β-Amyloid (Aβ) & Alzheimer’s Disease (AD)

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by the presence of extracellular amyloid plaques and intraneuronal neurofibrillary tangles in the brain. Biochemical analysis of amyloid plaques revealed that the main constituent is fibrillar aggregates of a 39-42 residue peptide referred to as the amyloid-β protein (Aβ). Several lines of evidence point towards a central role for the process of Aβ fibril formation in the etiology of AD. Transgenic animals overexpressing mutant forms of its precursor, the amyloid precursor protein (APP), develop amyloid plaques comprising fibrillar Aβ. Several pathogenic AD mutations have been shown to affect the processing of APP resulting in increased Aβ levels, in particular the more amyloidogenic variant Aβ42. These data implicate the process of amyloid fibril formation as the cause of disease progression and neurodegeneration in AD. The nature of the toxic species and the mechanism(s) by which fibrillization may cause neurodegeneration in AD remains controversial. in vitro studies have clearly demonstrated that Aβ fibril formation occurs via a complex multi-step nucleated polymerization mechanism that involves discrete soluble oligomeric intermediates termed ADDLS or protofibrils (PFs), which disappear upon fibril formation. Several lines of evidence suggest that Aβ PFs are a pathogenic species. First, there is a lack of a clear correlation between the amount of fibrillar Aβ deposits at autopsy and AD severity, whereas a correlation exists between soluble Aβ levels in the brain and early cognitive dysfunction. Second, transgenic animals that overproduce APP exhibit neuronal and behavioral abnormalities before amyloid plaques can be detected. Third, nonfibrillar, oligomeric forms of Aβ alter neuronal function and/or cause cell death. Fourth, in some models, inhibiting fibril formation does not attenuate Aβ associated toxicity towards cultured neurons. Fifth, an autosomal dominant mutation (APP(E693G), Aβ (E22G)) with a clinical phenotype similar to that of idiopathic AD was shown to decrease Aβ production in vivo and promote protofibril formation in vitro.


Amyloid Pores Formed by the Arctic Mutation (Aβ40ARC)

Most APP mutations associated with familial Alzheimer’s disease (FAD) are thought to cause early-onset AD by modulating the proteolytic processing of APP to increase the total concentration of Aβ in the plasma and cerebrospinal fluid and/or to produce an increase in the ratio Aβ42/Aβ40. One exception is the “Arctic” APP mutation (E22G), which causes a reduction in Aβ40 and Aβ42 levels in plasma. A reduction in Aβ42 was also observed in conditioned media from cells transfected with APPE693G. This data suggests that the Arctic mutation may predispose individuals to early-onset AD by promoting the formation of toxic aggregates, possibly protofibrils. With all the evidence mounting in support of the pathogenic PF hypothesis, the structure(s) of the toxic species and its mechanism of action are yet to be determined. Electron and atomic force microscopy studies have shown that Aβ self-assembles into PFs of heterogeneous morphology, including spheres, chain-like PFs, and amyloid pores, before forming amyloid fibrils. Studies of the biochemical and biological properties of Aβ PFs employ heterogeneous mixtures of PFs, making it difficult to decipher which of these species is the pathogenic species. We have developed an approach, which takes advantage of complementary biophysical techniques and allows the preparation, purification and characterization of α-synuclein PFs. We identified optimal conditions for PF formation and fractionation by size exclusion chromatography, and developed methods for their characterization. In collaboration with Peter Lansbury (Brigham and Women’s Hospital), we have extended this approach to prepare and characterize discrete Aβ assemblies, including monomer, PFs of different morphologies, and fibrils formed by wild type Aβ (Aβ40WT) and the Arctic variant (Aβ40ARC). The increased propensity of the Arctic variant to form PFs presented us with an opportunity to generate significant quantities of PFs to investigate the effect of the Arctic mutations on structural properties of Aβ PFs. We performed a detailed biophysical characterization of PFs formed by Aβ40WT and the Arctic variant (Aβ40ARC) as well as the biologically relevant mixtures of both proteins that may model the situation in the heterozygous patients.

To probe the effect of the Arctic mutation on the structural properties of Aβ, in particular Aβ protofibrillar intermediates, the aggregation of AβWT and AβARC was monitored by electron microscopy as a function of time. Aβ40WT and Aβ40ARC were incubated at room temperature. After eight hours, only Aβ40ARC showed the formation of predominantly spherical protofibrillar structures (top left). After 14 hours, spheres (I), short chain-like protofibrils (II) and annular protofibrils (III) were observed for Aβ40ARC (top right). The chain-like protofibrils appeared to be composed of spherical species. Large spherical species were also observed (IV). The amount of spherical and annular protofibrils started to diminish after 19 hours with short filaments and fibrils emerging as the predominant species (bottom left). Although amyloid fibril formation by Aβ40ARC did occur during the first 19 hours of incubation, the chosen images focus only on the changes in the structural properties of protofibrillar Aβ40ARC. Aβ40WT did not exhibit significant protofibril formation over the incubation period (at room temperature) of 14–48 hours. However, after 19 hours some non-fibrillar Aβ40WT aggregates and some very rare annular protofibrils could be detected by EM (bottom right). These observations suggest that the Aβ40WT can form annular pore-like protofibrils, but much more slowly and to a lesser extent than Aβ40ARC.

EM examination of the protofibrils formed by the Arctic variant revealed several morphologies. To facilitate further characterization, the different protofibrillar species were partially purified by size exclusion chromatography fractionation using the stable protofibrils formed in an equimolar mixture of Aβ40WT and Aβ40ARC. After a 16 hour incubation at room temperature, the mixture was separated. The oligomeric Aβ40 eluted as a broad peak between the void-volume peak and the Aβ40 monomer peak, suggesting the presence of protofibrils of different sizes and morphologies. The protofibrils were divided into seven fractions. The EM images revealed that early fractions (F1–F5; F2 top left, F4 top right, and F5 bottom left) contained predominately short filaments. Among the filaments were also spherical and ring-shaped structures. In contrast, very few filaments were evident in the late fraction (F7 bottom right), which contained numerous, ring-shaped structures. In addition, the filaments in fractions F5–F7 were shorter than those in the earlier fractions, suggesting that partial separation of filaments of different sizes was achieved by the size exclusion chromatography column. Examination of fraction F7 revealed that it contained a significant amount of annular protofibrils in addition to the spheres and large spherical oligomeric species.

Single particle averaging was employed to analyze fraction F7 obtained by gel-filtration fractionation. Multi-variate statistical analysis was used to divide the particle images into 100 classes. These 100 classes fell into four major groups. The first group consisted of annular structures (panels 1–3). These amyloid pores are similar to those formed by α-synuclein and reminiscent of those observed for bacterial pore-forming toxins. The second group showed rectangular particles of varying length (panels 4–6). The third group showed spherical aggregates (panel 7). The fourth group showed large spherical aggregates (panels 8 and 9). The first, third and fourth groups represent the majority of structures observed in F7.

» Lashuel et al. (2003) J. Mol. Biol. 332: 795-808.


Aβ Protofibrils Possess a Stable Core Structure

To better understand the roles of protofibrils in amyloid assembly and Alzheimer’s disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. In collaboration with Peter Lansbury (Brigham and Women’s Hospital) and Ronald Wetzel (University of Tennessee), we chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Aβ‚(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We showed that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We found that about 40% of the backbone amide hydrogens of Aβ‚ protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also found a surprising degree of specificity in amyloid assembly, in that wild type Aβ‚ is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides.

» Kheterpal et al. (2003) Biochemistry 42: 14092-14098.