Fig. 4. Disease pathology in mutant DNAJC6.

(A) Transient plots from multielectrode array with representative sorted neural signals and the firing rate of the neural signals over time recorded from WT and ΔDNAJC6 hMLO organoids using a 16-electrode silicon neural probe. (B and C) Measurement of intracellular reactive oxygen species (ROS) in hMLO (scale bars, 500 μm) and differentiated NSC cultures (scale bars, 50 μm) through dihydrodichlorofluorescein diacetate (DCF) DA staining (B); ROS level is presented as mean fluorescence intensity value (C). n = 3 independent experiments. (D) Quantification of DA release in hMLO DIV94 and differentiated NSC D15. n = 3 independent experiments. mnt, minutes. (E) Heatmap for the expressions of DA metabolism, DA transmission, and pacemaker-related genes in the RNA-seq data. Expression value is shown as log₂ fold change of ΔDNAJC6 versus WT hMLO DIV15 and DIV30, respectively. (F to I) WB analyses to determine endogenous α-syn protein levels from Triton X-100 (Tx-100)–soluble and –insoluble fractions of WT and ΔDNAJC6 hMLO at DIV55 and DIV130 (F and H). Intensities of the bands in (F) and (H) were quantified using ImageJ software, and the values were normalized to GAPDH (G and I). n = 3 independent experiments. (J to M) Intracellular detection of α-synuclein aggregation using bimolecular fluorescence complementation (BiFC) on hMLO DIV55 and hNSC D12 (I). Analysis on hMLO DIV55 was conducted using CLARITY imaging and further confirmation by sectioned organoid (thickness, 30 μm) from the same batch and analyzing it using confocal (J). α-Syn aggregation shown in terms of the percentage of BiFC⁺ cells, n = 3 independent experiments (K), number of BiFC⁺ puncta per cell (L), and number of BiFC⁺/pSer129-αSyn⁺ puncta per cell (M), n = 30 cells analyzed in each group. Scale bars, CLARITY, 200 μm; 30 μm for high-magnification image; hNSC, 50 μm. N.D., not detected. Data are presented as means ± SEM. Significance at *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.