Fig. 3: Analysis of the mitophagy-related parameters in CLN5 cell model.

a Mitochondrial network organization in SH-SY5Y KO model. When compared to the empty vector control line, CLN5 KO cells reveal an altered mitochondrial network with increased fragmentation both after Mitotracker red and VDAC staining. MT red CMXRos probe accumulation, which is dependent upon membrane potential, is reduced in KO lines suggesting a decreased ΔΨm. Figure shows representative images from three independent cell staining. Scale bar, 10 µm. Inserts show a 3x magnification. b Neuroblastoma cells were stained with TMRM probe revealing in CLN5 KO line a reduced mitochondrial membrane potential both in terms of probe accumulation and membrane potential maintenance. End-point assay indicates a mitochondrial membrane depolarization in KO cells reported as average TMRM relative fluorescence units RFU ± SD subtracting the fluorescence related to FCCP treatment. Data were normalized by DAPI staining as a function of cell number. Kinetic track demonstrates a differential ability between KO and control cells to maintain polarized the mitochondrial membrane particularly after oligomycin blocking proton transit through Complex V, highlighting any leakage of inner mitochondrial membrane. FCCP was added at the end of the experiments to fully depolarized mitochondrial to demonstrate specificity of the acquired measurements. c Redox state of cells lacking CLN5 using the fluorogenic dye H2DCFDA shows a significantly larger amount of ROS in SH-SY5Y CLN5 KO cells as compared to controls both in regular medium (RM) and under stress condition (short-term TBHP treatment). Data represent the mean (± standard deviation) of three independent experiments (n = 9). Asterisks indicate statistical significance of Ctrl versus KO cells in the presence/absence of TBHP treatment, as determined by the Student t test. ***p < 0.001.