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.
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 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.
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.
Head of Laboratory
Simon W.M. John, Ph.D.
Bethany Preble, B.S., Executive Administrative Assistant
Mimi de Vries, Ph.D. Lab Manager/Research Scientist
Richard Smith, M.D.,D.M.S Research Specialist
Gareth Howell, Ph.D. Research Scientist
Sai Nair, Ph.D. Postdoctoral Fellow/Research Scientist
Krish Kizhatil, Ph.D. Research Scientist
Mimi de Vries, Ph.D. Lab Manager/Research Scientist
Ileana Soto, Ph.D. Postdoctoral Fellow
Sai Nair, Ph.D. Postdoctoral Fellow/Research Scientist
Research Assistants & Students
Stephen Kneeland, M.S. Research Assistant
Margaret Ryan, M.S. Research Assistant
Amy Bell, B.S. Research Assistant
Katharine Harmon, B.A. Research Assistant
Catherine Braine, B.A.Research Assistant
Michael Sellarole, B.S. Research Assistant
Francis Ding, B.S. Research Assistant
Brianna Caddle, B.S. Graduate Student/Former Research Assistant
Jeffrey Marchant, Ph.D.
James Morgan, Ph.D.
Former Postdoctoral Fellows
Doug Gould, Ph.D.
Mike Anderson, Ph.D.
Rick Libby, Ph.D.
Xianjun Zhu, Ph.D.
Gould DB, Phalan FC, van Mil SE, Sundberg JP, Vahedi K, Massin P, Germaine Bousser M, Heutink P, Miner JH, Tournier-Lasserve E, John SWM. 2006. Role of COL4A1 in small-vessel disease and hemorrhagic stroke. N E J Med 354: 1489-1496Anderson MG, Haraszti T, Petersen GE, Wirick S, Jacobsen C, John SWM, 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 SWM. 2006. Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma. BMC Biol 4:20 Gould DB, Reedy M, Wilson LA, Smith RS, Johnson RL, John SWM. 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
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 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 565-78
Gray MP, Smith RS, Soules KA, John SWM. Link BA. 2009. The aqueous humor outflow pathway of zebrafish. Invest Ophthalmol Vis Sci 50:4 1515-21
Lee B, Kano K, Young J, John SWM, Nishina PM, Naggert JK, Naito K. 2009. A novel ENU-induced mutation, peewee, causes dwarfism in the mouse. Mamm Genome 20(7):404-13
Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SWM, Gregg RG, Link BA. 2011. Mutations in zebrafish lrp2 result in adult onset ocular pathogenesis that models myopia and other risk factors for glaucoma. Plos Genetics. (in press)
Mao M, Hedberg-Buentz A, Koehn D, John SWM, Anderson MG. 2011. Anterior segment dysgenesis and early-onset Glaucoma in mice with mutation Sh3pxd2b. IOVS doi:10.1167/iovs.10-5993
Howell GR, Macalinao DG, Sousa G, Walden M, Soto I, Kneeland S, Barbay J, King B, Marchant J, Hibbs M, Stevens B, Barres B, Clark A, Libby R, John SWM. 2011. Molecular clustering identified complement and endothelin induction as early events in a mouse glaucoma. J Clinical Invest. doi:10.1172/JCI44646
Gregory MS, Hackett CG, Abernathy EF, Lee KS, Saff RR, Hohlbaum AM, Moody KL, Hobson MW, Jones A, Kolovou P, Karray S, Giani A, John SWM, Chen DF, Marshak-Rothstein A, Ksander BS. 2011. Opposing roles for membrane-bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death. PLoS ONE (In press).
Lachke SA, Alkuraya FS, Kneeland SC, Ohn T, Aboukhalil A, Howell GR, Saadi I, Cavallesco R, Yue Y, Tsai ACH, Nair KS, Cosma MI, Smith RS, Hodges E, AlFadhli SM, Al-Hajeri A, Shamseldin HE, Behbehani AM, Hannon GJ, Bulyk ML, Drack AV, Anderson PJ, John SWM, Maas RL. 2011. Mutations in the RNA Granule Component TDRD7 Cause Cataract and Glaucoma. Science* 331(6024):1571-1576 * Joint corresponding author
Chow EY, Ha D, Lin TY, De Vries WN, John SWM, Chappell WJ, Irazoqui PP. 2010. Sub-cubic millimeter intraocular pressure monitoring implant to enable genetic studies on pressure-induced neurodegeneration. Conf Proc IEEE Eng Med Biol Soc. 1:6429-32
Lin TY, Ha D, deVries WN, Kim B, Chlebowski A, John SWM, Irazoqui PP, Chappell WJ. 2011. Ultra-thin Tag Fabrication and Sensing Technique using Third Harmonic for Implantable Wireless Sensors. IEEE MTT-S Int. Microwave Symp., Baltimore, MD. TU2B-3 (In press).