Abstract
Oligomers of β-amyloid 42 (Aβ42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aβ42 dimer than toward fibrils produced from WT Aβ42 or from a toxic, conformationally constrained Aβ42 variant, E22P-Aβ42. We obtained these RNA aptamers by using the preincubated dimer model of E22P-Aβ42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P-AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aβ42 than for less-toxic Aβ40 aggregates. Comparison of CD data from the full-length and random regions of E22P-AbD43 suggested that the preferential binding of E22P-AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P-AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P-AbD43 also significantly protected against the neurotoxicity of WT Aβ42 and E22P-Aβ42. Furthermore, in an AD mouse model, E22P-AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P-AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.
Highlights
Oligomers of -amyloid 42 (A42), rather than fibrils, drive the pathogenesis of Alzheimer’s disease (AD)
The current report describes a comprehensive study on the development of RNA aptamers with potent affinity for the toxic dimer model of A42 (E22P–V40DAP–A42 dimer), and we discuss their application to therapeutics and diagnostics based on experiments using an AD mouse brain
Given unsuccessful enrichment in the two trials stated above using the A-immobilized column, it should be noted that the use of membrane filtering (Fig. S2) may be more effective for the selection of aptamers targeting oligomeric assemblies including ordered structures such as PFs
Summary
An RNA aptamer with potent affinity for a toxic dimer of amyloid 42 has potential utility for histochemical studies of Alzheimer’s disease. We report RNA aptamers with higher affinity toward PFs derived from a toxic A42 dimer than toward fibrils produced from WT A42 or from a toxic, conformationally constrained A42 variant, E22P–A42 We obtained these RNA aptamers by using the preincubated dimer model of E22P–A42, which dimerized via a linker located at Val-40, as the target of in vitro selection. We recently developed a covalently linked dimer model of E22P–A42 using an L,L-2,6-diaminopimelic acid (DAP) linker at Val-40 in the C-terminal hydrophobic core (Fig. 1A, E22P–V40DAP–A42 dimer), which plays an important role in the formation of toxic oligomers This dimer model produced quasistable PFs, which were toxic oligomers, and had a neurotoxic effect on SH-SY5Y human neuroblastoma cells following incubation [20]. The current report describes a comprehensive study on the development of RNA aptamers with potent affinity for the toxic dimer model of A42 (E22P–V40DAP–A42 dimer), and we discuss their application to therapeutics and diagnostics based on experiments using an AD mouse brain
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