Fig 2: Decreased Cln5 protein amounts in Cln7 ko cells and brain tissue. (A) Primary bone macrophages isolated from 5-month-old Cln7 ko and age-matched wild-type mice (N = 3) were cultivated for 14 days. Whole cell lysates were analysed by immunoblotting using antibodies against Cln5, cathepsins S (Ctss) and B (Ctsb). Equal loading was verified by a-tubulin western blotting. The positions of the molecular mass markers and the precursor (open arrowhead) and mature (filled arrowhead) Cln5 and Ctsb proteins, respectively, are indicated. Densitometric quantification of the immunoreactive band intensities has been performed and the relative protein amounts are shown in a bar diagram (mean ± SD, n = 3–5). n.s.: not significant, *P < 0.05 (two-tailed Student’s t-test). (B) Lysosome-enriched fractions of CLN7 ko and wild-type HAP1 cells were analysed by western blotting using antibodies against CLN5, cathepsin D (CTSD), PPT1, glucocerebrosidase (GBA), SIDT2 and LAMP2. Equal loading was confirmed by a-tubulin western blotting. The positions of the molecular mass markers and the precursor (open arrowhead) and mature (filled arrowhead) forms of CLN5, CTSD and PPT1, respectively, are indicated. (C) Whole brain lysates from three 10-month-old Cln7 ko and age-matched wild-type mice were analysed by Cln5 immunoblotting. ß-Tubulin western blotting was used as loading control. The positions of the molecular mass markers are indicated. Bar diagram represents densitometric analysis of Cln5 protein levels normalized to the loading control (mean ± SD, n = 5). *P < 0.05 (two-tailed Student’s t-test).

Fig 3: Translational confirmations in patient’ skin fibroblasts.a CLN5 protein levels, measured by Western Blotting, show a full lacked CLN5-immunoreactivity in Pt 2/F and 11/M, whereas a different CLN5 expression pattern is shown in patient 6/M, likely due to different effects of CLN5 mutations exerted on protein synthesis. Immunochemical studies demonstrate that subunit c of mitochondrial ATP synthase (SCMAS), the characteristic hallmark in NCL5, is stored in intracellular aggregates in patients’ fibroblasts. Scale bar, 10 µm. b Micro-oxygraphy track shows a reduced OCR, which is clearly evident after FCCP injection, reflecting a deficient spare respiratory capacity. Moreover, under oxidative conditions (blocking glycolysis with 2-deoxy-d-glucose + pyruvate) a reduced ATP content and bioenergetics defects in patients with more severe mutations and less abundant protein levels are seen. G glucose used as source for ATP production; O oligomycin, used to block mitochondrial respiration; 2DG + P = 2-deoxy-d-glucose plus pyruvate, used to block glycolysis. c Fibroblast pellets were fixed with 1.25% glutaraldehyde and 0.5% paraformaldehyde in phosphate buffer, post-fixed in 1% osmium tetraoxide and stained with uranyl acetate and lead citrate. Abnormal cytoplasmic pattern is marked, as demonstrated by the increased amount of vacuoles, dense bodies and lysosomes (A and B). Osmiophilic figures with different ultrastructural arrangement, including honeycomb structures can be observed (insert A), but classical cytosomes are not detectable. Several vacuoles, outlined by a single membrane and containing osmiophilic material, are consistent with features of autophagic process. Osmiophilic inclusions featuring multilamellar structures can be detected within the vacuoles (insert B). Scale bar = 1.0 µm; insert A, scale bar = 0.2 µm; insert B, scale bar = 0.3 µm. d Western Blotting analysis reveals significantly increased expression of p62 in patients’ fibroblasts, consistent with a block of autophagosome-lysosome maturation. Data represent the mean (± standard deviation) of three independent experiments (n = 9). Statistical significance was determined by the Student t test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: 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 ??m. 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.

Fig 5: Increased turnover of Cln5 by cysteine proteases in Cln7 ko lysosomes. (A) Wild-type and Cln7 ko MEFs were incubated in Opti-MEM medium for 24 h. Lysosome-enriched fractions (LF, 50 µg protein), whole cell extracts (100 µg protein) and conditioned media (20%) were analysed by western blotting using anti-Cln5 and anti-Lamp1 antibodies. Equal loading in LF and total cell extracts was confirmed by Lamp1 and Gapdh immunoblotting, respectively. The positions of the molecular mass markers and the precursor (p, open arrowhead) and mature (m, filled arrowhead) Cln5 proteins are indicated. Densitometric quantification of the immunoreactive band intensities from four independent experiments has been performed and the percentage of secreted Cln5 precursor forms related to the total amount of Cln5 is shown in a bar diagram (mean ± SD). n.s.: not significant (two-tailed Student’s t-test). (B) Wild-type and Cln7 ko MEFs were incubated in the presence of leupeptin (Leu, 100 µm), pepstatin A (Pep A, 30 µm), E64 (50 µm) or bafilomycin A1 (Baf A1, 100 nm) for 20 h. Water- and DMSO-treated cells served as negative controls for leupeptin/E64- and pepstatin/bafilomycin-treated cells, respectively. Whole cell extracts (75 µg protein) were analysed by immunoblotting using antibodies against Cln5, cathepsin B (Ctsb) and Lamp1. Equal loading was confirmed by Gapdh western blotting. The positions of the molecular mass markers as well as the precursor (p, open arrowhead) and mature (m, filled arrowhead) Cln5 and Ctsb proteins, respectively, are indicated. Densitometric quantification of the immunoreactive band intensities from at least three independent experiments was performed, and the total Cln5 protein amount was calculated (mean ± SD) relative to water or DMSO controls (set as 1.0). n.s.: not significant, *P < 0.05 compared with a corresponding negative control (two-tailed Student’s t-test).