Abstract
Parkinson's disease (PD) is the second most common neurodegenerative disease, but molecular mechanisms underlying PD pathogenesis remain unclear. Alpha-synuclein (α-SYN) and its oligomeric species are implicated as a key player in Parkinson's disease. Thus, the determination of α-SYN expression levels and its oligomerization states is crucial to understand PD. However, it has been challenging to analyze them quantitatively because of the limited amount of specimens and the lack of techniques that are insensitive to the size and conformation of α-SYN oligomers. Here, we demonstrated quantitative analysis of endogenous α-SYN taken from postmortem human brain tissue using a single-molecule pull-down (SiMPull) assay. The SiMPull assay probes immunoprecipitated proteins, which are then tagged with a fluorescent molecule for imaging and detection. By utilizing in vivo crosslinking, we preserved the native oligomerization state of the α-SYN proteins. Our results showed that the tissue lysates from the substantia nigra of a human PD brain showed 3.3-fold higher number of α-SYN molecules and 2.4-fold higher oligomeric population compared to a healthy brain. Our technique is a powerful diagnostic tool for the detection of various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, and can be used to analyze various biospecimens not limited to post mortem brain tissue.
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