Molecular Mechanisms of Glaucoma Section

• Staff
• Selected Publications

From left to right: Heung Sun Kwon, Afia Sultana, Thomas Johnson, Naoki Nakaya, Myung Kuk Joe and Stanislav Tomarev

Current Research

Glaucoma is the second leading cause of blindness in developed countries. It is a group of optic neuropathies characterized by the death of retinal ganglion cells, leading to a specific deformation of the optic nerve head. Peripheral vision declines first in glaucoma, while central vision loss occurs much later. Elevated intraocular pressure (IOP) is one of the main risk factors in glaucoma, but it is not completely understood how elevated IOP kills ganglion cells. Several genes have implicated in glaucoma pathogenesis but the search for other contributing genes continues. This section conducts basic research on glaucoma and genes that might be essential for retinal ganglion cell development, function, and survival.

Our interests are concentrated on early changes in the retina and the optic nerve in the course of glaucoma. Since it is hard to study such changes in the retina and optic nerve on human subjects, we use existing animal models and develop genetic rodent models of glaucoma for our investigations; subsequently we plan to confirm and apply our results to humans. Another main area of research is the discovery of new genes involved in glaucoma. This requires parallel studies on genes that are important for the function of the retina, the optic nerve and the eye outflow system in the normal eye. We are particularly interested in genes encoding olfactomedin domain-containing proteins.

In addition to our own research, we collaborate extensively with other laboratories at the NEI, NIH, and the external community.

Below is a brief summary of MMGS research as of April 2009.

Development of rodent models of glaucoma

It is now well established that mutations in the MYOCILIN gene are associated with juvenile open-angle glaucoma, often exhibiting elevated IOP. Between 2.6% and 4.3% of cases of sporadic primary open-angle glaucoma are associated with mutations in this gene. We used a transgenic approach to express mutated human or mouse Myocilin in the eye of transgenic mice. We produced several lines of transgenic mice using BAC DNAs, containing the full length mouse or human MYOCILIN gene with point mutation.

Transgenic mice expressing human Tyr437His or mouse Tyr423His Myocilin mutants demonstrated a moderate elevation of IOP, the loss of about 20% of the retinal ganglion cells in the peripheral retina, and axonal degeneration in the optic nerve. Retinal ganglion cell RGC depletion was associated with the shrinkage of their nuclei and DNA fragmentation in the peripheral retina. Pathological changes observed in the eyes of transgenic mice are similar to those observed in glaucoma patients and that these transgenic mice represent a novel animal model of glaucoma. Current studies are directed toward the characterization of the molecular changes in the eye angle tissues and retina of knockout and transgenic mice and rats as well as the production of new lines of transgenic mice demonstrating more dramatic retinal damage.

We believe that genetic rodent models of glaucoma will be invaluable for addressing fundamental questions in glaucoma, including the identification of the signaling pathways in the retina and the optic nerve activated in the disease, the effects of modifier genes, and neuroprotection. In addition to in vivo transplantation, we are developing the in vitro technique of retinal explant tissue culture as a model to assess cellular transplantation.

Mechanisms of myocilin action

The functions of wild-type myocilin are still not well understood. To study functions of wild type and mutated myocilin we developed stably transfected cell lines expressing these proteins under an inducible promoter. We demonstrated that myocilin induced formation of stress fibers in primary cultures of human trabecular meshwork or NIH3T3 cells. The stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin as well as secreted inhibitors of Wnt-signaling, sFRP1 or sFRP3. We showed that myocilin interacts with sFRP3 and several frizzled receptors. Treatment of NIH3T3 cells with myocilin and its fragments induced intracellular re-distribution of ß-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of ß-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of ß-catenin in these tissues. We suggested that Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, as myocilin modulates Wnt signaling by interacting with components of this signaling pathway. Interaction of myocilin with components of the Wnt signaling pathway as well with a number of other proteins is under investigation.

Function of the olfactomedin domain-containing proteins

Myocilin belongs to a family of olfactomedin domain-containing proteins consisting of at least 13 members in mammals. Some family members, such as latrophilins and gliomedin, are membrane-bound proteins containing the olfactomedin domain in the extracellular N-terminal region, while the intracellular C-terminal domain of these proteins is essential for the transduction of extracellular signals to the intracellular signaling pathway. Others family members, similar to Myocilin, are secreted glycoproteins whose functions are mostly unknown. Available data suggest that the olfactomedin domain may be essential for the interaction with other proteins, including receptors and extracellular matrix proteins. Several genes encoding olfactomedin domain proteins are expressed in the eye. We focus our attention of the genes that are expressed in the retinal ganglion cells and the eye angle tissues and use several approaches to elucidate their functions:

We developed several stably transfected cell lines expressing olfactomedin 1 (Olfm1), Olfm2, and optimedin, also known as olfactomedin 3. Expression of optimedin changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. Olfm1 expression, on the contrary, induced neurite outgrowth in NGF-stimulated PC12 cells. We showed that Olfm1, similar to myocilin, may interact with several components of Wnt signaling pathway and may be a modulator of Wnt signaling. Several other proteins interacting with Olfm1 were identified and the functional significance of these interactions is under study.

