Eldon E. Geisert - US grants

The goals of this proposed research are to define the effects of monocular deprivation on area 18, determine if area 18 is capable of recovery from the abnormalities caused by monocular deprivation, and demonstrate how the connectivity pattern between the lateral geniculate nucleus (LGN) and the visual cortex is altered by monocular deprivation. Area 18 is an integral part of the geniculo-cortical system and is especially interesting since it is dominated by Y-relay cell input(1,2). Little is known about the effects of monocular deprivation on area 18(3,4) in contrast to the LGN(5-11) and area 17(12-16). Thus, the effects of monocular deprivation in a major portion of the developing geniculo-cortical system remain ill-defined. Furthermore, in monocularly deprived (MD) cats when the non-deprived eye is removed and the deprived eye is opened (allowing for normal pattern vision), there is a complete recovery of the Y-cell population 6. These recovered Y-cells do not drive neurons in area 17(13,17). However, they may have access to cortical processing through area 18, a structure dominated by Y-cell input in normal cats. Four specific sets of experiments are designed to define the functional and structural changes occurring in the geniculo-cortical system following monocular deprivation and subsequent recovery. The proposed experiments will: 1) define the projections of the lateral geniculate nucleus to area 18, allowing for between group comparisons; 2) demonstrate the physiological effects of suturing one eyelid closed on single area 18 neurons; 3) determine if single neurons in area 18 of monocularly deprived cats are capable of recovery, defining the conditions necessary and sufficient for recovery to occur; and 4) determine if the projections of the lateral geniculate nucleus to areas 17 and 18 are altered by visual deprivation, using double label transport methods.

nervous system regeneration; antiserum;

DESCRIPTION (Adapted from the applicant's abstract): This application focuses on the role of CD81 in the cellular response of the retina to injury. This group was the first to demonstrate that CD81 is expressed in the retina and that there is a significant up-regulation of the gene product following injury. The hypotheses is that TAPA is part of the molecular complex mediating contact inhibition for glial cells and retinal pigment epithelium (RPE). This application will address four questions regarding CD81 and the proliferative response of retinal cells: (1) whether CD81 regulates glial cell and RPE number during normal retinal development; (2) whether CD81 plays a role following injury; (3) whether the activation of CD81 down-regulates the proliferative response of retinal glia; and (4) what glial proteins associate with CD81 to regulate cell proliferation in the retina.

Hepatitis C is a major health problem within the United States and the world. Recently, considerable progress was made in understanding the nature of this disease. Recently considerable progress was made by identifying CD81 as a putative ligand of the Hepatitis C virus. For the past several years, our laboratory has focused on the role of CD81 in the regulation of cell growth. The overall goal of the present proposal is to investigate the molecular associations of CD81 in the liver that may contribute to binding or entry of the hepatitis C virus. We hypothesize the CD81 is part of a molecular complex which varies between different body tissues. By defining components of this complex in the liver and comparing this to other tissues, we will begin to reveal specific molecular interactions critical to the virus interactions with CD81. The present proposal is designed to answer three questions related to hepatitis C infections. 1) What tissues and cells within the human body express CD81? 2) Is CD81 part of a molecular complex within the plane of t he membrane, and does the components of this complex change from tissue to tissue? 3) Is it possible to block or alter the interaction of hepatitis C with CD81 by altering cis interactions within the plane of the membrane or trans interactions with the E2 protein on the hepatitis C virus. This information will define the potential targets for Hepatitis C. By comparing the cells expressing CD81 and t he cells capable of the binding the virus, we will use subtractive methods to identify potential co- receptor molecules on the surface of the cell. This information can then be used to examine potential allelic variation in populations of patients that either spontaneously recover from hepatitis C infection or that are predestined to develop chronic infections. Once we identify genetic differences that result in alterations in alterations in the progression of hepatitis C infection or that are predest8ned to develop chronic infections. Once we identify genetic difference that result in alterations in the progression of hepatitis C infections, we may be able to develop treatment protocols to better treat this disease.

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): Our group is interested in the response of the retina to injury. Recently we expanded our approach looking at a large segment of the transcriptome using microarrays and bioinformatics. During this process a unique compelling opportunity focused our collective interest on a project looking at retinal axon damage (optic nerve crush) in the mouse as a model system. In collaboration with the Rob Williams' group we are using the BXD recombinant inbred (Rl) strains to define genetic networks in the eye and retina. These interactions have put us in a unique position to study the early signature of retinal injury and genomic loci that may underlie susceptibility or resistance to optic nerve damage. The long-term goal of this project is to define genetic networks controlling susceptibility and resistance of the retina to optic nerve damage and to characterize the early molecular signatures associated with these changes. Our working hypothesis is that the differential vulnerability of the retina is modulated by network of polymorphic genes that contribute to susceptibility or resistance to neurodegeneration and ganglion cell death. We will use a unique set of isogenic strains of mice (the BXD Rl strains) to investigate an experimental model of optic nerve damage. This large set of strains is uniquely suited to study the genetics of optic nerve damage because one of the parental strains is susceptible to injury (DBA/2J strain) whereas the other strain is resistant (C57BL/6 strain). This work exploits the unique properties of this particular strain panel in three specific aims. Aim 1 will define the normal patterns of transcriptional activity in the retinas of recombinant inbred strains. The transcriptional networks identified in these uninjured mice will serve as a background to define the changes occurring after optic nerve damage. Aim 2 will test the hypothesis that there are a series of transcriptome signatures in the retina of the BXD Rl strains that are predictive of susceptibility or resistance of ganglion cell death after axon injury. These data will define the common and unique genetic networks that are activated following injury to the optic nerve axons and will define the early changes associated with optic nerve damage. Finally we will be able to identify the genetic networks controlling susceptibility and resistance to ganglion cell death. [unreadable] [unreadable] [unreadable]

