Robert W. Nickells - US grants

DESCRIPTION (Adapted from applicant's abstract): Retinal ganglion cells die in a variety of optic nerve diseases, the most prevalent of which is glaucoma. Previous studies conducted by the investigator and others showed that ganglion cells died in experimental glaucoma and other models of optic nerve disease with characteristics of a form of programmed cell death known as apoptosis. This form of cell death is genetically controlled and it is likely that a better understanding of the genes that regulate this process in ganglion cells will lead to better treatments that can be used to block the death process. This proposal is aimed at determining the role of three genes in the regulation of ganglion cell death. These genes, p53, bcl-x, and bax, appear to form a molecular switch that acts as one of the early control steps in regulating apoptosis in a variety of cell types. Early work has shown that these genes are expressed in ganglion cells. One set of specific aims in this proposal is to determine, using a combined quantitative and localization study, if the expression of these genes in ganglion cells is altered in a fashion predicted by the molecular switch hypothesis (e.g., that p53 expression increases causing a decrease in bcl-x expression and an increase in bax expression). The remaining specific aims involve direct tests of the functions of these genes in the ganglion cell death process. These direct tests will be carried out on genetically altered transgenic mice that have either defective p53 or bax expression or overexpress bcl-x, The basic experimental design of these experiments is to stimulate ganglion cell death in mice using two different approaches (thus the functions of these genes can be tested in diverse pathways leading to ganglion cell death), which include a partial crush of the optic nerve and intravitreal injection of varying doses of the glutamate analog N-methyl-D-aspartate, followed by a quantitative analysis of the rate of cell death.

DESCRIPTION: (Applicant's Abstract) Retinal ganglion cells die in a variety of optic nerve diseases, the most prevalent of which is glaucoma. The mechanism of cell death is a genetically controlled program with 4 distinct stages. These stages are (i) early changes in gene expression that includes both the upregulation and downregulation of genes, (ii) the activation of key regulatory proteins such as p53 and BAX, (iii) dysfunction of mitochondria, and (iv) the activation of caspase proteases and nucleases. A goal in developing a neuroprotective strategy for ganglion cells is to block one or more of these stages of cell death. Current attempts to do this have shown that blocking late events (ii-iv) is not completely neuroprotective. As a consequence, we propose to characterize and block the early change in gene expression, specifically the downregulation. of genes in these cells. Preliminary studies indicate that ganglion cells exhibit extensive decreases in the expression of all normally active genes prior to the commitment to the cell death pathway. This includes genes that could enhance cell survival including Bc1X and the TrkB receptor genes. We will characterize further 3 classes of genes that may be affected by this silencing event and examine if histone deacetylation is the mechanism underlying the phenomenon. These latter experiments will be conducted by monitoring the acetylated status of histones on the ganglion cell marker gene Thy1, and by assessing the effects of inhibitors of histone deacetylases on Thy1 transcription and the activation of downstream events in the apoptotic program of ganglion cells.

Glaucoma is a prevalent blinding disease characterized by the progressive loss of retinal ganglion cells. Previously, we used Bax knockout mice to show that this proapoptotic gene was essential for ganglion cell death stimulated by optic nerve crush and in a mouse model of spontaneous glaucoma. During this work, we found that mice heterozygous for the Bax knock out allele have dramatically different phenotypes depending on their genetic background. In some lines, Bax+/- mice have a wild type phenotype, (all ganglion cells die), while in other lines, Bax+/- mice have a mutant phenotype, (all the cells survive). This proposal will extend our studies on the effects of Bax in retinal ganglion cells in an optic nerve crush model. In the first aim, we will examine the basis for the variable effects of the Bax+/- genotype in different lines. Preliminary studies indicate that two distinct lines (DBA/2J and 129B6, which have all-or-none phenotypes as Bax heterozygotes), exhibit different levels of Bax gene expression (high and low levels), consistent with their phenotype. This phenomenon will be studied further by analyzing the sequence and strength of the Bax promoters in each line. Axon loss precedes soma death in glaucoma. In the second Aim, we will assess if regeneration can be stimulated in cells arrested in the cell death pathway at the level of Bax. Regeneration will be stimulated in Bax+/+ and Bax-/- ganglion cells using established methods to determine if the Bax deficient cells have a greater capacity for new growth. These experiments are critical to show that blocking cell death is practical, since a live cell with no axon is still non-functional. To complete the strategy of targeting Bax, in the third Aim we will examine several reagents that are designed to reduce Bax expression and/or function in wild type ganglion cells. These reagents interfere with either BAX translation, the cellular translocation of BAX to mitochondria, or prevent the down stream effects of BAX. Ultimately, the ideal treatments of Aims 2 and 3 will be combined to produce a workable therapy that will block soma death and stimulate it to regenerate a functional axon. The findings of Aim 1 will also enhance our treatment of glaucoma by helping to identify the genetic basis of the variable susceptibility to optic nerve damage at the level of Bax gene function.

