Amyloid -protein (A) assembly: A40 and A42 oligomerize through distinct pathways

Amyloid -protein (A) assembly: A40 and A42 oligomerize through distinct pathways
November 8, 2002
Published online before print December 27, 2002
Gal Bitan*, Marina D. Kirkitadze*, Aleksey Lomakin, Sabrina S. Vollers*, George B. Benedek, and David B. Teplow*,
PNAS | January 7, 2003
* Center for Neurologic Diseases, Brigham and Women's Hospital, and Department of Neurology, Harvard Medical School, Boston, MA 02115; and Department of Physics, Center for Material Science and Engineering, and Materials Processing Center, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by George B. Benedek
Amyloid -protein (A) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of A in AD is new evidence that oligomeric forms of A are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of A, A42, with the disease. Detailed knowledge of the structure and assembly dynamics of A thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that A oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of A40 and A42. Carefully prepared aggregate-free A40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, A42 preferentially formed pentamer/hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to A40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that A42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of A42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.
Amyloid -protein (A) fibril formation and deposition long have been linked to the neuropathogenesis of Alzheimer's disease (AD) (1-5). However, recent data have shown that oligomeric A assembly intermediates are potent neurotoxins, and that these intermediates may be the key effectors of neurotoxicity in AD (6). In transgenic mice expressing the human amyloid -protein precursor (APP) and A, neurologic deficits develop before and independently of the appearance of amyloid deposits (6, 7). Importantly, soluble oligomeric forms of A are neurotoxic in vitro (8-15) and in vivo (15). The main alloforms of A found in amyloid deposits are 40 and 42 amino acids long (designated A40 and A42, respectively). Despite the small structural difference between these two peptides, they display distinct clinical, biological, and biophysical behavior. The concentration of secreted A42 is 10% that of A40, yet the longer form is the predominant component in parenchymal plaques (16-18). An increase in the A42/A40 concentration ratio is associated with familial forms of early onset AD (19, 20). Treatments that reduce A42 levels have been shown to correlate with decreased risk for AD (21). In addition, A42 displays enhanced neurotoxicity relative to A40 (22-24). Studies of the kinetics of A fibril formation have shown that A42 forms fibrils significantly faster than A40 (25), leading to the oft-repeated statement "A42 is more amyloidogenic" than A40 (for a review, see ref. 26). However, the structural and thermodynamic meaning of this statement is not entirely clear. Moreover, if oligomeric assemblies, rather than fibrils, are the key effectors of neurotoxicity in AD, kinetic differences in fibril assembly may not underlie the differences in biological activity between the two alloforms. The distinct clinical results of elevated A42 levels may stem from qualitative differences, such as the formation of unique intermediates. Thus, understanding the differences between A40 and A42 with regard to the assembly of these peptides is biologically and clinically important. Here a combination of biochemical, spectroscopic, and morphologic methods was used to study the initial oligomerization and assembly of A40 and A42. The data show that these peptides have distinct behaviors at the earliest stage of assembly, monomer oligomerization. This finding may explain the particularly strong association of A42 with AD and suggest approaches for appropriate targeting of therapeutic agents for AD.
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