Lentiviral vector-mediated knockdown of the neuroglycan 2 proteoglycan or expression of neurotrophin-3 promotes neurite outgrowth in a cell culture model of the glial scar

Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up-regulated following spinal cord injury. Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin-3 (NT-3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT-3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over-expression of NT-3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS).