Research interests

Genetics of Pressure- and Non-Pressure-Induced Neurodegenerations

We use a multidisciplinary approach including genetics, genomics, proteomics, and physiology to identify the fundamental biologic processes that cause ocular disease. Our work spans developmental biology, melanosomal biology, immunology, and neurobiology. We have largely used the mouse but plan to integrate the use of mice with other model organisms such as flies, fish or worms.

Glaucoma affects 70 million people worldwide. In glaucoma, retinal ganglion cell death and optic nerve degeneration lead to blindness. Harmfully high intraocular pressure (IOP) is an important contributing factor in many cases. The genetic factors determining IOP and susceptibility to pressure-induced damage are largely unknown. Age-related macular degeneration (ARMD) is another common ocular disease involving retinal neurodegeneration (primarily photoreceptors). The genetic and molecular causes of ARMD are also poorly defined. Although we study ARMD-relevant phenotypes, and high IOP is not necessary for all glaucoma, the following summary focuses on our pressure-related glaucoma research.

Causes of High IOP

To understand the initial processes leading to glaucoma, it is important to identify the genes that cause elevated IOP. We have studied IOP in mice carrying mutated genes that are expressed in ocular tissue involved in aqueous humor production and drainage. Although these studies identified genes that control IOP, none of the tested mutations elevated IOP to levels that cause glaucoma. Thus, we are currently focusing on identifying and characterizing new mutants with greater elevations of IOP.

Genetics of Glaucoma in DBA/2J Mice

In pigmentary glaucoma, iris cells are damaged resulting in dispersal of iris pigment into the ocular drainage structures, and this induces high IOP. We demonstrated that DBA/2J mice develop a form of pigmentary glaucoma caused by mutations in the glyocoprotein (transmembrane) nmb gene, Gpnmb, and the tyrosinase-related protein 1 gene, Tyrp1. Since both genes encode melanosomal proteins, we hypothesized that their mutation somehow allows toxic intermediates of pigment production to leak from melanosomes, causing iris disease and pigmentary glaucoma. Supporting this, albino and hypopigmentation mutations prevent disease development. This suggests that mutant melanosomal protein genes may contribute to human pigmentary glaucoma, and that therapeutic strategies to decrease pigment production may be beneficial.

Adding a further layer of understanding, our experiments demonstrate that bone marrow-derived cells and inflammatory processes contribute to the depigmenting iris disease. The Gpnmb gene is expressed in dendritic cells that control immune responses. Current experiments suggest that the Gpnmb mutation disturbs ocular immune privilege and allows immune cells to attack the iris and propagate the iris disease that induces glaucoma.

Future experiments will focus on the nature of the immune system’s involvement in this glaucoma. Not all individuals with pigment dispersion develop high IOP. Thus, we will also assess the possibility that differences in immune responses determine whether the dispersed pigment induces high IOP.

Neurobiology of Pressure-Induced Damage in Glaucoma

The neurobiology of pressure-induced cell death in glaucoma is poorly understood. DBA/2J mice provide a tractable model for dissecting pathways of cell death in inherited glaucoma and for investigating neuroprotective strategies. The inherited nature of the DBA/2J disease, marked by a progressive, relatively mild onset of pressure insult, is an important feature of this model.

We use the DBA/2J model to address how and why retinal ganglion cells (RGCs) die in glaucoma. RGCs appear to die by apoptosis in glaucoma, but the damaging pathways and the location of the initial RGC insult(s) are not clear. It is now known that the pathways that destroy the soma and axon of the same neuron can differ. Using mutants in which somal and axonal degeneration are separately impeded, we have started experiments to determine whether high IOP insults the RGC soma, axon, or both in glaucoma. We recently demonstrated that the pro-apoptotic molecule BAX is required for RGC death in DBA/2J glaucoma. However, BAX is not required for RGC axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma.

Future efforts will focus on understanding the different somal and axonal degeneration pathways. To understand these processes, we are using a variety of approaches including genomics, proteomics, and the cloning of mutant genes that underlie mouse axonopathy phenotypes. When studying axon degeneration pathways, we plan to integrate the use of mice and flies. These studies will provide new insights into RGC death in glaucoma and will identify potential therapeutic targets.

