Recently, propagation of α-synuclein is of interest within the field of synucleinopathies, including Parkinson;;s disease, dementia with Lewy bodies, and multiple system atrophy. However, little is known about membrane proteins which influence cellular uptake in the neuronal propagation of α-synuclein. Thus, to identify those membrane proteins, I have established a cell-based assay and screened membrane proteins. α-Synuclein was purified from E. coli and incubated in vitro for 21 days with sonication. Fibril forms of α-synuclein aggregates were confirmed by transmission electron microscope and labeled with Alexa Fluor 488 fluorescent dye. Cellular uptake of Alexa Flour 488-labeled α-synuclein aggregates in SH-SY5Y cells was diminished by the treatment with hypertonic sucrose for inhibiting clathrin-mediated endocytosis, and 5-(N-Ethyl-N-isopropyl) amiloride for inhibiting macropinocytosis, supporting that some membrane proteins may regulate cellular uptake of α-synuclein aggregates. The cDNA expression library that encodes thousands of membrane proteins was prepared. SH-SY5Y cells grown on multi-well tissue culture plate were transfected with each of those cDNA and then exposed to α-synuclein aggregates. Nine putative cDNA clones were isolated to stimulate cellular uptake of α-synuclein aggregates. Among them, ectopic expression of STM1D9 or MTM7A8 was effective to stimulate cellular uptake of α-synuclein aggregates. On the other hand, overexpression of STM1D9 and MTM7A8 did not affect cellular uptake of tau aggregates and amyloid β oligomers. Moreover, ectopic expression of STM1D9 or MTM7A8 significantly increased cell-to-cell transmission of Venus1-α-synuclein and α-synuclein-Venus2 inSH-SY5Y/BiFC system. Conversely, knockdown of STM1D9 and MTM7A8 expression by sgRNA reduced cellular uptake of α-synuclein aggregates. These results suggest that STM1D9 and MTM7A8 might play a role in cellular uptake of α-synuclein aggregates during neuronal transmission.
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