The goal of this project is to determine how elevated levels of homocysteine (Hcy) (hyperhomocysteinemia, HHcy)) contribute to retinal neurovascular disease. HHcy is implicated in retinal vascular occlusive disease, optic neuropathies (e.g. pseudoexfoliation glaucoma), macular degeneration, and diabetic retinopathy. Mild HHcy occurs in ~5%-12% of the population;hence understanding how HHcy contributes to retinal disease is critically important. Hcy is a key intermediate in methionine metabolism. HHcy is due to genetic defects involving cystathionine synthase (CBS), methylenetetrahydrofolate reductase (MTHFR) or methionine synthase as well as vitamin deficiencies (folate/B12/B6). During the previous funding period we analyzed retinas of mice with endogenous HHcy due to deficiency/lack of CBS and observed profound loss of visual function, marked disruption of retinal neuronal layers and florid vasculopathy in cbs-/- mice, and a milder phenotype with vascular alterations, progressive ganglion cell loss, dysregulation of axonal mitochondrial dynamics and delayed visual evoked potentials in cbs+/- mice. These data suggest that HHcy compromises retinal structure/function, the severity of which correlates with Hcy levels;and that HHcy alters retinal vasculature initially, subsequently triggering retinal neuron death. The phenotype may be due to increased ER stress, oxidative stress, and/or excitotoxicity;however since CBS irreversibly converts Hcy to cystathionine via the """"""""transsulfuration pathway"""""""" leading to formation of cysteine, which is then converted to glutathione (GSH), hydrogen sulfide (H2S), and/or taurine, the observed disrupted retinal structure/function may be due to decreased levels of these downstream factors critical for retinal health. To investigate this, we will analyze retinas of mice lacking/deficient in MTHFR. mthfr mice have a defect in the """"""""remethylation pathway"""""""" with concomitant HHcy;however their transsulfuration pathway is intact. Using these models we can dissect the role of HHcy in retina. Upon completion of the studies we will be poised to investigate whether folate/B12/B6 supplementation reverses/retards retinal neurovasculopathy associated with HHcy due to mutations in CBS or mthfr. There are three aims proposed for this study:
Aim 1 : Test the hypotheses that (A) HHcy compromises retinal structure/function regardless whether due to a defect in the transsulfuration or remethylation pathway;and (B) severe HHcy alters retinal vasculature initially, which subsequently triggers widespread death of retinal neurons (vasculopathy precedes neuropathy) while mild HHcy induces modest vascular changes disrupting only the inner retina;
Aim 2 : Test the hypothesis that HHcy due to CBS deficiency induces retinal neurovascular disease not only by inducing oxidative stress, excitotoxicity, and/or ER stress, but also by altering levels of GSH, H2S and taurine, whereas HHcy due to MTHFR deficiency induces retinal neurovascular disease through the oxidative stress, excitotoxicity, and/or ER stress pathways;
Aim 3 : Test the hypothesis that folate/B12/B6 supplementation will retard/reverse HHcy-induced retinal neurovasculopathy.

Public Health Relevance

Homocysteine is an amino acid that plays a role in methionine metabolism. When elevated (as occurs in ~5- 12% of the population), homocysteine has dire consequences on the cardiovascular and nervous systems. There is an emerging clinical literature that implicates elevated homocysteine in retinal neurovascular diseases such as optic neuropathies (e.g. pseudoexfoliation glaucoma), retinal vascular occlusive diseases, macular degeneration, and diabetic retinopathy. The mechanism(s) by which excess homocysteine disrupts retinal structure/function is poorly understood and is the focus of this research project. We will use mouse models with endogenous elevation of homocysteine, due to two different enzymatic defects, to investigate the molecular mechanisms responsible for the retinal phenotype and we will determine whether supplementation with folate/B12/B6, vitamins that are related to the homocysteine/methionine metabolic pathways, can retard/reverse the retinal disruption.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012830-11
Application #
8619632
Study Section
Special Emphasis Panel (ZRG1-BDCN-H (02))
Program Officer
Shen, Grace L
Project Start
1999-12-01
Project End
2017-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
11
Fiscal Year
2014
Total Cost
$367,500
Indirect Cost
$122,500
Name
Georgia Regents University
Department
Biology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Markand, Shanu; Saul, Alan; Tawfik, Amany et al. (2016) Mthfr as a modifier of the retinal phenotype of Crb1(rd8/rd8) mice. Exp Eye Res 145:164-72
Markand, Shanu; Saul, Alan; Roon, Penny et al. (2015) Retinal Ganglion Cell Loss and Mild Vasculopathy in Methylene Tetrahydrofolate Reductase (Mthfr)-Deficient Mice: A Model of Mild Hyperhomocysteinemia. Invest Ophthalmol Vis Sci 56:2684-95
Ibrahim, Ahmed S; Tawfik, Amany M; Hussein, Khaled A et al. (2015) Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids. Biochim Biophys Acta 1851:290-8
Ibrahim, Ahmed S; Elshafey, Sally; Sellak, Hassan et al. (2015) A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase. J Lipid Res 56:599-611
Tawfik, Amany; Markand, Shanu; Al-Shabrawey, Mohamed et al. (2014) Alterations of retinal vasculature in cystathionine-β-synthase heterozygous mice: a model of mild to moderate hyperhomocysteinemia. Am J Pathol 184:2573-85
Tawfik, Amany; Smith, Sylvia B (2014) Increased ER stress as a mechanism of retinal neurovasculopathy in mice with severe hyperhomocysteinemia. Austin J Clin Ophthalmol 1:1023
Tawfik, Amany; Gnana-Prakasam, Jaya P; Smith, Sylvia B et al. (2014) Deletion of hemojuvelin, an iron-regulatory protein, in mice results in abnormal angiogenesis and vasculogenesis in retina along with reactive gliosis. Invest Ophthalmol Vis Sci 55:3616-25
Tawfik, Amany; Al-Shabrawey, Mohamed; Roon, Penny et al. (2013) Alterations of retinal vasculature in cystathionine-Beta-synthase mutant mice, a model of hyperhomocysteinemia. Invest Ophthalmol Vis Sci 54:939-49
Markand, Shanu; Tawfik, Amany; Ha, Yonju et al. (2013) Cystathionine beta synthase expression in mouse retina. Curr Eye Res 38:597-604
Farook, J M; Shields, J; Tawfik, A et al. (2013) GADD34 induces cell death through inactivation of Akt following traumatic brain injury. Cell Death Dis 4:e754

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