Fig. 6

Dysregulations at the synapses might precede other phenotypes in CLN3^Q352X cerebral organoids. a Representative confocal images of synaptic protein staining for Synaptophysin (red) and PSD95 (green). Scale bars, 200 μm. Zoomed regions with TUJ1 (white) neuronal staining. b Quantification of the Synaptophysin and PSD95 positive particles normalized to nuclear (Hoechst positive) count. Two regions of interest (ROI) per organoid section were imaged. Following automatic thresholding, the particle analyzer tool from ImageJ (NIH) was used to quantify punctate stainings -PSD95 and Synaptophysin- and the ITCN nuclear count tool to quantify Hoechst positive nuclei. Each data point represents a region of interest (ROI). Total n per group equals 10 sections taken from organoids generated in at least three independent derivations. Comparison between groups was performed with a Mann-Whitney test (**p < 0.01; ns, not significant). c Heatmap representing hierarchical clustering of deregulated metabolites between the Control and the CLN3^Q352X mutant organoids. 5 different pools of 3 organoids were analyzed and 3 technical replicates per measurement were performed. d Table containing cerebral tissue metabolites. Arrows indicate relative increase or decrease in the CLN3^Q352X mutant organoids compared to the controls. Asterisks mark significantly deregulated metabolites in the mutant organoids, corresponding to p values. e Neurotransmitter GABA is particularly downregulated in the CLN3^Q352X mutant organoids. Significant differences were evaluated with a Mann-Whitney test comparison (**p < 0.01). Data points represent the average of technical replicates per organoid pool. Data in B and E is shown as mean ± SEM. f Representative confocal images of inhibitory GABAergic neurons pre-synaptic protein vGAT staining (green) and MAP 2 positive neuronal areas (red). Single vGAT channel is displayed inside a square dashed box for better visualization. Scale bars, 20 μm. g Quantification of the vGAT positive particles normalized to Hoechst positive nuclei. Several regions of interest (ROI) per organoid section were imaged. Following automatic thresholding, the particle analyzer tool from ImageJ (NIH) was used to quantify vGAT punctate staining and the ITCN nuclear count tool was used to quantify Hoechst positive nuclei. Each data point represents a region of interest (ROI). Total n per group is 4 sections, one per organoid and each belonging to independent derivations. Comparison between groups was performed with an unpaired t test with Welch’s correction (*p < 0.05)