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 are continuing our investigations on the role of myocilin in the central (optic nerve) and peripheral (sciatic nerve) nervous systems. We have demonstrated that myocilin affects myelination in both CNS and PNS but acts through different signaling pathways. In the sciatic nerve, myocilin is expressed in Schwann cells, binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. In the optic nerve, myocilin is expressed in astrocytes. We have demonstrated that secreted myocilin interacts with the Lingo-1/Nogo receptor complex. This interaction affects differentiation of oligodendrocytes and induces elongation of oligodendrocyte processes in the optic nerve. We have demonstrated that myocilin plays an important role regulating cell growth/survival. Myocilin-containing conditioned medium increased cell proliferation and myocilin-expressing cells were more resistant to serum starvation-induced apoptosis. Myocilin-defective mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis. To find the signaling pathway(s) induced by myocilin, we screened phospho-MAPK antibody arrays and identified increased phospho ERK1/2 in myocilin-expressing cells. We confirmed that phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells. In addition, the elevated phosphorylation of ERK1/2 was observed in the trabecular meshwork of transgenic mouse expressing 15-fold higher levels of myocilin. Our results suggest that myocilin promotes cell proliferation and apoptosis resistance via ERK1/2 MAPK signaling pathways. Our data also implies that myocilin may have functions not only in cells of the eye, but also in adult stem cells outside the eye. 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. Mesenchymal stem cell (MSC) transplantation appears to be protective in a variety of neurodegenerative disorders of the brain and spinal cord, in part, via neurotrophic factor secretion. We identified several factors that were secreted by MSCs and provided protection of retinal ganglion cells (RGCs) in retinal explants. Among identified factors, the strongest effect was seen for members of the PDGF family of proteins, which increased RGC survival. Blockade of PDGF signalling with anti-PDGF antibody or with small molecule inhibitors of PDGF receptor kinase or downstream phosphatidylinositol 3 kinase eliminated RGC neuroprotection conferred by MSC co-culture. We expanded these observations to animal model of glaucoma. In collaboration with Dr. Keith Martin (Cambridge University, UK), we demonstrated that intravitreal injection of PDGF-AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data suggest that targeting PDGF signalling pathways may represent a new approach to RGC neuroprotection and glaucoma treatment. Another possible approach for myocilin-induced glaucoma treatment was developed in collaboration with Dr. Chad Dickey (University of South Florida, Tampa). We have previously shown that myocilin is a secreted protein that is typically transported through the endoplasmic reticulum/Golgi network. Mutations in myocilin leading to severe forms of juvenile glaucoma lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, cell death. It was shown that glucose-regulated protein 94 (Grp94) specifically recognizes mutant myocilin, triaging it through endoplasmic reticulum-associated degradation. However, depletion of Grp94 led to efficient mutated myocilin degradation through an alternative autophagic clearance pathway. These data suggest that selective inhibition of Grp94 could be beneficial for patients suffering from myocilin-induced glaucoma.

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National Eye Institute (NEI)
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Mead, Ben; Amaral, Juan; Tomarev, Stanislav (2018) Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Promote Neuroprotection in Rodent Models of Glaucoma. Invest Ophthalmol Vis Sci 59:702-714
Mead, Ben; Tomarev, Stanislav (2018) Retinal ganglion cell neuroprotection by growth factors and exosomes: lessons from mesenchymal stem cells. Neural Regen Res 13:228-229
Joe, Myung Kuk; Lieberman, Raquel L; Nakaya, Naoki et al. (2017) Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3. Invest Ophthalmol Vis Sci 58:5308-5318
Takahama, Shokichi; Adetunji, Modupe O; Zhao, Tantai et al. (2017) Retinal Astrocytes and GABAergic Wide-Field Amacrine Cells Express PDGFR?: Connection to Retinal Ganglion Cell Neuroprotection by PDGF-AA. Invest Ophthalmol Vis Sci 58:4703-4711
Mead, Ben; Tomarev, Stanislav (2017) Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms. Stem Cells Transl Med 6:1273-1285
Mead, Ben; Tomarev, Stanislav (2016) Evaluating retinal ganglion cell loss and dysfunction. Exp Eye Res 151:96-106
Morgan, Joshua T; Kwon, Heung Sun; Wood, Joshua A et al. (2015) Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells. Exp Eye Res 135:127-33
Joe, Myung Kuk; Nakaya, Naoki; Abu-Asab, Mones et al. (2015) Mutated myocilin and heterozygous Sod2 deficiency act synergistically in a mouse model of open-angle glaucoma. Hum Mol Genet 24:3322-34
Joe, Myung Kuk; Kwon, Heung Sun; Cojocaru, Radu et al. (2014) Myocilin regulates cell proliferation and survival. J Biol Chem 289:10155-67
Johnson, Thomas V; DeKorver, Nicholas W; Levasseur, Victoria A et al. (2014) Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome. Brain 137:503-19

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