Transthyretin Anti-Amyloidogenic and Fibril Disrupting Activities of Bacopa monnieri (L.) Wettst (Brahmi) Extract

Abstract

The homotetrameric plasma protein transthyretin (TTR), is responsible for a series of debilitating and often fatal disorders in humans known as transthyretin amyloidosis. Currently, there is no cure for TTR amyloidosis and treatment options are rare. Thus, the identification and development of effective and safe therapeutic agents remain a research imperative. The objective of this study was to determine the effectiveness of Bacopa monnieri extract (BME) in the modulation of TTR amyloidogenesis and disruption of preformed fibrils. Using aggregation assays and transmission electron microscopy, it was found that BME abrogated the formation of human TTR aggregates and mature fibrils but did not dis-aggregate pre-formed fibrils. Through acid-mediated and urea-mediated denaturation assays, it was revealed that BME mitigated the dissociation of folded human TTR and L55P TTR into monomers. ANS binding and glutaraldehyde cross-linking assays showed that BME binds at the thyroxine-binding site and possibly enhanced the quaternary structural stability of native TTR. Together, our results suggest that BME bioactives prevented the formation of TTR fibrils by attenuating the disassembly of tetramers into monomers. These findings open up the possibility of further exploration of BME as a potential resource of valuable anti-TTR amyloidosis therapeutic ingredients.

Keywords: Bacopa monnieri; amyloidosis; antioxidants; bioactive compounds; degenerative diseases; fibrillation; transthyretin.

Figure 1

The effect of BME on human TTR aggregation and fibril formation. TEM images of human TTR subjected to acid-mediated aggregation assay in the absence (A) or presence (B) of BME after 14 days of incubation at 37 °C.

Figure 2

The effect of BME on native TTR dissociation under urea mediated denaturation stress. Bar charts (A,B) represent folded human TTR (hTTR) and L55P TTR, respectively, in percentage left after 72 h of incubation in the presence or absence of BME. Images (C,D) are representative of Tricine-SDS-PAGE images of human TTR and L55P TTR, respectively, after denaturation.

Figure 3

(A) Quantitative analysis of the effect of BME on human TTR (hTTR) tetrameric resistance to moderate acidic denaturation conditions. (B) Representative Tricine-SDS-PAGE gel image of human TTR (hTTR) resistance to acid mediated denaturation without (Lane 3) or with (Lane 4) BME. Lane 1—protein molecular weight marker.

Figure 4

The effect of BME on human TTR (hTTR) quaternary structural stability and its dose-dependent effect. Representative Tricine-SDS-PAGE gel images of glutaraldehyde cross-linked protein samples with or without BME after acid-mediated denaturation (A) and urea-mediated denaturation (B) assays. (C) Quantitative analysis of dose-dependent activity of BME on urea-mediated denaturation of human TTR. (D) Representative Tricine-SDS-PAGE gel image of the effect of increasing BME amounts on human TTR tetramer stability. M—molecular weight marker; 1—human TTR in DMSO, without urea; 2—human TTR in DMSO, 6M urea; 3—human TTR in 1 µg/µL BME, 6M urea; 4—human TTR in 5µg/µL BME, 6M urea; 5—human TTR in 10µg/µL BME, 6M urea; 6—10µg/µL curcumin, 6M urea.

Figure 5

Images of pre-formed human TTR amyloid fibrils in the absence (A) or presence of BME (B) after incubation for 24 h. (C) Tricine-SDS-PAGE gel representation of human TTR fibril disruption activity of BME. Lane 1—protein molecular weight marker; Lane 2—human TTR pre-formed fibrils without BME; Lane 3—human TTR pre-formed fibrils with BME.

Figure 6

Human TTR-ANS fluorescence intensity without or with increasing concentrations of BME for the determination of ANS displacement from human TTR T4-binding sites by BME.