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. 2019 Sep 13;13:399. doi: 10.3389/fncel.2019.00399

FIGURE 8.

FIGURE 8

Neurotoxicity of primary cultured SN dopaminergic neurons caused by miR-204-5p is mediated by the increased level of DYRK1A protein. (A) Cotransfection of shRNAs of DYRK1A or treatment of DYRK1A inhibitor harmine (1μM) attenuated miR-204-5p mimic-induced upregulation of α-Syn, phospho-α-Syn, tau, or phospho-tau in primary cultured dopaminergic neurons. (B) In the presence of DYRK1A shRNAs and DYRK1A inhibitor harmine, miR-204-5p mimic failed to upregulate the expression of ER stress-related proteins, including Grp78, IRE1α, CHOP, and active caspase-12, in primary cultured dopaminergic neurons. (C) The miR-204-5p mimic-induced decrease in protein expression of autophagy markers, including Beclin-1, Atg7, Atg16L1, and LC3-II/I ratio, in primary cultured dopaminergic neurons was prevented by cotransfection of shRNA of DYRK1A or treatment of DYRK1A inhibitor harmine. (D) Knockdown of DYRK1A expression or treatment of harmine in primary cultured dopaminergic neurons attenuated miR-204-5p mimic-induced activation of JNK, c-Jun, caspase-9, and caspase-3 in primary cultured SN dopaminergic neurons. (E) In the presence of DYRK1A shRNA or DYRK1A inhibitor harmine, the miR-204-5p mimic failed to significantly upregulate the mRNA level of Bim in primary cultured dopaminergic neurons. Each qRT-PCR experiment was performed in triplicate. (F) Cotransfection of DYRK1A shRNA or treatment of DYRK1A inhibitor harmine reversed miR-204-5p mimic-induced cell death of primary cultured dopaminergic neurons. For the assays of cell viability, each experiment was performed in triplicate. Each bar indicates the mean ± SEM value of four independent experiments. ∗p < 0.05, ∗∗p < 0.01 compared to scramble miR control (SC)-transfected primary cultured dopaminergic neurons.