Neuroprotection

We are also interested in mechanisms of neuroprotection that may shield RGCs from glaucoma. Importantly, we have discovered a profound neuroprotective effect of a radiation and bone marrow treatment. The protective effect is long-lasting and prevents glaucoma in almost all treated mice. The magnitude of the protection is unprecedented and we are eager to understand the underlying biology, which may involve stem cells, trophic factors, neuronal-glial interactions or simply neuronal mechanisms. Due to the robust protective effect, this treatment offers a new tool for studying mechanisms of protection against glaucoma and possibly other neurodegenerative disease.

Anterior Segment Dysgenesis and Developmental Glaucoma

Dysgenesis of the ocular drainage structures is associated with elevated IOP and glaucoma. To facilitate mouse studies, we determined the anatomy and development of the anterior chamber angle drainage structures. We have characterized genes that cause angle dysgenesis, including the forkhead box C2 trancription factor (Foxc2) and bone morphogenetic protein 4 (Bmp4), and the type IV procollagen α1 (Col4a1) genes. Additionally, we characterized the angles of mice with mutations in the Foxc1 and cytochrome p450 (Cyp1b1) genes, whose orthologs cause human developmental glaucoma. Together, our work demonstrates that angle dysgenesis presents as a complex, quantitative trait, which elevates IOP after reaching a certain severity.

We also tested mouse strains for modifier genes that enhance or suppress angle abnormalities in Cyp1b1 and Foxc1 mutants. These experiments identified the tyrosinase gene as a modifier whose deficiency exacerbates defects in both Cyp1b1 and Foxc1 mutant mice. Finally, we demonstrated that the severe dysgenesis in eyes lacking CYP1B1 and tyrosinase is alleviated by administration of L-DOPA, a normal product of tyrosinase. Thus, a pathway involving tyrosinase is important in angle development.

