Section on Retinal Ganglion Cell Biology
First row from left to right: Heung Sun Kwon, Afia Sultana, and Victoria Levasseur. Second row from left to right: Thomas Johnson, Stanislav Tomarev, Myung Kuk Joe, Nicholas DeKorver, and Naoki Nakaya
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 (RGCs), 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 RGCs. Several genes have been implicated in glaucoma pathogenesis but the search for other contributing genes continues. This section conducts basic research on glaucoma. We study genes, proteins and signaling pathways that might be essential for RGC and optic nerve development, function, survival, and regeneration.
Our interests are concentrated on early changes in the retina and the optic nerve during 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 our research is the identification 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 aqueous humor outflow system in the normal eye. We are particularly interested in genes encoding olfactomedin domain-containing proteins. To study function of these proteins we also use zebrafish as a model system.
Treatments currently available for glaucoma exert their effects by reducing IOP, the most important risk factor for the onset and progression of the disease, but have no direct effects on RGCs or the optic nerve and are not always optimally effective in slowing the progression of the disease. Thus, the development of novel, neuroprotective glaucoma therapies are of great importance. We are interested in investigating the potential neuroprotective benefits of stem cell transplantation, which has produced encouraging results in different models of CNS degeneration.
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 our research as of February 2012.
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 a 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 RGCs 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. Mice expressing myocilin mutants were mated to different modifier lines. More dramatic upregulation of IOP and damage to the retina and optic nerve were observed in some of the produced lines compared with the lines expressing myocilin mutants or modifiers alone. Pathological changes observed in the eyes of transgenic mice are similar to those observed in glaucoma patients and these transgenic mouse lines represent animal models of glaucoma. Current studies are directed toward the characterization of molecular changes in the eye angle tissues and retina of these new mouse lines.
We believe that genetic rodent models of glaucoma will be invaluable for addressing fundamental questions in glaucoma, including the identification of signaling pathways in the retina and the optic nerve activated in the disease, the effects of modifier genes, and neuroprotection.
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 expression of mutated myocilin caused an unfolded protein response and made cells more sensitive to oxidative stress. These observations were confirmed in our mouse models of glaucoma.
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 beta-catenin and its accumulation on the cellular membrane, but did not induce nuclear accumulation of beta-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of beta-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. Our recent data demonstrate that myocilin may be also involved in the regulation of several other signaling pathways. These observations are currently 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. Other family members, similar to myocilin, are secreted glycoproteins whose functions are mostly unknown. Available data suggest that the olfactomedin domain may be essential for 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 on the genes that are expressed in retinal ganglion cells and 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 the 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 the 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, 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 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 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 studying their functions will lead to a better understanding of the molecular mechanisms of their actions.
Neuroprotection in Glaucoma
As one of the possible approaches to neuroprotection, we use stem cell transplantation. In addition to in vivo transplantation, we use the in vitro technique of retinal explant tissue culture as a model to assess cellular transplantation. We have demonstrated strong neuroprotection derived from mesenchymal stem cell (MSC) transplantation. However, the mechanism underlying this effect is unclear. Our future work aims to identify the precise mechanism(s) that contribute to the neuroprotective effect we observed following MSC transplantation in glaucoma. We investigate the neuroprotective factors that MSCs secrete and analyze changes in gene expression in the retina after cultivation with MSCs. These experiments have already identified several molecules and pathways likely to be involved in MSC-mediated neuroprotection. The role of these candidate genes and pathways is under investigation.
MSCs may provide a temporal protective effect but are not the best candidates for RGC replacement therapy. The primary requirement for cell replacement therapy for glaucoma is the development of a reliable protocol capable of differentiating precursor cells into mature, functional RGCs. While a convincing in vitro differentiation of a mature RGC from a suitable precursor cell type has yet to be demonstrated, advances in driving stem cells, including induced pluripotent stem cells, towards an RGC-like fate are continually being made. Our future experiments will be directed towards the generation of RGC precursors. We believe that production of such cells is a necessary step for subsequent RGC replacement therapy in glaucoma.
|Stanislav Tomarev, Ph.D.
||Section Headfirstname.lastname@example.org||(301) 496-8524|
|Naoki Nakaya, Ph.D.||Staff Scientistemail@example.com||(301) 402-4534|
|Myung Kuk Joe, Ph.D.||Visiting Fellowfirstname.lastname@example.org||(301)451-1983|
|Thomas Johnson, Ph.D.||Special Volunteeremail@example.com||(443) 977-7459|
|Victoria Levasseur||Post-Bac Fellowfirstname.lastname@example.org||(301) 402-3677
|Heung Sun Kwon, Ph.D.||Visiting Fellowemail@example.com||(301) 435-6241|
|Afia Sultana, Ph.D.||Visiting Fellowfirstname.lastname@example.org||(301) 402-0506|
Recent Selected Publications
Sultana, A., Nakaya, N., Senatorov, V.V., and Tomarev, S.I. (2011) Olfactomedin 2: expression in the eye and interaction with other olfactomedin domain-containing proteins. Invest. Ophthalmol. Vis. Sci. 52, 2584-2592.
Bull, N.D., Johnson, T.V., Welespar, G., DeKorver, N., Tomarev, S.I. and Martin, K.R. (2011) Use of an adult retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies. Invest. Ophthalmol. Vis. Sci. 52, 3309-3320.
Kwon, H.-S. and Tomarev, S.I. (2011) Myocilin promotes cell migration through activation of integrin-focal adhesion kinase-serine/threonine kinase signaling pathway. J. Cell. Physiol. 226, 3392-3402.
Chi, Z.-L, Akahori1, M., Obazawa1, M., Minami1, M., Noda1, T., Nakaya, N., Tomarev, S., Kawase, K., Yamamoto, T., Noda, S., Sasaoka, M., Shimazaki, A., Takada, Y., and Iwata, T. (2010) Overexpression of optineurin E50K disrupts Rab8 interaction and leads to a progressive retinal degeneration in mice. Hum. Mol. Genet. 19, 2606-2615.
Li, L., Nakaya, N., Chavali, V.R.M., Ma, Z., Jiao, X., Sieving, P., Riazuddin, S., Tomarev, S.I., Ayyagari, R., Riazuddin, S.A., and Hejtmancik, J.F. (2010) A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal recessive Retinitis Pigmentosa. Am. J. Hum. Genet. 87, 1-10.
Johnson, T.V., Bull, N.D., Tomarev, S.I., Hunt, D.P., and Martin, K.R. (2010) Local mesenchymal stem cell transplantation confers neuroprotection in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 51, 2051-2059.
Joe, M.-K and Tomarev, S.I. (2010) Expression of myocilin mutants sensitizes cells to oxidative stress-induced apoptosis: implication for glaucoma pathogenesis. Am. J. Pathol. 176, 2880-2890.
Tomarev, S.I. (2010) Animal models of glaucoma. In: Encyclopedia of the Eye, 1st Edition (Dartt. D. A., Beshrse. J. C. and Dana, R., Eds.). vol.1, pp. 106-111.
Johnson, T.V. and Tomarev, S.I. (2010) Rodent models of glaucoma. Brain Res. Bull. 81, 349-358.
Tomarev, S.I. and Nakaya, N. (2009) Olfactomedin domain-containing proteins: Possible mechanisms of action and functions in normal development and pathology. Mol. Neurobiol. 40, 122-138.
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.
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.
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.
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.
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.
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. 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.
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.
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.
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.
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.
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.
Tomarev, S.I. (2001) Eyeing a new route to glaucoma along an old pathway. Nature Med. 7, 294-295.
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.
Last Updated: February 2012