It is now well established that a 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 heavily expressed in and secreted by the trabecular meshwork, one of the key components in the aqueous humor outflow system of the eye. Although this gene is also expressed in both ocular and non-ocular tissues, the function of the encoded protein, myocilin, are still not fully understood. To better understand the functions of myocilin, we performed a proteomic analysis of proteins interacting with wild-type and mutated myocilin in the mouse eye. Eyes were dissected from mouse lines expressing wild-type or mutated myocilin, as well as from Myoc null line. After removal of the lens, eye lysates were prepared and proteins interacting with myocilin were precipitated using affinity-purified antibodies against mouse myocilin. Mass spectrometry analyses of the isolated complexes identified several proteins specifically interacting with wild-type myocilin, mutated myocilin or both. The functional significance of these interactions is now being tested experimentally. We continued our investigations on the role of myocilin in the central (optic nerve) and peripheral (sciatic nerve) nervous systems. In the sciatic nerve, myocilin is expressed in Schwann cells and localized at the nodes of Ranvier. In the optic nerve, myocilin is expressed mainly by astrocytes and is not associated with the nodes. We demonstrated that myocilin interacts with Lingo-1 and the N-terminal domain of myocilin is critical for this interaction. Myoc null mice showed defects in the myelination of the optic nerve leading to changes in the visual evoked potential. These data suggest that myocilins action is not limited to the trabecular meshwork. Furthermore, its expression in the optic nerve may reflect a direct role in the pathological changes observed in glaucomatous eyes. 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. Transplantation of human MSCs onto retinal explants resulted in 6711% greater RGC survival (p<0.001) compared to controls;treatment of explants with MSC conditioned medium increased RGC survival by only 258% (p<0.05). Sephacryl gel fractionation of MSC conditioned media yielded one fraction with improved neuroprotective capacity, which increased RGC survival by 4811% (p<0.05). To avoid confounding effects of unidentified secreted factors, explants were treated with a cocktail of 13 purified proteins, identified as being enriched in the MSC secretome. These factors increased RGC survival by 296% (p<0.05). Subgrouping proteins according to signaling pathway revealed that factors acting through gp130 (IL-6, IL-11, and LIF) reduced RGC survival by 152% (p<0.01) whereas other subgroups significantly protected RGCs;the strongest effect was seen for members of the PDGF family of proteins, which increased RGC survival by 629% (p<0.001). MSC gene expression analysis revealed that 1,351 genes were significantly (p<0.05) up- or down-regulated more than 2-fold in MSCs following co-culture with retinal explants. Ingenuity Pathway Analysis of differentially regulated genes revealed overrepresentation of genes involved in growth factor signaling, inflammation, endocytosis and axon guidance that could potentially contribute to neuroprotection by MSCs. Ongoing studies aim to identify the most important factors mediating this neuroprotection and to determine how changes in gene expression induced by exposure to retinal tissue promote the neuroprotective effects of MSCs. We also demonstrated that myocilin is expressed in MSCs derived from mouse, rat and human bone marrow with human MSCs exhibiting the highest level of myocilin expression. Expression of myocilin was increased during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous myocilin further enhanced osteogenesis. MSCs derived from Myoc null mice had a reduced ability to differentiate into an osteoblastic lineage, which was partially rescued by exogenous extracellular myocilin treatment. Myocilin-stimulated osteogenic differentiation of MSC was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as upregulated expression of the osteogenic transcription factors, Runx2 and Dlx5. Finally, we used micro computed tomography (microCT) to measure three-dimensional trabecular bone structure in wild-type and Myoc null mice. We demonstrated that cortical bone thickness as well as trabecular volume was reduced in the Myoc null mice compared with wild-type mice. These data suggest that myocilin should be considered as a target for improving the bone regenerative potential of MSCs, and may suggest that myocilin plays a role in bone formation or maintenance in vivo.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000318-15
Application #
8556817
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$949,683
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Mead, Ben; Tomarev, Stanislav (2016) Evaluating retinal ganglion cell loss and dysfunction. Exp Eye Res 151:96-106
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
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
Kwon, Heung Sun; Nakaya, Naoki; Abu-Asab, Mones et al. (2014) Myocilin is involved in NgR1/Lingo-1-mediated oligodendrocyte differentiation and myelination of the optic nerve. J Neurosci 34:5539-51
Chou, Tsung-Han; Tomarev, Stanislav; Porciatti, Vittorio (2014) Transgenic mice expressing mutated Tyr437His human myocilin develop progressive loss of retinal ganglion cell electrical responsiveness and axonopathy with normal iop. Invest Ophthalmol Vis Sci 55:5602-9
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
Kwon, Heung Sun; Johnson, Thomas V; Joe, Myung Kuk et al. (2013) Myocilin mediates myelination in the peripheral nervous system through ErbB2/3 signaling. J Biol Chem 288:26357-71
Kwon, Heung Sun; Johnson, Thomas V; Tomarev, Stanislav I (2013) Myocilin stimulates osteogenic differentiation of mesenchymal stem cells through mitogen-activated protein kinase signaling. J Biol Chem 288:16882-94
Joe, Myung Kuk; Kee, Changwon; Tomarev, Stanislav I (2012) Myocilin interacts with syntrophins and is member of dystrophin-associated protein complex. J Biol Chem 287:13216-27

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