Opposing Roles of Voltage-Gated Ca2+ Channels in Neuronal Control of Regenerative Patterning

There is intense interest in developing methods to regulate proliferation and differentiation of stem cells into neuronal fates for the purposes of regenerative medicine. One way to do this is through in vivo pharmacological engineering using small molecules. However, a key challenge is identification of relevant signaling pathways and therein druggable targets to manipulate stem cell behavior efficiently in vivo. Here, we use the planarian flatworm as a simple chemical-genetic screening model for nervous system regeneration to show that the isoquinoline drug praziquantel (PZQ) acts as a small molecule neurogenic to produce two-headed animals with integrated CNSs following regeneration. Characterization of the entire family of planarian voltage-operated Ca2+ channel alpha subunits (Ca-v alpha), followed by in vivo RNAi of specific Ca-v subunits, revealed that PZQ subverted regeneration by activation of a specific voltage-gated Ca2+ channel isoform (Ca(v)1A). PZQ-evoked Ca2+ entry via Ca(v)1A served to inhibit neuronally derived Hedgehog signals, as evidenced by data showing that RNAi of Ca(v)1A prevented PZQ-evoked bipolarity, Ca2+ entry, and decreases in wnt1 and wnt11-5 levels. Surprisingly, the action of PZQ was opposed by Ca2+ influx through a closely related neuronal Ca-v isoform (Ca(v)1B), establishing a novel interplay between specific Ca(v)1 channel isoforms, Ca2+ entry, and neuronal Hedgehog signaling. These data map PZQ efficacy to specific neuronal Ca-v complexes in vivo and underscore that both activators (Ca(v)1A) and inhibitors (Ca(v)1B) of Ca2+ influx can act as small molecule neurogenics in vivo on account of the unique coupling of Ca2+ channels to neuronally derived polarity cues.