Dominic M. Walsh

Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.

Although extensive data support a central pathogenic role for amyloid β protein (Aβ) in Alzheimers disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Aβ and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Aβ are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Aβ monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Aβ species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aβ monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Aβ oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with γ-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Aβ oligomers can be targeted therapeutically.

Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.

Alzheimers disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimers disease has not been achieved. We extracted soluble amyloid-β protein (Aβ) oligomers directly from the cerebral cortex of subjects with Alzheimers disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Aβ from Alzheimers disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Aβ dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Aβ N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimers disease cortex did not impair LTP unless they were first solubilized to release Aβ dimers, suggesting that plaque cores are largely inactive but sequester Aβ dimers that are synaptotoxic. We conclude that soluble Aβ oligomers extracted from Alzheimers disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

Aβ Oligomers – a decade of discovery

Converging lines of evidence suggest that progressive accumulation of the amyloid β‐protein (Aβ) plays a central role in the genesis of Alzheimer’s disease, but it was long assumed that Aβ had to be assembled into extracellular amyloid fibrils to exert its cytotoxic effects. Over the past decade, data have emerged from the use of synthetic Aβ peptides, cell culture models, β‐amyloid precursor protein transgenic mice and human brain to suggest that pre‐fibrillar, diffusible assemblies of Aβ are also deleterious. Although the precise molecular identity of these soluble toxins remains unsettled, accumulating evidence suggests that soluble forms of Aβ are indeed the proximate effectors of synapse loss and neuronal injury. Here we review recent progress in understanding the role of soluble oligomers in Alzheimer’s disease.

Natural oligomers of the amyloid-β protein specifically disrupt cognitive function

A central unresolved problem in research on Alzheimer disease is the nature of the molecular entity causing dementia. Here we provide the first direct experimental evidence that a defined molecular species of the amyloid-β protein interferes with cognitive function. Soluble oligomeric forms of amyloid-β, including trimers and dimers, were both necessary and sufficient to disrupt learned behavior in a manner that was rapid, potent and transient; they produced impaired cognitive function without inducing permanent neurological deficits. Although β-amyloidosis has long been hypothesized to affect cognition, the abnormally folded protein species associated with this or any other neurodegenerative disease has not previously been isolated, defined biochemically and then specifically characterized with regard to its effects on cognitive function. The biochemical isolation of discrete amyloid-β moieties with pathophysiological properties sets the stage for a new approach to studying the molecular mechanisms of cognitive impairment in Alzheimer disease and related neurodegenerative disorders.

Natural Oligomers of the Alzheimer Amyloid-β Protein Induce Reversible Synapse Loss by Modulating an NMDA-Type Glutamate Receptor-Dependent Signaling Pathway

Alzheimers disease (AD) is characterized by decreased synapse density in hippocampus and neocortex, and synapse loss is the strongest anatomical correlate of the degree of clinical impairment. Although considerable evidence supports a causal role for the amyloid-β protein (Aβ) in AD, a direct link between a specific form of Aβ and synapse loss has not been established. We demonstrate that physiological concentrations of naturally secreted Aβ dimers and trimers, but not monomers, induce progressive loss of hippocampal synapses. Pyramidal neurons in rat organotypic slices had markedly decreased density of dendritic spines and numbers of electrophysiologically active synapses after exposure to picomolar levels of soluble oligomers. Spine loss was reversible and was prevented by Aβ-specific antibodies or a small-molecule modulator of Aβ aggregation. Mechanistically, Aβ-mediated spine loss required activity of NMDA-type glutamate receptors (NMDARs) and occurred through a pathway involving cofilin and calcineurin. Furthermore, NMDAR-mediated calcium influx into active spines was reduced by Aβ oligomers. Partial blockade of NMDARs by pharmacological antagonists was sufficient to trigger spine loss. We conclude that soluble, low-n oligomers of human Aβ trigger synapse loss that can be reversed by therapeutic agents. Our approach provides a quantitative cellular model for elucidating the molecular basis of Aβ-induced neuronal dysfunction.

