Effects Of Secondary Metabolite Extract From Nodulisporium Sp. On Aβ42 Aggregation Under Different Conditions

Alzheimer’s disease(AD) is one of the neurodegenerative diseases. There are several pathogenesis mechanisms for AD and β-amyloid cascade hypothesis is important one. Based on the hypothesis, β-amyloid peptides consist of 38~43 amino acid residues from amyloid precursor protein(APP) and aggregate to oligomers and fibrils which would result in synaptic damage and inflammatory to cause Alzheimer’s disease. Aβ42 oligomers are the primary toxic species to neuronal cells.Therefore, inhibiting and clearing Aβ42 oligomers are the most useful strategies for preventing Alzheimer’s disease.Thioflavin T(Th T) assay, SDS-PAGE and atomic force microscopy(AFM) was utilized to study the Aβ42 aggregation procedure. Aβ42 aggregated into dimer, tetramer and high polymer in 10 m M PBS(p H=7.4) solution at 25℃ and the fibril was chiefly induced in 10 m M HCl(p H2.0) solution at 37℃. Th T assay is convenient and sensitive to apply to high throughput screening of active compounds to inhibit Aβ42 aggregation. However, it was not able to distinguish Aβ42 monomer, dimer, tetramer, high polymer and fibril in solution. The different Aβ42 aggregates were separated and quantified by SDS-PAGE which was an important supplement to the Th T assay. The height, dimensional size and area of Aβ42 aggregates were determined by AFM. Therefore, the three experimental techniques were able to reveal comprehensively the variation tendency of Aβ42 aggregation, and provided useful methods to study Aβ42 aggregation in vitro.In the previous study, two 4-methyl-progesteroids,Demethoxyviridin(Dmv) and Inoterpene B(Inp B),were isolated from the extract of an endolichenic fungal strain Nodulisporium sp.(No. 65-17-2-1). Their Aβ42 aggregation inhibitory activity was evaluated by standard Th T assay with EGCG as a positive control. Dmv displayed anti-Aβ42 aggregation activity with IC50 value of 13.4μM while Inp B showed low inhibitory activity. In this study, we further investigated the effect of Dmv and Inp B on Aβ42 oligomerization by using SDS-PAGE and AFM. Dmv was able to depress the Th T fluorescence of Aβ42 aggregates in 10 m M PBS solution at 25℃. Aβ42 monomer, dimer, tetramer and high polymer were decreased while dyeing substances gradually increased in the samples loading well of the stacking-gel. Meanwhile, AFM revealed that the amount of amorphous aggregates over 8nm height increased significantly. Compared to Dmv, Inp B with similar structure had hardly effect on Aβ42 oligomerization. Therefore, Dmv was able to accelerates Aβ42 monomer, dimer, tetramer, high polymer to form amorphous aggregates in 10 m M PBS solution at 25℃.The effects of Dmv and Inp B on Aβ42 fibrillization were also investigated. In 10 m M HCl(p H2.0) solution at 37℃, Dmv debased the Th T fluorescence of Aβ42 aggregation. SDS-PAGE revealed that Aβ42 monomer, dimer, tetramer were decreased while high polymer increased slightly, but there was not significant increasing of the dyeing substances in the loading wells. AFM result also showed Dmv didn’t change the fibril’s length and quantity. In pure water at 37℃, Dmv debased the Th T fluorescence of Aβ42 aggregation too; SDS-PAGE showed that Aβ42 monomer, dimer, tetramer were decreased and high polymer increased at first and then decreased, but the amount of dyeing substances increased gradually. A lot of short fibrils and amorphous aggregates were observed by AFM. Therefore, Dmv was able to promote Aβ42 oligomers to form amorphous aggregates and short fibrils in pure water at 37℃.In summary, Dmv was able to promote cytotoxic Aβ42 oligomers to form amorphous aggregates or short fibrils and may be a potential candidate to prevent Alzheimer’s disease.