Mueller, Brett H.


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Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) that affects over 70 million individuals worldwide. Glaucoma pathology is manifested as cupping of the optic disk, damage to the nerve fiber layer, and visual field deficits. The final pathological step of this disease contributing to visual field loss is the apoptosis of retinal ganglion cells (RGCs). Currently, the only therapeutic agents that are used to treat glaucoma are IOP lowering drugs. However, even when IOP is brought within normal range, a significant number of patients still have progression of visual deficits. Currently, there are no treatment options that have the ability to sustain the viability of RGCs during the disease process of glaucoma. Therefore, neuroprotective drugs that protect RGCs need to be developed as adjunct therapeutic agents to IOP lowering drugs. The sigma-1 receptor (σ-1r) is a non-opioid receptor that has been shown to have the ability to bind to benzomorphans, steroids, and psychotropic drugs. This receptor is ubiquitously expressed throughout the entire body; however, the endogenous ligand and function of σ-1r is not yet known. Several in vitro and in vivo studies have demonstrated the neuroprotective effects of σ-1r stimulation in several models of retinal neurodegenerative diseases including glaucoma and diabetic retinopathy. Numerous studies have linked the neuroprotective effects of σ-1r to its ability to block cytotoxic calcium ion influx through ligand gated and voltage gated ion channels, modulation of ER stress, maintenance of mitochondrial homeostasis, and stimulation of pro-survival intracellular signaling pathways. However in primary RGCs, there have been no studies demonstrating σ-1 receptor mechanism of action. The only proposed neuroprotective mechanism of action of σ-1r that has been performed in retinal flat mounts is blockage of calcium ion influx through activated NMDA receptors. This present research project investigated the mechanism of neuroprotective effects of σ-1rs in primary RGCs, particularly involving L-type voltage gated calcium channels (VGCCs) and activation of extracellular-signal-regulated kinases (ERK 1/2). We demonstrated that VGCCs were activated using KCl (20mM). Pre-treatment with a known L-type VGCC blocker produced a 57% decrease in calcium ion influx through activated VGCCs (following depolarization by KCl). In addition, calcium imaging showed that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Treatment with a σ-1r antagonist, BD1047, produced a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs. This confirms that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms of action of both these agents. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in primary RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in primary RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in primary RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in primary RGCs implies that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs Another signaling pathway that was studied as a potential target of σ-1r mediated neuroprotection was the MAP kinase pathway, in particular, ERK phosphorylation as an index of cell survival. RGCs subjected to oxygen and glucose deprivation (OGD) for 6 hours induced 50% cell death in primary RGCs and inhibited pERK1/2 expression by 65%. Cell death was attenuated when RGCs were treated with pentazocine under OGD and pERK1/2 expression was increased by 1.6 fold compared to OGD treated RGCs without pentazocine treatment. The co-treatment of with an ERK1/2 inhibitor PD098059 with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. These results established a link between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. In conclusion, we have established two novel mechanisms underlying σ-1 receptor mediated neuroprotection in primary RGCs. These findings suggest that activation of the σ-1 receptor in RGCs has a role in calcium regulation and the activation of the ERK1/2 pathway. In addition, this study also demonstrates the robust neuroprotective effects of σ-1 receptor in RGCs when subjected to OGD. These data also provide evidence suggesting that σ-1 receptor may be a therapeutic target to protect RGCs during ocular neurodegenerative diseases like glaucoma.