• Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

      Markand, Shanu; Department of Cellular Biology and Anatomy (2015-05)
      Homocysteine (hcy), a sulfur containing amino acid, is an integral part of methionine metabolism. Elevated plasma level of hcy (Hhcy) is identified as a risk factor for cardiovascular disorders and implicated in various retinal diseases such diabetic retinopathy, glaucoma, age related macular degeneration and central retinal vein occlusion. Cystathionine β-synthase (CBS) and methylene tetrahydrofolate reductase (MTHFR) are key enzymes of hcy metabolism. CBS catalyzes the transsulfuration pathway yielding beneficial downstream products such as taurine, H2S and glutathione (GSH). MTHFR is required for methylation of hcy. Mutations in MTHFR are the most common genetic cause for Hhcy. Murine models of CBS and MTHFR are an invaluable tools to understand Hhcy pathophysiology in humans. Our lab has reported the retinal phenotype of CBS mutant mice. Depending upon the loss of one or both alleles, mild to marked retinal neurovascular and functional alterations are observed. The data from CBS mutant mice raise an important question: is the retinal neurovasculopathy observed in absence/deficiency of CBS attributed to excess hcy levels or is it due to decline in availability of taurine, H 2S and GSH? This can be addressed by studying the retinal phenotype of MTHFR mutant mice which have an intact CBS pathway. No information is available is currently available about the retinal expression of MTHFR and current data regarding CBS in the mouse retina is contentious. This thesis work tested the hypothesis that CBS and MTHFR are expressed in the mouse retina at gene and protein levels and that Hhcy would induce retinal functional and neurovascular alterations in MTHFR-deficient mice. For gene and protein expression studies, RNA and protein were isolated from retinas for analysis of Cbs and Mthfr gene expression by RT-PCR and protein expression by Western blotting. Eyes were harvested from C57BL6 mice and used for immunodetection of CBS and MTHFR in the retina. RT-PCR revealed robust Cbs and Mthfr expression in retina. Western blotting detected CBS and MTHFR protein in mouse retina. In immunohistochemical studies of the intact retina, CBS was present most abundantly in the ganglion cell layer of WT retina while MTHFR showed widespread retinal expression. Our immunofluorescence studies revealed presence of CBS and MTHFR in retinal ganglion, Müller and RPE cells. Taken together, we have compelling molecular evidence that CBS and MTHFR are expressed in mouse retina at gene and protein levels. These data indicate the underlying importance of hcy metabolism in the retina. For characterization of the retinal phenotype in MTHFR deficient mice, we employed tools such as ERG, Fundus and FA, OCT, HPLC, morphometric, immunohistochemistry (IHC) and PCR arrays. ERG revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/- mice at 24 wks. FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12-16 wks and 60% by 24 wks suggesting potential vascular damage mediated by Hhcy. SD-OCT revealed a significant decrease in NFL thickness at 24 wks in Mthfr+/- compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 wks in Mthfr+/- mice by HPLC and IHC. Morphometric analysis revealed ∼20% reduction in cells in the ganglion cell layer of Mthfr+/- mice at 24 wks. IHC indicated significantly-increased GFAP labeling suggestive of Müller cell activation. The similar loss of ganglion cells, focal vascular leakage, 2-fold increase in retinal hcy, gliosis and functional abnormities were reported in Cbs+/- mice. Taken together, these data support our hypothesis that Hhcy induces retinal neurovascular and functional alterations in MTHFR deficient mice. In addition, we explored retinal mitochondrial gene alteration as a possible mechanism of Hhcy mediated retinal alterations. PCR array data analysis revealed upregulation of pro-apoptotic genes and downregulation of genes associated with normal mitochondrial transport function. Future studies will validate these results at protein and functional levels. To conclude, our data support the hypothesis that Hhcy may be causative in certain retinal neurovasculopathies. These data contribute to our understanding of the potential effects of Hhcy on the retina and may prove useful in other disease model systems of Hhcy.
    • Characterization of the Thioredoxin System in the Diabetic Retina

      Lamoke, Folami; Department of Cellular Biology and Anatomy (2013-07)
      Diabetes is a group of diseases, which are characterized by high blood glucose levels that are a consequence of the inability to produce and/or utilize insulin. Type 1 diabetes (T1D, previously referred to as juvenile diabetes) is typically diagnosed in children and young adults. In this form, the body does not produce insulin primarily due to autoimmune-mediated destruction of pancreatic - cells, leading to insulin deficiency. Type 2 diabetes (T2D, adult onset or noninsulin dependent diabetes) is a chronic condition where the body either resists the effects or does not produce sufficient insulin. This form is more common in African Americans, Latinos, Native Americans, Asian Americans, Pacific Islanders, as well as the aged population.
    • The Effects of pp60v-src Expression on the Development of the Chicken Optic Tectum

