Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro.

Ganglion cells are the only cells from the retina that physically interact with the central nervous system, and they play a critical role in transmitting light signals to visual processing centers in the brain. In a number of neurodegenerative conditions including glaucoma,1–3 these terminally differentiated retinal ganglion cells (RGCs) undergo cell death.4–7 However, the mechanisms underlying the death of RGCs are still poorly understood. We have previously reported that under certain glaucoma-related degenerative conditions, increased levels of two plasminogen activators (PAs), the tissue plasminogen activator (tPA) and the urokinase type plasminogen activator (uPA), promoted the death of RGCs. First, we reported that retinal ischemia, induced by the ligation of the optic nerve in mice, leads to elevated levels of tPA and uPA in the ganglion cell layer of the retina, and that elevated levels of these proteases, in turn, promote the death of RGCs.8 Second, we reported that hyperstimulation of retinal glutamate receptors in mice (excitotoxicity) also leads to elevated levels of tPA and uPA and that these proteases, in turn, promote the death of RGCs.9 In addition, recent studies from other laboratories have reported that the same proteases promote the death of RGCs in response to hyperstimulation of glutamate receptors.10,11 Despite this evidence, it is still unclear whether these proteases directly cause the death of RGCs. Therefore, we explored an in vitro system in which RGCs are devoid of tPA and uPA and in which levels of both these proteases can be controlled. Despite the fact that primary cultures of RGCs are appropriate for in vitro studies, primary RGCs survive for few generations in vitro and their isolation from retinas is cumbersome. In addition, normal adult RGCs express low levels of tPA proteolytic activity constitutively,9 whereas our study required ganglion cells devoid of PAs. Previous studies have used PC12 cells, in which tPA levels can be induced, but these cells are originally derived from a rat pheochromocytoma,12 not from the normal retina, and these cells do not share characteristic features of RGCs. Recent studies indicated that a rat transformed ganglion cell line, RGC-5, has a number of characteristic features of normal RGCs, including the expression of Thy-1, Brn-3c, and glutamate receptors.13 Although RGC-5 cells can be used as a substitute for primary RGCs, these cells proliferate in vitro and exhibit morphology similar to that of fibroblasts. RGC-5 cells, nevertheless, can be differentiated into neuronlike (and nonmitotic) cells by treatment with a nonspecific kinase inhibitor, staurosporine.14 Furthermore, we observed that undifferentiated RGC-5 cells do not express tPA proteolytic activity constitutively; therefore, these cells seemed suitable for our study. Because previous studies on PC12 cells indicated that staurosporine induces tPA proteolytic activity15 and because undifferentiated RGC-5 cells do not express tPA proteolytic activity, we used RGC-5 cells to modulate proteolytic activities of tPA and uPA and determined whether these proteases play a direct role in differentiation and in cell death. Here, we report that elevated levels of tPA and uPA, indeed, play a direct role in the death of RGC-5 cells.