Differential Effects of Myocilin and Optineurin, Two Glaucoma Genes, on Neurite Outgrowth

Abstract

Myocilin and optineurin are two genes linked to glaucoma , a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells , we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC WT -EGFP, pMYOC P370L -EGFP, pMYOC 1-367 -EGFP, pOPTN WT -EGFP , and pOPTN E50K -EGFP. PC12 cells transfected with pEGFP-N1 produced , as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast , neither transfection of the optineurin constructs pOPTN WT -EGFP and pOPTN E50K -EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct , but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes , myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.

Processing of Optineurin in Neuronal Cells

“…4, C and D) as was seen in neurodegenerative diseases (38,50,51). In addition, overexpression of both wild type and E50K optineurin leads to apoptosis in cultured cells (40), and the toxicity can be rescued by rapamycin treatment (Fig. 8C).…”

“…After transfection with wild type or E50K optineurin, the optineurin foci are observed to distribute in the perinuclear region in proximity to the Golgi complex (17,18,40). Following precedent of those described for aggresomes, inclusion bodies, or Lewy bodies, the optineurin foci are formed in a microtubule-dependent manner (17,18).…”

“…The total number of DAPI-stained transfected cells (green) and the number of CR(DEVD) 2 -stained transfectants (displaying both green and red fluorescence) in 20 of randomly selected 10ϫ fields were counted (40). The percentage of caspase 3/7-positive apoptotic cells in ϳ100 transfectants (number of green and red cells/number of green only cells) was calculated.…”

Current concepts on primary open-angle glaucoma genetics: a contribution to disease pathophysiology and future treatment

“…On the contrary, Koga et al 73 recently showed that overexpression of wild-type OPTN results in increased apoptosis of RGCs and that OPTN (wild type or the E50K mutant form), unlike MYOC, is devoid of effects on neurite outgrowth in neuronal cells. They concluded that MYOC and OPTN appear to have different functions and may contribute to the development of neurodegenerative glaucoma via distinct mechanisms, which are still largely unknown.…”

“…Koga et al 73 demonstrated that overexpression of wild-type MYOC or P370L and Q368X mutants, but not OPTN, caused an inhibition of neurite outgrowth and thus may contribute to the development of neurodegenerative glaucoma. 73 OPTN at the GLC1E locus The second gene associated with POAG was identified in 2002 and it was given the name OPTN (OPTN for optic neuropathy-inducing protein) by Rezaie et al 9 An adult-onset POAG locus had been previously mapped on chromosome 10p14-p15, the GLC1E locus, in a linkage study of a large British family in which 15 of 46 family members were affected with NTG. 10 After excluding four genes, Rezaie et al 9 selected OPTN as a candidate gene on the basis of its physical location in this region and its expression in retina.…”