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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis

Fig. 2

The CLN3 c.1054C > T introduction generated classical JNCL phenotypes in vitro. a Protocol for endothelial cell (EC) derivation from hiPSCs and representative immunofluorescence pictures of CD31 positive endothelial cells for characterization. Scale bar, 200 μm. Quantification of CD31 positive cells performed in Control and CLN3Q352X ECs. Mean fluorescence intensity of the CD31 channel normalized to the nuclear (Hoechst positive) signal was calculated for 12 random fields per condition using ImageJ (NIH) (Welch’s test; ns, not significant). Data is shown as mean ± SEM. b Representative TEM pictures highlighting increased presence of autophagic vacuoles (AVs, yellow dashed line) in the CLN3Q352X ECs. Other organelles (n, nuclei; g, Golgi; l, lysosome; m, mitochondria). Scale bars, 1 μm. b Quantification of the percentage of AVs evaluated as the number of events in 56 Control and 52 CLN3Q352X random cell profiles. Area of the AVs was calculated on ImageJ (NIH) by manually delineating the perimeter as regions of interest (ROI). AV areas were normalized to the total area of the cell containing them. All measurements were corrected for the magnification of the pictures if needed. Comparison between genotypes was performed by a Mann-Whitney test (*p < 0.05; ns; not significant). Data is shown as mean ± SEM. d Representative TEM pictures of fingerprint inclusions (FPPs) seen in CLN3Q352X ECs (Gly, glycogen deposits). Scale bar, 500 nm. Zoomed in picture (yellow dashed square) for better visualization of membrane stacks. Scale bar, 200 nm. e High-content imaging of SCMAS (green), LAMP1 (red) structures, and co-localization events (yellow) in Control and CLN3Q352X ECs. Scale bar, 200 μm. f SCMAS, LAMP1 and colocalization counts normalized to the nuclear (Hoechst positive) area of each field. Differences were evaluated using Mann-Whitney test (*p < 0.05, ****p < 0.0001). Data is represented as mean ± SEM of three technical replicates. g Representative TEM images of structural differences in the Golgi compartment in the Control (stacked) and the CLN3Q352X (dilated) ECs (g, Golgi; m, mitochondria; gly, glycogen). Scale bars, 500 nm. h High-content imaging of Golgi (GM130 positive) structures (red). Scale bar, 200 μm. i Quantification of GM130 counts, normalized to the nuclear (Hoechst positive) area of each field. GM130 positive structures present an increased ramification in terms of skeleton pixels and average nodes per Golgi structure. Comparison performed with Mann-Whitney test (****p < 0.0001). Data is represented as mean ± SEM of three technical replicates

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