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. Identification of proteins interacting with myocilin is one of the possible approaches to elucidating its functions since interacting proteins are often involved in the same physiological processes and pathologies. TIMP3 was identified by a shotgun proteomic analysis as a protein that was co-immunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by co-immunoprecipitation of myocilin and TIMP3 from eye lysates using anti-TIMP3 antibodies. The olfactomedin domain of myocilin is essential for interaction with TIMP3 as shown using HepG2 cells transiently transfected with corresponding constructs. In the eye, the main sites of myocilin and TIMP3 co-localization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene. We also used the CRISPR technology to produce null mutations in the Myocilin gene in zebrafish. These fish are viable. Different aspects of eye physiology in Myocilin null mutants are currently analyzed. In addition to studying the pathophysiology of glaucoma, we are also interested in potential treatments for this disease. Glaucoma is associated with impairment in retrograde transport of neurotrophic factors to retinal ganglion cell bodies. Previously we demonstrated that mesenchymal stem cell transplantation is protective in a rat model of glaucoma and that factors secreted by mesenchymal stem cells are essential for neuroprotection. To elucidate the nature of neuroprotective factors we began to use exosomes produced by mesenchymal stem cells and demonstrated that they provide neuroprotection of retinal ganglion cells. The identification of exosomes components responsible for neuroprotective effects is under way. We also used intravitreal injection of adeno-associated viral vectors to express a number of candidate proteins that may provide retinal ganglion cell neuroprotection and/or axon regeneration. Expression of some of these proteins led to a profound neuroprotective affects and the molecular mechanisms involved in neuroprotection are currently under investigation.
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