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Keywords:

  • cAMP ;
  • chloride current;
  • CLIC1;
  • neurite growth;
  • PKA;
  • retinal ganglion cells

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation.

Using a combination of electrophysiology and immunohistochemistry, we found that the chloride intracellular channel 1 (CLIC1) protein modulates the speed of neurite growth. The chloride current mediated by CLIC1 is essential for maintaining growth cone morphology and is required for sustaining cAMP-stimulated neurite elongation in dissociated immunopurified neurons. The presence of either the CLIC1 current blocker IAA94 or the anti-CLIC1 antibody inhibits neurite growth of Retina Ganglion Cells cultured in the presence of 10 micromolar forskolin for 24 h.

Abbreviations used
AcS

actin-covered surface

BDNF

brain-derived neurotrophic factor

BSA

bovine serum albumin

CLIC1

chloride intracellular channel 1

N2a

neuro-2a

PKA

protein kinase A

Several external stimuli and intrinsic factors regulate axonal elongation during neuronal differentiation, including neurotrophic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability (Goldberg et al. 2002). In particular, neuronal activity plays a key role in regulating axonal outgrowth (Goldberg et al. 2002). Indeed, in cultured retinal ganglion cells (RGCs), depolarizing stimuli (e.g., KCl) and electrical stimulation of RGCs potentiate axonal outgrowth induced by brain-derived neurotrophic factor (BDNF) in a cAMP-dependent manner (Goldberg et al. 2002). Within this context, ion channels may play a crucial role. It has been demonstrated that in cerebellar neurons, the inhibition of tetrodotoxin-sensitive voltage-gated sodium channels impairs axon outgrowth by reducing cell excitability (Brackenbury et al. 2010). Transient receptor potential channels control growth cone (GC) morphology through their specific localization (Greka et al. 2003). In addition, calcium channels have a key role in calcium waves in GC and affect neurite growth in motor neurons (Jablonka et al. 2007). It has also been shown that ligand-gated chloride channels play a key role in neuronal maturation (Sernagor et al. 2010). However, the involvement of voltage-gated and other chloride channels in axonal elongation remains still unclear.

Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance (Novarino et al. 2004; Milton et al. 2008; Averaimo et al. 2010). In fact, when inserted in the plasma membrane, it constitutes a chloride-selective ion channel (Valenzuela et al. 1997; Tonini et al. 2000; Warton et al. 2002). CLIC1 insertion into the plasma membrane is driven by different stimuli, including cell oxidation (Novarino et al. 2004; Milton et al. 2008), intracellular pH (Warton et al. 2002), and signaling molecules like, for instance, Wnt proteins (Yang et al. 2009). Singh and Ashley have shown that CLIC1 insertion in the plasma membrane is favored by a specific lipid composition, comprising mainly phosphatidylethanolamine, phosphatidylserine, and cholesterol (Singh et al. 2007). Moreover, there is evidence that the actin cytoskeleton modulates the activity of CLIC proteins. CLIC5 was first identified as a protein associated with the actin cytoskeleton (Berryman and Bretscher 2000). It has also been shown that F-actin and CLIC proteins (i.e., CLIC1 and CLIC5) specifically interact in vitro and that such interaction modulates CLIC ion channel activity (Singh et al. 2007). Indeed, electrophysiological studies in artificial lipid bilayers have shown that the presence of F-actin at the intracellular side inhibits CLIC ion channel activity, while the soluble form G-actin restores the ion channel activity (Singh et al. 2007).

We have previously demonstrated that CLIC1 is expressed in the brain, more specifically in microglia, where its activation is required to set the membrane potential to the optimal values necessary for efficient reactive oxygen species (ROS) production (Novarino et al. 2004; Milton et al. 2008). Here, we investigated the expression of CLIC1 in primary hippocampal neurons and RGCs and examined how the functional expression of CLIC1 chloride channel specifically modulates neurite outgrowth of neonatal murine RGCs. We found that CLIC1 is expressed in primary hippocampal neurons and in neonatal murine RGCs and that the chloride current mediated by CLIC1 is required for both maintaining GC morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In fact, inhibition of CLIC1 reduces the rate of neurite outgrowth when RGCs are cultured in cAMP-elevating conditions. Furthermore, CLIC1-mediated chloride current is detected only when cAMP is elevated. Protein kinase A (PKA) inhibition prevents CLIC1 current, which results from an increased open probability of the channel. These results indicate that CLIC1 functional expression is regulated by cAMP via PKA and is required for stimulated neurite outgrowth during neuronal differentiation.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Animals

C57BL/6J mice (Charles River Laboratories Inc., Wilmington, MA, USA) were housed in filtered cages in a temperature-controlled room with a 12 : 12-h dark/light cycle with ad libitum access to water and food. Animal health and comfort were veterinary controlled. All the animal experiments were performed in accordance with the European Community Council Directive dated November 24, 1986 (86/609/EEC) and were approved by the Italian Ministry of Health and by the IIT Ethical Committee.

