Impact of alternating amino acid sequences on beta-amyloid-induced neurotoxicity and neuroinflammation in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative disease and the common cause of dementia. The aggregation of beta-amyloid (Aβ peptide) leading to excessive neuroinflammation is considered to be the neuropathological hallmark of AD, although the precise mechanisms remain unclear. Oligomerization of these peptides may be associated with their 42 amino acid residue arrangement. However, the process of amyloid plaque formation is still not well known. The protein folding-shape code (PFSC) method is a powerful tool to analyze protein confirmation which could exhibit the local structural folding features in detail. In our study, we utilized the PFSC to analyze Aβ peptide in humans and mice and found that mouse Aβ42 is less likely to polymerize than human's. Subsequently, we used the PFSC method to analyze the 42 amino acids of Aβ, transformed some species in human Aβ42 and obtained 7 mutants. We showed that it was not easy to aggregate Aβ in mutants. Herein, inflammatory responses were decreased, as indicated by the expression of cytokines. We confirmed that the neurotoxicity of mutant human Aβ was decreased by preventing peptide aggregation. This may represent a new therapeutic approach for treating AD.

Keywords: Alzheimer’s disease; alternating amino; beta-amyloid; neuroinflammation; neurotoxicity.

Figure 1

(A) The protein conformation fingerprint change matrix of the amino acid sequence of amyloid protein in mice and humans, (B) The protein conformation fingerprint change curve of the amino acid sequence of amyloid protein in mice and humans,(C) The amyloid protein oligomers in mice and humans, (D) The percentage area occupied by microglia in wild-type (n = 6) and AD (n = 8) samples (^**P < 0.01), (E) The cell bodies of microglia stained with Iba1 antibodies (green) in wild–type and transgenic mice as shown by confocal microscopy. Scale bar = 200 μm for left and 20 μm for right pictures.

Figure 2

(A) The expression of interferon-γ (IFNγ), IL-6, interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) in the hippocampus and cortex in 3-month-old 5XFAD and wild-type mice (n = 4~8), (B) The expression of IFN-γ and IL-1β in 6-month-old 5XFAD and wild-type mice (n = 7~8), (C) The expression of inflammatory cytokines (IL-2, IL-13, IL-10, TNFα, IL-1b, IL-6, MCP-1, IL-17A, IL-1a) in the serum of 8-month-old 5XFAD and wild-type mice (n = 3) (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 3

(A) The expression of IL-1β and IL-6 after 2 days of stimulation, (B) The expression of IL-6 after 1 hour of stimulation, (C) The secretion of IL-6 in the culture medium, and (D) The inflammatory responses in microglia stimulated by monomers compared with human Aβ oligomers (^*P < 0.05).

Figure 4

The protein conformation fingerprint change curves of unmutated compared with seven mutated amino acid sequences. Five carbon atoms were chosen as a universal folder and all possible folding shapes of local folding variations for all permutation were collected by PFSC method.

Figure 5

(A) Western blot analysis of non-mutants and mutants, (B) The expression of IL-6 and TNFα in response to non-mutants and mutants, (C, D) The inflammatory response of microglial cells to non-mutant and mutants from C57BL/6 mice and bv2 cells respectively, and (E, F) The concentration of IL-6 in RAW 264.7 cells and TNFα in bv2 cells in response to non-mutant and mutants, as determined by ELISA kits.