Enhancing Structural Plasticity of PC12 Neurons During Differentiation and Neurite Regeneration with a Catalytically Inactive Mutant Version of the zRICH Protein

Abstract Background Adult humans, as warm-blooded vertebrates, do not regenerate axons in their central nervous system (CNS) spontaneously. Conversely, cold-blooded vertebrates demonstrate remarkable abilities for nerve regeneration in their CNS. Studies of the molecular mechanisms of nerve regeneration have led to the discovery of several proteins that are induced during successful nerve regeneration. RICH proteins were identified as proteins induced during the regeneration of the optic nerve of teleost fish. These proteins are 2’,3’-cyclic nucleotide, 3’-phosphodiesterases that can bind to cellular membranes through a carboxy-terminal membrane localization domain. They interact with the tubulin cytoskeleton and are able to enhance neuronal structural plasticity by promoting the formation of neurite branches. Results PC12 stable transfectant cells expressing a fusion protein combining a red fluorescent protein with a catalytically inactive mutant version of zebrafish RICH protein were generated. These cells were used as a model to analyze effects on neuritogenesis. Computer-assisted morphometric analysis methods were developed to determine the effects of expressed proteins on neuritogenesis with this model system. Conclusions Two different methods indicated that the catalytically inactive RICH protein induced the formation of branching points and secondary neurites both during differentiation and neurite regeneration. The use of a fluorescent fusion protein facilitates detection of expression levels, and a procedure based on analysis of random field images was developed to increase the efficiency for determining effects of expressed proteins on neuronal structural plasticity, providing comparable results to classic neurite tracing methods.