Antibodies

Anti-CLIC1 polyclonal antibody [in rabbit, kindly provided by Dr Mark Berryman at Ohio University College of Osteopathic Medicine (Athens, OH, USA)] was diluted 1 : 400; mouse anti-β-tubulin monoclonal antibody (Sigma-Aldrich, Milan, Italy) was diluted 1 : 300. Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used for detection. Antibody specificity was tested on recombinant CLIC1, CLIC4, and CLIC5 mouse proteins (Fig. 1a), which have been previously shown to have ion channel activity. CLIC2 and CLIC3 have never been associated with ion channel activity (Littler et al. 2010) and were not tested. The antibody recognizes a 27-kDa band, corresponding to recombinant CLIC1 as shown in previous work (Valenzuela et al. 1997), but not bands corresponding to CLIC4 (29 kDa) and CLIC5 (28 kDa) (Fig. 1a). Anti-NH2 CLIC1 antibody (in sheep) was kindly provided by Dr Samuel Breit (St Vincent's Centre of Applied Medical Research, Sydney, Australia).

image

Figure 1. Chloride intracellular channel 1 (CLIC1) is expressed in neurons of the CNS. (a) Western blot analysis of CLIC1, CLIC4, and CLIC5 recombinant protein (RP) blotted with a custom-made anti-CLIC1 antibody. The antibody specifically recognizes CLIC1, but not CLIC4 or CLIC5. Whole-retina homogenate was run alongside recombinant proteins and revealed CLIC1 expression in the retina. (b and c) Cultured primary mouse hippocampal neuron (b) and retinal ganglion cells (RGCs) (c) stained with anti-βIII-tubulin antibody (left panel), phalloidin (middle panel), and with the anti-CLIC1 antibody (on the right). Scale bar is 10 μm.

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Immunohistochemistry

For immunohistochemistry on mouse retina, adult mice were anesthetized and killed by cervical dislocation. The eyes were enucleated and fixed in 4% paraformaldehyde at 4°C for 24 h, washed extensively in phosphate-buffered saline (PBS), and cryo-preserved in 30% sucrose. The eyes were included in optimal cutting temperature compound and cryosectioned with a Leica CM3050 S cryostat (Leica Microsystem, Wetzlar, Germany). Slices of 20-μm retina were blocked in 5% bovine serum albumin (BSA) and 0.5% Triton X-100 for 2 h at 25°C and incubated overnight with primary antibody in PBS with 1% BSA, 0.2% Triton X-100 at 4°C. Nuclei were counter-stained with Hoechst 33342, and slides were mounted with Fluoromount G (Southern Biotech, Birmingham, AL, USA).

Primary cultures of immunopurified retinal ganglion cells

Murine RGCs were isolated from post-natal day 1 (P1) mouse retinas by immunopanning (Goldberg et al. 2002) through a modified protocol. Briefly, the retinas were isolated from mouse pups and digested with 16 U/mL papain (Roche Molecular Biochemicals, Indianapolis, IN, USA), 0.04 mg/mL DNAse (Roche Molecular Biochemicals), in presence of 1.3 mg/mL L-cysteine (Sigma-Aldrich) at 37°C for 30 min. The digestion was terminated by buffer removal and washed in 15 mg/mL ovomucoid trypsin inhibitor (Roche Molecular Biochemicals), 15 mg/mL BSA, and 0.04 mg/mL DNAse. Tissue fragments were gently triturated in ovomucoid solution to obtain a single cell suspension. The cells were pelleted at 400 g for 10 min at 25°C, resuspended in a pre-warmed separation buffer containing 0.5 mg/mL BSA and 2 mM EDTA, in PBS, and incubated with 1 : 100 anti Thy1.1 antibody conjugated to magnetic microbeads (Milteny Biotech, Bergisch Gladbach, Germany) for 10 min at 4°C. After excess antibody removal, the cells were rinsed in separation buffer, spun at 300 g for 10 min at 25°C, resuspended in fresh separation buffer, and isolated by magnetic separation using MACS columns (Milteny Biotech) according to the manufacturer's protocol. Eluted RGCs were counted, spun at 300 g for 10 min at 25°C, resuspended in culture medium, and plated in 4-well plates containing poly-d-lysine/laminin-coated coverslips (55 000 cells/well). The cells were cultured in neurobasal medium containing 100 μg/mL transferrin (Sigma-Aldrich), 100 μg/mL BSA (Sigma-Aldrich), 60 ng/mL progesterone (Sigma-Aldrich), 16 μg/mL putrescine (Sigma-Aldrich), 40 ng/mL sodium selenite (Sigma-Aldrich), B27 supplement (Invitrogen), 40 ng/mL T3 (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 ng/mL insulin, 10 μM forskolin, 50 ng/mL BDNF, and 100 ng/mL Ciliary neurotrophic factor (Invitrogen). To examine the role of cAMP, in selected experiments, forskolin was omitted from the growing medium.

The immunopurification protocols yield cultures containing neurons (94.85 ± 1.84%), most of which are also immunoreactive for the RGC marker NeuN (85.84 ± 7.65%). The remaining cells are either glial fibrillary acidic protein-positive glia or microglia immunoreactive for the microglia markers Ionized calcium binding adaptor molecule 1 and CD45.

Primary cultures of hippocampal neurons

Hippocampal neurons were cultured from P1 mouse neonates. Meninges-free hippocampus were isolated and digested trypsin (Sigma) in HIBERNATE-A media (BrainBits, Springfield, IL, USA) supplemented with B27 (Invitrogen) and 0.5 mM glutamine (Sigma-Aldrich) for 20 min at 30°C. Hippocampus were transferred to Neurobasal-A/B27 (NBA/B27) medium (Invitrogen), triturated, and plated onto poly-l-lysine (0.01 mg/mL)-coated tissue culture wells. Neurons were grown in NBA/B27 for 5 days in vitro before the experiment. The use of NBA/B27 provided highly purified cultures of neurons, with very low contamination of glial cells (Brewer et al. 1993).

