular models of memory, in the dentate gyrus of rat hippocampal slices. Compared with vehicle controls, slices pre-incubated 60 min in the presence of Aβ-derived diffusible ligands (ADDLs) showed no differences in threshold intensity to evoke a synaptic response, slope of field excitatory post-synaptic potentials (EPSPs), or the input/output function. Tetanus-induced LTP and reversal of LTD were strongly inhibited in ADDLs-treated slices whereas LTD was unaffected. These data suggest that soluble non-fibrillar amyloid may contribute to the pathogenesis of AD both by impairing LTP/memory formation at the cellular level and by creating ‘neuroplasticity imbalance’ manifested by unopposed LTD in the setting of impaired capacity for neural repair via reversal of LTD or LTP.

Protection of synapses against Alzheimer's-linked toxins: Insulin signaling prevents the pathogenic binding of Aβ oligomers

Synapse deterioration underlying severe memory loss in early Alzheimers 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.

Amyloid beta oligomers induce impairment of neuronal insulin receptors

Recent studies have indicated an association between Alzheimers disease (AD) and central nervous system (CNS) insulin resistance. However’ the cellular mechanisms underlying the link between these two pathologies have not been elucidated. Here we show that signal transduction by neuronal insulin receptors (IR) is strikingly sensitive to disruption by soluble Aβ oligomers (also known as ADDLs). ADDLs are known to accumulate in AD brain and have recently been implicated as primary candidates for initiating deterioration of synapse function, composition, and structure. Using mature cultures of hippocampal neurons, a preferred model for studies of synaptic cell biology, we found that ADDLs caused a rapid and substantial loss of neuronal surface IRs specifically on dendrites bound by ADDLs. Removal of dendritic IRs was associated with increased receptor immunoreactiv‐ity in the cell body, indicating redistribution of the receptors. The neuronal response to insulin, measured by evoked IR tyrosine autophosphorylation, was greatly inhibited by ADDLs. Inhibition also was seen with added glutamate or potassium‐induced depolarization. The effects on IR function were completely blocked by NMDA receptor antagonists, tetrodotoxin, and calcium chelator BAPTA‐AM. Downstream from the IR, ADDLs induced a phosphorylation of Akt at serine473, a modification associated with neurodegenerative and insulin resistance diseases. These results identify novel factors that affect neuronal IR signaling and suggest that insulin resistance in AD brain is a response to ADDLs, which disrupt insulin signaling and may cause a brain‐specific form of diabetes as part of an overall pathogenic impact on CNS synapses.— Zhao, W. Q., De Felice, F. G., Fernandez, S., Chen, H., Lambert, M. P., Quon, M. J., Krafft, G. A., Klein, W. L. Amyloid beta oligomers induce impairment of neuronal insulin receptors. FASEB J. 22, 246–260 (2008)

Temporal Profile of Amyloid-β (Aβ) Oligomerization in an in Vivo Model of Alzheimer Disease A LINK BETWEEN Aβ AND TAU PATHOLOGY

Accumulation of amyloid-β (Aβ) is one of the earliest molecular events in Alzheimer disease (AD), whereas tau pathology is thought to be a later downstream event. It is now well established that Aβ exists as monomers, oligomers, and fibrils. To study the temporal profile of Aβ oligomer formation in vivo and to determine their interaction with tau pathology, we used the 3xTg-AD mice, which develop a progressive accumulation of plaques and tangles and cognitive impairments. We show that SDS-resistant Aβ oligomers accumulate in an age-dependent fashion, and we present evidence to show that oligomerization of Aβ appears to first occur intraneuronally. Finally, we show that a single intrahippocampal injection of a specific oligomeric antibody is sufficient to clear Aβ pathology, and more importantly, tau pathology. Therefore, Aβ oligomers may play a role in the induction of tau pathology, making the interference of Aβ oligomerization a valid therapeutic target.

Monoclonal antibodies that target pathological assemblies of Aβ

Amyloid beta (Aβ) immunotherapy for Alzheimers disease has shown initial success in mouse models of Alzheimers disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Aβ oligomers (amyloid β‐derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimers disease brain. Clones were selected for the ability to discriminate Alzheimers disease from control brains in extracts and tissue sections. These antibodies recognized Aβ oligomers and fibrils but not the physiologically prevalent Aβ monomer. Discrimination derived from an epitope found in assemblies of Aβ1–28 and ADDLs but not in other sequences, including Aβ1–40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL‐induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer‐dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimers disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Aβ assemblies, these antibodies provide tools by which pathological Aβ assemblies from Alzheimers disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimers disease therapeutics.

Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies

In recent studies of transgenic models of Alzheimers disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1–42 (Aβ1−42) solutions (mixtures of Aβ monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non‐fibrillar forms of Aβ. It is now known that Aβ toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Aβ1−42 oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Aβ1−42 solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Aβ in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell‐attached oligomers to receptor‐like puncta, and for immunoblots that show the presence of SDS‐stable oligomers in Alzheimers brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno‐neutralization of soluble Aβ‐derived toxins. Analogous immuno‐neutralization of oligomers in humans may be a key in AD vaccines.

Alzheimer's disease-type neuronal tau hyperphosphorylation induced by Aβ oligomers

Alzheimer’s disease (AD) is characterized by presence of extracellular fibrillar Aβ in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble Aβ 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 Aβ oligomers from AD brains, but not by an extract from non-AD brains. Aβ 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.