title: Depletion of optineurin in RGC-5 cells derived from retinal neurons causes apoptosis and reduces the secretion of neurotrophins.
abstract: Optineurin is a Golgi complex-associated ubiquitous protein with high expression levels in retinal ganglion cells (RGCs). Mutations in optineurin have been observed in rare hereditary cases of primary open-angle glaucoma and in amyotrophic lateral sclerosis. We explored the possibility that optineurin deficiency will compromise neuronal exocytosis leading to a diminished secretion of neurotrophic factors that are critically required for neuronal survival. To this end, we used RNA interference to induce depletion of optineurin in RGC-5 cells derived from retinal neurons. SiRNA specific for optineurin was transiently transfected. Moreover, a stable cell line with constitutive optineurin deficiency (RGC-5 pSilencer OPTN) was generated. In addition, we investigated the subcellular localization of optineurin in primary RGCs in retinal cell cultures isolated from eyes of mature mice. In RGC-5 cells, optineurin localized to the periphery of the Golgi complex and was observed in vesicular structures throughout the cytoplasm and close to the plasma membrane. A comparable Golgi-associated localization of optineurin was observed in cultured primary RGCs that were identified by TUJ1 labeling. Optineurin deficiency caused a marked increase in the number of RGC-5 cells with fragmented Golgi complex. RGC-5 pSilencer OPTN with stable optineurin deficiency showed a pronounced increase in the number of cells undergoing apoptotic cell death. Furthermore, the amounts of secreted neurotrophin-3 (NT-3) and ciliary neurotrophic factor were significantly lower in culture medium of RGC-5 pSilencer OPTN cells when compared to controls. Adding exogenous NT-3 to the culture medium to achieve amounts seen in control cultures completely prevented the increase in apoptotic cell death. We propose that lack of neurotrophic support due to impaired secretion of neurotrophic proteins is a critical factor that causes or contributes to RGC or motor neuron death in patients with mutated optineurin.
authors: Institute of Human Anatomy and Embryology, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.
authors: Christiane
authors: C
authors: Sippl
authors: Anja K
authors: AK
authors: Bosserhoff
authors: Dietmar
authors: D
authors: Fischer
authors: Ernst R
authors: ER
authors: Tamm
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D026901:Membrane Transport Proteins; D051379:Mice; D009414:Nerve Growth Factors / Q000378:metabolism*; D020933:Neurotrophin 3 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D014162:Transfection
chemical_list: D018797:Cell Cycle Proteins; D020934:Ciliary Neurotrophic Factor; D017931:DNA Primers; D005136:Eye Proteins; D026901:Membrane Transport Proteins; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; C501288:Optn protein, mouse; D034741:RNA, Small Interfering