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.

Amyloid beta (Ab) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's 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 Ab oligomers (amyloid b-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Ab oligomers and fibrils but not the physiologically prevalent Ab monomer. Discrimination derived from an epitope found in assemblies of Ab1-28 and ADDLs but not in other sequences, including Ab1-40. 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 Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Ab assemblies, these antibodies provide tools by which pathological Ab assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.

Synaptic loss underlies the memory deficit of Alzheimer's disease (AD). The molecular mechanism is elusive; however, excitatory synapses organized by the postsynaptic density (PSD) have been used as targets for AD treatment. To identify pathological entities at the synapse in AD, synaptic proteins were screened by quantitative proteomic profiling. The critical proteins were then selected for immunoblot analysis. The glutamate receptors N-methyl-D-aspartate (NMDA) receptor 1 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2 (GluR2) were substantially lost; specifically, the loss of GluR2 was up to 40% at PSD in AD. Shank proteins, the organizers of these glutamate receptors at excitatory synapses, were dramatically altered in AD. The level of Shank2 was increased, whereas the protein level of Shank3 was decreased. Further, the Shank3 protein was modified by ubiquitin, indicating that abnormal activity of the ubiquitinproteasome system may lead to Shank3 degradation in AD. Our findings suggest that disruption of glutamate receptors at the Shank-postsynaptic platform could contribute to destruction of the PSD which underlies the synaptic dysfunction and loss in AD.

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer's disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.

Introduction: β-Amyloid protein (Aβ) putatively plays a seminal role in synaptic loss in Alzheimer's disease (AD). While there is no consensus regarding the synaptic-relevant species of Aβ, it is known that Aβ oligomers (AβOs) are noticeably increased in the early stages of AD, localizing at or within the synapse. In cell and animal models, AβOs have been shown to attach to synapses and instigate synapse dysfunction and deterioration. To establish the pathological mechanism of synaptic loss in AD, it will be important to identify the synaptic targets to which AβOs attach.Methods: An unbiased approach using far western ligand blots has identified three synaptic proteins to which AβOs specifically attach. These proteins (p100, p140, and p260) were subsequently enriched by detergent extraction, ultracentrifugation, and CHT-HPLC column separation, and sequenced by LC-MS/MS. P100, p140, and p260 were identified. These levels of AβOs targets in human AD and aging frontal cortexes were analyzed by quantitative proteomics and western-blot. The polyclonal antibody to AβOs was developed and used to block the toxicity of AβOs. The data were analyzed with one-way analysis of variance.Results: AβOs binding proteins p100, p140, and p260 were identified as Na/K-ATPase, synGap, and Shank3, respectively. α3-Na/K-ATPase, synGap, and Shank3 proteins showed loss in the postsynaptic density (PSD) of human AD frontal cortex. In short term experiments, oligomers of Aβ inhibited Na/K-ATPase at the synapse. Na/K-ATPase activity was restored by an antibody specific for soluble forms of Aβ. α3-Na/K-ATPase protein and synaptic β-amyloid peptides were pulled down from human AD synapses by co-immunoprecipitation. Results suggest synaptic dysfunction in early stages of AD may stem from inhibition of Na/K-ATPase activity by Aβ oligomers, while later stages could hypothetically result from disrupted synapse structure involving the PSD proteins synGap and Shank3.Conclusion: We identified three AβO binding proteins as α3-Na/K-ATPase, synGap, and Shank3. Soluble Aβ oligomers appear capable of attacking neurons via specific extracellular as well as intracellular synaptic proteins. Impact on these proteins hypothetically could lead to synaptic dysfunction and loss, and could serve as novel therapeutic targets for AD treatment by antibodies or other agents.

The most common causes of neurodegenerative dementia include Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). We believe that, in all 3, aggregates of pathogenic proteins are pathological substrates which are associated with a loss of synaptic function/plasticity. The synaptic plasticity relies on the normal integration of glutamate receptors at the postsynaptic density (PSD). The PSD organizes synaptic proteins to mediate the functional and structural plasticity of the excitatory synapse and to maintain synaptic homeostasis. Here, we will discuss the relevant disruption of the protein network at the PSD in these dementias and the accumulation of the pathological changes at the PSD years before clinical symptoms. We suggest that the functional and structural plasticity changes of the PSD may contribute to the loss of molecular homeostasis within the synapse (and contribute to early symptoms) in these dementias.

Synaptic loss is the structural basis for memory impairment in Alzheimer's disease (AD). While the underlying pathological mechanism remains elusive, it is known that misfolded proteins accumulate as β-amyloid (Aβ) plaques and hyperphosphorylated Tau tangles decades before the onset of clinical disease. The loss of Pin1 facilitates the formation of these misfolded proteins in AD. Pin1 protein controls cell-cycle progression and determines the fate of proteins by the ubiquitin proteasome system. The activity of the ubiquitin proteasome system directly affects the functional and structural plasticity of the synapse. We localized Pin1 to dendritic rafts and postsynaptic density (PSD) and found the pathological loss of Pin1 within the synapses of AD brain cortical tissues. The loss of Pin1 activity may alter the ubiquitin-regulated modification of PSD proteins and decrease levels of Shank protein, resulting in aberrant synaptic structure. The loss of Pin1 activity, induced by oxidative stress, may also render neurons more susceptible to the toxicity of oligomers of Aβ and to excitation, thereby inhibiting NMDA receptor-mediated synaptic plasticity and exacerbating NMDA receptor-mediated synaptic degeneration. These results suggest that loss of Pin1 activity could lead to the loss of synaptic plasticity in the development of AD.