Effects of Myocilin and Optineurin on Neurite Outgrowth in PC12 Cells

Abstract

Purpose:: To investigate the effects of forced expressed wild-type (WT) myocilin (MYOC), WT optineurin (OPTN), and their mutants on neurite outgrowth in rat neuronal PC12 cells.

Methods:: PC12 cells were seeded onto collagen I (10 µg/ml) coated plates in triplicates. The attached cells were transiently transfected with pEGFP-N1, pMYOC_WT-EGFP, pMYOC_P370L-EGFP, pMYOC_Q368X-EGFP, pOPTN_WT-EGFP, or pOPTN_E50K-EGFP. After 2 days, fresh medium containing 100 ng/ml nerve growth factor (NGF) was added to the cells to induce differentiation. Photographs of transfected cells were taken using fluorescence microscope 5 days after the NGF induction. The neurite length was measured and the number of neurites was counted in at least 50 transfected cells. Data were statistically analyzed by Student’s t tests.

Results:: PC12 cells transfected with pEGFP-N1 empty vector produced long and extensive neurites upon NGF induction. The neurite length of those transfected with pMYOC_WT-EGFP was approximately one half of that in pEGFP-N1controls although the number of neurites was comparable. pMYOC_P370L-EGFP and pMYOC_Q368X-EGFP elicited an inhibitory effect on neurite outgrowth similar to that seen with the WT MYOC. By contrast, transfection of neither of the OPTN constructs, pOPTN_WT-EGFP and pOPTN_E50K-EGFP induced significant alterations in the length or the number of neurites.

Conclusions:: MYOC and OPTN exhibit differential effects on neurite outgrowth in PC12 cells. While overexpression of both WT and mutant MYOC causes a significant inhibition of neurite outgrowth, forced expressed WT and E50K mutant OPTN do not result in any changes. MYOC and OPTN may be involved in development of neurodegenerative glaucoma via different mechanisms.

Keywords: pathobiology • mutations • imaging/image analysis: non-clinical