Overview
Our research investigates the molecular features of complex diseases that lead to the death of neural cells (neurodegenerations). Most of our projects focus on glaucoma. Glaucoma is a major cause of human blindness and is often associated with elevated pressure within the eye itself (intraocular pressure, or IOP). The harmfully high pressure damages retinal ganglion cells (RGCs) resulting in a pressure-induced neurodegeneration. We use mouse models to study glaucoma. We combine genetics with genomics, cell/molecular biology and physiology to understand glaucoma. We are identifying new genes and pathways that cause glaucoma. We study how abnormal ocular development and other processes lead to high IOP and glaucoma and are determining how high intraocular pressure damages retinal neurons. We are also investigating possible treatments and are currently experimenting with a new radiation treatment we discovered that completely prevents glaucomatous neurodegeneration in the vast majority of treated animals.
Scientific report
Genetics of Pressure- and Non-Pressure-Induced Neurodegenerations
We use genetics, genomics, cell biology and physiology to understand the fundamental biologic processes that cause disease, with a focus on neurodegeneration and glaucoma. Our work spans developmental biology, melanosomal biology, immunology, and neurobiology. In addition to glaucoma, we study stroke and age-related macular degeneration-relevant phenotypes. The following summary focuses on our glaucoma research.
Glaucoma
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.
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. We are characterizing several interesting mutants that appear to affect different pathways.
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. 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. Current experiments suggest that the Gpnmb mutation of DBA/2J disturbs ocular immune privilege and allows immune cells to attack the iris and propagate the iris disease that induces glaucoma.
Ongoing experiments 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. We have produced a useful tool for these studies by backcrossing the Gpnmb and Tyrp1 mutant alleles from DBA/2J into the C57BL/6J strain. This mutant C57BL/6J congenic strain develops the same iris disease as DBA/2J mice, but is much less susceptible to IOP elevation. We are studying this strain difference, in susceptibility to IOP elevation. An understanding of the genes and pathways that are involved may lead to new treatments to prevent IOP elevation in human glaucoma.
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. 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 genetics and genomics. We are completing a comprehensive genomic study of gene expression changes at different stages of glaucoma and are identifying very early changes. 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. Experiments are underway to understand this neuroprotection. Additionally, we are testing the ability of focal ocular irradiation to prevent glaucoma.
Anterior segment dysgenesis and developmental glaucoma
In developmental glaucomas, abnormal formation of the ocular drainage structures in the angle of the eye is a cause of IOP elevation. We have characterized angle malformation genes, including the forkhead transcription factor (Foxc2), bone morphogenetic protein (Bmp4), and collagen type IV alpha 1 (Col4a1). We have also studied mice with mutations in the genes for cytochrome P450 1b1 (Cyp1b1) and forkhead box transcription factor C1 (Foxc1), which cause human developmental glaucoma. Because of extensive variability in the human disease, we used mice to identify a modifier gene that alters ocular abnormalities due to mutations in Cyp1b1 and Foxc1. Genetic deficiency of tyrosinase exacerbates defects in both Cyp1b1 and Foxc1 mutant mice. Tyrosinase provides L-DOPA that protects against angle malformation. Future work will be aimed at further understanding the role of L-DOPA in ocular development and glaucoma, evaluating if and how dietary DOPA modulates the severity of human glaucoma, and identifying further genes that modify the phenotypic effects of Cyp1b1 mutations.
Lab staff
Principal Investigator/Professor Former Postdoctoral Fellows
Simon W.M. John, Ph.D. Mike Anderson, Ph.D.
Doug Gould, Ph.D.
Medical Fellows Rick Libby, Ph.D.
Mihai Cosma
Research Scientist/Postdoctoral Fellows Laboratory Staff
Richard Smith, M.D., D.M.S. Michael Walden, Lab Manager
Gareth Howell, Ph.D. Amy Bell
Sai Nair, Ph.D. Xianjun Zhu, Ph.D.
Stephen Kneeland Danilo Macalinao
Margaret Ryan
Visiting Investigators Administrative
Jeff Marchant, Ph.D. Bethany Preble
James Morgan, Ph.D.
