Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan‐dependent inhibition of neurite extension

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Abstract
The central nervous system (CNS) fails to regenerate after injury. A glial scar forms at the injury site, contributing to regenerative failure partly resulting from the chondroitin sulfate proteoglycans (CSPGs) in the glial scar. The family of Rho GTPases, which includes Cdc42, Rac1, and RhoA, is involved in growth cone dynamics. Although the response of neural cells to the inactivation of Rho when contacting myelin‐related substrates, or CSPG, has been investigated, Rac1's and Cdc42's abilities to modulate CSPG‐dependent inhibition have yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108‐15 cells and E9 chick dorsal root ganglia (DRGs) were cultured on the lanes. By using the protein delivery agent Chariot, the neuronal response to exposure of constitutively active (CA) and dominant negative (DN) mutants of the Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percentage ratio of neurites crossing the CSPG lanes. CA‐Cdc42, CA‐Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared with the negative controls for both the NG108‐15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac, as well as inhibition of Rho, helps overcome the CSPG‐dependent inhibition of neurite extension.