Western blot

Retinas from adult mice were lysed in lysis buffer containing 50 mM Tris pH 7.4, 1% sodium dodecyl sulfate 1%, 50 mM NaCl, 1 mM EDTA, protease inhibitor cocktail for 30 min on ice. After sonication, the samples were spun at 400 g for 10 min at 4°C to remove the insoluble fraction and the supernatant was loaded on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. Recombinant CLIC1, CLIC4, and CLIC5 proteins were loaded on a 10% acrylamide gel and run at 130 V for 2 h. Then, the gel was blotted on a 0.2-μm nitrocellulose membrane for 2 h at 4°C at constant 200 mA current. After blocking for 1 h with tris-buffered saline (TBS) containing 5% milk and 0.2% tween-20, by shaking at 25°C, the blot was incubated overnight at 4°C with anti-CLIC1 antibody (kindly provided by Dr Sam Breit, University of Sidney) diluted 1 : 800 in TBS with 1% BSA and 0.2% Tween-20, followed by Alexa 488-conjugated goat anti sheep secondary antibody (1 : 1000) for 2 h at 25°C in the dark. The membrane was extensively rinsed in TBS and dried up for 30 min. The signal was detected using Typhoon (GE Healthcare Life Sciences, Uppsala, Sweden) by exciting with 488 laser. Anti-actin antibody (1 : 500; Sigma-Aldrich) followed by Alexa 546-anti-rabbit secondary antibody was used as a control.

Cell viability assay

Cell viability was assessed as described previously (Gasparini et al. 2004). Briefly, immunopurified RGCs were seeded on 96-well plates at 20 000 cells/well and grown in complete medium or in medium without forskolin. After 24 h, the media were replaced with serum-free medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the cells were further incubated at 37°C for 90 min. The medium was discarded and the crystals of violet formazan were dissolved in acidic isopropanol (isopropanol/1M HCl 1/4 24: 1 v/v) by shaking at 25°C for 30 min. Absorbance was read at 595 nm. Cell viability after drug treatments was expressed as percentage of vitality of untreated cells.

Pharmacological treatments

All the treatments were performed by diluting the compounds in the culture medium. IAA94 [(R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] was dissolved in ethanol at 50 mM and diluted in the culture medium to 50 μM final concentration. Anti-NH2-CLIC1 antibody was diluted 1 : 100 in culture medium. For neurite length quantification, the cells were treated for 24 h, while for GCs analysis they were cultured and treated for 3 days.

Immunocytochemistry

Cultured RGCs were fixed in pre-warmed 4% paraformaldehyde for 45 min, blocked in 5% BSA and 0.1% Triton X-100 in PBS for 2 h at 25°C, and incubated overnight with primary antibodies (in rabbit) in PBS with 1% BSA and 0.1% Triton X-100 followed by Alexa-conjugated secondary anti-rabbit antibodies (Invitrogen). For F-actin detection, Alexa 488-conjugated phalloidin (Invitrogen) was added to coverslips for 30 min at 37°C. Nuclei were counter-stained with Hoechst 33342 and coverslips were mounted with Fluoromount G (Southern Biotech).

To quantify neurite length, RGCs were immunostained for βIII-tubulin. RGCs were imaged with an Olympus BX51 Neurolucida microscope equipped with a dry 20× objective and microfire Color Optronic camera. Neurites were traced on acquired images using the MBF bioscience Neurolucida V 8.0 software (MBF Bioscience, Williston, VT, USA). Only neurites longer than the cell body diameter were considered. Only the longest neurite of each neuron was measured and averaged.

For colocalization analysis, RGCs were immunostained with anti-βIII-tubulin antibody (in mouse) and with Alexa 488-phalloidin to label F-actin. Images were acquired using a Leica confocal SP5 microscope (Leica Microsystem) with optimized pinhole and sectioning step. The analysis was performed with the ImageJ software (Schneider et al. 2012) using the colocalization plug-in. The degree of colocalization between actin and βIII-tubulin was measured as percentage of volume for each optical section and averaged for all the sections.

For the quantification of altered GC morphology, NIH ImageJ software was used (Schneider et al. 2012). The areas of the masks created on the phalloidin (actin-covered surface, AcS) and on the βIII-tubulin immunoreactivity (tubulin covered surface, TcS) were measured (Davis et al. 2009). GCs were defined collapsed when the AcS/TcS ratio value was reduced about 50% or lower than 3.76, which represent the mean value obtained for IAA94-treated GCs.

Time-lapse video microscopy

Time-lapse experiments were accomplished by DIC microscopy using an Olympus ScanR inverted microscope IX81 (Olympus, Hamburg, Germany) equipped with a dry 20× objective and an OKOlab incubator for temperature and CO2 control. Cells were imaged every 10 min for up to 24 h. For calculation of neurite growth rate, the length of the neurite in each frame during growing periods was measured and plotted as a function of time. The m value of the linear fit of this plot was considered as a parameter of the neurite growing speed.

Neuronal cell line cultures

Neuro-2a (N2a) cells were cultured in advanced Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum, 2 mM glutamine, 1× penicillin-streptomycin, at 37°C and 5% CO2 in a humidified incubator.

Electrophysiology recordings

RGCs and N2a cells were bathed in a solution containing (in mM): 90 NaCl, 40 Tetraethylammonium chloride, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 Hepes, 10 Glucose, and pH 7.4. For perforated patch experiments, the pipette solution contained 2.5 μg/mL gramicidin and (in mM) 140 KCl, 10 Hepes, 10 Glucose, pH 7.2. For whole-cell patch-clamp experiments, forskolin and the cell-permeable cAMP analogue, 8-Br-cAMP, were added to the external solution to a final concentration of 10 μM. The PKA inhibitor 6–22 amide (Millipore Corporation, Bedford, MA, USA) was included in the pipette solution at a final concentration of 4 μM. From a holding potential of −40 mV, steps at +60 mV were applied every 5 s and the evoked current was measured. In cell-attached experiments, the pipette solution contained (in mM): 130 N-methyl-d-glucamine-chloride, 5 Tetraethylammonium chloride, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 Hepes, 10 Glucose, 200 μM DIDS, and 1 μM 4-Aminopyridine, pH 7.4.

