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Abstract #45895 Published in IGR 13-2
Functional role of Optineurin (OPTN) in the morphology of the Golgi apparatus and in endocytosis of neurotrophic factors
Sippl C; Tamm ERMedizinische Genetik 2011; 23: 153
Purpose: It has been shown that mutations in the OPTN gene can cause hereditary forms of open-angle glaucoma whereas most of the patients suffer from normal-tension glaucoma (Rezaie et al., 2002). Optineurin is a 74 kDa protein implicated in several cellular processes like signal trans-duction of the tumor necrosis factor pathway (Li et al., 1998), vesicular trafficking (Sahlender et al., 2002) and regulation of transcription (Moreland et al., 2000). In the present study we seeked to determine the subcellular function of Optineurin at the Golgi apparatus and in secretion of neurotrophic factors in the retinal ganglion cell line RGC-5 after knock-down of the endogenous protein by RNA-interference. Methods: The expression of the OPTN gene was silenced in the RGC-5 cell line applying a shRNA expression vector to generate a stable knockdown as well as transient transfection of siRNA. The localization of the endogenous protein and the morphology of the Golgi apparatus was compared to cells treated with scrambled siRNA using a confocal microscope by immunostaining of Optineurin and the Golgi marker GM130. In addition, the ultrastructure of the Golgi apparatus was analyzed with a transmission electron microscope. To study the secretion of neurotrophic factors the concentrations of CNTF and Neurotrophin 3 were quantified after knock-down in the culture medium and expression levels of CNTF and Neurotrophin 3 were analyzed by RT-PCR. In order to observe the cell growth and volume of cells the CASY((registered trademark)) Cell Counter and Analyser TT was used and apoptosis was quantified by an ELISA for the detection of cytoplasmic histone-associated DNA fragments. Cell cycle analyses were performed using a FACS. Results: In control cells Optineurin was located in diffuse distributed vesicles and in close proximity to the Golgi complex. After knock-down the cells showed a fragmentation of the Golgi complex and an increase in cells containing abnormal and multiple nuclei. This was also observed by electron microscopy. The Golgi stacks were dispersed and had swollen and irregular cisternae. Multinucleated cells exhibited an abnormal number of centrosomes. In stable knock-down with decreasing amounts of Optineurin the cells became bigger in diameter and volume and showed a slower increase in cell number. After transient transfection, there was a dramatic decrease in cell numbers compared to cells treated with scrambled siRNA. In addition, a significant number of apoptotic cells was observed using an ELISA in Optineurin-deficient cells when compared to controls. In contrast no evidence was found for an arrest in mitosis by FACS analyses. In culture media of Optineurin-deficient cells the amounts of Neuro-trophin 3 and CNTF were smaller than in controls. Conclusions: Optineurin is critically required for maintenance of the Golgi apparatus and the secretion of neurotrophic factors in retinal ganglion cells. We conclude that a lack of neurotrophic factors leads to apoptotic cell death in Optineurin-deficient cells. A comparable scenario could account for RGC death in glaucoma patients with mutated Optineurin.
C. Sippl. University of Regensburg, Regensburg, Germany.
Classification:
3.4.2 Gene studies (Part of: 3 Laboratory methods > 3.4 Molecular genetics)
3.6 Cellular biology (Part of: 3 Laboratory methods)
