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Sanja B. Turturro, Hongyu Ying, Xiang Shen, Rajalekshmy Shyam, Beatrice Y. Yue; Mapping of Functional Consequence Sites in the Human Optineurin Gene. Invest. Ophthalmol. Vis. Sci. 2012;53(14):777.
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© ARVO (1962-2015); The Authors (2016-present)
Optineurin is a gene associated with normal tension glaucoma (NTG) and amyotrophic lateral sclerosis (ALS). Foci formation and functional consequences including Golgi fragmentation, apoptosis and impairment of vesicle trafficking were demonstrated previously with the wild type and E50K optineurin. The purpose of the present study was to determine whether similar phenotypes could also be observed with various other optineurin mutations and deletion fragments in retinal ganglion RGC5 cells.
A total of 16 GFP tagged constructs that included NTG (E50K and 2bp-AG), ALS (exon 5_deletion, R96L, Q398X, and E478G) and non-disease (L157A and D474N) associated mutants and various deletion fragments were cloned into mammalian expression vectors and transfected for 24-48 h into RGC5 cells. The cells were subsequently monitored for foci formation and stained by immunofluorescence with anti-GM130 to analyze the Golgi integrity. Apoptosis was examined with the active caspase 3/7 detection kit. In addition, transferrin uptake experiments were preformed to evaluate the protein trafficking process. Differences between the transfected and non-transfected cells as well as GFP mock transfected controls were evaluated. The phenotypes observed with the mutants and deletion fragments were categorized based on the severity against those displayed by the wild type and E50K optineurin.
A point mutation R96L, found in patients with ALS, exhibited a strong tendency, similar to E50K, to form foci. The Golgi was fragmented, apoptosis was at a high level, and the transferrin uptake was inhibited. Little or no more than mild extent of foci formation was observed with L157A mutation in the leucine zipper (LZ) region, E478G and D474N mutations in the ubiquitin binding domain (UBD), and deletion fragments that lack either or both of the LZ and UBD sequences. Fragments 1-209, 1-424, 210-424, and 217-398 as well as exon 5_deletion showed none to mild phenotypes. E478G, Q398X and deletion fragments 425-577 and 217-577, on the other hand, caused fragmentation of the Golgi, apoptosis, and/or reduction of the transferrin uptake despite minor foci formation.
Expression of disease associated mutations including E50K, R96L, Q398X, and E478G resulted in moderate to severe extent of alterations in the Golgi integrity, apoptosis level, and transferrin uptake process. Foci formation appeared to require both the LZ and UBD sequences in the optineurin gene. Such a formation however was not necessarily predictive as to manifestation of the optineurin phenotypes.
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