      Mogan, John C.; Department of Biology and Anatomy (1999-03)
      The chicken optic tectum (OT) develops from the dorsal mesencephalon (midbrain) and processes crossed input from each retina. Previous experiments using a replicationdeficient retrovirus that contained the marker gene lacZ have demonstrated the normal pattern of development for the OT. Clonal cohorts derived from a single neuroepithelial stem cell migrate both radially and tangentially and differentiate into many types of neurons and at least three types of glia (radial glia and two types o f astrocytes). The goal of our laboratory is to identify important proteins involved in tectal development by: (1) directly altering expression of endogenous proteins through senseor antisense-containing retroviruses, or (2) indirectly altering endogenous protein expression or function by retroviral expression of an exogenous protein. These two approaches will allow us to determine which proteins are important in the normal and abnormal development of tectal clones. Many processes are involved in the development of the OT: proliferation, migration, differentiation, and synapse formation. Four non-receptor tyrosine kinases of the Src family (c-src, c-src+, fyn, and yes) are expressed in a spatially and temporally regulated manner in the nervous system. Their expression patterns in neural cells in vivo and in vitro have implicated these Src family members in all four of the major developmental processes mentioned above. Knockout mice of these three Src family members individually (c-src, fyn, or yes), however, show few or no overt neural developmental abnormalities. These unexpected results indicated that other members of the Src family can assume the roles of the missing kinase. Knockout mice for the major known negative regulator of Src family kinases, Csk (c-src kinase), however, show severe developmental abnormalities and defects in neural tube closure. These mice died around E9-E10 and showed elevated kinase activity for at least three Src family members (c-src, fyn, and Iyn). This fact makes it impossible to conclude that the overactivity of any one Src family tyrosine kinase is responsible for the developmental defects observed, and the early death of these embryos prevents the study of neural cell lineage, migration and differentiation in vivo. Given these results, the use of antisense to reduce expression of c-src would yield little information about the role of this kinase in neural development due to functional redundancy among Src family kinases. I decided to express in tectal clones an unregulated member of the Src family, pp60“'src, to determine how its expression alters normal tectal development. The v-src oncogene of Rous sarcoma virus was the first member of the Src family to be discovered, v-src encodes an activated tyrosine kinase (pp60,'"irc) which has lost a critical regulatory tyrosine present in the carboxy terminus of all other Src family members. Consequently, pp60'fcsrc expression affects the proliferation, migration and differentiation of many cell types in vitro and in vivo, but the effects of its expression on neural development in vivo are not well characterized. Expression of this kinase in tectal clones will provide an excellent system to study how a single unregulated Src kinase influences development of the nervous system. Expression o f a protein (pp60v'src) known to affect many different processes (proliferation, migration/cell adhesion and differentiation) in tectal clones will allow us to answer many questions of biological significance: (1) Is the proliferative potential of neuroepithelial stem cells restricted in vivo, or can stem cells generate clones of larger size?, (2) If multiple cell adhesion systems are presumptively inactivated in pp60*N,rc - expressing tectal neuroblasts, then how will clonal migration patterns differ from the norm?, (3) Is clonal differentiation in the OT controlled by only extracellular influence (e.g., growth factors, gradients) or can the developmental fate of stem cell progeny be altered by expression of pp60*’'src. In the first set of experiments I wanted to determine how wild-type pp60w'5rc expression alters the development of tectal clones in vivo. I used a replication-deficient retrovirus (LZIS), which efficiently coexpresses both LacZ and pp60*fc*rc, to determine the effects of pp60,,'*rc expression on several clonal parameters: cell number, migration pattern, and differentiation. In the next set of experiments I constructed and tested retroviral vectors which efficiently coexpress LacZ and mutated pp60w‘src proteins with deleted SH2 or SH3 domains (LZISASH2 and LZISASH3). These domains normally allow the pp60u'*rc tyrosine kinase to associate with certain cellular proteins which contain phosphotyrosines or a proline-rich stretch of amino acids, respectively. Mutation or deletion of these domains alters the biochemical and biological function of pp60^rc. I hoped to determine if the SIC or SID domains of pp60v'src are necessary for the wild-type pp60ltsrc phenotype, and to determine if they afford a unique but altered clonal phenotype compared to wild-type pp60v^rc. These experiments are novel in that they demonstrate that the overexpression of activated forms of Src family kinases influences development of the vertebrate brain. I conclude from my results that: (1) the proliferative potential of neuroepithelial stem cells in the OT is not restricted, (2) tangential migration of neuroblasts in the developing OT appears enhanced with pp60*fc*rc expression, and (3) the proper differentiation of radial glia is hindered but not prevented by pp60lHirc
    • Regulation of Reduced-Folate Transporter-1 in Retinal Pigment Epithelium

