It is now well established that genetic factors contribute to glaucoma, and several glaucoma-associated genes have been identified. The first identified and the most studied gene is MYOCILIN, which is highly expressed in the trabecular meshwork, one of the key components in the aqueous humor outflow system of the eye. Although MYOCILIN is expressed in both ocular and non-ocular tissues, the function of the encoded protein, myocilin, is still not fully understood. We continued our investigations on the role of myocilin in the central (optic nerve) nervous system. 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 myelin-associated glycoprotein (MAG)-induced degeneration as well as reduced MAG-stimulated growth cone collapse. The direct physical interaction of myocilin and MAG was demonstrated by co-immunoprecipitation 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. These data suggest that myocilin protects axons from MAG-induced degeneration and this protection could be achieved through interaction with axons, soma or both. We continued our investigations of the molecular mechanisms involved in the protection of retinal ganglion cells by PDGF-AA. Using mice expressing EGFP under the control of PDGFR-alpha promoter, we identified and isolated retinal cells expressing PDGFR-alpha. In collaboration with Dr. Paul Russell (Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, USA) we showed that myocilin could induce alkaline phosphatase activity in adipose-derived mesenchymal stem cells indicating osteogenic differentiation. Although myocilin is detected in aqueous humor, its osteogenic-inducing activity was lower than the corresponding activity of a 5% aqueous humor treatment. Myocilin could only partially rescue the osteogenic-inducing effect of aqueous humor after heat treatment of aqueous humor, indicating that there were additional thermally labile constituents of aqueous humor involved in the osteogenic response. Increased osteogenic potential upon exposure to aqueous humor represents a potential challenge to developing adipose-derived mesenchymal stem cell-based therapies directed at the eye.
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