CLN3 Deficient Cells Display Defects in the ARF1-Cdc42 Pathway and Actin-Dependent Events

Abstract

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects. We discover that active Cdc42 (Cdc42-GTP) is elevated in endothelial cells from CLN3 deficient mouse brain, and correlates with enhanced PAK-1 phosphorylation, LIMK membrane recruitment, and altered actin-driven events. We also demonstrate dramatically reduced plasma membrane recruitment of the Cdc42 GTPase activating protein, ARHGAP21. In line with this, GTP-loaded ARF1, an effector of ARHGAP21 recruitment, is depressed. Together these data implicate misregulated ARF1-Cdc42 signaling as a central defect in JNCL cells, which in-turn impairs various cell functions. Furthermore our findings support concerted action of ARF1, ARHGAP21, and Cdc42 to regulate fluid phase endocytosis in mammalian cells. The ARF1-Cdc42 pathway presents a promising new avenue for JNCL therapeutic development.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo

“…Cln3 expression was stably restored in Cln3 −/− MBEC using a lentivirus vector as described previously (Tecedor et al, 2013), creating the Cln3 R , sister cell line. Cln3 R and Cln3 −/− MBECs behave similarly to primary wildtype and Cln3 −/− MBECs (Schultz et al, 2014; Tecedor et al, 2013). Immortalized MBECs are positive for Von Willebrand factor and ZO-1 (Tecedor et al, 2013).…”

“…Briefly, 0.8 mg/ml of snap frozen lysate was incubated in the ELISA plate and the manufactures instructions were followed for detection and analysis as previously described (Schultz et al, 2014). Data were normalized to the protein standard and significance was tested by one-way ANOVA with Tukey post-hoc analysis.…”

“…Then cells were briefly washed 3× with 37°C PBS to remove unbound dextran and immediately fixed with 4% PFA at 37 °C. Membrane impermeable 200 mM Red-40 was added to quench extra cellular dextran signal allowing quantification of only internalized dextran (Schultz et al, 2014). Cells were immediately imaged on an Olympus IX81 microscope and fluorescent intensity was calculated by ImageJ.…”

“…Work in model systems over the last two decades suggest that CLN3 impacts multiple cellular functions including lysosomal pH (Golabek et al, 2000; Holopainen et al, 2001; Pearce et al, 1999; Pearce and Sherman, 1998), vesicular trafficking (Cao et al, 2006; Codlin and Mole, 2009; Fossale et al, 2004; Kama et al, 2011; Metcalf et al, 2008), palmitoyl desaturase activity (Narayan et al, 2006), endocytosis (Codlin et al, 2008; Fossale et al, 2004; Luiro et al, 2001; Luiro et al, 2004; Schultz et al, 2014; Tecedor et al, 2013; Vidal-Donet et al, 2013), and membrane microdomain formation or stability (Tecedor et al, 2013). …”

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…Although the primary function of CLN3 has not yet been fully uncovered it is proposed to play a role in vesicular trafficking because its deficiency leads to altered dis-tribution of endosomal and lysosomal proteins and phospholipids (7)(8)(9)(10)(11)(12), abnormal morphology of endocytic and lysosomal organelles (7,11), lysosomal pH dyshomeostasis (13)(14)(15), and amino acid transport defects (16). The hallmark JNCL storage material containing subunit c accumulates in both autophagosomes and lysosomes implicating further impact of CLN3 deficiency on the autophagy pathway (17).…”