Fig. 1: LUHMES-derived DA neuronal spheroids treated with MPP⁺ recapitulate α-synuclein upregulation and pathology.

A Working hypothesis. Our previous results indicate that ZSCAN21 increases the transcription of the SNCA gene, TRIM41 is an E3 ubiquitin-ligase for ZSCAN21 that induces its proteasomal degradation, and TRIM17 inhibits TRIM41-mediated ubiquitination and degradation of ZSCAN21. B A spheroid of LUHMES-derived DA neurons after 7 days of differentiation. The spheroid was cleared, stained with antibodies against synuclein (green), TH (red) and Hoechst 33342 (blue), and analysed by confocal microscopy coupled with a spinning disk (Dragonfly). The scale bar is 50 μm. C After 6 days of differentiation, LUMHES spheroids were treated or not with 2 μM or 5 μM MPP⁺ for 24 h. Then, total RNA was extracted, and the pre-mRNA and mRNA levels of SNCA were estimated by quantitative RT-PCR, using YWHAZ and TBP as reference genes. The graph shows mean ± SEM and individual results of five independent experiments. ^*Significantly different from non-treated cells (0) (one-way ANOVA followed by Dunnett’s multiple comparison test). D LUMHES spheroids were treated or not with 2 μM MPP⁺ for 24 h. ChIP assays were performed using an antibody against H3K27ac or rabbit IgG used as a negative control. Quantitative PCR was carried out on the immunoprecipitates and the input chromatin using primers specific for the promoter region and different enhancers. Data are expressed as the percentage of the input chromatin used for each immunoprecipitation and are the individual values and means ± SEM of four independent experiments. The ratios of enrichment in MPP⁺ vs control conditions with the H3K27ac antibody are indicated for each DNA sequence above the MPP⁺ columns. NB: the data are scattered because the enrichment rate is quite different from one ChIP experiment to another. However, in all experiments, a higher enrichment of all DNA sequences was seen in MPP⁺ vs control conditions, indicating a real reproducibility. Below the graph, a schematic representation of the human SNCA gene from the UCSC genome browser is shown. It depicts a single representative transcript (NCBI RefSeq NM_000345.4) and the epigenetic marks measured in ChIP-seq experiments on 7 cell lines from the ENCODE project. H3K27ac and H3K4me1 marks are often found near active regulatory elements, and H3K4me3 marks near promoters. The regulatory elements studied by ChIP are indicated. E LUMHES spheroids were treated or not with 2 μM MPP⁺ for 24 h. Then, total protein extracts were analysed by western blot using antibodies against α-synuclein, Ser129-phosphorylated α-synuclein and GAPDH. The intensity of the α-synuclein bands was quantified, normalised by the intensity of the GAPDH bands and expressed relative to the values obtained with non-treated cells. The graph shows mean ± SEM and individual points from three to five independent experiments. ^*Significantly different from non-treated cells (unpaired t-test). F LUMHES spheroids were treated or not with 2 μM MPP⁺ for 29 h. Then, Triton-X100-soluble and SDS-soluble protein fractions were analysed by western blot using antibodies against α-synuclein and GAPDH. These data are representative of two independent experiments.