Liquid-liquid Phase Separation Of Alzheimer’s Disease Associated β-amyloid Peptide

Alzheimer’s disease(AD)is a typical neurodegenerative disease and one of the most famous dementia.It is characterized by mental impairment,including memory loss,language ability,spatial discrimination,and cognitive decline.The two pathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular amyloid plaques.Neurofibrillary tangles are caused by the hyperphosphorylation of Tau protein,while amyloid plaque deposition is caused by the misfolding and aberrant aggregation of β-Amyloid peptide(Aβ).Amyloid proteins is prone to aggregate from disordered monomeric state into highly ordered amyloid fibrils and this process is known as protein misfolding and aberrant aggregation.There are several hypotheses about the causes of AD.However,a generally accepted hypothesis has not been settled on.Amyloid hypothesis proposes that the amyloid formation of Aβ,leading to the excessive phosphorylation of Tau protein,and further causing the loss of neuronal function is the main pathogenesis of AD.Amyloid hypothesis not only explains the link between these two pathological features,but also illustrates the development and progression of the disease.Therefore,based on this hypothesis,the aggregation mechanism of Aβ and various types of aggregates formed during the aggregation have attracted much attention in related scientific and research fields.This project aims to the molecular mechanism of Aβ misfolding and aberrant aggregation.The aggregation process of Aβ from monomeric state to oligomeric state,and the final aggregates amyloid fibrils was investigated in combination of several biophysical and biochemical techniques.Due to the highly dynamic nature and structural heterogeneity of Aβ oligomers,a low concentration of sodium dodecyl sulfate(SDS)was used to stabilize the assembly of oligomers.The molecular weight,secondary structure and morphology of Aβ42 oligomer were characterized by protein electrophoresis,circular dichroism(CD),transmission electron microscopy(TEM).We also analyzed the chemical exchange between monomer and oligomers by using nuclear magnetic resonance(NMR).According to the results,Aβ42 tetramers were formed during the sample preparation and containing α-helix secondary structure.Liquid-liquid phase separation(LLPS)of biomacromolecules is an emerging hot research topic in the field of life science.Aberrant phase separation and phase transition of amyloid proteins are relevant to the pathogenesis of several neurodegenerative diseases.Through systematic screening and optimization by using confocal laser scanning microscope imaging system,the liquid droplets were formed of Aβ42 oligomers in vitro at the early stage of Aβ42 aggregation.The phase separation process is regulated by salt ions and correspond to Hofmeister Series,suggesting the hydrophobic interactions play a key role in LLPS of Aβ42oligomers.In addition,the destruction of 1,6-hexadiol into the droplets formed by Aβ42 oligomers further confirmed the important role of hydrophobic interaction in regulating the LLPS of Aβ42 oligomers.Hydrophobic interactions are also driving forces of amyloid aggregation and fibrillization.Previous studies have demonstrated that LLPS can accelerate the amyloid fibrillization.Therefore,the link between amyloid oligomerization,LLPS,and fibrillization was further investigated in this study.Combined with confocal laser scanning microscope,TEM,western blot and fluorescence spectroscopy,we found that oligomerization plays an important role in regulating the liquid-liquid phase separation of Aβ42,and LLPS will further accelerate the fibrillization of Aβ42.In addition,the main active and water-soluble component of green tea epigallocatechin gallate(EGCG),as a typical amyloid inhibitor,destroyed the droplets formation suggesting EGCG enables to modulate LLPS of Aβ42 oligomers and further demonstrates the relationship between phase separation and amyloid aggregation.We focused on the aggregation process of Aβ42 and performed the characterization on Aβ42 oligomers.The results showed that Aβ42 oligomers underwent LLPS in vivo.Furthermore,the role of oligomerization in LLPS of Aβ42 was analyzed.The aggregation of Aβ42 from monomer,oligomer,droplet to fibrils was analyzed to obtain better understanding of the molecular mechanism of amyloid aggregation,the relationship between the oligomerization LLPS and amyloid aggregation,further to provide insights into the pathogenesis research and treatment for AD.