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.
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
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.