      Naggar, Hany A.; Department of Cellular Biology and Anatomy (2003-04)
      (First Paragraph) The purpose of these studies was to analyze the regulation of the folate transport protein, reduced-folate transporter (RFT-1) in the retinal pigment epithelium (RPE) under conditions o f hyperglycemia, hyperhomocysteinemia and folate deficiency. A detailed description o f the retina, followed by information regarding folate and regulation o f RFT-1, is provided below.
    • Signaling Mechanism of Blood-Retinal Barrier Regulation: Role of Mitogen-Activated

      Yang, Jinling; Department of Cellular Biology and Anatomy (2011-03)
      Breakdown of the blood-retinal barrier (BRB) is an early hallmark of diabetic retinopathy. A critical component in retinal vascular hyper-permeability is increased production of vascular endothelial growth factor (VEGF). VEGF is a potent permeability factor that activates mitogen-activated protein (MAP) kinases. Pigment epithelium-derived factor (PEDF), an endogenous anti-permeability factor, blocks VEGF-induced vascular permeability increase. However, the mechanisms underlying the actions of VEGF and PEDF in regulating endothelial permeability are not yet clear. Previous studies in our laboratory have shown that VEGF induces paracellular permeability via beta-catenin nuclear translocation/transcriptional activation and subsequent upregulation of urokinase plasminogen activator receptor (uPAR). This current study tests the role of two MAP kinases, p38 and extracellular-signal regulated kinase (ERK), in regulating VEGFinduced beta-catenin signaling, uPAR expression and BRB breakdown. We also evaluate the effects of PEDF on this VEGF permeability inducing pathway. The role of MAP kinase in this VEGF permeability inducing pathway was first evaluated using inhibitors of p38 and ERK. These inhibitors preserve the endothelial barrier function upon VEGF treatment. In confluent endothelial cells, cytosolic beta-catenin is phosphorylated by glycogen synthase kinase (GSK) then ubiquitinated and degraded. With VEGF treatment, GSK is phosphorylated/inactive followed by beta-catenin cytosolic accumulation, nuclear translocation and subsequent uPAR expression. These effects were blocked by MAP kinases inhibitors. This indicates p38 and ERK as mediators of VEGF-induced beta-catenin signaling, uPAR expression and endothelial barrier breakdown. Next, it was found that PEDF not only blocks VEGF-induced endothelial permeability increase and MAP kinase activation but also prevents the activation of GSK/beta-catenin signaling as well as uPAR expression. However, PEDF did not block VEGF receptor-2 (VEGFR-2) phosphorylation suggesting that PEDF acts downstream of VEGFR-2 and upstream of MAP kinase level. To further evaluate the role of p38 in regulating VEGF-induced permeability, adenovirusmediated delivery of p38alpha mutants was used. One p38alpha mutant has an altered ATP-binding site thus looses its activity. It is more efficient in blocking VEGF-induced GSK/beta-catenin signaling, uPAR expression and paracellular permeability increase. This study identifies p38alpha and ERK as mediators of VEGF permeability-inducing signaling. They could also serve as potential therapeutic targets for diseases featured by blood-retinal barrier dysfunction.
    • Use of Sigma Receptor Ligands to Prevent Retinal Ganglion Cell Apoptosis Characteristic of Diabetic Retinopathy

      Martin, Pamela M; Department of Cellular Biology and Anatomy (2003-04)
      (First Paragraph)Diabetic retinopathy is a major sight-threatening disease and is the leading cause of blindness among working-aged Americans, affecting approximately 10 to 12 million persons (Wu, 1995). Although retinal vasculature is particularly vulnerable to damage in diabetes, other retinal cells are at risk. Very recently, Barber et al. (1998) documented increased apoptosis of neural retinal cells in experimental diabetes in rats and diabetes mellitus in humans. Notably, retinal ganglion cells (RGCs) were found to be at particular risk. Ganglion cell death in diabetic retinopathy is thought to be mediated via overstimulation o f N-methyl-D-aspartate (NMDA) receptors by glutamate. oRl is a nonopiate and nonphencyclidine-binding site that has numerous pharmacological and physiological functions. In some studies, agonists for aR l have been shown to afford neuroprotection against overstimulation of the NMDA receptor. The purpose of these studies was to evaluate the potential use of aR ligands, particularly those that bind specifically to o R l, as neuroprotective agents in the treatment of RGC apoptosis characteristic of diabetic retinopathy. A detailed description of the retina, followed by information about diabetes and the mechanisms thought to be involved in the pathogenesis of diabetic retinopathy, particularly the apoptotic death of RGCs associated with diabetic retinopathy, is provided below.