EGCG Inhibited Lipofuscin Formation Based on Intercepting Amyloidogenic β-Sheet-Rich Structure Conversion

Abstract

Background: Lipofuscin (LF) is formed during lipid peroxidation and sugar glycosylation by carbonyl-amino crosslinks with biomacrolecules, and accumulates slowly within postmitotic cells. The environmental pollution, modern dietary culture and lifestyle changes have been found to be the major sources of reactive carbonyl compounds in vivo. Irreversible carbonyl-amino crosslinks induced by carbonyl stress are essentially toxiferous for aging-related functional losses in modern society. Results show that (-)-epigallocatechin gallate (EGCG), the main polyphenol in green tea, can neutralize the carbonyl-amino cross-linking reaction and inhibit LF formation, but the underlying mechanism is unknown.

Methods and results: We explored the mechanism of the neutralization process from protein, cell, and animal levels using spectrofluorometry, infrared spectroscopy, conformation antibodies, and electron microscopy. LF demonstrated an amyloidogenic β-sheet-rich with antiparallel structure, which accelerated the carbonyl-amino crosslinks formation and disrupted proteolysis in both PC12 cells and D-galactose (D-gal)-induced brain aging mice models. Additionally, EGCG effectively inhibited the formation of the amyloidogenic β-sheet-rich structure of LF, and prevented its conversion into toxic and on-pathway aggregation intermediates, thereby cutting off the carbonyl-amino crosslinks.

Conclusions: Our study indicated that the amyloidogenic β-sheet structure of LF may be the core driving force for carbonyl-amino crosslinks further formation, which mediates the formation of amyloid fibrils from native state of biomacrolecules. That EGCG exhibits anti-amyloidogenic β-sheet-rich structure properties to prevent the LF formation represents a novel strategy to impede the development of degenerative processes caused by ageing or stress-induced premature senescence in modern environments.

Fig 1. (-)-Epigallocatechin gallate (EGCG) prevented β-sheet-rich amyloidogenesis of lipofuscin (LF).

(A) Effect of EGCG on LF formation as measured by emission of LF-like fluorescence intensity at 460 nm in MDA-modified HSA artificial LF (1 mg/mL) reaction system. (B) Effect of EGCG on LF β-sheet-rich structure formation by measuring Thioflavin T (ThT) fluorescence emission at 485 nm. (C) Analysis of sample aggregation reactions by transmission electron microscopy (TEM) in different groups after 96 h of incubation. Scale bars represent 100 nm. Data are expressed as means ± SD, n = 3.

Fig 2. FTIR analysis of lipofuscin (LF) β-sheet-rich amyloidogenesis structure.

(A) The FTIR spectra of the protein products in different treatment groups. (B-D) The peaks from a Gaussian curve fitting of the FTIR spectra of the amide I band (normalization processing) of protein products in different treatment groups. Sparse shading is β-sheet structure (1625 to 1640 cm^-1), and dense shading is antiparallel β-sheet/aggregated strands structure (1675 to 1695 cm^-1). LF and LF+EGCG represent LF and EGCG (300 μM) treated LF groups after 96 h of incubation, respectively.

Fig 3. (-)-Epigallocatechin gallate (EGCG) stimulated assembly of nontoxic, off-pathway oligomers.

(A) Detection of amyloid oligomers by A11 antibody and fiber-specific OC antibody in HSA, lipofuscin (LF) or EGCG LF oligomers. EGCG LF oligomers were produced with 100 or 300 μM concentrations of EGCG for 96 h at 37°C. (B) Western blot detection of ubiquitin-conjugated proteins (UBI), A11, and p62 expression of PC12 cell which incubated with HSA, LF or EGCG LF oligomers (100 or 300 μg/mL) for 48 h. (C) The inhibitory effect of EGCG (10 or 20 μg/kg/d) on the formation of carbonylated proteins in the brain aging cortex. (D) Western blot detection of A11 and p62 expression from the brain cortex lysate of different treatments. (E) Comparison of LF and mitochondria of cortical neurons in different treatment groups. ^##P<0.01 represents the statistical significance vs. control group; **P<0.01 represents statistical significance vs. D-gal-treated group, n = 3.

Fig 4. Working models of effects of (-)-Epigallocatechin gallate (EGCG) on lipofuscin (LF) amyloidogenesis.

Native proteins tend to aggregate to form β-sheet-rich amyloidogenesis structures (A11-positive and OC-positive) by carbonyl-amino crosslinking under carbonyl stress. The carbonyl-amino crosslinks and misfolded β-sheet-rich amyloid structures activated each other, a pathological feedback loop resulting in LF formation and accumulation which continuously supplied by native protein as raw material. EGCG prevented β-sheet-rich amyloidogenesis formation by inducing off-pathway oligomers protein modifications, in this way to inhibit LF formation.