BDNF Secretion is Dependent on Optineurin Expression

Abstract

Purpose: : Variants of the optineurin gene have been associated with certain forms of glaucoma, while BDNF has been shown to be an important survival factor for retinal ganglion cells. An interaction between optineurin, myosin VI, and Rab8 proteins suggests that optineurin may be involved in the BDNF secretory pathway. The purpose of these experiments was to determine if BDNF secretion was dependent on optineurin expression.

Methods: : RGC-5 (retinal neuronal precursor cells) were terminally differentiated with staurosporine for 48 hrs. Stable expression of miRNAs (miRNA.NS=non targeting; miRNA.OPT=optineurin targeting) in RGC-5 cells was acheived for at least 2 weeks using BLOCK-iT^TM miR RNAi Select (Invitrogen). Optineurin expression in wild-type (wt) RGC-5 cells, or RGC-5 cells transfected with control miRNA.NS, or miRNA.OPT was determined using immunohistochemistry and western immuoblotting to confirm knockdown only in miRNA.OPT RGC-5 cells. Each cell type was stressed with glutamate (48 hrs; 2x10^-5M=LD₅₀ wt-RGC-5). Some cells were pre-treated with hrBDNF(0 to 1x10^-6 gm/ml; BioShop). BDNF secretion was measured using ELISA using the BDNF E_max ImmunoAssay System (Promega).

Results: : 48hr exposure to glutamate killed 50% wt-RGC-5 and miRNA.NS RGC-5 cells, and 99% miRNA.OPT RGC-5 cells. This result demonstrates that optineurin expression protected RGC-5 cells from glutamate-mediated stress. Treatment with 3x10^-7 g/ml hrBDNF provided 100% protection for wt-RGC-5 and miRNA.NS RGC-5 cells and only 68% for miRNA.OPT RGC-5 cells. hrBDNF pretreatment increased BDNF secretion (5 fold increase) in wt-RGC-5 and miRNA.NS RGC-5 cells. This increase in BDNF secretion was greater (9 fold increase) when wt-RGC-5 and miRNA.NS RGC-5 cells were challenged with glutamate. No increase in BDNF secretion was observed in miRNA.OPT RGC-5 cells, either treated with glutamate, or left untreated.

Conclusions: : This study suggests that one mechanism of BDNF mediated neuroprotection is through recycling of exogenous BDNF. BDNF secretion was dependent on optineurin expression. Optineurin's involvment in BDNF secretion may explain why non-functional mutations of this protein have been implicated in the onset of glaucoma.

Keywords: ganglion cells • growth factors/growth factor receptors • protective mechanisms