Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis) is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney) cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm), human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

Mutations in the CLN3 gene cause a fatal neurodegenerative disorder, juvenile CLN3 disease. Exploring the cause of the motor coordination deficit in the Cln3−/− mouse model of the disease we have previously found that attenuation of AMPA receptor activity in 1-month-old Cln3−/− mice significantly improves their motor coordination [20]. To elucidate the mechanism of the abnormally increased AMPA receptor function in Cln3−/− mice, we examined the surface expression of AMPA receptors using surface cross-linking in brain slices from 1-month-old wild type (WT) and Cln3−/− mice. In surface cross-linked brain samples, Western blotting for AMPA receptor subunits revealed significantly increased surface levels of GluA1 and GluA2 in the cerebellum, and of GluA2 in the cortex and striatum of Cln3−/− mice as compared to WT mice. Expression levels of the GluA4 subunit were similar in the cerebellum of WT and Cln3−/− mice. While intracellular GluA1 levels in the WT and Cln3−/− cerebellum or cortex were similar, the intracellular expression of GluA1 in the Cln3−/− striatum was decreased to 56% of the WT level. Our results show a prominent increase in AMPA receptor surface expression in the brain of Cln3−/− mice and suggest that CLN3 is involved in the regulation of AMPA receptor surface expression.