Delivery of Cdc42, Rac1, and Brain-derived Neurotrophic Factor to Promote Axonal Outgrowth After Spinal Cord Injury

Injury severs the axons in the spinal cord causing permanent functional loss. After injury, a series of events occur around the lesion site, including the deposition of growth cone inhibitory astroglial scar tissue containing chondroitin sulfate proteoglycan (CSPG)- rich regions. It is important to encourage axons to extend through these inhibitory regions for regeneration to occur. The work presented in this dissertation investigates the effect of three proteins, constitutively active (CA)-Cdc42, CA-Rac1, and brain-derived neurotrophic factor (BDNF) on axonal outgrowth through CSPGs-rich inhibitory regions after spinal cord injury (SCI). Cdc42 and Rac1 are members of the Rho GTPase family and BDNF is a member of the neurotrophin sub-family. These three proteins affect the actin cytoskeleton dynamics. Therefore, Cdc42, Rac1, and BDNF promote axonal outgrowth. The effect of CA-Cdc42 and CA-Rac1 on neurite extension through CSPG regions was determined in an in vitro model. Rac1 and Cdc42 s ability to modulate CSPG-dependent inhibition has 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 ganglions (DRGs), were cultured on the lanes. Using the protein delivery agent Chariot, the neuronal response to exposure of CA and dominant negative (DN) Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percent 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 to 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 helps overcome the CSPG-dependent inhibition of neurite extension. In an in vivo study, CA-Cdc42 and CA-Rac1 were locally delivered into a spinal cord cavity. Additionally, BDNF was delivered to the lesion site, either individually or in combination with either CA-Cdc42 or CA-Rac1. The dorsal over-hemisection model was utilized, creating a ~2mm defect that was filled with an in situ gelling hydrogel scaffold containing lipid microtubules loaded with the protein(s) to encourage axons. The lipid microtubules enable slow release of proteins while the hydrogel serves to localize them to the lesion site and permit axonal growth. The results from this study demonstrate that groups treated with BDNF, CA-Cdc42, CA-Rac1, BDNF/CA-Cdc42, and BDNF/CA-Rac1 had significantly higher percentage of axons from the corticospinal tract (CST) that traversed the CSPG-inhibitory regions, as well as penetrate the glial scar compared to the untreated and agarose controls. Although axons from the CST tract did not infiltrate the scaffold-filled lesion, NF-160+ axons were observed in the scaffold. Treatment with BDNF, CA-Cdc42, and CA-Rac1 also reduced the inflammatory response, quantified by analyzing GFAP and CS-56 intensity for reactive astrocytes and CSPGs, respectively, at the interface of the scaffold and host tissue. Therefore, the local delivery of CA-Cdc42, CA-Rac1 and BDNF, individual and combination demonstrated the ability of axons to extend through CSPG inhibitory regions, as well as reduce the glial scar components.Ph.D.Committee Chair: Bellamkonda, Ravi V.; Committee Member: Brady-Kalnay, Susann M.; Committee Member: LaPlaca, Michelle C.; Committee Member: McDevitt, Todd C.; Committee Member: McKeon, Robert J