Glucocorticoids (GCs) are commonly used anti-inflammatory and immunosuppressive therapeutic agents for a plethora of diseases and conditions. Over 1% of our population receives GC prescriptions annually. Despite the very broad and potent anti-inflammatory effects, prolonged GC therapy can cause serious side effects, including damage to the eye. Between 30-75% of individuals receiving prolonged GC therapy develop GC- induced ocular hypertension (OHT), which if unrecognized can lead to iatrogenic open-angle glaucoma and permanent vision loss. Despite recognition of this significant GC side effect for more than six decades, we still do not understand the reason for differences in susceptibility to GC-induced OHT or the mechanism(s) of action responsible for GC-OHT. We have previously shown that the alternative spliced dominant negative isoform of the glucocorticoid receptor (GRb) inhibits GC activity in cultured human TM cells. TM cells isolated from glaucoma donor eyes (GTM) have low GRb levels and are therefore more sensitive to GCs. Although a number of studies have examined the DEX-induced transcriptome in TM cells and tissues, there is no indication which of the differentially expressed genes or molecular pathways are involved in GC-OHT. Several studies have shown that susceptibility to develop GC-OHT is genetically inherited, but no genes have been definitively linked to GC-OHT. Our overall hypothesis is that GC-OHT is: (a) determined by the ratio of endogenous GRa to GRb expression in the TM; (b) mediated by specific molecular pathways that can be differentiated from GC-responder and non-responder eyes; and (c) genetically determined so that GC-OHT genes can be mapped and identified. This overall hypothesis will be tested in 3 specific aims. Specific Aim #1: Determine the role of endogenous GRb in regulating GC-OHT in human anterior segment ex vivo perfusion culture and in vivo in mice. Specific Aim #2: Determine the TM transcriptome in GC-OHT resistant and sensitive strains of mice and in anterior segment perfusion cultured human eyes in order to identify the molecular pathways that are responsible for GC-OHT. Specific Aim #3: Map and identify the genes responsible for GC-OHT using QTL of the BXD recombinant inbred mouse lines. This research is innovative in that we will evaluate the role of endogenous GRb in mouse strains (with our new mouse model of GC-OHT) and in ex vivo perfusion cultured human anterior segments that differ in sensitivity to GC-OHT, use mouse strains and human perfusion cultured anterior segments that are differentially responsive to GC-OHT to molecularly dissect the pathway responsible for GC-OHT, and map GC-OHT genes using BXD recombinant inbred mice. This work is essential and significant because our experimental results will help determine the role of endogenous GRb in regulating responsiveness to GC-OHT, the molecular mechanisms responsible for GC-OHT, and the best and most effective way to predict steroid responders, which still is an important unmet clinical need as GC-OHT is becoming increasingly prevalent.

Summary The leading cause of blindness in Americans over the age of 60 is glaucoma. One risk factor for glaucoma is central corneal thickness. The thinner the cornea, the greater the risk of developing glaucoma. Recently, we have identified a transcription factor, POU6F2, that modulates central corneal thickness in the mouse and that is a risk factor for human glaucoma. The Pou6f2 knockout mouse has a thinner cornea than its wild-type littermates, while there is no apparent change in intraocular pressure. We propose that POU6F2 is expressed in novel subclasses of retinal ganglion cells (RGCs) that are sensitive to glaucomatous injury. Premise: Our four findings lead us to hypothesize that POU6F2 is in a transcriptional cascade responsible for the susceptibility and early death of a novel collection of ON-OFF directionally selective RGC subtypes. To test this hypothesis, we will focus on the role of POU6F2 in the response of RGCs in models of experimentally-induced and naturally- occurring glaucoma. The proposed experiments will uncover the POU6F2 molecular cascade that induces glaucomatous damage. We will relate our findings in the mouse to the human, through a collaboration with Dr. Janey Wiggs and the NEIGHBORHOOD consortium, interrogating the NEIGHBORHOOD meta-dataset to define molecular pathway associated with POU6F2 targets. We will determine if any of these downstream targets and their associated pathways represent factors for glaucoma risk. This basic understanding of the molecular interactions of POU6F2 will inform the rational design of strategies to improve detection of and therapy for glaucoma.