Processing of Optineurin in Neuronal Cells

Abstract

Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory ␤5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.

From the Cover: Alterations in Optineurin Expression and Localization in Pre-clinical Parkinson’s Disease Models

“…OPTN itself is typically degraded by the proteasome, and is a known autophagy cargo adaptor (Shen et al, 2011). Thus, our data showing LC3 colocalization strongly suggest puncta stem from autophagic vesicles, instead of aberrant OPTN degradation (Shen et al, 2011). Overall, our findings are consistent with the observations that autophagy is impaired in PD brains and models, where alpha-synuclein accumulation has been intensely investigated (Mader et al, 2012;Tan et al, 2014).…”

“…Both autophagic dysfunction and neuroinflammation occur in PD and are linked to pathogenesis (Macchi et al, 2015;Tan et al, 2014). OPTN itself is typically degraded by the proteasome, and is a known autophagy cargo adaptor (Shen et al, 2011). Thus, our data showing LC3 colocalization strongly suggest puncta stem from autophagic vesicles, instead of aberrant OPTN degradation (Shen et al, 2011).…”

Toward an integrative view of Optineurin functions

“…4B). 54,55 Thus, Wild et al identified a putative LC3-interacting region (LIR) located in the N-terminal domain of Optn (Fig. 2B).…”

Autophagy dysregulation and the fate of retinal ganglion cells in glaucomatous optic neuropathy

“…Furthermore, OPTN has been recently identified as an autophagy receptor involved in the clearance of cytosolic Salmonella (Wild et al, 2011). The mechanisms by which OPTN causes the optic pathology are still unclear; however, it has been shown that overexpression of wild-type optineurin or its Glu50Lys (E50K) mutant in RGC-5 cultures activates autophagy and treatment with rapamycin reduced the level of apoptosis elicited in these conditions (Shen et al, 2011). Those data were also validated by in vivo finding showing an induction of autophagy in the retina of transgenic mice carrying the optineurin E50K mutation (Shen et al, 2011).…”