Distribution and localization of abnormally expressed OPTN proteins in RGC5 retinal ganglion cells and their effects on subcellular morphology

Abstract

ABSTRACT. The objectives of this study were to investigate the distributions of abnormally expressed optineurin (OPTN) proteins in retinal ganglion cells (RGC5s) of transgenic rats and their effects on subcellular morphological structures. Green fluorescent protein labeled EGFP wildtype (OPTN WT ), E50K mutant type (OPTN E50K ), and OPTN siRNA (si-OPTN) eukaryotic expression plasmids were constructed and transfected into RGC5s. Intracellular structures were labeled with organelle specific fluorescent dyes. Construct localization and cell morphologies were visualized by confocal fluorescence microscopy. OPTN WT was observed to be distributed as fine punctate fluorescent particles in the cytoplasm around the nucleus, along with exhibiting nuclear expression. OPTN E50K exhibited similar distribution but with non-uniform fluorescence particle size. si-OPTN distribution was similar to that of EGFP: uniform across the cytoplasm and nucleus. Compared with the negative control group, OPTN WT , and OPTN E50K and to a lesser degree pEGFP-transfected cells exhibited fracture and loss of myofilament proteins and mitochondrial swelling and cytoplasmic accumulation, along with abnormal lysosomal distribution and increased volume, and Golgi fragmentation. However, si-OPTN transfected cells exhibited no significant damage. Therefore, we demonstrated that the E50K mutation disrupts the uniformity of OPTN protein distribution upon exogenous overexpression. Furthermore, these results suggested that si-OPTN transfection, and thus potentially OPTN knockdown, did not impact subcellular morphology of RGC5 cells, whereas transfection, especially when combined with wild-type or mutant OPTN expression, led to substantial abnormalities in subcellular morphological structures. These findings lay a foundation for further research into the function of the OPTN protein.

Rhodopsin Replacement in a I307N Mouse Model of ADRP by the Homology Independent Transgene Insertion Method

“…The formation of indels for targeted Rho suppression resulted in a 45% knock-out rate [24]. This approach can be very useful for mutations such as E50K OPTN which accounts for 80% of all OPTN mutations [25]. However, the allele-specific approaches are poorly suited to genes such as Rho with a high degree of mutation heterogeneity.…”

Immunomodulatory and Antioxidant Drugs in Glaucoma Treatment

“…Specifically, they focused on the most common OPTN mutation in NTG, known as E50K, and highlighted its impact in triggering abnormal aggregation of intracellular vesicles [ 68 , 69 ]. Moreover, the E50K mutation was associated with a disruption of the Golgi structure, leading to cellular toxicity [ 70 , 71 , 72 , 73 ]. In addition, mutations in the optic atrophy type 1 (OPA1) gene, which is crucial for mitochondrial dynamics, have also been implicated in the pathogenesis of NTG.…”