Figure 5

Sertraline inhibits the neuron-to-neuron and neuron-to-oligodendroglia transmission of α -synuclein in co-culture models. A, SH-SY5Y cells overexpressing mCherry or N-terminally mCherry-tagged αSYN (donor cells) were co-cultured with PC12 neuronal or MO3.13 oligodendroglial cells stably expressing EGFP (acceptor cells) in the presence or absence of 10 μM sertraline. Vehicle-only-transfected cells were treated with solvent (0.1% DMSO) only. The mCherry fluorophore was used to trace the donor-derived αSYN protein. After 72 hours of co-culture, the transmission of mCherry-αSYN from the donor cells to the acceptor PC12 as well as MO3.13 cells was detected, confirming the uptake of mCherry-αSYN secreted from the donor cells. The arrowhead indicates the transferred mCherry-αSYN-positive aggregates in the acceptor cells. The crossed lines indicate the positions of the xz- and yz-planes. B, A co-culture experiment using donor cells expressing mCherry-αSYN showed that the percentage of acceptor cells with inclusions were 4.2% (PC12) and 3.8% (MO3.13), which is much higher than the percentage of inclusion-positive cells (under 0.2% in both cell lines) co-cultured with mCherry-expressing donor cells. The addition of sertraline to the culture medium reduced the incorporation of mCherry-αSYN into the acceptor cells (*p < 0.05), whereas this effect was not observed in the acceptor cells that were co-cultured with the mCherry-expressing donor cells. To quantify the mCherry-positive inclusions in the acceptor cells, the total number of cells containing aggregates was counted per 250–300 cells within five randomly selected fields. From this value, the percentages of cells with red-fluorescent inclusions were calculated. Pooled data from four independent experiments were statistically analyzed. Data are presented as the mean ± standard errors. C, Western blot analysis demonstrated that sertraline treatment did not affect the secretion of the mCherry-αSYN from the donor SH-SY5Y cells. BSA and Hsp90 were used as markers of the culture medium and the cytosolic proteins, respectively. Each immunoblot was performed at least three times and the replicates yielded similar results.