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.