Objective: Utilize cyclic amplification methods to facilitate detection of aggregation prone forms of a-syn from submandibular or esophageal tissue of patients with syncleinopathies.
Background: In synucleinopathies such as Parkinson’s disease, phosphorylated alpha-synuclein (a-syn) has been detected by immunohistochemistry in remote sites including peripheral nerves of the skin and in the gastrointestinal tract. More recently, cyclic amplification assays have been developed including protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC) which have demonstrated the ability to amplify a-syn aggregated forms from CSF and brain tissue. Detection of aggregation-prone forms of a-syn from submandibular gland or esophagus may be useful for diagnostic and treatment purposes.
Method: PD (n=6) and MSA (n=4) and control (n=4) tissue from post-mortem submandibular gland and esophagus was obtained through the Banner Health Bank. Tissue was homogenized and serially-extracted through buffer and detergent. Immunoblot for the presence of a-syn/phosphorylated a-syn was performed. Resultant samples underwent amplification via QuIC. Aliquots were tested via lipofectamine-induced transduction into HEK cells overexpressing a-syn-A53T-CFP/YFP  and evaluated via confocal microscopy.
Results: a-Syn was detected via immunoblot from tissue extract, but phosphorylated forms were not seen. Direct incubation of tissue extract yielded minimal seeding activity (just one positive MSA submandibular sample) in a-syn-A53T-CFP/YPF cells . Immunoprecipitation of tissue extract with an antibody for a-syn and subsequent amplification produced aggregate positivity on exposure to cells in approximately 50% of the PD and MSA samples, both esophageal and submandibular. There was more robust detection in insoluble fractions than soluble extract.
Conclusion: Likely due to small amounts of aggregation-prone a-syn present in these tissues (as compared to brain), a-syn seeding activity was inconsistently detected in PD and MSA submandibular and esophageal extracts. However, these results suggest that aggregation-prone forms of a-syn are present in these tissues, and further optimization of the assay and extraction method may boost detection.
References:  Yamasaki, T.R., Holmes, B.B., Furman, J.L. Dhavale, D.D., Su, B.W., Song E.S., Cairns, N.J., Kotzbauer, P.T., and Diamond M.I. (2019). Parkinson’s disease and multiple system atrophy have distinct alpha-synuclein seed characteristics. Journal of Biological Chemistry 18:294(3) 1045-1058. PMCID: PMC6341389
To cite this abstract in AMA style:T. Yamasaki, T. Sudhakar, E. Ostrakhovitch, E. Song. Detection of alpha-synuclein aggregation from submandibular and esophageal tissue of patients with synucleinopathies [abstract]. Mov Disord. 2020; 35 (suppl 1). https://www.mdsabstracts.org/abstract/detection-of-alpha-synuclein-aggregation-from-submandibular-and-esophageal-tissue-of-patients-with-synucleinopathies/. Accessed December 7, 2023.
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