Axonal growth regulation of fetal and ES-derived dopaminergic neurons by Netrin-1 and Slits

¹ Harvard University Udall Parkinson's Disease Research Center of Excellence and Neuroregeneration Laboratories, Harvard Medical School/McLean Hospital, Belmont, Massachusetts

^* To whom correspondence should be addressed. E-mail: llin{at}mclean.harvard.edu.

	Abstract

The physical restoration of dopamine circuits damaged or lost in Parkinson's disease by implanting embryonic stem (ES) derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors, and Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here we also demonstrate that ES-differentiated neurons including DA neurons express Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the co-culture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the co-cultures of ES-derived DA neurons with Netrin-1, Slit1 and Slit3 producing cells. The findings suggest that ESderived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells, but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning and conditioning during ES cell differentiation.

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