Reduction of Chondroitin Sulfate Proteoglycans to Increase Axonal Outgrowth In a Neuronal Co-Culture Model
Hannah M. Tuinstra1, Amy C. Sebeson2, and Lonnie D. Shea1. (1) Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd, Tech E136, Evanston, IL 60208, (2) Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208
Regeneration after spinal cord injury is limited in part by an environment that inhibits axonal outgrowth, and overcoming this barrier is a necessary step. In particular, deposition of an inhibitory molecule, chondroitin sulfate proteoglycan (CSPG), is increased through the activation of astrocytes at the injury site. Our laboratory is developing an in vitro neuronal-astrocyte co-culture model to investigate methods to degrade and prevent the production of CSPGs after spinal cord injury. Neu7, the astrocyte cell line used in this model was derived from rat cerebral cortex astrocytes, produces CSPGs and inhibits axon growth. Several approaches have been investigated to decrease CSPGs associated with Neu7 cells, including enzyme degradation, non-viral delivery of shRNAs, and siRNA. These approaches can be implemented by either encapsulating factors within poly(lactide-co-glycolide) bridges for localized release or by immobilization to the culture surface. Initial studies targeted degradation of CSPGs by treatment with the enzyme chondroitinase ABC. CS-56 staining for CSPGs revealed a 20% decrease in average fluorescence intensity relative to untreated cells. Subsequent experiments attempted to limit the production of CSPGs using shRNA. Neu7s with shRNA vectors against enzymes involved in assembling CSPGs (such as chondroitin polymerizing factor) could lead to a reduction in CSPGs and allow for greater axonal outgrowth from the co-cultured neurons. Neu7 cells were transfected with Transfast-pEGFPLuc lipoplexes resulting in luciferase transgene expression of 300 RLU per μg protein, with 5% of the cells transfected. This low percentage of transfected cells was expected to have minimal effect on axonal outgrowth. The final approach was to employ siRNA to target enzymes involved in CSPG assembly, as siRNA is active within the cytoplasm and was expected to have greater efficacy. Preliminary results with siRNA transfection indicate that transcript levels can be reduced by 85%. Results will be presented for axonal outgrowth in the neuronal-astrocyte co-culture model. These studies will identify the most effective ways to overcome the CSPG inhibition of axonal outgrowth.