Publication listings
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.
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.
Anderson MG, Haraszti T, Petersen GE, Wirick S, Jacobsen C, John SW, Grunze M. 2006. Scanning transmission X-ray microscopic analysis of purified melanosomes of the mouse iris. Micron 37:689-698.
Anderson MG, Libby RT, Mao M, Cosma IM, Wilson LA, Smith RS, John SW. 2006. Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma. BMC Biol 4:20.
Breedveld G, de Coo IF, Lequin MH, Arts WF, Heutink P, Gould DB, John SW, Oostra B, Mancini GM. 2006. Novel mutations in three families confirm a major role of Col4a1 in hereditary porencephaly. J Med Genet 43:490-495.
Gould DB, Phalan FC, van Mil SE, Sundberg JP, Vahedi K, Massin P, Bousser MG, Heutink P, Miner JH, Tournier-Lasserve E, John SWM. 2006. Role of Col4a1 in small-vessel disease and hemorrhagic stroke. N Engl J Med 354:1489-1496.
Gould DB, Reedy M, Wilson LA, Smith RS, Johnson RL, John SW. 2006. Mutant myocilin non-secretion in vivo is not sufficient to cause glaucoma. Mol Cell Biol 26:8427-8436.
Gould DB, Marchant JK, Savinova OV, Smith RS, John SWM. 2007. Col4a1 mutation causes endoplasmic reticulum stress and genetically modifiable ocular dysgenesis. Hum Mol Genet 16: 7798-807
Fox MA, Sanes JR, Borza, DB, Eswarakumar VP, Fassler R, Hudson B, John SWM, Ninomiya Y, Pedchenko V, Rheault M, Sado Y, Segal Y, Werle MJ, Umemori H. 2007. Distinct targeted-derived signals organize formation, maturation and maintenance of motor nerve terminals. Cell 129: 179-193
Howell GR, Libby RT, Marchant JK, Wilson LA, Cosma IM, Smith RS, Anderson MG, John SWM. 2007. Absence of glaucoma in DBA/2J mice homozygous for wild-type versions of Gpnmb and Tyrp1. BMC Genet 8:45 http://www.biomedcentral.com/1471-2156/8/45
Brooks BP, Larson DM, Chan C-C, Kjellstrom S, Smith RS, Crawford MA, Lamoreux L, Huizing M, Hess R, Jiao X, Hejtmancik FJ, Maminishkis A, John SWM, Bush R, Pavan WJ. 2007. Analysis of ocular hypopigmentation in Rab38cht/cht mice. Invest Ophthalmol Vis Sci 48:9
Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow A, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith SJ, John SWM, Barres BA. 2007. The Classical Complement Cascade Mediates CNS Synapse Elimination. Cell 131: 1164-1178
Libby RT, Howell GR, Pang I-H, Savinova OV, Barter J, Smith RS, Clark AF, John SWM. 2007. Inducible nitric oxide synthase, Nos2, does not mediate optic neuropathy and retinopathy in the DBA/2J glaucoma model. BMC Neurosci 8:108 doi:10.1186/1471-2202-8-108
Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, Whitmore AV, Masland RH, John SWM. 2007. Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Biol 179:1523-1537.
Anderson MG, Nair KS, Amonoo LA, Mehalow A, Trantow C, Masli S, John SWM. 2008. GpnmbR150X allele must be present in bone marrow derived cells to mediate DBA/2J glaucoma. BMC Genet Apr 10; 9:30
Anderson MG, Hawes NL, Trantow CM, Chang B, John SWM. 2008. Iris phenotypes and pigment dispersion caused by genes influencing pigmentation.
Pigment Cell & Melanoma Research PMID
Books
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.
Book chapters and reviews
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.
Smith RS, Nishina PM, Sundberg JP, Zwaan J, John SWM. In press. The mouse in biomedical research. In: Eye Research. Fox J, Newcomer C, Smith A, Barthold S, Quimby F, Davisson M, (Eds.) Elsevier, San Diego, CA.