Publications

• Anderson MG, Smith RS, Hawes NL, Zabaleta A, Chang B, Wiggs JL, John SWM. 2002. Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat Genet 30:81-85.
• Gould DB, John SWM. 2002. Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum Mol Genet 11:1185-1193.
• Kim HS, Lee G, John SWM, Maeda N, Smithies O. 2002. Molecular phenotying for analyzing subtle genetic effects in mice: application to an angiotensinogen gene titration. Proc Natl Acad Sci USA 99:4602-4607.
• Smith RS, Korb D, John SWM. 2002. A goniolens for clinical monitoring of the mouse iridocorneal angle and optic nerve. Mol Vis 8:26-31
• Sugiyama F, Churchill GA, Li R, Libby LJM, Carver T, Yagami K-I, John SWM, Paigen B. 2002. Quantitative trait loci associated with blood pressure, heart rate and heart weight in CBA/CaJ and BALB/cJ mice. Physiol Genomics. 10:5-12.
• Lehmann OJ, Tuft S, Brice G, Smith R, Blixt A, Bell R, Johansson B, Jordan T, Hitchings RA, Khaw PT, John SW, Carlsson P, Bhattacharya SS. 2003. Novel anterior segment phenotypes resulting from forkhead gene alterations: evidence for cross-species conservation of function. Invest Ophthalmol Vis Sci 44:2627-2633.
• Libby RT, Smith RS, Savinova OV, Zabaleta A, Martin JE, Gonzalez FJ, John SWM. 2003 Modification of ocular defects in mouse developmental glaucoma models by tyrosinase. Science 299:1578-1581.
• Mo JS, Anderson MG, Gregory M, Smith RS, Savinova OV, Serreze DV, Ksander BR, Streilein JW, John SWM. 2003. By altering ocular immune privilege, bone marrow-derived cells pathogenically contribute to DBA/2J pigmentary glaucoma. J Exp Med 197:1335-1344.
• Wang D, Oparil S, Feng JA, Li P, Perry G, Chen LB, Dai M, John SWM, Chen YF. 2003. Effects of pressure overload on extracellular matrix expression in the heart of the atrial natriuretic peptide-null mouse. Hypertension 42:88-95.
• Gould DB, Miceli-Libby L, Savinova OV, Torrado M, Tomarev SI, Smith RS, John SWM. 2004. Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma. Mol Cell Biol 24:9019-9025.
• Gould DB, Smith RS, John SW. 2004. Anterior segment development relevant to glaucoma. Int J Dev Biol 48:1015-1029.
• Link BA, Gray MP, Smith RS, John SWM. 2004. Intraocular pressure in zebrafish: comparison of inbred strains and identification of a reduced melanin mutant with raised IOP. Invest Ophthalmol Vis Sci 45:4415-4422.
• Anderson MG, Libby RT, Gould DB, Smith RS, John SWM. 2005. High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma. Proc Natl Acad Sci USA 102:4566-4571. http://www.pnas.org/cgi/content/full/102/12/4566
• Breedveld G, de Coo RF, Lequin MH, Arts WF, Heutink P, Gould DB, John SWM, Oostra B, Mancini GM. 2005. Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly. J Med Genet. doi: 10.1135/jmg.2005.035584
• Gould DB, Phalan FC, Breedveld GJ, van Mil SE, Smith RS, Schimenti JC, Aguglia U, van der Knaap MS, Heutink P, John SWM. 2005. Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly. Science 308:1167-1171.
• Hagaman JR, John SWM, Xu L, Smithies O, Maeda N. 2005. An improved technique for tail-cuff blood pressure measurements with dark-tailed mice. Contemp Top Lab Anim Sci 44:43-46.
• Jakobs TC, Libby RT, Ben Y, John SW, Masland RH. 2005. Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice. J Cell Biol 171:313-325.
• Libby RT, Anderson MG, Pang I-H, Robinson Z, Savinova OV, Cosma IM, Snow A, Wilson LA, Smith RS, Clark AF, John SWM. 2005. Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration. Vis Neurosci 22:637-648.
• Libby RT, Li Y, Savinova OV, Barter J, Smith RS, Nickells RW, John SWM. 2005. Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage. PLoS Genet 1:17-26.
• Smith RS, John SWM, Nishina PM, Sundberg JP. 2002. Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida.
• Scriver CR, John SWM, Rozen R, Eisensmith R, Woo SLC. 1993. Associations between populations, PKU mutations and RFLP haplotypes at the PAH locus: an overview. Dev Brain Dysfunct 6:11-25.
• John SWM, Anderson MG, Smith, RS. 1999. Mouse Genetics: A tool to help unlock the mechanisms of glaucoma. Journal of Glaucoma 8: 400-412.
• Smith RS, Nishina PM, Ikeda S, Jewett P, Zabaleta A, John SWM. 2000. Interpretation of Ocular Pathology in Genetically-Engineered and Spontaneous Mutant Mice. In Pathology of Genetically Engineered Mice. Ward J, Sundberg J, (Eds.) University of Iowa Press, Iowa City, Iowa, 217-231.
• Gould DB, John SWM. 2002. Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum Mol Genet 11: 1185-1193.
• John SWM, Savinova OV. 2002. Intraocular pressure measurement in mice: Technical aspects. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 313-319.
• Smith RS, Hawes NL, Miller J, Sundberg JP, John SWM. 2002. Necrospy and photography. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 251-264.
• Smith RS, John SWM, Nishina PM. 2002. The posterior segment and orbit. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 25-45.
• Smith RS, John SWM, Sundberg JP. 2002. Optic nerve and orbit. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 227-250.
• Smith RS, Kao W, John SWM. 2002. Ocular development. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 45-66.
• Smith RS, Sundberg JP, John SWM. 2002. The anterior segment. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 111-160.
• Smith RS, Sundberg JP, John SWM. 2002. The anterior segment and ocular adnexae. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods, Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 3-24.
• Smith RS, Zabaleta A, John SWM. 2002. Light microscopy. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 266-271.
• Sundberg JP, Smith RS, John SWM. 2002. Selection of controls. In Systematic Evaluation of the Mouse Eye: Anatomy, Pathology and Biomethods. Smith RS, John SWM, Nishina PM, Sundberg JP, (Eds.) CRC Press, Boca Raton, Florida, 77-80.
• Gould DB, Smith RS, John SWM. 2004. Anterior segment development relevant to glaucoma. Int J Dev Biol 49:1015-1029.
• John SWM. 2005. Mechanistic insights to glaucoma provided by experimental genetics: The Cogan Lecture. Invest Ophthalmol Vis Sci 46:2650-2661.
• Whitmore AV, Libby RT, John SWM. 2005. Glaucoma: thinking in new ways - a role for autonomous axonal self-destruction and other compartmentalized processes? Prog Retin Eye Res 24:639-662.
• Libby RT, Gould DB, Anderson MG, John SWM. 2006. Complex genetics of glaucoma susceptibility. Annu Rev Genomics Hum Genet 6:15-44.

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