%0 Online Multimedia %A Y., Huang %A Z., Liu %A B.-B., Cao %A Y.-H., Qiu %A Y.-P., Peng %D 2017 %T Supplementary Material for: Treg Cells Protect Dopaminergic Neurons against MPP+ Neurotoxicity via CD47-SIRPA Interaction %U https://karger.figshare.com/articles/media/Supplementary_Material_for_Treg_Cells_Protect_Dopaminergic_Neurons_against_MPP_Neurotoxicity_via_CD47-SIRPA_Interaction/4729801 %R 10.6084/m9.figshare.4729801.v1 %2 https://karger.figshare.com/ndownloader/files/7721299 %K Regulatory T cells %K Parkinson's disease %K Dopaminergic neurons %K CD47 %K Signal regulatory protein α %X Background/Aims: Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). Methods: Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-β1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. Results: Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-β1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. Conclusion: Treg cells protect dopaminergic neurons against MPP+neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation. %I Karger Publishers