Elsevier

Experimental Cell Research

Volume 314, Issue 15, 10 September 2008, Pages 2895-2905
Experimental Cell Research

Research Article
Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin–Na+, K+ ATPase complex

https://doi.org/10.1016/j.yexcr.2008.06.016Get rights and content

Abstract

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na+, K+ ATPase. The ion pumping activity of Na+, K+ ATPase was unchanged in Cln3−/− mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3−/− mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na+, K+ ATPase were remarkably affected in Cln3−/− mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na+, K+ ATPase. Most of the processes regulated by multifunctional fodrin and Na+, K+ ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na+, K+ ATPase may play a role in the neuronal degeneration in JNCL.

Introduction

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of recessively inherited neurodegenerative disorders characterised by intracellular accumulation of autofluorescent lipopigment. Typical clinical features are epileptic seizures, mental impairment, progressive loss of vision and motor skills and ultimately, premature death. Different NCL subtypes are classified by age of onset, defective gene and distinct ultrastructure of the accumulated material [reviewed in 1].

The juvenile onset NCL (JNCL, Batten disease) is the most common form of NCL and also inherited neurodegenerative disorders of childhood [1]. JNCL results from mutations in the CLN3 gene [2]. The topology of the human CLN3 has been studied extensively both experimentally and by computational methods. Experimental analyses favor a model of six transmembrane domains and cytoplasmic N- and C-termini [3], [4], [5]. This has recently been confirmed by the extensive computational prediction analysis [6] thus, amending the earlier topology predictions of the protein containing lumenal N-terminus and five transmembrane domains [7]. Although CLN3 is highly conserved across species and is evidently important for fundamental cellular processes, it is not homologous to any known protein. The membrane topology is suggestive for a transporter function, and distant relationships between CLN3 and equilibrative nucleoside transporters as well as fatty acid desaturases have been reported [8], [9]. The current view favors endosomal/lysosomal localisation of CLN3 [4], [10]. However, it has been reported that CLN3 may also exist at the plasma membrane or traffic via the plasma membrane to lysosomal/vacuolar compartments [11], [12].

JNCL has many physiological consequences and CLN3 is proposed to have multiple functional roles affecting several cellular pathways, including autophagy, apoptosis, proteolipid modification and lipid metabolism [9], [13], [14], [15]. Recently, the association to apoptosis was further supported by the finding that CLN3 interacts with calsenilin, a protein associated with Ca2+-induced cell-death [16]. However, most of the functional findings point to disturbances in cytoskeletal regulation and endocytic events or nutrient transport across the membrane. Studies with patients fibroblasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe yeasts with deleted CLN3 ortholog BTN1 (btn1-Δ strain) as well as human embryonic kidney cells with inhibited CLN3 synthesis have shown disturbances in the maintenance of lysosomal/vacuolar pH [12], [17], [18], [19]. The S. cerevisiae studies, as well as subsequent mammalian cell analyses have also implicated that Btn1p/CLN3-deficiency results in defects in basic amino acid homeostasis with decreased intracellular levels of arginine, lysine and glutamic acid and decreased inward transport of arginine across the vacuolar/lysosomal membrane [20], [21]. Human JNCL cells and Cln3-deficient mouse models show defects in endocytosis and abnormalities in microtubular motor components as well as in fast axonal transport [22], [23], [24], [25]. Finally, mammalian protein interaction studies have brought us closer to the direct role of CLN3 by demonstrating an interaction between CLN3 and a novel endocytic micotubulus-associated protein Hook1, further highlighting the cytoskeletal and endocytic association of CLN3 [23].

This study was undertaken to gain deeper insight into the cytoskeletal and endocytic connections of CLN3. Yeast two-hybrid and subsequent co-immunoprecipitation analyses revealed novel interactions of CLN3 with the plasma membrane cytoskeleton component fodrin (dimer of α-II and β-II spectrins) and the associated plasma membrane protein Na+, K+ ATPase which led to further functional studies of this complex in the context of Cln3 deficiency.

Section snippets

Antibodies

Goat polyclonal β-fodrin (β-II-spectrin, 1:50 in immunohistochemistry), mouse monoclonal α-fodrin (α-II-spectrin, 1:100 in immunofluorescence), mouse monoclonal Na+, K+ ATPase α1 (clone α6F, 1:5 and 1:200 in immunofluorescence and Western blotting, respectively), mouse monoclonal GRP78/BiP (1:1000 in Western blotting) and mouse monoclonal β-tubulin (1:500 in Western blotting) antibodies were from Santa Cruz Biotechnology, Inc., Chemicon, Hybridoma Bank (University of Iowa, IA, USA), BD

Yeast two-hybrid screening reveals putative cytoskeletal and plasmalemmal/endosomal interaction partners for CLN3

In order to obtain further understanding of the cytoskeletal and endocytic roles of CLN3, we utilised the yeast-two-hybrid assay to screen for cytoplasmic CLN3 interaction partners. We used two cytoplasmic CLN3 domains as baits, amino acids 1–40 of the N-terminus and amino acids 232–280 of the major cytoplasmic loop, and screened a human fetal brain cDNA library by the GAL4 yeast two-hybrid method. The cytoplasmic C-terminus of CLN3 was excluded from the screening process due to its autonomous

Discussion

In the present study, we show that CLN3, defective in juvenile NCL, interacts with the proteins of the plasma membrane/endosomes and cytoskeleton beneath the cell surface, namely fodrin and Na+, K+ ATPase. Fodrin is composed of α-II and β-II spectrins and represents a plasma membrane-associated pool of the spectrin protein family. Spectrins are a major component of membrane-associated cytoskeleton and are connected to membrane proteins either directly or via ankyrins [reviewed in [35], [36]].

Acknowledgments

Kaija Antila, Auli Toivola and Elina Maaranen are thanked for their technical assistance. Anna-Elina Lehesjoki (Folkhälsan Institute of Genetics, Department of Medical Genetics and Neuroscience Center, Helsinki, Finland) is acknowledged for support in yeast two-hybrid assay. We thank the Molecular Imaging Unit (Biomedicum Helsinki, Finland) for providing the facilities for the image analysis. This work was financially supported by the European Commission 6th Framework Research Grant

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