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Y. Dun, M. Thangaraju, V. Ganapathy, S. B. Smith; Analysis of the Mechanism by Which the Sigma Receptor (R1) Ligand (+)-pentazocine ((+)-PTZ) Protects Against Excitotoxic Death in Primary Retinal Ganglion Cells (1ºRGC). Invest. Ophthalmol. Vis. Sci. 2008;49(13):2068. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We reported recently that (+)-PTZ, a σR1 ligand, afforded marked protection against glutamate (GLU)-induced apoptosis of 1ºRGCs (Dun, et al, IOVS, 2007) and inhibited RGC death in diabetic Ins2Akita mice (Smith et al, ARVO, 2007). As a first step in understanding the mechanism by which (+)-PTZ is neuroprotective, we screened for pro- and anti-apoptotic genes using the GLU-induced 1ºRGC death as a model system.
1ºRGCs were isolated by immunopanning from 1-3 day mouse retinas (Dun et al, Cell Tiss Res, 2006), maintained 3 days in culture, exposed to 25 µM GLU for 18 h in the presence or absence of (+)-PTZ (3.0 µM). Total RNA was isolated using TRIzol and a wide array of pro-apoptotic genes (FOXO3a, FasL, TNFR, TRAIL, Bak, p85, p53, Bad) and anti-apoptotic genes (Survivin, p21, Bcl-2, Bcl-xL, c-myc) were analyzed by semiquantitative RT-PCR.
The most profound changes were observed in FOXO3a and Survivin. Expression of FOXO3a, a pro-apoptotic transcription factor that regulates the extrinsic apoptotic pathway, was increased 212% in GLU-treated cells, but was eliminated by (+)-PTZ. Expression of the FOXO3A-regulated gene FasL was elevated 53 fold by GLU and markedly reduced by (+)-PTZ; TRAIL and TNFR expression were affected similarly, but to a lesser degree. Survivin, a conserved member of the inhibitor of apoptosis protein (IAP) family, was not expressed in GLU-treated cells, but was expressed significantly in (+)-PTZ-treated cells. Expression of p21, a cyclin-dependent kinase (CDK)-binding protein that may protect against apoptosis, was reduced by GLU, but restored with (+)-PTZ.
(+)-PTZ appears to mediate neuroprotection in GLU-induced 1ºRGC death by inhibiting expression of pro-apoptotic FOXO3a and downstream components of the extrinsic death receptor pathway, especially FasL and TRAIL. Additionally (+)-PTZ upregulates key genes in the anti-apoptotic pathway, notably Survivin and p21. Future studies will evaluate mechanisms of (+)-PTZ neuroprotection using in vivo models.
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