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The Journal of Neuroscience, July 11, 2007, 27(28):7447-7458; doi:10.1523/JNEUROSCI.4266-06.2007

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Cellular/Molecular
Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with 5ß1 Integrin Initiates Neurite Outgrowth

Gusheng Wu,¹ Zi-Hua Lu,¹ Alexander G. Obukhov,² Martha C. Nowycky,² and Robert W. Ledeen^1,2

Departments of ¹Neurology and Neurosciences and ²Physiology and Pharmacology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

Correspondence should be addressed to Dr. Gusheng Wu, Department of Neurology and Neurosciences, MSB-H506, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. Email: gwu{at}umdnj.edu

Previous studies demonstrated that cross-linking of GM1 ganglioside with multivalent ligands, such as B subunit of cholera toxin (CtxB), induced Ca²⁺ influx through an unidentified, voltage-independent channel in several cell types. Application of CtxB to undifferentiated NG108-15 cells resulted in outgrowth of axon-like neurites in a Ca²⁺ influx-dependent manner. In this study, we demonstrate that CtxB-induced Ca²⁺ influx is mediated by TRPC5 channels, naturally expressed in these cells and primary neurons. Both Ca²⁺ influx and neurite induction were blocked by TRPC5 small interfering RNA (siRNA). Pretreatment of NG108-15 cells with neuraminidase increased cell-surface GM1 and greatly enhanced the signal. GM1 was not directly associated with TRPC5 but rather with 5ß1 integrin, which opened the channel through a signaling sequence after cross-linking of the GM1/integrin complex. This cascade included autophosphorylation of focal adhesion kinase and subsequent activation of phospholipase C (PLC) and phosphoinositide-3 kinase [PI(3)K]. Pharmacological blockers that inhibited tyrosine kinase, PLC, and PI(3)K suppressed both CtxB-induced Ca²⁺ influx and neurite outgrowth. These were also suppressed by SK&F96365, a nonspecific transient receptor potential channel blocker. Confocal immunocytochemistry revealed that GM1 cross-linking induced colocalization of GM1 with these signaling elements in sprouting regions of plasma membrane. In primary cerebellar granular neurons (CGNs), TRPC5 was detected at 2 d in vitro (2 DIV), a stage corresponding to CtxB-stimulated Ca²⁺ influx. Neurite outgrowth in CGNs, determined at 3 DIV, was accelerated by CtxB and suppressed by TRPC5 siRNA and the above blockers. The crucial role of GM1 was indicated with CGNs from ganglio-series null mice, in which growth of axons was significantly retarded.

Key words: TRPC5 channel; GM1 ganglioside; calcium signaling; integrin; neuritogenesis; focal adhesion kinase

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Received Sept. 29, 2006; revised May 18, 2007; accepted May 21, 2007.

Correspondence should be addressed to Dr. Gusheng Wu, Department of Neurology and Neurosciences, MSB-H506, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. Email: gwu{at}umdnj.edu

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