Probing and trapping a sensitive conformation: amyloid-β fibrils, oligomers, and dimers.
Alzheimer's disease (AD) is a devastating neurodegenerative disease with pathological misfolding of amyloid-β protein (Aβ). The recent interest in Aβ misfolding intermediates necessitates development of novel detection methods and ability to trap these intermediates. We speculated that two regions of Aβ may allow for detection of specific Aβ species: the N-terminal and 22-35, both likely important in oligomer interaction and formation. We determined via epitomics, proteomic assays, and electron microscopy that the Aβ(42) species (wild type, ΔE22, and MetOx) predominantly formed fibrils, oligomers, or dimers, respectively. The 2H4 antibody to the N-terminal of Aβ, in the presence of 2% SDS, primarily detected fibrils, and an antibody to the 22-35 region detected low molecular weight Aβ species. Simulated molecular modeling provided insight into these SDS-induced structural changes. We next determined if these methods could be used to screen anti-Aβ drugs as well as identify compounds that trap Aβ in various conformations. Immunoblot assays determined that taurine, homotaurine (Tramiprosate), myoinositol, methylene blue, and curcumin did not prevent Aβ aggregation. However, calmidazolium chloride trapped Aβ at oligomers, and berberine reduced oligomer formation. Finally, pretreatment of AD brain tissues with SDS enhanced 2H4 antibody immunostaining of fibrillar Aβ. Thus we identified and characterized Aβs that adopt specific predominant conformations (modified Aβ or via interactions with compounds), developed a novel assay for aggregated Aβ, and applied it to drug screening and immunohistochemistry. In summary, our novel approach facilitates drug screening, increases the probability of success of antibody therapeutics, and improves antibody-based detection and identification of different conformations of Aβ.
Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M Health Science Center, Bryan, TX 77807, USA.
Janelle N
JN
Fawver
Karen T
KT
Duong
Olivia
O
Wise-Scira
Rachel
R
Petrofes Chapa
Hayley E
HE
Schall
Orkid
O
Coskuner
Xiongwei
X
Zhu
Luis V
LV
Colom
Ian V J
IV
Murray
D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western; D003198:Computer Simulation; D019281:Dimerization; D044127:Epigenesis, Genetic; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D009412:Nerve Fibers / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D040901:Proteomics; D057165:Proteostasis Deficiencies / Q000473:pathology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D016229:Amyloid beta-Peptides; D000906:Antibodies; D012967:Sodium Dodecyl Sulfate