Extended Data Fig. 4: Testing the effects of CLN3 loss on lysosomal pH and cellular lipid metabolism.
From: CLN3 is required for the clearance of glycerophosphodiesters from lysosomes
a to d, CLN3 loss does not increase lysosomal pH. a, A standard calibration curve of the ratiometric dye LysoSensor Yellow/Blue DND-160. See methods for experimental details. b, Lysosomal pH in wild-type and CLN3 KO HEK293T cells as calculated using the standard curve in a. Data are presented as mean ± s.d., n = 12 biologically independent samples. c, Targeted analyses of the fold changes in whole-cell and lysosomal levels of GPDs upon treatment with 500 nM Bafilomycin A1 for 6 h in CLN3 expressing HEK293T cells. Data are presented as mean ± s.d., n = 3 biologically independent samples, (Two-tailed unpaired t-test). d, The levels of several amino acids whose egress from lysosomes is sensitive to the proton gradient across the lysosomal membrane are not affected by CLN3 loss. The levels of proline, alanine, and glutamate in whole cells and lysosomes were compared between CLN3 KO cells with and without CLN3 cDNA addback and upon treatment with 500 nM Bafilomycin A1 (BafA1) for 6 h. Data are presented as mean ± s.d., n = 3 biologically independent samples, (Two-tailed unpaired t-test). e, The recombinant CLN3 protein does not have glycerophosphodiesterase activity. Deuterated GPC (D9-GPC) was incubated with recombinant 3×Flag–CLN3 or the positive control glycerophosphodiesterase1 (3×Flag–GDE-1) for the indicated time points. The extent of D9-GPC hydrolysis was determined by measuring the decline in its level and the increase in the levels of the product D9-choline. Data presented as mean ± s.e.m. of n = 3 biologically independent samples. f, CLN3 loss does not decrease tracer uptake. Fold change in tracer levels (D9-16:0-16:0 PC) normalized to total protein for samples measured in Fig. 4f and g. Data presented as mean ± s.e.m. of n = 4 biologically independent samples. g, CLN3 loss does not affect the biosynthesis or turnover of PC and sphingomyelin (SM) in cells. Free D9-choline was used as a tracer. Data are presented as fold changes in the whole-cell molar percent enrichment (MPE) of D9-choline-containing lipids in cortical neuron cultures prepared from Cln3−/− mice relative to those from Cln3+/− animals (mean ± s.e.m., n = 3 biologically independent samples, (Two-tailed unpaired t-test)).
