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C. Sippl, E. R. Tamm; Depletion of Optineurin in RGC-5 Cells Leads to Fragmentation of the Golgi Apparatus and to a Decrease in Secretion of Neurotrophic Factors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6115.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in OPTN are causative for GLC1E-linked primary open angle glaucoma (POAG). Optineurin is a 74 kDa protein which is involved in several cellular processes such as tumor necrosis factor signaling, vesicular trafficking or transcriptional regulation. Optineurin is expressed in retinal ganglion cells (RGC) and affected patients mainly suffer from normal tension glaucoma. Here, we wanted to determine the subcellular function and localization of optineurin in RGC-5 cells after knock-down of the endogenous protein by RNA-interference.
The expression of optineurin was silenced in RGC-5 cells applying a shRNA expression vector to generate a stable knock-down as well as by transient transfection of siRNA. The cells were differentiated with staurosporine, and Thy1, CNTF and GDNF expression was quantified by real time RT-PCR. The localization of optineurin and the morphology of the Golgi apparatus were compared to that of cells treated with scrambled siRNA using confocal fluorescence microscopy and immunostaining of optineurin and the Golgi marker GM130. The ultrastructure of the cells was analyzed by transmission electron microscopy (TEM). The amounts of ciliary neurotrophic factor (CNTF) and neurotrophin 3 (NT-3) in culture medium were quantified by ELISA.
Following staurosporine treatment, an upregulation of CNTF, GDNF and Thy1 was observed indicating differentiation. Optineurin was located in diffusely distributed vesicles and in close proximity to the Golgi apparatus. After knock-down of optineurin, the cells showed fragmentation of the Golgi complex and an increase in cells containing abnormal and multiple nuclei. Multinucleated cells exhibited an abnormal number of centrosomes. By TEM, the Golgi stacks were found to be dispersed and had swollen and irregular cisternae. Cells with stable knock-down of optineurin increased their volume, while overall cell number decreased. After transient transfection, there was a dramatic decrease in cell number compared to cells treated with scrambled siRNA. Knock-down of optineurin resulted in the decrease of NT-3 and CNTF secretion in the culture medium.
Optineurin is critical for the maintenance of the Golgi apparatus and the secretion of retinal ganglion cells. Diminished CNTF and NT-3 secretion in patients with GLC1E-linked glaucoma might contribute to RGC cell death and POAG.
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