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.

Figure 1. The role of Cdc42 GTP to GDP cycling in fluid phase endocytosis.

Fluid-phase endocytosis requires Cdc42 cycling from the GTP to the GDP bound state, which is controlled in part by GAPs and GEFs, and upstream of that, ARF1. Defects in Cdc42 cycling negatively influence endocytosis. In the absence of CLN3, faulty recruitment or function of a regulator (for example ARF1, GAP or GEF) can create an imbalance toward either Cdc42-GDP (left) or Cdc42-GTP (right), which would in turn impair endocytosis.

Figure 2. Fluid-phase endocytosis is impaired in CLN3-null MBECs.

Primary (A) and immortalized (B) MBECs of the indicated genotypes were incubated with Hoechst 33342 to label cell nuclei (blue) followed by incubation in Alexa 488 conjugated dextran (green). A488-Dextran uptake was evaluated by fluorescence microscopy and intensity quantified using ImageJ. Data are the mean of three independent experiments. Error bars ± s.e.m. (t-test, *, p<0.05, ***, p<0.001). Scale bars are 10 µm (A) and 20 µm (B).

Figure 3. Cdc42-GTP is elevated in CLN3-null MBECs.

(A) Cdc42-GTP activity was quantified from primary (WT and Cln3 ^−/−) or immortalized (Cln3 ^R and Cln3 ^−/−) MBEC lysates. (B) Total Cdc42 protein levels were quantified by western blot, expressed as band intensity normalized to the actin loading control. (C) Cln3 ^R MBECs were transfected with WT-Cdc42-GFP, GFP (negative control), dominant negative (DN)-Cdc42-GFP, or constitutively active (CA)-Cdc42-GFP constructs. Endocytosis of Rhodamine conjugated dextran (red) was imaged by epifluorescence and quantified in transfected cells (green). Data represent the mean of four (A, B) and three (C) independent experiments. Error bars ± s.e.m. ((A,B) t-test, (C) 1-way ANOVA with Tukey post-hoc, *, p<0.05, **, p<0.005, n.s. =  not significant). (A) Scale bar is 10 µm. Dashed lines indicate the outline of transfected cells.

Figure 4. PAK-1 phosphorylation and LIMK recruitment are elevated in CLN3-null MBECs.

(A) Total cell lysates or (B) membrane fractions were analyzed by western blot for PAK-1, P-PAK-1, and LIMK. Actin and β-catenin were used as total lysate loading controls, and transferrin receptor (TFNR) as the loading control for membrane enriched samples. Normalized band intensity was calculated as in Fig. 3. Results are the mean of three independent experiments. Error bars represent ± s.e.m. (t-test, *, p<0.05).

Figure 5. Cln3 ^−/− MBECs have defects in actin dependent processes.

(A) SEM images of immortalized MBECs of the indicated genotypes were taken, compiled, and filopodial length and number was measured. White arrows indicate filopodia. Filopodia were counted on 32 Cln3 ^R and 36 Cln3 ^−/− MBECs. (B) A scratch was made across a confluent monolayer of cells and migration was assessed via live cell microscopy and quantified. Representative images from three time-points are shown. The graph shows data from three independent experiments ± s.e.m. ((A) t-test, (B) 2-way ANOVA with Bonferroni post-hoc correction, *, p<0.05). Scale bars are 5 µm (A) or 100 µm (B).

Figure 6. CLN3-null MBECs have reduced endogenous ARHGAP21 plasma membrane recruitment.

Fixed MBECs were immuno-stained for endogenous ARHGAP21 (green). (A) ARHGAP21 was analyzed by confocal microscopy (cell nuclei, blue). (B) Basal membrane localization of ARHGAP21 was analyzed by TIRFM and quantified (≥70 cells per group). (C) Total endogenous ARHGAP21 protein levels were quantified by western blot, expressed as band intensity normalized to the actin loading control. Results represent the data from three independent experiments. Error bars are ± s.e.m. ((B,C) t-test, ****, p<0.0001, n.s. =  not significant). Scale bars are (A) 10 µm and (B) 25 µm.

Figure 7. The impact of ARHGAP21 overexpression on endocytosis.

(A) Cln3 ^−/− and Cln3^R MBECs were transfected with GFP, WT-Cdc42, dominant negative Cdc42 (DN-Cdc42), or ARHGAP21 expressing plasmids and Cdc42-GTP levels measured. (B) MBECs were transfected with GFP negative control, WT-Cdc42, or ARHGAP21(GAP21) constructs and rhodamine-conjugated dextran uptake was assessed as in Fig. 2. Results represent the mean of three independent experiments. Error bars are ± s.e.m. ((A left panel and B) t-test, (A right panel), ANOVA with multiple comparison test, *, p<0.05, ***, p<0.0001).

Figure 8. ARF1-GTP is reduced in CLN3-null MBECs.

(A) ARF1-GTP levels were quantified in Cln3 ^R and Cln3 ^−/− MBEC lysates. (B) Total ARF1 protein levels were quantified in Cln3 ^R and Cln3 ^−/− MBEC lysates by western blot, expressed as band intensity normalized to the actin loading control. Data represent the mean of (A) five and (B) three independent experiments. Error bars ± s.e.m. (t-test, *, p<0.05, n.s. =  not significant).