Fig. 5.

miR-221 prevents MPP⁺-induced neurite retraction and cell loss in DJ-1 KD ReNcell VM dopaminergic neurons. ReNcell NPCs were made to stably express either control non-targeting shRNA or pooled DJ-1 shRNA, then differentiated into post-mitotic dopaminergic neurons. (A) Immunoblotting shows that DJ-1 is knocked down effectively by DJ-1 shRNA. (B) Quantification of (A). (C) As seen previously in SH-SY5Y cells, DJ-1 KD decreases miR-221 levels in differentiated ReNcell VM neurons. (D) Control KD (Ctrl KD) or DJ-1 KD cells were transduced with either lenti-Ctrl miR or lenti-miR-221 and exposed to 0.5 mM MPP⁺ or vehicle (DMEM) for 24 h. MPP⁺ exposure resulted in cell loss and neuritic retraction (Scale bar: 100 µm). (E) Measurements of neurite length indicated that miR-221 protects DJ-1 KD neurites from MPP⁺. (F) Compared to control KD cells, long neurites (≥ 80 µm, longer than 75th percentile of all neurites) were less preserved among DJ-1 KD cells treated with MPP⁺. This effect was reversed when DJ-1 KD cells were made to over-express miR-221. (G) MPP⁺ treatment significantly decreased the cell count in Ctrl miR transduced neurons, whereas miR-221 transduced neurons showed a smaller, non-significant decrease in cell count. For (E, G), n = 450 neurites per group, representative of length measurements taken from 15 microscopic fields per experimental group across three biological replicates. For (F), mean values of percent neurites ≥ 80 µm long were generated with bootstrap resampling, where each sample run contained n = 100 neurites per group. Resampling runs were repeated 500 times to generate confidence intervals for mean values of percent neurites ≥ 80 µm. For (G), Average cell count was assessed by counting DAPI-positive nuclei in these fields. For (B, C, E, G), data are presented as means ± S.E.M. For (F), data are presented as means ± Confidence Interval. Asterisks denote statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) relative to control. ns = not significant. (B, C) analyzed using two-tailed student's t-test. (E, F, G) Analyzed using one-way ANOVA with Bonferroni post-hoc multiple comparisons test.