A zebrafish model is used to study function of Olfm1 in development. There are two olfm1 genes in zebrafish, each producing four different transcripts. Our data suggest that Olfm1 might be essential for axon growth in zebrafish, supporting observations obtained with olfactomedin-expressing PC12 cells. Over-expression of full length Olfm1, and especially its BMY form lacking the olfactomedin domain, increased the thickness of the optic nerve and produced a more extended projection field in the optic tectum as compared with control embryos. In contrast, injection of olfm1-morpholino oligonucleotide reduced the eye size, inhibited optic nerve extension, and increased the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers.

Mutations in the OLFM2 gene were implicated in glaucoma in humans. We study possible functions of this protein using different approaches. We produced knockout mice for some olfactomedin-domain containing genes and we are in the process of producing knockouts for other genes belonging to this family. These animals will be used to study possible functions of olfactomedin domain-containing proteins in different mammalian tissues with emphasis on the brain and retina.

We believe that olfactomedin domain-containing proteins may play important roles in normal eye development, function and pathology, including glaucoma, and that our multifaceted approach to their functions will lead to a better understanding of the molecular mechanisms of their actions.

Staff

Recent selected publications

1. Kwon, H.-S., Lee, H.-S., Ji, Y., Rubin, J.S., and Tomarev, S.I. (2009) Myocilin is a modulator of Wnt signaling. Mol. Cell. Biol. 29, 2139-2154.
2. Nakaya, N., Lee, H.-S., Takada, Y., Tzchori, I., and Tomarev, S. I. (2008) Zebrafish olfactomedin 1 regulates retinal axon elongation in vivo and is a modulator of Wnt signaling pathway. J. Neurosci. 28, 7900-7910.
3. Zhou, Y., Grinchuk, O., and Tomarev, S. (2008) Transgenic mice expressing the Tyr437His mutant of human myocilin protein develop glaucoma. Invest. Ophthamol. Vis. Sci. 49, 1932-1939.
4. Lee, H.-S. and Tomarev, S.I. (2007) Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells. Exp. Cell Res. 313, 98-108.
5. Senatorov, V., Malyukova, I., Fariss, R., Wawrousek, E., Swaminathan, S., Sharan, S., and Tomarev, S. (2006) Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. J. Neurosci. 26, 11903-11914.
6. Wilting, J., Aref, Y., Huang, R., Tomarev, S.I., Schweigerer, L., Christ, B., Valasek, P., and Papoutsi, M. (2006) Dual origin of avian lymphatics. Dev. Biol. 292, 165-173.
7. Grinchuk, O, Kozmik, Z., Wu, X., and Tomarev, S.I. (2005) The Optimedin gene is a downstream target of Pax6. J. Biol. Chem. 280, 35228-35237.
8. Gould, D.B., Miceli-Libby, L., Savinova, O.V., Torrado, M., Tomarev, S.I., Smith, R.S., and John, S.W.M. (2004) Genetically increasing Myoc expression supports a necessary pathological role of abnormal proteins in glaucoma. Mol. Cell. Biol. 24, 9019-9025.
9. Steffenson, K.R., Holter, E., Bavner, A., Tobin, K.-A., Tomarev, S., and Treuter, E. (2004) Functional conservation of interactions between a homeodomain cofactor and a mammalian nuclear receptor FTZ-F1 homologue. EMBO Rep. 5, 613-619.
10. Ahmed, F., Brown, K.M., Stephan, D.A., Morrison, J., Johnson, E., and Tomarev, S.I. (2004) Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure. Invest. Ophthamol. Vis. Sci. 45, 1247-1258.
11. Tomarev, S.I., Wistow, G., Raymond, V., Dubois, S., and Malyukova, I. (2003) Gene expression profile of the human trabecular meshwork. Invest. Ophthamol. Vis. Sci. 44, 2588-2596.
12. Torrado, M., Trivedi, R., Zinovieva, R., Karavanova, I., and Tomarev, S.I. (2002) Optimedin: a novel olfactomedin-related protein that interacts with myocilin. Hum. Mol. Genet. 11, 1291-1301.
13. Tomarev, S.I. (2001) Eyeing a new route to glaucoma along an old pathway. Nature Med. 7, 294-295.
14. Kim, B-S., Savinova, O.V., Reedy, M.V., Martin, J, Lun, Y., Gan, L., Smith, R., Tomarev, S.I., John, S.W.M., and Johnson, R.L. (2001) Targeted disruption of myocilin (Myoc) suggests that human glaucoma-causing mutations are gain of function. Mol. Cell. Biol. 21, 7707-7713.