Abstract
The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.
Highlights
Neuron development and differentiation are complex processes that involve dynamic cell morphology changes
We found that ADP ribosylation factor 6 (ARF6) knockdown resulted in increased glucosylceramide content and glucosylceramide synthase activity in Neuro-2a neuronal cells
It has previously been observed that inactivation of ARF6 by overexpression of a GAP or a dominant-negative ARF6 promotes neuron differentiation, as shown by increased neuronal outgrowth, in various neuronal cell systems [9,19]
Summary
Neuron development and differentiation are complex processes that involve dynamic cell morphology changes. We investigated whether ARF6-dependent neuron differentiation is regulated by alterations in lipid composition. We found that ARF6 knockdown resulted in increased glucosylceramide content and glucosylceramide synthase activity in Neuro-2a neuronal cells. We found that ARF6-dependent neuron differentiation is regulated by the altered glucosylceramide synthase activity in neuronal cells.
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