Revisiting the neuronal localization and trafficking of CLN 3 in juvenile neuronal ceroid lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis, the most common neurodegenerative disease affecting children, is caused by mutations of the CLN 3 gene encoding CLN 3, a transmembrane protein with so far undefined function. The embryonic expression of the gene has not been studied in detail before. Moreover, the protein CLN 3 was mostly localized on the subcellular level to lysosomes but the exclusiveness is still under debate. Here, we analyze the expression pattern of murine CLN 3 at different developmental stages by in situ hybridizations. We observe expression maxima in the developing thalamus and cerebral cortex and outside of the central nervous system in the gastrointestinal tract and other peripheral organs. In differentiated primary neurons, the protein CLN 3 shows mainly a somatodendritic localization. In primary neurons, we thoroughly revisit the subcellular localization of CLN 3 and find a predominant localization in late endosomal–lysosomal compartments. Moreover, we expressed the major mutant form of CLN 3 – CLN 3deltaExon7/8 – in neurons and demonstrate that it is retained in the endoplasmatic reticulum. Time‐lapse microscopy analysis of neurons revealed co‐trafficking of CLN 3 with the late endosomal marker Rab7, but not with the early endosomal marker Rab5. Furthermore, a constitutive active mutant of Rab7 traps CLN 3 in enlarged endosomes. Our subcellular localization study in neurons refines the localization and subcellular targeting of CLN 3 to late endosomal–lysosomal compartments and provides information on the velocity of CLN 3 in living neurons which has not been investigated before.