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

Abstract

A␤1-42 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 ⑀-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of A␤1-42. Methyglyoxal is commonly used for the unspecific glycation of A␤1-42, 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 A␤1-42 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 A␤1-42 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 A␤1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of A␤1-42 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 A␤1-42. A␤1-42 peptides are implicated in the pathogenesis of Alzheimer's disease (AD). 5 A␤1-42 peptides are released extracellularly after being enzymatically processed from the amyloid precursor protein and are prone to aggregation. Aggregated A␤1-42 eventually forms amyloid plaques and causes neuronal dysfunction (1). As aging is a major risk factor for AD development, this natural process of aging also ubiquitously produces advanced glycated end products (AGEs), which are a type of post-translational modification (glycation). Several seminal studies provide evidence of increased AGE modifications that are associated with AD brains (2-4). Other disease states, which affect cerebral sugar homeostasis as a consequence of aging, such as Type II diabetes mellitus, may also contribute to the formation of AGEs within the brain (5). Glycation is an irreversible and nonenzymatically driven process that occurs through a Maillard reaction that conjugates sugars and proteins (6, 7). A particular in vivo sugar derivative, methylglyoxal (MG), is a glycating agent capable of forming different AGEs on A␤1-42 at lysine residues 16 (Lys-16) and 28 (Lys-28) (8, 9). The increase in in vivo MG production was observed in AD patients (10). Several ke...

Reduced Influence of apoE on Aβ43 Aggregation and Reduced Vascular Aβ43 Toxicity as Compared with Aβ40 and Aβ42

“…Another, frequently used, method to obtain monomeric Aβ solutions is pre-treatment with alkaline reagents such as NH 4 OH or NaOH, which prevents the Aβ solution of reaching the isoelectric point of 5.5 at which Aβ aggregation is maximal [78][79][80]. We followed a previously established protocol based on this latter method [30,[81][82][83][84][85] for the preparation of aggregate-free Aβ solutions and found that it tremendously increased reproducibility of our findings as compared with HFIP pre-treatment. Furthermore, we carefully controlled experimental conditions by determination and normalization of Aβ concentrations before every experiment, as we observed a high variation in actual protein content of commercially available Aβ peptides, often not consistent with the indicated amounts.…”

Potential Therapeutic Approaches for Cerebral Amyloid Angiopathy and Alzheimer’s Disease

“…To further clarify the pathological significance of glycated Aβ 42 , a recent study examined the characteristics of three glycated Aβ 42 variants compared to non-glycated Aβ 42 . The glycated variants possessed site-specific glycation at Lys16, Lys28, or Lys16 and Lys28 [84]. Transmission microscopy and in silico modeling revealed that double Aβ 42 glycation at Lys16 and Lys28 reduced free energy change and destabilized fibril structures, thereby increasing the rate of Aβ 42 aggregation [84].…”

“…The glycated variants possessed site-specific glycation at Lys16, Lys28, or Lys16 and Lys28 [84]. Transmission microscopy and in silico modeling revealed that double Aβ 42 glycation at Lys16 and Lys28 reduced free energy change and destabilized fibril structures, thereby increasing the rate of Aβ 42 aggregation [84]. They also showed that a single glycation at Lys16 slowed down fibril formation, whereas a single glycation at Lys28 had no significant effect [84].…”

“…Transmission microscopy and in silico modeling revealed that double Aβ 42 glycation at Lys16 and Lys28 reduced free energy change and destabilized fibril structures, thereby increasing the rate of Aβ 42 aggregation [84]. They also showed that a single glycation at Lys16 slowed down fibril formation, whereas a single glycation at Lys28 had no significant effect [84]. These results suggest that double-glycated (Lys16 and Lys28) and single-glycated (Lys16) Aβ 42 tended to maintain a low rate of fibrils and a high rate of oligomers, whereas single-glycated (Lys28) and non-glycated Aβ 42 had a high rate of fibrils and a low rate of oligomers [84].…”

“…They also showed that a single glycation at Lys16 slowed down fibril formation, whereas a single glycation at Lys28 had no significant effect [84]. These results suggest that double-glycated (Lys16 and Lys28) and single-glycated (Lys16) Aβ 42 tended to maintain a low rate of fibrils and a high rate of oligomers, whereas single-glycated (Lys28) and non-glycated Aβ 42 had a high rate of fibrils and a low rate of oligomers [84]. Non-glycated and single-glycated Aβ 42 exhibited cytotoxic effects on the retinoic-acid-differentiated human neuroblastoma cell line SH-SY5Y, which was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay [84].…”