Statistical analysis

Data were presented as mean ± SEM. The significance was calculated with two sample Student's t-test for the electrophysiology data and anova test for the axon growth rate using Origin software (Origin Lab Corporation, Northampton, MA, USA). Differences were considered statistically significant when p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

CLIC1 is expressed in retinal ganglion cells and its functional expression affects growth cone morphology

CLIC1 expression in microglia of the CNS has been previously demonstrated (Novarino et al. 2004; Milton et al. 2008). However, the expression of CLIC1 is not restricted to microglia. We initially investigated CLIC1 expression in mouse hippocampal primary neurons and mouse RGCs by immunohistochemical and biochemical analyses with a polyclonal antibody specific for CLIC1 (Fig. 1). CLIC1 was expressed in both primary hippocampal neurons (Fig. 1b) and RGCs (Figs 1c and 2a–c). Antibody specificity was tested on recombinant CLIC1, CLIC4, and CLIC5 proteins (Fig. 1a). Indeed, CLIC1 expression was detected also in homogenate of mouse retina, confirming its expression in this neural tissue (Fig. 1a). Next, we investigated the localization of CLIC1 in the C57BL/6J mouse retina. Strong immunoreactivity for CLIC1 was detected in the RGC layer (RGL) and, to a lesser extent, in the nerve fiber, inner and outer plexiform layers. Specifically, CLIC1 was mainly detected in the soma of RGCs (Fig. 2a), with only weak signal in axons and dendrites. To analyze the subcellular localization of CLIC1, we isolated RGCs from mouse neonatal retinas and immunostained them after culturing for 3 days in vitro (Fig. 2b and c). Consistently, CLIC1 was mainly localized in the soma of RGCs (Fig. 2b) and, to a lesser extent, in neurites. Notably, CLIC1 immunoreactive puncta were also detected in GCs of RGCs (Fig. 2c).

image

Figure 2. Chloride intracellular channel 1 (CLIC1) is expressed in murine retina and retinal ganglion cells (RGCs). (a) Representative images of the immunoreactivity for CLIC1 (red) in the mouse retina. Nuclei were counter-stained with Hoechst 33342 (blue), while βIII-tubulin (green) staining highlights the structure of the retina. RGL, RGC layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar is 50 μm. (b) Representative images of CLIC1 (red) and βIII-tubulin (green) immunoreactivity in cultured dissociated RGCs. Scale bar is 10 μm. (c) Representative images of CLIC1 (red) and actin (green) immunoreactivity in RGC growth cones (GCs). Scale bar is 5 μm.

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The GC plays a key role in axon elongation. During axon outgrowth, it interacts with the extracellular matrix and, in response to different stimuli, it undergoes deep changes in its morphology (Geraldo and Gordon-Weeks 2009). Therefore, we initially investigated whether CLIC1 functional expression was required to maintain GC morphology, and we incubated RGC cultures in the absence or presence of 100 μM IAA94, which is a widely used inhibitor of CLIC1-mediated current. (Novarino et al. 2004; Milton et al. 2008). The GCs of untreated (Fig. 2c) and vehicle-treated RGCs (Fig. 3a) showed the classical fan-like morphology, with the central tubulin-rich area surrounded by the actin network, with very low degree of colocalization between the two proteins. CLIC1 immunoreactivity localized throughout the entire GC (Figs 2c and 3a), with maximal immunoreactivity in the tubulin-rich core. When CLIC1 current was inhibited by bath application of IAA94, most of the GCs collapsed. The ratio of the AcS and βIII-tubulin (TcS) was reduced by 46% in GCs of RGCs cultured in the presence of IAA94 (Fig. 3e) and the percentage of GCs with collapsed actin cytoskeleton was three-fold higher compared to that of vehicle-treated RGCs (61% of total GCs in IAA94-treated vs 22% in vehicle-treated RGCs). Consistently, GCs of IAA94-treated RGCs displayed increased colocalization of actin with βIII-tubulin compared to vehicle-treated cells (Fig. 3d). The colocalization of CLIC1 with actin was significantly reduced in IAA94-treated RGCs in the absence of changes in CLIC1-βIII-tubulin colocalization (Fig. 3f and g), in agreement with the collapse of actin cytoskeleton upon CLIC1 inhibition.

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Figure 3. Chloride intracellular channel 1 (CLIC1)-mediated current modulates the morphology of retinal ganglion cell (RGC) growth cones (GCs). (a and b) Representative immunofluorescence images of RGC cultured in the presence of vehicle (VEH; a) or IAA94 (b) for 24 h. Immunoreactivity for βIII-tubulin (green), actin (magenta), CLIC1 (red), and merge images are shown. Scale bar is 5 μm. (c) Scheme of the analysis of GC morphology. Masks were created at the rim of the actin (yellow line) and βIII-tubulin immunoreactive signals (white line). Scale bar is 5 μm. (d) Quantification of βIII-tubulin and actin colocalization in VEH- and IAA94-treated RGC. Bars represent the average percentage of colocalization ± SEM. **p < 0.01, Student's t-test. (e) Quantification of GC morphology. βIII-tubulin- (TcS) and actin-covered surface (AcS) were measured in RGCs treated with vehicle (VEH; n = 35) or IAA94 (n = 40) and expressed as AcS/TcS ratio. Bars represent the average ratio ± SEM of three independent experiments. **p < 0.01, Student's t-test. (f and g) Quantification of colocalization of CLIC1 with actin (f) and βIII-tubulin (g) in RGCs treated with VEH or IAA94. Bars represent the average percentage of colocalization ± SEM. **p < 0.01, Student's t-test.

