Site-specific glycation of Aβ1–42 affects fibril formation and is neurotoxic

A1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). N-E-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of A1-42. Methyglyoxal is commonly used for the unspecific glycation of A1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on A1-42 at Lys-16 (A-CEL16), Lys-28 (A-CEL28), and Lys-16 and -28 (A-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of A1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that A-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of A1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of A1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only A-CEL16 and A-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas A-CEL16&28 showed no neurotoxicity. Interestingly, A-CEL16&28 had depolarized the mitochondrial membrane potential, whereas A-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated A1-42.