Extended Data Fig. 2: Generation and validation of LysoTag mouse model for Batten disease studies.

a, Schematic depicting the breeding strategy to generate LysoTag models for Batten disease studies. Cln3^−/− mice were crossed with heterozygous constitutive LysoTag mice and their progeny were then crossed back to Cln3^−/− animals to generate the indicated experimental mouse genotypes. Mouse drawing was created with BioRender. b, Expression and localization of the TMEM192–3×HA fusion protein to lysosomes in brain neurons of Cln3^+/+, Cln3^+/− and Cln3^−/− LysoTag mice. TMEM192–3×HA, lysosomes and neurons were detected in an immunofluorescence assay using antibodies to the HA epitope the lysosomal marker, LAMP1 and the neuronal marker Neu-N, respectively. Scale bar = 5 µm, and magnified insets are labelled with white boxes in the main image. Percentage of colocalization between TMEM192–3×HA and LAMP1 shown in the right panel was measured in n = 7 cells per genotype. Data are shown as mean ± s.e.m. Micrographs are representative of at least three independent experiments. c , Volcano plot comparing the lipidomic data from whole brain homogenates of Cln3^+/− and Cln3^−/− mice shows that only few lipids change significantly and they belong to phospholipids (yellow) and lysophosphatidylglycerol (LPG) in red. Loss of CLN3 also led to a decrease in the brain of bis(monoacylglycero)phosphate (BMP), a class of lysosomal lipids (blue). BMP/PG (cyan) annotation was used when MS/MS fragmentation was not acquired (see Supplementary Table 3 and methods for details). Horizontal line indicates a p-value of 0.05, and vertical dotted lines a fold change of 2. Each dot represents a lipid species. Data were acquired in negative ion mode and normalized to internal lipid standards for the best-matched lipid class (n = 5 and 4 for Cln3^+/− and Cln3^−/− mice, respectively). Female mice with an average age of 7 months were used. Data are in Supplementary Table 3. Two-tailed unpaired t-test was used.

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