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CLIC1-mediated current is required for RGC neurite elongation

The localization of CLIC1 at the GCs and the effects of its inhibition on GC morphology prompted us to investigate whether chloride permeability could affect RGC axonal outgrowth. To test this hypothesis, we cultured dissociated RGCs in the presence of BDNF, Ciliary neurotrophic factor, and forskolin, which have been previously reported to induce maximal axonal outgrowth in immunopurified RGCs (Goldberg et al. 2002) and retinal explants in vitro (Gasparini et al. 2011). To confirm the inhibition of CLIC1-mediated current by IAA94 or anti-NH2-CLIC1 antibody, we performed whole-cell perforated patch experiments on RGCs. Consistent with previous findings in other cells (Setti et al. 2013), RGCs membrane permeability showed to be sensitive to both CLIC1 ionic channel blocker and anti-NH2-CLIC1 antibody (Fig. 4a and b). To evaluate the effects of CLIC1 inhibition on neurite outgrowth, the IAA94 inhibitor was added to RGC cultures 30 min after seeding and neurite length was measured 24 h later. We found that neurite length was significantly reduced by 49.9% when CLIC1-mediated current was inhibited by IAA94 (Fig. 4d and f). To confirm that CLIC1-mediated current was specifically required, we analyzed neurite elongation upon application of an antibody directed against its N-terminal extracellular domain (i.e., anti-NH2-CLIC1 antibody) that inhibits CLIC1-mediated current (Tonini et al. 2000; Milton et al. 2008). We found that incubation of RGCs with the anti-NH2-CLIC1 antibody significantly reduced by 41.6% neurite length compared to untreated cultures (Fig. 4c, e and g) indicating that CLIC1-mediated current is required for RGC neurite outgrowth.

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Figure 4. Chloride intracellular channel 1 (CLIC1)-mediated current is required for axon outgrowth of cultured retinal ganglion cells (RGCs). (a and b) Representative time course membrane current recording in RGCs in perforated patch configuration, upon treatment with IAA94 (a) or AB NH2 (b) (n = 4). A voltage step of + 60 mV was applied every 5 s and the evoked current was measured. (c–e) Representative immunofluorescence images of RGCs treated for 24 h with vehicle (VEH; c), IAA94 (d) or anti-NH2 CLIC1 antibody (AB NH2; e) and stained for βIII-tubulin for axonal length determination. Scale bar is 10 μm. (f) Axon length of RGCs treated with IAA94. Axons from a total of 153 RGCs from three independent experiments were analyzed (VEH average length: 378.3 ± 43.5 μm, n = 68; IAA94 average length: 188.7 ± 11.5 μm, n = 84). **p < 0.01, Student's t-test. (g) Axon length of RGCs treated with AB NH2. Box plot represents the length of axons. Axons from a total of 177 RGCs from three independent experiments were analyzed (untreated average length: 346.7 ± 35.6 μm, n = 91; AB NH2 average length: 144.5 ± 18.2 μm, n = 86). **p < 0.01, Student's t-test.

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Consistent evidence indicates that cAMP elevation enhances RGC axonal outgrowth in response to neurotrophic stimuli and it does so through PKA activation (Goldberg et al. 2002). To investigate whether the raise of cAMP was required for CLIC1-mediated neurite outgrowth, we omitted forskolin from the growing medium while maintaining all others growth factors and we measured neurite outgrowth in the absence or presence of IAA94. Consistent with previous findings (Goldberg et al. 2002; Gasparini et al. 2011), RGCs cultured in the absence of forskolin grew shorter neurites (mean length ± SEM: 123.9 ± 12.2 μm) than those grown in presence of forskolin (mean length ± SEM: 452.5 ± 66.5 μm). Moreover, we found that, in the absence of forskolin, inhibition of CLIC1 did not affect RGC neurite outgrowth (Fig. 5), indicating that CLIC1 functional expression affects axonal elongation only when cAMP levels are elevated.

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Figure 5. Chloride intracellular channel 1 (CLIC1) inhibition does not affect axon outgrowth in the absence of forskolin. (a and b) Representative immunofluorescence images of retinal ganglion cells (RGCs) treated for 24 h with vehicle (a) or IAA94 (b) in forskolin-free medium and stained for βIII tubulin for axonal length determination. Scale bar is 10 μm. (c) Axon length of RGCs treated with IAA94. Axons from a total of 122 RGCs from three independent experiments were analyzed (VEH average length: 123.9 ± 12.2 μm, n = 60; IAA94 average length: 102.4 ± 12.6 μm, n = 62).

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To investigate whether the effects on neurite elongation and GC morphology depend on altered RGC viability, we examined the RGC survival in the selected experimental conditions using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell viability was similar in RGCs cultured in the presence or absence of forskolin (cell viability ± SEM. RGC with forskolin 1.0 ± 0.1; without forskolin 1.0 ± 0.2), IAA94 (0.9 ± 0.1), or vehicle (1.1 ± 0.2) for 24 h. No significant alterations of RGC viability were observed even after exposure to anti-CLIC1 antibody for 24 h (1.0 ± 0.01) or 3 days (1.1 ± 0.2) or after exposure to IAA94 for 3 days (0.9 ± 0.1). These findings indicate that the effects on neurite elongation and GC morphology are not a consequence of reduced RGC survival.

CLIC1 regulates the rate of RGC neurite outgrowth

To investigate how CLIC1 inhibition modulates neurite outgrowth, we performed time-lapse video microscopy on RGCs cultured for 24 h in the absence or presence of forskolin and/or IAA94. Cells were imaged every 10 min, starting 1 h after plating and the growing speed was calculated as described in the methods. Neurites of RGCs cultured in the presence of forskolin grew significantly faster than those of cells cultured without forskolin, consistent with previous data showing increased neurite length in forskolin-treated RGCs (Goldberg et al. 2002). IAA94 alone did not significantly affect the rate of neurite growth that remained similar to that of RGCs grown in forskolin-free medium. However, IAA94 significantly slowed down neurite outgrowth when coapplied with forskolin, indicating that CLIC1-mediated current regulates the speed of neurite growth upon cAMP elevation (Fig. 6a and b).

image

Figure 6. Axonal outgrowth speed depends on chloride intracellular channel 1 (CLIC1) functional expression. (a) Time-lapse video microscopy images of axonal outgrowth in retinal ganglion cells (RGCs) cultured for 24 h in the absence or presence of forskolin and/or IAA94. Images for the present figure were collected every 6 h. Scale bar is 50 μm. (b) Quantification of axonal growth rate. The growth rate was determined as described in the 'Methods'. Data were normalized on the average growing speed measured in the absence of forskolin. A total of 114 RGCs from three independent experiments were analyzed (VEH, n = 27; IAA94, n = 28; Fsk, n = 30; Fsk+IAA94, n = 29). *p < 0.05 by anova.

