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.

These findings suggest that superoxide generated in the mitochondrial electron transport chain could be a parallel system to neurotrophic deprivation for signaling cell death after axonal injury.

No abstract

Retinal ganglion cells (RGCs) are central neurons that undergo apoptosis after axonal injury. As the relationship between mitochondrial and oxidative signaling of apoptosis in neuronal systems is unclear, we sought to achieve a better understanding of the interplay of these two pathways by investigating the effect of direct oxidative stress on mitochondrial membrane potential in cultured RGCs, as measured with the dual-emission probe JC-1. Treatment with hydrogen peroxide caused RGC mitochondrial depolarization. Several pharmacological treatments were used to define the mechanism. Whereas cycloheximide, tris(2-carboxyethyl)phosphine, and cyclosporin A were unable to prevent the depolarization, bongkrekic acid significantly reduced the severity of the depolarization. This suggests that the hydrogen peroxide-induced depolarization may act through mitochondrial permeability transition pore opening independent of thiol oxidation, and may be preventable under certain conditions.

The reactive oxygen species superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. Based on this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1), promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including ASK1, JNK or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the ERK1/2 pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.