Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay

Abstract

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.

Small soluble α-synuclein aggregates are the toxic species in Parkinson’s disease

“…While many previous studies characterizing α-synuclein aggregates by size have been conducted in vitro, more research remains to be done on identifying the soluble aggregates present in the human brain. Endogenous levels of α-synuclein aggregates were measured in the substantia nigra in control and PD post-mortem tissue by Je et al at the single-molecule level 42 . They reported that the oligomeric species accounted for 56% of the α-synuclein species in the PD samples but only 23% in non-PD control samples.…”

Single molecule imaging of protein aggregation in Dementia: Methods, insights and prospects

“…(e) oligomers and fibrils of amyloid aggregates super resolved using DNA-PAINT (reproduced with permission from ( Whiten et al, 2018a )). (f) SiMPull of αS aggregates in postmortem brain punches from patients with and without PD (reproduced with permission from ( Je et al, 2017 )). …”

“…We have previously discussed SiMPull as a simple, yet powerful, tool for measuring the composition of protein complexes with single molecule precision. In a recent study, αS SiMPull was applied to post mortem brains of humans diagnosed with PD as well as healthy controls to image endogenous αS aggregates ( Je et al, 2017 ). To make these measurements, aggregates were sandwiched between 2 antibody systems: a primary and secondary capture antibodies which hook the aggregates to a Polyethylene glycol (PEG) coated coverslip and, another, primary and secondary detection antibodies of which the latter is conjugated to a synthetic fluorophore as a reporter.…”

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

“…SiMPull can offer a potential means to clarify the behavior of these proteins while ensemble methods such as SDS‐PAGE or gel‐filtration can yield controversial results (Burré et al, ). By analyzing the intensity of captured single‐molecule spots, SiMPull performed on lysate solutions from human brain tissue showed higher levels of both overall α‐SYN and oligomeric α‐SYN in PD patients (Je et al, ). This method is applicable to diverse biospecimens including cerebrospinal fluid, blood and tear fluid.…”

“…Recently, SiMPull has further extended its application areas to neurodegenerative diseases (Je et al, 2017). Parkinson's disease (PD) and Alzheimer's disease are notoriously difficult to diagnose without a postmortem brain biopsy, and the proteins involved in their pathogenesis have been challenging to study.…”