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CLIC1-mediated current is modulated by cAMP via PKA

To investigate how cAMP regulates CLIC1 activity, we performed whole-cell patch-clamp experiments to monitor the change in membrane permeability upon forskolin application in RGCs and neuronal N2a cells, which also express CLIC1 (Figure S2). In N2a cells, whole-cell perforated patch current recordings showed a sustained increase in current upon forskolin application, which was reduced upon IAA94 treatment (Fig. 7a). This demonstrated the presence of an IAA94-sensitive CLIC1-mediated current upon forskolin stimulation, which was calculated by subtracting the residual current after IAA94 perfusion to the forskolin-induced whole-cell current (Fig. 7b). The current/voltage (I/V) curve of the IAA94-sensitive CLIC1-mediated current showed a reversal potential of CLIC1-mediated current between −20 and −40 mV (Fig. 7e), which is the putative chloride reversal potential at external chloride concentration of 140 mM and internal chloride concentration around 40–60 mM, as it has been previously shown for N2a cells (Bettendorff et al. 2002).

image

Figure 7. Forskolin treatment elicits a chloride intracellular channel 1 (CLIC1)-mediated current. (a) Representative time course current recording in neuro-2a (N2a) cells in perforated patch configuration, upon forskolin treatment. A voltage step of + 60 mV was applied every 5 s and the evoked current was measured. The current amplitude decreased upon IAA94 perfusion. (b) Representative examples of current family upon forskolin treatment (upper traces), and upon IAA94 treatment (middle traces) in N2a cells. The lower traces represent the IAA94-sensitive current. (c) The initial IAA94 perfusion did not affect the current amplitude in naive N2a cells. However, after IAA94 washout, forskolin perfusion caused a clear increase of the current amplitude that was partially blocked by IAA94. (d) Quantification of the whole-cell current in N2a cells. The average current from all the experiments was normalized to the current baseline. Forskolin current amplitude and the residual current after IAA94 perfusion are shown as percentage (n = 8). **p < 0.01 by anova. (e) I/V curve of the IAA94-sensitive current in N2a cells. (f and g) Representative time course current recording in retinal ganglion cells (RGCs) in perforated patch configuration, upon forskolin (f) and 8-Br-cAMP (g) treatment. A voltage step of +60 mV was applied every 5 s and the evoked current was measured. The current amplitude decreased upon IAA94 perfusion (n = 3).

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The increase in membrane current upon forskolin perfusion occurred within minutes (Fig. 7a–c). Forskolin-elicited current was reduced by perfusion of the cells with IAA94, suggesting that the current is partially mediated by CLIC1. The same behavior was observed in RGCs. Both forskolin and the cell-permeable cAMP analogue, 8-Br-cAMP, increased membrane current within minutes from application (Fig. 7f and g). Such current was reduced by perfusion of IAA94 (Fig. 7f and g). These results indicate that cAMP elevation is associated with CLIC1 activation in both N2a and RGCs.

We next investigated whether CLIC1-mediated current was specifically prompted by activation of membrane-resident CLIC1 upon forskolin treatment. Indeed, IAA94 perfusion of N2a cells before forskolin treatment (Fig. 7c) did not affect the whole-cell current, demonstrating the lack of CLIC1-mediated current in the absence of forskolin. After washout of the channel blocker, forskolin perfusion increased the membrane current, which was reduced by subsequent IAA94 application. IAA94 only partially reverted forskolin-induced current (Fig. 7c and d), suggesting that forskolin activates additional ionic permeabilities. In fact, forskolin stimulation doubled the current, but only about 50% of the forskolin-induced current is IAA94 sensitive (Fig. 7d). Importantly, CLIC1-mediated current was detected also using whole-cell configuration. Patch-clamp in whole-cell configuration causes a partial dialysis of the cell, diluting the cytoplasmic components and thus reducing the concentration of soluble CLIC1. In such experimental setting, forskolin treatment was still able to induce an increase of the whole-cell current that was partially inhibited by IAA94 (Fig. 8a). These results indicate that forskolin activates the CLIC1 already resident in the plasma membrane.

image

Figure 8. Forskolin affects chloride intracellular channel 1 (CLIC1) functional expression via protein kinase A (PKA). (a and b) Whole-cell recording in neuro-2a (N2a) cells in the absence (a) or presence of 4 μM PKA inhibitor (PKI) (b) in the pipette solution. (c) Quantification of the average current elicited by forskolin in the absence or presence of PKI and IAA94 (n = 5). **p < 0.01 by anova.

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The increase of cAMP elicited by forskolin treatment may affect CLIC1 activity either via PKA activation, or through a direct binding to CLIC1. To discriminate between these two possibilities, we specifically inhibited PKA with the 6–22 peptide PKA inhibitor (PKI), which mimics the active portion of the heat-stable PKA inhibitor protein (Kubler et al. 1989). PKI was added to the pipette solution and, after the seal formation, the achievement of the whole-cell configuration allowed its diffusion into the cell cytoplasm. Five minutes later, the cell was challenged with forskolin. In PKA-inhibited cells, forskolin treatment failed to elicit any current increase (Fig. 8b). PKA inhibition abolished CLIC1-mediated current elicited by forskolin (Fig. 8c), suggesting that the effects of cAMP on CLIC1 ion channel are most likely mediated by PKA rather than by a direct interaction of cAMP with CLIC1.

