Kinase-dependent differentiation of a retinal ganglion cell precursor.
OBJECTIVE
Cell lines are frequently used to elucidate mechanisms of disease pathophysiology. Yet extrapolation of results with cell lines to neurodegenerative disorders is difficult because they are mitotic and usually have other non-neuronal properties. The RGC-5 cell line has many features of retinal ganglion cells (RGCs). Despite its expression of Thy-1 and NMDA receptors, as found in primary RGCs, this line's ability to proliferate and non-neuronal appearance differentiate it from other central neurons, complicating its use for the study of neuronal survival, electrophysiology, or neurite extension.


METHODS
A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 muM staurosporine and assessed for a variety of differentiation markers.


RESULTS
Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors.


CONCLUSIONS
This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.
Frassetto
Laura J
LJ
Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, WI 53792, USA.
Schlieve
Christopher R
CR
Lieven
Christopher J
CJ
Utter
Amy A
AA
Jones
Mathew V
MV
Agarwal
Neeraj
N
Levin
Leonard A
LA
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D007473:Ion Channels / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000201:enzymology; D019311:Staurosporine / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology; D018800:Thy-1 Antigens / Q000378:metabolism
D015415:Biomarkers; D004791:Enzyme Inhibitors; D007473:Ion Channels; D011493:Protein Kinase C; D016194:Receptors, N-Methyl-D-Aspartate; D019311:Staurosporine; D018800:Thy-1 Antigens