Deciphering the Molecular Basis of Memory Failure in Alzheimer's Disease

Acutely developing lesions of the brain have been highly instructive in elucidating the neural systems underlying memory in humans and animal models. Much less has been learned from chronic neurodegenerative disorders that insidiously impair memory. But the advent of a detailed molecular hypothesis for the development of Alzheimers disease and the creation of compelling mouse models thereof have begun to change this situation. Experiments in rodents suggest that soluble oligomers of the amyloid beta protein (Abeta) may discretely interfere with synaptic mechanisms mediating aspects of learning and memory, including long-term potentiation. In humans, memory impairment correlates strongly with cortical levels of soluble Abeta species, which include oligomers. Local inflammatory changes, neurofibrillary degeneration, and neurotransmitter deficits all contribute to memory impairment, but available evidence suggests that these develop as a consequence of early Abeta accumulation. Accordingly, attempts to slow memory and cognitive loss by decreasing cerebral Abeta levels have entered human trials.

Amyloid β-Protein Fibrillogenesis STRUCTURE AND BIOLOGICAL ACTIVITY OF PROTOFIBRILLAR INTERMEDIATES

Alzheimer’s disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid β-protein (Aβ). Fibrils, both in vitro andin vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Aβ production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Aβ fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364–22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Aβ (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer’s disease and for our understanding of fibril assembly are discussed.

Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate.

Fibrillogenesis of the amyloid β-protein (Aβ) is a seminal pathogenetic event in Alzheimer’s disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Aβ fibrillogenesis. We report results of studies designed to examine the initial stages of Aβ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 m Tris-HCl, pH 7.4, and removal of pre-existent seeds, Aβ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ± 0.2 nm, consistent with the predicted size of dimeric Aβ. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6–8 nm in diameter and <200 nm in length, which we have termed “protofibrils.” The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of Aβ-(1–40), Aβ-(1–42), and Aβ-(1–40)-Gln22, peptides associated with both sporadic and inherited forms of Alzheimer’s disease, suggesting that protofibril formation may be a general phenomenon in Aβ fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host

Protein aggregation is an established pathogenic mechanism in Alzheimers disease, but little is known about the initiation of this process in vivo. Intracerebral injection of dilute, amyloid-β (Aβ)–containing brain extracts from humans with Alzheimers disease or β-amyloid precursor protein (APP) transgenic mice induced cerebral β-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. The seeding activity of brain extracts was reduced or abolished by Aβ immunodepletion, protein denaturation, or by Aβ immunization of the host. The phenotype of the exogenously induced amyloidosis depended on both the host and the source of the agent, suggesting the existence of polymorphic Aβ strains with varying biological activities reminiscent of prion strains.

Soluble oligomers of amyloid β-protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake

In Alzheimers disease (AD), the impairment of declarative memory coincides with the accumulation of extracellular amyloid-beta protein (Abeta) and intraneuronal tau aggregates. Dementia severity correlates with decreased synapse density in hippocampus and cortex. Although numerous studies show that soluble Abeta oligomers inhibit hippocampal long-term potentiation, their role in long-term synaptic depression (LTD) remains unclear. Here, we report that soluble Abeta oligomers from several sources (synthetic, cell culture, human brain extracts) facilitated electrically evoked LTD in the CA1 region. Abeta-enhanced LTD was mediated by mGluR or NMDAR activity. Both forms of LTD were prevented by an extracellular glutamate scavenger system. Abeta-facilitated LTD was mimicked by the glutamate reuptake inhibitor TBOA, including a shared dependence on extracellular calcium levels and activation of PP2B and GSK-3 signaling. In accord, synaptic glutamate uptake was significantly decreased by soluble Abeta. We conclude that soluble Abeta oligomers perturb synaptic plasticity by altering glutamate recycling at the synapse and promoting synapse depression.