Forskolin modulates CLIC1 ion channel activation

Whole-cell patch-clamp experiments suggest that cAMP-induced PKA activates plasma membrane-resident CLIC1. To investigate whether cAMP elicited its effects by altering the biophysical properties of CLIC1 ion channel, we performed single-channel recordings in cell-attached patch-clamp configuration (Fig. 9). CLIC1 single-channel recordings were elicited by voltage steps from −20 to +40 mV either in control conditions (Fig. 9a) or after forskolin stimulation (Fig. 9b). The single-channel conductance was calculated as the linear regression of the channel amplitudes at different membrane potentials and did not differ between control and forskolin-stimulated cells (Fig. 9c). In fact, the slope conductance was 9.56 ± 0.1 pS and 9.45 ± 0.1 pS in untreated and forskolin-stimulated cells, respectively. To compare the open probability of CLIC1 before and after forskolin treatment, we set the membrane potential at 0 mV and recorded the single-channel activity for 5 min both in control conditions and in forskolin-stimulated N2a cells (Fig. 9d and e). CLIC1 channel had a basal open probability (Po) ranging from 4 ± 0.1% at −20 mV to 7 ± 0.1% at 40 mV membrane potential (Fig. 8f). In forskolin-treated cells, Po was significantly higher at all voltages, increasing slightly from 15.5 ± 0.2% to 18.2 ± 0.2% between 0 mV and 40 mV, respectively (Fig. 9f).

image

Figure 9. Forskolin perfusion affects chloride intracellular channel 1 (CLIC1) biophysical properties by increasing the open probability of CLIC1 chloride channel. (a and b) Selected traces of CLIC1 cell-attached single-channel activity recorded at −40, −20, 0, and +20 mV membrane potential applied in control conditions (a) and upon application of forskolin (b). (c) Current/voltage relationships. Single-channel conductance was 9.56 ± 0.1 and 9.45 ± 0.1 pS for control (open circles) and forskolin stimulated cells (open squares), respectively (n = 5). (d and e) Continuous recording (2 min) of CLIC1 activity in cell-attached configuration in control conditions (d) or after 15-min perfusion of 10 μM forskolin (e) at 0 mV of cell membrane potential. The insets in each panel on a faster time scale reveal the single-channel openings, which are increased upon forskolin perfusion. (f) Channel open probability. The channel open probability changes from 6.5 ± 0.6% in control conditions (open circles) to 17.2 ± 0.9% upon forskolin perfusion (open squares) at 0 mV resting membrane potential.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Our findings demonstrate that CLIC1 is expressed in hippocampal neurons, in the adult murine retina, and in neonatal RGCs (Figs 1 and 2), where it plays a key role during neurite outgrowth induced by cAMP. In fact, when cAMP levels are elevated by forskolin, the specific inhibition of CLIC1-mediated current disrupts GC morphology (Fig. 3) and reduces neurite elongation of immunopurified RGCs (Fig. 4) by decreasing neurite growth speed (Fig. 6). The effects of CLIC1 require its ion channel permeability, are achieved by the plasma membrane-resident protein through cAMP-induced activation of PKA, and rely on increased open probability of the channel in the absence of changes in its conductance (Figs 7-9).

cAMP is a critical second messenger mediating neuronal survival and axonal elongation in response to neurotrophic stimuli. In cultured immunopurified RGCs, combinations of neurotrophic stimuli achieve maximal survival and axon extension when cAMP is elevated by the transmembrane adenylate cyclase activator forskolin, cell-permeable analogues of cAMP, electrical stimulation, or depolarizing conditions (Meyer-Franke et al. 1995; Goldberg et al. 2002). More recently, it has been shown that cAMP produced by concomitant activation of soluble adenylate cyclase and transmembrane adenylate cyclase achieves synergic actions on RGC survival and axonal elongation, possibly through effects in different subcellular compartments (Corredor et al. 2012). We now report that CLIC1 is a downstream effector of cAMP and PKA for inducing neurite outgrowth in RGCs. Our findings indicate that CLIC1 contributes to neurite outgrowth through modulation of its functional expression by cAMP. Indeed, forskolin application induces a CLIC1-mediated current in the plasma membrane of neuronal cells, which is prevented by inhibition of PKA (Figs 7-9). Consistently, neurite outgrowth in cultured RGCs is reduced when CLIC1 is inhibited by application of IAA94 (Fig. 4) or the anti-NH2-CLIC1 antibody (Fig. 4) that specifically inhibit CLIC1-mediated current (Tonini et al. 2000; Milton et al. 2008).

The mechanisms by which CLIC1-mediated current sustains cAMP-induced neurite elongation remain unclear. We speculate that CLIC1 activity might modulate the neuronal membrane potential. Previous data indicate that the internal chloride concentration is high in neonatal RGCs and decreases during development (Zhang et al. 2006). According with our single-channel measurements the chloride reversal potential in cultured RGCs oscillates from −40 to −30 mV, implying an intracellular chloride concentration between 40 and 60 mM. RGC resting potential oscillates between −70 and −80 mV membrane voltage (−74 ± 4.3 mV; n = 18), meaning that at rest chloride current is inward (chloride flowing out). Therefore, cAMP-induced increase in chloride channel activity would favor depolarization, which, according to previous findings, contributes to axon development (Goldberg et al. 2002). Conversely, the inhibition of CLIC1-mediated current by IAA94 treatment would cause hyperpolarization and result in impaired axonal outgrowth, which is consistent with our present findings.

