Pauline T. Velasco

Aβ Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease

The basis for memory loss in early Alzheimers disease (AD) seems likely to involve synaptic damage caused by soluble Aβ-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 Alzheimers 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.

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.

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.

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.

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.

Deleterious Effects of Amyloid β Oligomers Acting as an Extracellular Scaffold for mGluR5

Soluble oligomers of amyloid beta (Abeta) play a role in the memory impairment characteristic of Alzheimers disease. Acting as pathogenic ligands, Abeta oligomers bind to particular synapses and perturb their function, morphology, and maintenance. Events that occur shortly after oligomer binding have been investigated here in live hippocampal neurons by single particle tracking of quantum dot-labeled oligomers and synaptic proteins. Membrane-attached oligomers initially move freely, but their diffusion is hindered markedly upon accumulation at synapses. Concomitantly, individual metabotropic glutamate receptors (mGluR5) manifest strikingly reduced lateral diffusion as they become aberrantly clustered. This clustering of mGluR5 elevates intracellular calcium and causes synapse deterioration, responses prevented by an mGluR5 antagonist. As expected, clustering by artificial crosslinking also promotes synaptotoxicity. These results reveal a mechanism whereby Abeta oligomers induce the abnormal accumulation and overstabilization of a glutamate receptor, thus providing a mechanistic and molecular basis for Abeta oligomer-induced early synaptic failure.

Alzheimer's-associated Aβ oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal

It now appears likely that soluble oligomers of amyloid-beta1-42 peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimers disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt Abeta oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble Abeta species, when assayed with both sequence- and conformation-specific Abeta antibodies, indicating changes in oligomer structure. Analysis of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (Abeta-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.

Why Alzheimer's is a disease of memory: the attack on synapses by A beta oligomers (ADDLs).

Individuals with early-stage Alzheimer’s disease (AD) suffer from profound failure to form new memories. A novel molecular mechanism with implications for therapeutics and diagnostics is now emerging in which the specificity of AD for memory derives from disruption of plasticity at synapses targeted by neurologically active Aβ oligomers (1). We have named these oligomers “ADDLs” (for pathogenic Aβ-Derived Diffusible Ligands). ADDLs constitute metastable alternatives to the disease-defining Aβ fibrils deposited in amyloid plaques. In AD brain, ADDLs accumulate primarily as Aβ 12mers (2) (∼54 kDa) and can be found in dot-like clusters distinct from senile plaques (3). Oligomers of equal mass have been reported to occur in tg-mouse AD models where they emerge concomitantly with memory failure (4), consistent with ADDL inhibition of LTP (1). In cell biology studies, ADDLs act as pathogenic gain-of-function ligands that target particular synapses, binding to synaptic spines at or near NMDA receptors (5,6). Binding produces ectopic expression of the memory-linked immediate early gene Arc. Subsequent ADDL-induced abnormalities in spine morphology and synaptic receptor composition (7) are predicted consequences of Arc overexpression, a pathology associated with memory dysfunction in tg-Arc mice. Significantly, the attack on synapses provides a plausible mechanism unifying memory dysfunction with major features of AD neuropathology; recent findings show that ADDL binding instigates synapse loss, oxidative damage, and AD-type tau hyperphosphorylation. Acting as novel neurotoxins that putatively account for memory loss and neuropathology, ADDLs present significant targets for disease-modifying therapeutics in AD.

Towards Non-Invasive Diagnostic Imaging of Early-Stage Alzheimer’s Disease

One way to image the molecular pathology in Alzheimer’s disease (AD) is by positron emission tomography using probes that target amyloid fibrils. However, these fibrils are not closely linked to the development of the disease. It is now thought that early stage biomarkers that instigate memory loss comprise of Aβ oligomers (AβOs). Here we report a sensitive molecular magnetic resonance imaging (MRI) contrast probe that is specific for AβOs. We attach oligomer-specific antibodies onto magnetic nanostructures and show the complex is stable and it binds to AβOs on cells and brain tissues to give a MRI signal. When intranasally administered to an AD mouse model, the probe readily reached hippocampal AβOs. In isolated samples of human brain tissue, we observed an MRI signal that distinguished AD from controls. Such nanostructures that target neurotoxic AβOs are potentially useful for evaluating the efficacy of new drugs and ultimately for early-stage AD diagnosis and disease management.