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Small Molecule Inhibitors of Aggregation Indicate That Amyloid β Oligomerization and Fibrillization Pathways Are Independent and Distinct

2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês

10.1074/jbc.m608207200

ISSN

1083-351X

Autores

Mihaela Necula, Rakez Kayed, Saskia Milton, Charles Glabe,

Tópico(s)

Parkinson's Disease Mechanisms and Treatments

Resumo

Alzheimer disease is characterized by the abnormal aggregation of amyloid β peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid β oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid β aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid β monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms. Alzheimer disease is characterized by the abnormal aggregation of amyloid β peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid β oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid β aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid β monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms. 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Biol. 1997; 4: 119-125Abstract Full Text PDF PubMed Google Scholar). These prefibrillar soluble oligomers are specifically recognized by a polyclonal antibody, A11, 3Nomenclature is as follows. A11 is an anti-oligomer-specific antibody (58Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3228) Google Scholar). Inhibition of oligomerization refers to inhibition of formation of A11-immunoreactive oligomers. An inhibitor of oligomerization refers to a molecule that inhibits the formation of A11-immunoreactive oligomers. "Oligomerization conditions" refer to conditions that favor A11-reactive oligomer visualization. "Fibrillization conditions" refer to conditions that favor fiber formation in the absence of contamination with A11-positive oligomers. Fibrillization conditions and oligomerization conditions represent a nomenclature used for the sole purpose of describing conditions where Aβ42 forms oligomer-free fibers or fiber-free oligomers (at early time points), respectively. These conditions allow separate screening of effects of compounds on fiber or oligomer formation. This nomenclature is not meant to indicate that these are the best or the only conditions to prepare fibers and oligomers. that recognizes a generic backbone epitope that is common to the oligomeric state independent of the protein sequence (58Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3228) Google Scholar). A11 does not recognize Aβ monomer, Aβ dimer, trimer, or tetramer or Aβ fibrils (58Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3228) Google Scholar). A11 positive oligomers display the same intermediate kinetics as observed for soluble oligomers and protofibrils by electron microscopy and atomic force microscopy and A11 blocks the toxicity of Aβ oligomers, indicating that they represent the primary toxic species. Aβ*56 is a soluble oligomeric form of Aβ that is closely associated with pathogenesis in the Tg2576 mouse model of AD, and Aβ*56 is specifically recognized by A11 anti-oligomer antibody (59Lesne S. Koh M.T. Kotilinek L. Kayed R. Glabe C.G. Yang A. Gallagher M. Ashe K.H. Nature. 2006; 440: 352-357Crossref PubMed Scopus (2279) Google Scholar). Although ADDLs were originally described as low molecular weight trimeric or tetrameric species, more recent investigations indicate that native masses of ADDLs are the same as previously reported for other Aβ-soluble oligomers (60Hepler R.W. Grimm K.M. Nahas D.D. Breese R. Dodson E.C. Acton P. Keller P.M. Yeager M. Wang H. Shughrue P. Kinney G. Joyce J.G. Biochemistry. 2006; 45: 15157-15167Crossref PubMed Scopus (157) Google Scholar). A11 and anti-ADDL antibodies identify the same time course of soluble oligomer accumulation in the 3xTg-AD mouse (61Oddo S. Caccamo A. Tran L. Lambert M.P. Glabe C.G. Klein W.L. LaFerla F.M. J. Biol. Chem. 2006; 281: 1599-1604Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Therefore, A11 antibody recognizes a significant and important class of oligomers associated with AD that are distinct from amyloid fibrils. Analysis of the mechanism of action of amyloid aggregation inhibitors holds the promise of clarifying the relationship between oligomers and fibrils, because if oligomers are obligate intermediates on the pathway to fibril formation, then all inhibitors that block oligomer formation would be expected to block fibril formation as well. Alternatively, if fibrils and oligomers represent distinct aggregation pathways, then it would be expected that some inhibitors would block oligomerization but not fibril formation. Here we utilize the anti-oligomer antibody, A11, as a primary read out for oligomer formation, and we analyze the mechanism of action of small molecules that have been reported to inhibit the aggregation of different amyloidogenic proteins or their toxicity. The results demonstrate that some inhibitors specifically target oligomers, whereas others specifically inhibit fibrillization. These data indicate that soluble oligomers are not an obligate intermediate for fibril formation and that oligomers and fibrils represent separate and distinct aggregation pathways. The results further indicate that screening for aggregation inhibitors using fibril-specific assays, like thioflavin T (ThT) fluorescence, will not necessarily identify inhibitors of oligomerization. Materials—Synthetic Aβ42 was prepared as previously described (57Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar). 200-mesh formvar/carbon-coated nickel grids were obtained from Electron Microscopy Sciences (Ft. Washington, PA), and 4–20% Tris-HCl gels were from Bio-Rad. 96-well clear, flat bottom microplates were from Nalge Nunc International (Rochester, NY), and 3,3′,5,5′-tetramethylbenzidine was from KPL (Gaithersburg, MD). A11 anti-oligomer antibody is available from Invitrogen. Horseradish peroxidase-conjugated anti-rabbit IgG (AR) was purchased from Promega (Madison, WI), 6E10 and 4G8 antibodies were from Signet (Dedham, MA), and the ECL chemiluminescence kit was from Amersham Biosciences. 0.2-μm nitrocellulose membranes were form Schleicher & Schuell. Small molecule compounds and all other reagents were from Sigma or Calbiochem. 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