Alternatively, CLIC1 may achieve its effects on neurite outgrowth by regulating the transient or chronic status of oxidative stress of the system. Our previous findings in cultured microglia indicate that CLIC1-mediated current contributes to determine the oxidative status of the cells (Milton et al. 2008). Transient conditions of oxidative stress are cyclically present in active GCs and physiological levels of ROS are critical for maintaining a dynamic actin cytoskeleton and controlling neurite outgrowth (Cheung et al. 2000; Munnamalai and Suter 2009). Notably, oxygen levels affect axon pathfinding in Caenorhabditis elegans (Pocock and Hobert 2008) and oxidized proteins induce elongation and regeneration of transected RGC axons in the cat in vivo (Okada et al. 2005; Watanabe 2010). The effects of oxidized proteins are to some extent mimicked by cAMP elevation induced by forskolin (Okada et al. 2005), suggesting a possible involvement of CLIC1-mediated mechanisms. However, further investigation is warranted to evaluate whether CLIC1-mediated current is required for axonal elongation and axon regeneration in vivo.

We found that the cytosolic levels of cAMP serve as a regulatory mechanism for CLIC1 activity in the plasma membrane. Patch-clamp experiments in whole-cell configuration indicate that cAMP induces CLIC1-mediated current and it possibly does so by modulating the biophysical properties (i.e., open probability) of the channel that is already resident in the plasma membrane, rather than inducing its translocation from the cytosol (Figs 7-9). A direct interaction of cAMP with the ion channel may underlie these effects. However, several lines of evidence indicate that cAMP indirectly affects CLIC1-mediated current via PKA activation. First, we showed here that the PKA inhibitor, PKI, abolishes CLIC1-mediated current elicited by forskolin treatment in cultured neuronal cells (Fig. 8). Second, in silico studies suggest that CLIC1 has a putative PKA phosphorylation site (Valenzuela et al. 1997), suggesting that the channel may be directly phosphorylated by cAMP-activated PKA. Finally, it has been reported that CLIC1 interacts with AKAP350 (Shanks et al. 2002), which can scaffold several protein kinases and phosphatases, including PKA (Schmidt et al. 1999).

Another possibility is that cAMP-mediated PKA activation regulates CLIC1-mediated current by acting on CLIC1 interacting proteins, such as F-actin (Singh et al. 2007). Mammalian CLIC proteins interact extensively with cytoskeletal components. For example, rat brain CLIC4 (p64H1) associates with actin in a multiprotein complex (Suginta et al. 2001). Moreover, human CLIC5 is present in an actin-rich complex in placental microvilli (Berryman and Bretscher 2000) and associates with cortical actin (Berryman et al. 2004). Notably, studies of the recombinant proteins reconstituted in planar lipid bilayers in vitro demonstrated that F-actin strongly and reversibly inhibits CLIC1 and CLIC5, but not CLIC4, in the absence of other proteins, unraveling potential effects of the actin cytoskeleton on the chloride channel activity (Singh et al. 2007). We found that inhibition of CLIC1 by bath application of IAA94 disrupts the morphology of actin cytoskeleton in GC of cultured RGCs. Indeed, IAA94 treatment reduces the colocalization of CLIC1 with actin. CLIC1 inhibition increased the proportion of collapsed GCs, thereby reducing the speed of neurite outgrowth (Figs 3 and 6). Overall, these data suggest a possible bidirectional regulation between actin and CLIC1 via cAMP-induced PKA-mediated mechanisms and CLIC1-mediated current to sustain GC activity and axonal outgrowth. However, further studies are required to validate this hypothesis.

It has been previously hypothesized that cAMP increases the responsiveness to trophic factors. Here, we showed that, once activated, cytoplasmic cAMP, through a modulation of CLIC1 activity, results in neurite outgrowth modulation (Figs 5 and 6). Although more than 15 years have passed since CLIC1 was cloned (Valenzuela et al. 1997), a defined physiological role of the protein in the brain, and especially in neurons, remains still uncertain. A possible role of CLIC1 functional expression as a chloride ion channel has been proposed in β-amyloid (Aβ)-stimulated microglia cells, where it has been suggested to have a crucial role in ROS production during neurodegenerative processes (Novarino et al. 2004; Milton et al. 2008). The present results now propose that CLIC1 functional expression as a chloride-selective ion channel on the plasma membrane plays a key role in sustaining neurite outgrowth in the presence of cAMP (Figs 4 and 6) and maintaining GC morphology (Fig. 3). This action of CLIC1 is regulated by PKA (Fig. 8), which affects the biophysical properties of the channel increasing its open probability (Fig. 9). Based on these findings, we propose that, in RGCs, CLIC1 may act as a transient modulator of neurite outgrowth depending on growth factors balance, sustaining the elongation of axons during optic nerve formation. Moreover, we speculate that CLIC1-mediated current might play a role during axonal regeneration in injured optic nerve. However, future studies are warranted to analyze the role of CLIC1 in axon outgrowth in vivo and the potential exploitation of CLIC1-mediated mechanisms in neuronal regeneration.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

We thank Dr Samuel Breit (St Vincent's Centre of Applied Medical Research, Sydney, Australia) for the kind gift of the anti-NH2 CLIC1 antibody. This work was supported by the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), PRIN to MM, and by IIT Intramural funds to LG (grant number PRIN 2007).

The authors have no conflict of interest to declare.

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  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12832-sup-0001-FigS1.tifimage/tif178K

Figure S1. Fitting method to quantify neurites' growth rates in time-lapse microscopy experiments.

jnc12832-sup-0002-FigS2.tifimage/tif181K

Figure S2. Western blot analysis of CLIC1 in N2A cells untransfected or transfected with M2-tagged CLIC1 (arrow). Arrowhead indicates endogenous CLIC1 protein.

jnc12832-sup-0003-FigS1-S2.pdfapplication/PDF292K 

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