A differential association of Apolipoprotein E isoforms with the amyloid-ß oligomer in solution.

The molecular pathogenesis of disorders arising from protein misfolding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates, whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex misfolding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease (AD), we have developed a 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled amyloid-ß (Aß) peptide to observe its isoform-dependent interaction with the apoE protein. Although most individuals carry the E3 isoform of apoE, ∼15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for AD. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aß peptide processing and hence Aß toxicity. We used electron paramagnetic resonance (EPR) spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aß in solution. EPR spectra of the spin-labeled side chain report on side chain and backbone dynamics as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aß oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aß.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aß peptide.

BACKGROUND: The deposition and oligomerization of amyloid ß (Aß) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aß peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by ß and γ secretases. Several lines of evidence point to the soluble Aß oligomer (AßO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AßO species. METHODOLOGY/PRINCIPAL FINDINGS: To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aß peptide to investigate the binding and influence of fluorene compounds on AßO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AßO toxicity and a route to directly observe the binding of the fluorene to the AßO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AßO toxicity, scavenge free radicals and diminish the formation of intracellular AßO species. CONCLUSIONS: Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aß peptide toxicity, because they possess both antioxidant properties and the ability to disrupt AßO species.