Extensive data supporting a central role for Aβ in the genesis of Alzheimer's disease notwithstanding, the amyloid hypothesis has its weaknesses. One is that a specific neurotoxic Aβ species and the nature of its effects on neuronal function in vivo have not been defined. In a symposium titled "Protein Aggregation and Neurodegenerative Disease-an Unfolding Story" (#674), Bruce Yankner reviewed studies with synthetic Aβ peptides that have shown fibrils like those present in amyloid plaques to be neurotoxic in vitro. Such studies have supported the concept that extracellular plaques of amyloid fibrils are principally responsible for neuronal dysfunction and loss in AD.
Changiz Geula (#584.6 and #584.7) reconfirmed the toxicity of fibrils in vivo in an elegant series of experiments, in which the authors injected small amounts of fibrillar Aβ (200 pg) into the cerebral cortex of aged rhesus monkeys. This produced a dense core similar to that of native Aβ plaques, causing altered tau phosphorylation, neuronal loss, microglial activation, and proliferation. The lesion size and extent of neuronal loss increased with survival time after injection, as did the number and activation state of microglia. Coinjecting microglial inhibitory factor reduced the number of activated microglia and the lesion size by half. In addition, Dr. Geula noted that activated microglia were associated with naturally occurring compact plaques present in the aged rhesus cortex. Together, these findings indicate that an important component of plaque toxicity results from microglial activation.
Another problem that dogs the amyloid theory is that the severity of dementia and the density of amyloid plaques correlate poorly. However, levels of soluble Aβ and the extent of synaptic loss correlate strongly with the severity of cognitive impairment, suggesting that an Aβ species preceding plaques causes early damage. Several groups (including Bruce Yankner's, # 674) have confirmed that protofibrils are neurotoxic. Dean Hartley (#584.11) presented compelling evidence, gained by whole cell patch-clamp analysis , that protofibrils alter the electrical activity of neurons independent of the fibrils' effect. The NMDA antagonist D-APV caused a 30% inhibition of fibril-induced activity, whereas it blocked protofibril-induced activity by 60%-70%. These effects were not simply a result of the "gumming up" of neuronal membranes but appeared to involve specific interactions, since removal of protofibrils allowed the electrical activity of neurons to return to normal.
These findings, together with behavioral and morphological alterations found in several strains of AβPP-transgenic mice prior to plaque formation, suggest that soluble Aβ oligomers are important effectors of neurotoxicity. But which Aβ species mediates these changes in vivo? Although discrete electrophysiological and morphological alterations have been detected in young AβPP transgenic mice prior to amyloid deposition (Mucke, #674), it is not possible to define whether monomers, soluble oligomers, protofibrils, or dispersed amyloid fibrils cause these early alterations in synaptic form and function. Our demonstration that cell-derived oligomers of human Aβ inhibit the late phase of LTP addresses this issue (#128.3 and #920.1). We used conditioned medium from CHO cells that overexpress human AβPP and secrete SDS-stable, low n-oligomers similar in size and concentration to those detected in both human brain and CSF. Upon microinjection of this medium into the lateral cerebral ventricle of a live anesthetized rat, recordings from the CA1 detected a dramatic decrease in the late phase of LTP. Biochemical and immunological manipulations revealed that the LTP block was not attributable to fibrillar or monomeric Aβ but was mediated by the oligomers. This approach overcomes limitations of using synthetic peptides or transgenic mice. It shows clearly that physiological levels of stable Aβ oligomers can alter a sensitive and validated measure of synaptic plasticity in the absence of effects by monomers or fibrils. We only examined the effects of secreted oligomers, yet we have detected oligomers in Golgi-like vesicles.
It seems likely-as Andrea Le Blanc suggested (#128.10)-that intracellular Aβ oligomers might also play a role in Aβ-mediated neurotoxicity. Le Blanc found that microinjection of Aβ1-42 into the cytoplasm of primary human neurons lead to a 50% loss of cells within two days, whereas extracellular application of the same peptide solution, or micro-injection of Aβ42-1, did not. She also reported that cell loss was similar for peptide preparations that had formed fibrils and for preparations containing monomers, dimers, and trimers.
Bruce Yankner (#674) also reported that Aβ42 accumulated in Down's syndrome astrocytes in a vesicular pattern similar to that reported for adult human brain (Gouras #584.1). Moreover, Aβ accumulation in these astrocytes was associated with altered mitochondrial membrane potential and an increase in tunnel-positive cells. However, not all astrocytes and neurons that showed evidence of Aβ accumulation were tunnel-positive, suggesting that Aβ accumulation induces apoptosis in a subset of neurons but is unlikely to be an early event in AD pathogenesis. After many years of painstaking work, it now seems clear that multiple species of Aβ are neurotoxic. Thus, rather than fixate on an individual species, one must consider the whole process of fibrillogenesis from the dimer on up.
This is a complex proposition since Aβ may begin to oligomerize intracellularly. Oligo- and polymerization reactions are highly concentration-dependent, therefore, limiting monomer production should target production of toxic assemblies. But as Claudio Soto's presentation (#584.9) made evident, anti-aggregation approaches may also prove viable.
Soto et al. are testing the usefulness of their "β-sheet breaker peptide iAβ5" in a mouse model of AD. The mice, which are transgenic for both human AβPP (London mutation V717I) and PS-1 (A246T mutation), develop plaques by six months of age and show signs of neuronal dystrophy and microgliosis. Chemically blocking the N- and C-termini with added acetyl and amide groups dramatically improved the stability of the iAβ5 peptide. Intracerebroventricular or intraperitoneal administration of the blocked peptide reduced amyloid burden by 67.3% and 46.5%, respectively, while increasing neuronal survival and decreasing astrogliosis and microglial activation. Further efforts to improve the bioavailability of iAβ5 and peptidomimetics are under way.
While the increased survival of neurons, the decreased inflammatory response, and the clearance of plaques are welcome news, the real test of this therapeutic approach will lie in whether it can destabilize neurotoxic species other than fibrils, as well. Will it destabilize fibrils only to stabilize protofibrils or oligomers? Only time will tell.—Dominic Walsh, Center for Neurologic Diseases, Harvard Institutes of Medicine, Boston
(Note: The author codiscovered amyloid protofibrils and is closely involved with some of the research discussed here.)
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