Transient Receptor Potential Canonical 5 Channels Activate Ca²⁺/Calmodulin Kinase Iγ to Promote Axon Formation in Hippocampal Neurons

Abstract

Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca 2ϩ -signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca 2ϩ /calmodulin kinase kinase (CaMKK) and its downstream target Ca 2ϩ / calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the ␥-isoform of CaMKI as shRNA to CaMKI␥, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKI␥, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKI␥ as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKI␥ and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKI␥, which subsequently promote axon formation.

CaMKK–CaMKI Signaling Pathways Differentially Control Axon and Dendrite Elongation in Cortical Neurons: Figure 1.

“…In this study, transient receptor potential canonical 5 channel (TRPC5) activation stimulated axon elongation via CaMKI␥ (Davare et al, 2009). Some of the findings of Davare et al (2009) are consistent with those of Ageta-Ishihara et al (2009); both groups observed that pharmacological inhibition of CaMKK or knockdown of CaMKI or CaMKK blocked axon elongation and that deficits in axon elongation could be rescued by expression of constitutively active CaMKI. However, the studies disagree on the specific CaMKI isoform responsible for axon elongation.…”

“…In contrast to Ageta-Ishihara et al (2009), another recent publication in The Journal of Neuroscience reported that CaMKI␥ is responsible for axonal rather than dendritic elongation in hippocampal cultures (Davare et al, 2009). In this study, transient receptor potential canonical 5 channel (TRPC5) activation stimulated axon elongation via CaMKI␥ (Davare et al, 2009).…”

“…However, the studies disagree on the specific CaMKI isoform responsible for axon elongation. Davare et al (2009) observed that knockdown of CaMKI␥ and to a lesser extent CaMKI␣ each significantly affected axonogenesis, but only CaMKI␥ activity affected total axon length.…”

“…This scheme incorporates combined results from Takemoto of CaMKI␣ on axon elongation. However, apparent conflict with the results of Davare et al (2009) calls for examination of whether CaMKK-CaMKI signaling pathways differ in cortical versus hippocampal neurons due to differences in CaMKI␥ signaling or cell density-dependent network activity.…”

Calcium transporters and their role in the development of neuronal disease and neuronal damage

“…The mechanism of activation is dependent on activation of PLC by guidance cues, which in turn leads to the production of IP 3 and Ca 2+ release from ER. Depletion of these stores opens the TRPC and Ca 2+ influx occurs (Davare et al 2009). …”