Abstract
BackgroundAmyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.ResultsWe immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.ConclusionSince the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.
Highlights
Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue
OC recognizes a generic epitope that is associated with the fibrillar amyloid state regardless of the sequence because it reacts well with α-synuclein fibrils and islet amyloid polypeptide (IAPP) fibrils
The size distribution of Aβ aggregates that are recognized by OC and A11 was determined by western blotting of fibrillar and prefibrillar oligomer Aβ samples (Fig. 2)
Summary
Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. The accumulation of misfolded proteins and peptides as amyloid deposits is a characteristic feature of many degenerative diseases, such as Alzheimer disease (AD), the pathological significance of these deposits remains unclear. The pathological significance of the fibrillar plaques is a matter of debate, since cognitively normal aged individuals frequently have large amounts of fibrillar deposits [9,10] and soluble oligomeric forms of Aβ correlate better with dementia [11,12]. Thioflavin dyes have served as the basis for the development of contrast agents to image amyloid accumulation in vivo in humans, these dyes do not label diffuse plaques in human AD brain nor the amyloid deposits that accumulate in transgenic mouse models of AD
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