Optogenetic Stimulation of Ca2+ Influx via Channelrhodopsin CapChR2 in Schwann Cells Promotes Neurite Outgrowth in...

Schwann cells (SCs) are essential players in peripheral nerve regeneration, contributing to both myelination and neuronal support via paracrine and contact-mediated mechanisms. Ca²⁺ influx is a critical regulator of cellular activation with diverse cells; however, the potential of optogenetic manipulation of Ca²⁺ signals in SCs to influence neuronal growth has not been thoroughly investigated. This study aimed to evaluate whether light-induced Ca²⁺ entry through a Ca²⁺-permeable channelrhodopsin (CapChR2) in SCs could enhance neurite outgrowth in co-cultured PC12 cells.

Immortalized Fischer rat SCs (IFRS1) were genetically modified to express CapChR2 and exposed to blue light. Intracellular Ca²⁺ dynamics were monitored using fura-2-based ratiometric imaging. Whole-cell patch-clamp recordings were used to assess light-gated cation influx through CapChR2 and characterize photocurrent properties. For functional assays, PC12 cells were co-cultured with IFRS1 under four different conditions: with or without light stimulation, and with or without CapChR2 expression. Neurite outgrowth was assessed by calculating the percentage of cells with neurites longer than ten micrometers and by measuring the average neurite length.

CapChR2-expressing IFRS1 showed an increase in intracellular Ca²⁺ levels upon light stimulation (ΔRatio: 0.13 ± 0.05) compared to dark controls (0.04 ± 0.02). In contrast, vector-transfected cells did not respond to light stimulation. Whole-cell patch-clamp recordings confirmed light-evoked inwardly rectifying photocurrents in CapChR2-expressing cells, whereas no photocurrent was detected in vector-transfected control cells. Ion selectivity analysis indicated variable Ca²⁺ permeability with the duration of light exposure, and the PCa²⁺/PNa⁺ ratio was estimated to be approximately 1.2 at 10 s and 0.7 at 30 s. In functional co-culture assays, PC12 cells grown with CapChR2-expressing IFRS1 displayed a significantly higher proportion of neurite-bearing cells (59.6-73.6 %) under both with and without light stimulation, compared to control conditions (41.4-47.3%). Moreover, a significant increase in neurite length was observed only in PC12 cells co-cultured with CapChR2-expressing IFRS1 under light-stimulated conditions.

Optogenetic activation of CapChR2-expressing IFRS1 successfully induced light-dependent Ca²⁺ influx and enhanced their capacity to promote neurite outgrowth in co-cultured PC12 cells. These results highlight the utility of glial optogenetic modulation as a novel and controllable approach to facilitate axonal regeneration. This strategy may hold therapeutic potential for the treatment of peripheral nerve injuries and demyelinating disorders by enhancing the regenerative microenvironment through targeted glial activation.