Abstract
Purpose:
To investigate the role of myocilin, a glaucoma-associated protein, in axon growth and degeneration.
Methods:
Changes in gene expression were evaluated by RNA sequencing. Retinal explant cultures were prepared from postnatal day 4 (P4) mice. Dorsal root ganglion (DRG) cultures were prepared from P6 mice. Microfluidic two-compartment chambers were used to study myocilin effects on soma and axons. Axon growth and degeneration were evaluated with a Zeiss 700 confocal microscope. Binding of myocilin to myelin-associated glycoprotein (MAG) was assessed by co-immunoprecipitation. Changes in the levels of different mRNAs in DRG axons after addition of myocilin were analyzed by 96 well RT2 Profiler PCR arrays.
Results:
Sequencing of RNA isolated from 2 month-old optic nerves of wild-type and Myocilin null mice demonstrated changes in components of the signaling pathway governing axon guidance. Addition of 1 μg/ml of purified myocilin to DRG cultures protected neurites from MAG-induced degeneration as well as reduced MAG-stimulated growth cone collapse. The direct physical interaction of myocilin and MAG was demonstrated by co-immunoprecipiatation from mouse optic nerve lysates. This interaction as well as the modulation of RhoA-GTPase level by myocilin may contribute to the protective effects of myocilin. Addition of myocilin to P4 mouse retinal explants similarly protected neurites from MAG-induced degeneration and reduction of neurite length. Myocilin provides protective action through interaction with soma or axons alone as was shown by cultivating DRGs in microfluidic two-compartment chambers. Addition of myocilin to the axonal compartment did not lead to changes in the number of axons or their total length compared with untreated samples, but protected axons from MAG-induced degeneration. Addition of myocilin to the axonal compartment induced changes in the levels of several mRNAs in axons as was shown first by PCR array analysis and confirmed by quantitative RT-PCR.
Conclusions:
Myocilin protects axons from MAG-induced degeneration and this protection could be achieved through interaction with axons, soma or both.<br />