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. 2019 May 31;294(22):8806-8818.
doi: 10.1074/jbc.RA118.006846. Epub 2019 Apr 17.

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

Jin Ng  1   2 , Harveen Kaur  3   4 , Thomas Collier  5   6 , Kevin Chang  7 , Anna E S Brooks  3   6 , Jane R Allison  3   6 , Margaret A Brimble  3   2   4   6 , Anthony Hickey  8 , Nigel P Birch  3   2
Affiliations

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

Jin Ng et al. J Biol Chem. .

Abstract

Aβ1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). Nϵ-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of Aβ1-42. Methyglyoxal is commonly used for the unspecific glycation of Aβ1-42, 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 Aβ1-42 at Lys-16 (Aβ-CEL16), Lys-28 (Aβ-CEL28), and Lys-16 and -28 (Aβ-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of Aβ1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that Aβ-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 Aβ1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of Aβ1-42 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 Aβ-CEL16 and Aβ-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas Aβ-CEL16&28 showed no neurotoxicity. Interestingly, Aβ-CEL16&28 had depolarized the mitochondrial membrane potential, whereas Aβ-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 Aβ1-42.

Keywords: Alzheimer disease; N-ϵ-(carboxyethyl)lysine (CEL); advanced glycated end product (AGE); amyloid-β (Aβ); fibril; glycation; mitochondria; neurodegeneration; protein aggregation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Double-CEL modifications at Lys-16 and Lys-28 differentially affect Aβ1–42 aggregation over 5 days. A, chemical structure of CEL. B, peptide sequences of Aβ1–42 and the three CEL variants on Aβ1–42. C–F, Aβ-CEL16&28 do not form extensive fibrils and have a high abundance of protofibrils and oligomers after 5 days in a fibril-promoting buffer relative to all CEL-glycated peptides and unglycated Aβ1–42. Representative TEM micrographs at day 5 (left panel) (n = 2–3; scale bar = 100 nm) with the graph of % aggregate species (right panel) showing proportions of fibrils, protofibrils, and oligomers over 5 days. *, p < 0.05. p values for oligomers and protofibrils are relative to day 0 and fibrils are relative to day 1 using Bonferroni's post hoc test in a one-way analysis of variance (ANOVA). See Fig. S1 for synthesis of peptide and Fig. S2 for examples of fibrils, protofibrils, and oligomers.
Figure 2.
Figure 2.
Double-CEL modifications at Lys-16 and Lys-28 decrease peptide stability and rate of fibril aggregation without affecting peptide secondary structure. A, Aβ-CEL16&28 has the least negative free energy for binding of a model monomer to a pentameric stack relative to other CEL-glycated peptides and unglycated Aβ1–42. Free energies of binding were computed using umbrella sampling with reported uncertainties determined through bootstrap analysis. B, Aβ-CEL16&28 has the slowest rate of fibril aggregation and Aβ-CEL28 has a higher threshold for maximum rate of fibril aggregation (>3 μm) relative to the CEL-glycated peptides and unglycated Aβ1–42. The rate of fibril aggregation was determined using the aggregation kinetics generated using the ThT assay. The log half-time (t½) is plotted against log starting monomer concentration (micromolars). A plateau at high starting monomer concentrations indicate a saturated (maximum) rate of fibril aggregation. Fitted lines are from 2 to 3 independent experiments and the numeric gradient of each fitted line is displayed. C, Aβ-CEL16&28 has an unchanged secondary structure profile relative to Aβ-CEL28 and Aβ1–42, however, Aβ-CEL16 has a decrease in β-sheet content with an increase in other secondary structures. Peptide secondary structure was estimated using CD (inset) and the spectra deconvoluted using BeStSel. Results are presented as mean ± S.E. (n = 2–3). *, p < 0.05 calculated using Bonferroni's post hoc test in a one-way ANOVA. See Fig. S3 for potential of mean force curves for free energy release. See Fig. S4 for aggregation kinetics of the ThT assay.
Figure 3.
Figure 3.
Single-CEL modifications decrease MTT reduction independent of a change in basal levels of necrosis and apoptosis. All experiments performed after 24 h of 10 μm peptide treatment in RA-differentiated SH-SY5Y human neuroblastoma cells. A, Aβ1–42, Aβ-CEL16, and Aβ-CEL28 inhibit MTT reduction, however, Aβ-CEL16&28 does not inhibit MTT reduction. MTT reduction was measured after 24 h of 10 μm peptide treatment. Results are presented as mean ± S.E. (n = 3–4). B and C, no significant difference in LDH release and caspase 3/7 activity was observed for all peptide treatments relative to the vehicle (Veh). Results are presented as mean ± S.E. (n = 4). *, p < 0.05 relative to the vehicle. p value for all experiments were calculated using Bonferroni's post hoc test in a one-way ANOVA from randomized complete block design.
Figure 4.
Figure 4.
Double-CEL modifications at Lys-16 and Lys-28 depolarizes ΔΨm independent of mitochondrial swelling and mitochondrial O2˙̄ production. All experiments performed by flow cytometry after 24 h of 10 μm peptide treatment in RA-differentiated SH-SY5Y human neuroblastoma cells. A, Aβ-CEL16&28 as a decrease in cell population of polarized ΔΨm. ΔΨm was assessed by JC-10. Results are presented as mean ± S.E. (n = 3) with representative flow cytometry plots available in Fig. S5. B, treatment with unglycated Aβ1–42 increased mitochondrial swelling relative to Aβ-CEL16 and the vehicle (Veh). This Aβ1–42 induced mitochondrial swelling is marginally different from Aβ-CEL28 (p = 0.07) and Aβ-CEL16&28 (p = 0.06). Mitochondrial volume was assessed by MitoTracker Green. Results are presented as mean ± S.E. (n = 4) with representative flow cytometry plots in the corresponding histogram (right). A shift of the histogram toward the right indicates an increase in MitoTracker Green within the cell. C, treatment with unglycated Aβ1–42 has increased mitochondrial O2˙̄ production relative to all peptides and the vehicle. Mitochondrial O2˙̄ production was assessed by MitoSOX. Results are presented as mean ± S.E. (n = 3–4) with representative flow cytometry plots shown in Fig. S5. *, p < 0.05; **, p < 0.01. p values were calculated in a one-way ANOVA using a completely randomized block design blocked by different experiments. See Fig. S5 for flow cytometry gating strategy.
Figure 5.
Figure 5.
CEL modification at Lys-16 increases mitochondrial respiration at complex II and increases maximum respiration. All experiments were performed by oximetry after 24 h of 10 μm peptide treatment in RA-differentiated SH-SY5Y human neuroblastoma cells. Aβ-CEL16 increases mitochondrial respiration at CII and CI+CII under OXPHOS and CI+CII under uncoupled (maximum) respiration. Aβ1–42 decreases mitochondrial respiration at CI under OXPHOS and CI+CII under uncoupled respiration. Shown here is oxygen consumption from intact cells (endogenous) and mitochondria of permeabilized cells at different states of respiration. The two main states of respiration are coupled respiration (OXPHOS) and uncoupled (maximum) respiration. Results are presented as mean ± S.E. (n = 5); CIV (n = 4). *, p < 0.05 relative to vehicle. p value was calculated using Bonferroni's post hoc test in a one-way ANOVA from randomized complete block design. See Fig. S6 for an example of oxygen consumption trace and the mitochondrial substrates added.
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