Osmotic Stress Changes the Expression and Subcellular Localization of the Batten Disease Protein CLN3

Abstract

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.

Mutation del 1,02kb in the CLN3 gene and extrapyramidal syndrome

“…Основная часть CLN3 расположена в мембранах комплекса Гольджи, лизосом и эндосом [9]. Его функции до конца не известны, однако в литературе имеются све-дения, что он участвует в формировании лизосом, их транспорте и созревании, адаптации клетки к осмотиче-скому стрессу [10], нейровоспалении (мутантный белок способствует активации микроглии и синтезу провоспа-лительных цитокинов) [11], транспорте галактозилцера-…”

Role of the Lysosomal Membrane Protein, CLN3, in the Regulation of Cathepsin D Activity

“…The pUB6-B plasmid expressing an N-terminally myc-tagged human CLN3 under the control of the human ubiquitin C promoter was generated previously [Getty et al, 2013]. The pUB6-B empty plasmid (Life Technologies, Grand Island, NY) served as a negative control.…”

“…The two baby hamster kidney (BHK) cell lines, one stably transfected with the pUB6-B plasmid expressing an N-terminally myc-tagged human CLN3 (C19), the other stably transfected with the empty pUB6-B plasmid (C2), were generated previously [Getty et al, 2013]. The pUB6-B expression vector provides resistance against the antibiotic, blasticidin.…”

“…Because of the lack of an antibody that specifically detects endogenous CLN3, an N-terminally myc-tagged human CLN3 was stably expressed in baby hamster kidney (BHK) cells. In this cell model, hyperosmolarity (800 mOsm), achieved by either NaCl/urea or sucrose, dramatically increased mRNA and protein levels of myc-CLN3 as determined by quantitative real-time PCR and Western blotting, and changed the subcellular localization of myc-CLN3 as showed by immunofluorescence [Getty et al, 2013]. …”

A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease

“…Porcine organs and skin fibroblasts in vitro were lysed in ice-cold RIPA buffer and used for immunoblotting as previously described (68). In brief, a total of 30 µg of protein was subjected to NuPAGE 3% gel electrophoresis.…”