Molecular Mechanisms of Glaucoma Section

• Staff
• Selected Publications

Current Research

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

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 February 2007.

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 and rats. We produced several lines of transgenic mice and rats 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. RGC depletion was associated with the shrinkage of their nuclei and DNA fragmentation in the peripheral retina. We concluded that 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. Several lines of transgenic rats were produced using the same BAC DNAs with mutated mouse or human MYOCILIN genes. Current studies are directed toward the characterization of the molecular changes in the eye angle tissues and retina of 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.

Function of the olfactomedin domain-containing proteins

Myocilin belongs to a family of olfactomedin domain-containing proteins consisting of more than 10 family members. 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:

1. We developed several stably transfected cell lines expressing olfactomedin, optimedin, also known as olfactomedin 3, and Myocilin. After stimulation with the nerve growth factor, optimedin-expressing PC12 cells demonstrated elevated levels of N-cadherin, ß-catenin, a-catenin, and occluding as compared with stimulated, control PC12 cells. The expression of optimedin induced Ca2+-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. The expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. Olfactomedin expression, on the contrary, induced neurite outgrowth in NGF-stimulated PC12 cells. Signaling pathways activated by the expression of different olfactomedin domain-containing proteins are under study.
2. Purified olfactomedin domain-containing proteins (olfactomedin 1, optimedin, Myocilin) are used to study their effects on cell morphology, cell signaling and interactions with other proteins.
3. A zebrafish model is used to study function of olfactomedin 1 in development. There are two olfactomedin 1 genes in zebrafish each producing four different transcripts. Our data suggest that olfactomedin 1 might be essential for axon growth in zebrafish, supporting observations obtained with olfactomedin-expressing PC12 cells. The inhibition of olfactomedin 1 expression by morpholino oligonucleotides inhibits the optic nerve extension. The regulation of the olfactomedin 1 genes in the course of zebrafish development is studied using injection of promoter constructs fused to a GFP reporter.
4. We are producing olfactomedin 1 and olfactomedin 3 knockout mice. They will be used to study possible functions of these proteins in different mammalian tissues with emphasis on the brain and retina.
5. Stem cells are used to study the effects of olfactomedin domain-containing proteins on cell differentiation.

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 (2001 - present)

1. Lengler, J., Krausz, E., Tomarev, S., Prescott, A., Quinlan, R., and Graw, J. (2001) Antagonistic action of Six3 and Prox 1 at the -crystallin promoter. Nucleic Acids Res. 29, 515-526.
2. Savinova, O.V., Sugiyama, F., Martin, J.E., Tomarev, S.I., Paigen, B.J., Smith, R.S., and John, S.W.M. (2001) Intraocular pressure in genetically distinct mice: an update and strain survey. BMC Genetics 2, 12.
3. 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.
4. Tomarev, S.I. (2001) Eyeing a new route to glaucoma along an old pathway. Nature Med. 7, 294-295.
5. Ahmed, F., Torrado, M, Johnson, E., Morrison, J., and Tomarev, S.I. (2001) Changes in mRNA levels for the Myoc/Tigr gene in the rat eye after experimental elevation of intraocular pressure or optic nerve transection. Invest. Ophthalmol. Vis. Sci. 42, 3165-3172.
6. Duncan, M.K., Cui, W., Oh, D.-J., and Tomarev, S.I. (2002) Prox1 is differently localized during lens development. Mech. Dev. 112, 195-198.
7. Surgucheva, I., McMahan, B., Ahmed, F., Tomarev, S., Wax, M., and Surguchov, A. (2002) Synucleins in glaucoma. J. Neurosci. Res. 68, 97-106.
8. 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.
9. Wilting, J., Papoutsi, M., Christ, B., Nicolaides, K.H., von Kaisenberg, C.S., Borges, J., Stark, G.B., Alitalo, K., Tomarev, S.I., Niemeyer, C., and Rossler, J. (2002) The transcription factor Prox1 is a marker of lymphatic endothelial cells in normal and diseased tissues. FASEB J. 16, 1271-1273.
10. Stone, J., Shang, J.L., and Tomarev, S.I. (2003) Expression of cProx1 defines sensorigenic and neurogenic regions of the avian otocyst. J. Comp. Neurol. 460, 487-502.
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. Cui, W., Tomarev, S.I., Piatigorsky, J., Chepelinsky, A.B., and Duncan, M.K. (2004) Mafs, Prox1 and Pax6 cooperate to direct spatiotemporal expression of chicken ßB1-crystallin. J. Biol. Chem. 279, 11088-11095.
13. 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.
14. 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.
15. Stone, J.S., Shang, J.L., and Tomarev, S.I. (2004) Levels of Prox1 distinguish progenitor cells and predict hair cell fate during avian hair cell regeneration. Dev. Dynamics 230, 597-614.
16. Ahmed, F., Torrado, M., Zinovieva, R.D., Senatorov, V.V., Wistow, G., and Tomarev, S.I. (2004) Gene expression profile of the rat eye irido-corneal angle. NEIBank expressed sequence tag analysis. Invest. Ophthamol. Vis. Sci. 45, 3081-3090.
17. 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.
18. Torrado, M., Senatorov, V.V., Trivedi, R., Farris, R., and Tomarev, S.I. (2004) Interaction of Pdlim2 protein with a-actinins and filamin A in the rat cornea and lung. Invest. Ophthamol. Vis. Sci. 45, 3955-3963.
19. Wistow, G., Wyatt, K., David, L., Gao, C., Bateman, O., Bernstein, S., Tomarev, S., Segovia, L., Slingsby, C., and Vihtelic, T. (2005) ?N-crystallin and the evolution of the ?-crystallin family in vertebrates. FEBS J. 272, 2276-2291.
20. 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.
21. Surgucheva, I, Park, N.-C., Yue, B., Tomarev, S., and Surguchov, A. (2005) Interaction of myocilin with -synuclein affects its secretion and aggregation. Cell. Mol. Neurobiol. 25, 1009-1033.
22. Malyukova, I., Lee, H.-S., Fariss, R.N, and Tomarev, S.I. (2006) Mutated mouse and human myocilins have similar properties and do not block general secretory pathway. Invest. Ophthamol. Vis. Sci. 47, 206-212.
23. 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.
24. 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.
25. 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.
26. Galeeva, A., Treuter, E., Tomarev, S., and Pelto-Huikko, M. (2007) A prospero-related homeobox gene Prox-1 is expressed during postnatal brain development as well as in the adult rodent brain. Neuroscience, in press.
27. Iwata, T. and Tomarev, S. (2007) Chapter 33: Animal Models for Eye Diseases and Therapeutics. Subtitle: Animal models of age-related macula degeneration and glaucoma. In: Source Book of Models for Biomedical Research. The Humana Press Inc., in press.