158. Real-Time Visualization of Neuronal Differentiation and Neurite Projection Using an Indicator HSV Amplicon Vector

Abstract

Top of pageAbstract Stem cell-based replacement therapy has great potential in treating neurological disorders, including brain and spinal cord injuries. Although several studies have demonstrated neuronal and glial differentiation of transplanted neural stem cells (NSCs) in vivo, the efficacy of the transplanted NSCs to generate neurons and of the newly generated neurons to form new synaptic connections with the remaining host neurons still needs clarification. To address these issues, we have constructed an HSV amplicon-based indicator vector that allows visualization of neuronal differentiation and neurite projection. Synapsin I (SYN) is a well characterized gene that expresses selectively in neurons. Thiel and colleagues found that a 400-bp 5' untranslated promoter region of SYN directed prominent expression of a reporter gene in various neuronal cell lines, but that no expression was detected in cell lines derived from non-neuronal tissue (Thiel G et al., PNAS 1991). On the other hand, microtubule- associated protein tau-reporter fusion protein has been used as a genetic reporter to visualize neuronal processes in vivo (Callahan CA et al., PNAS 1994). We, therefore, combined the two genetic elements in the context of HSV amplicon vectors, producing an indicator herpes simplex virus (HSV) amplicon vector, HSV-SYN-tauR, that carries tau fused with a monomeric red fluorescent protein (mRFP, Campbell et al., PNAS 2002) under the control of the 400- bp SYN promoter. When a mixed culture of mouse cortical neurons and glia was transduced with the HSV-SYN-tauR amplicon vector, RFP reporter was expressed only in neurons along their neurites, allowing visualization of every axon, dendrite, and spine. Time-lapse imaging of mixed cortical cultures transduced with the HSV-SYN-tauR amplicon vector allowed noninvasive real-time monitoring of extension and retraction of the neurites of live neurons. This indicator HSV amplicon vector could prove useful in studying neuronal differentiation, neurite projections, and regeneration of neuronal networks in vitro and in vivo.