[unreadable] DESCRIPTION: Glaucoma can be considered a complex genetic disease. One of the striking features of glaucoma is the variable susceptibility to elevated intraocular pressure (IOP) that is present in the general population. Some individuals become ocular hypertensive without ever developing an optic neuropathy, while others develop severe disease with normal or low lOPs. When considering the genetic components of glaucoma, it is likely that allelic variation can affect both the ability of retinal ganglion cells to respond to an apoptotic stimulus and their efficiency of executing the cell death program. To test this possibility, we screened 15 lines of inbred mice to determine if their genetic background affected the loss of retinal ganglion cells after a standardized optic nerve crush procedure. This screen led to the identification of a resistant strain (DBA/2J) and susceptible strain (BALB/cByJ). F1 progeny of an intercross of these strains acquire the resistant phenotype. Analysis of the means and variance of the parental and F1 populations using the Wright formula suggests that DBA/2J animals contribute a dominant allele for resistance to the crush stimulus. We propose to conduct an analysis of this quantitative trait locus. We will generate a mapping population from F2 mice and perform linkage analysis using a selective genotyping approach with informative polymorphic markers that span the genome at -20 cM intervals. Simple linkage will be determined by calculating LOD scores using interval mapping and select regions of significance will be subject to fine mapping. The relevance of this study is that it will provide important information on the genetic mechanisms involved in the process of ganglion cell death in response to an apoptotic stimulus and may identify a critical allele/gene that affects susceptibility to IOP in humans. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glaucoma is a chronic blinding neurodegenerative disease characterized by the progressive loss of retinal ganglion cells and degeneration of the optic nerve. Although significant progress has been made in the field of the genetics of this disease, the majority of these studies have so far examined rarer forms of the disease that exhibit Mendelian inheritance patterns. The majority of glaucomas, however, are complex genetic diseases that have multiple interacting loci that affect an individual's susceptibility. One possible area of susceptibility is the genetically controlled cell death program that is executed by dying ganglion cells. We have used experimental genetics in mice to help identify potential susceptibility alleles that could affect this process. A screen of 15 inbred mouse lines for the amount of ganglion cell loss after optic nerve crush revealed 2 lines with varying resistance to this procedure. The resistant phenotype was found to be inherited as a dominant trait, and genome wide mapping has identified the responsible gene to be located within a 25 cM interval of chromosome 5 (Chr5.loc34-59). This locus has been named Retinal ganglion cell susceptible 1 (Rgcs1). We propose to extend these observations by continuing to fine map the Rgcs1 locus to narrow the region of interest. Fine mapping will begin using SNP analysis to refine the interval to a predicted 10 cM region, followed by the generation of Interval Specific Congenic Lines (ISCLs) to dissect the region into 1 cM overlapping intervals. In addition, we will use in silico mapping to help define candidate genes of interest in the interval as it becomes smaller. To date, we have used this approach to identify 7 potential candidates from among the 578 genes known to exist in this region. Candidate genes will be characterized by quantitative and qualitative analyses between the two parental inbred strains. During the course of generating ISCLs, we will also create a substrain of DBA/2J mice to examine the role that the Rgcs1 locus plays in susceptibility to glaucoma. Wild type DBA/2J mice carry the resistant allele. Interestingly, these mice also develop chronic inherited glaucoma, which may seem like a paradox, but could also be consistent with a true susceptibility allele. Our test of the role of Rgcs1 in glaucoma, will be to cross the susceptible Rgcs1 locus from BALB/cByJ animals onto the DBA/2J background to generate the substrain DBA/2J.BALBRgcs1. We expect these mice to develop elevated intraocular pressure, and by virtue of carrying a susceptible Rgcs1 allele, a more severe form of glaucoma. Lastly, as we acquire more information on the mouse Rgcs1 locus, we will begin to interrogate the syntenic region of the human genome by examining for SNP differences in key candidate genes using a data set of human glaucoma patients housed at the Center for Human Genetics at Duke University.

DESCRIPTION (provided by applicant): Glaucoma is a prevalent blinding disease characterized by the progressive loss of retinal ganglion cells. Previously, we used Bax knockout mice to show that this proapoptotic gene was essential for ganglion cell death stimulated by optic nerve crush and in a mouse model of spontaneous glaucoma. Further study also showed that early atrophy of ganglion cells occurred in Bax-deficient cells. This observation poses an important caveat to neuroprotective strategies; it is possible to block cell death while at the same time lose normal cell function. Several of the early atrophic events are linked to the activity of Histone Deacetylases (HDACs). In dying cells, HDAC3 translocates to the nucleus and appears to be critical for global histone deacetylation, nuclear atrophy, and cell death, but not ganglion cell-specific gene silencing. We are proposing a series of experiments to directly test the role of Hdac3 in these early events, using a combined genetic approach (conditional knock-out of Hdac3 in mouse ganglion cells) and selective HDAC inhibitors. These experiments will evaluate Hdac3 function in ganglion cell death in both acute and chronic (glaucoma) optic nerve damage paradigms. It is also important to explore the function of HDAC3 mechanistically, in precipitating cell death. This will be conducted in vitro using a novel approach of comparing the differential effects of exogenous HDAC3 on pre- and post-differentiated neurons. The atrophic event of gene silencing is also dependent on HDAC activity, but preliminary evidence suggests that this does not include HDAC3. The other prominent HDACs in the mouse retina are HDAC1 and HDAC2. We will use selective inhibitors of all three HDACs to help tease out the relative contributions of each in the silencing process. We hypothesize that the critical playe is HDAC2, and the inhibitor studies will be complemented using Hdac2 conditional knock-out mice to specifically interrogate the role that this specific gene plays. In addition, we will also extend these studies to monitor the contribution of a protein involved in chromatin remodeling (CBX5) and the recruitment of HDAC2 co- repressor complexes in the processes of ganglion cell atrophy and death. The ultimate objective of these studies is to determine if powerful HDAC inhibitors will someday be useful therapeutics to treat ganglion cell loss in glaucoma. Not only do they hold promise in preventing ganglion cell death, but they may act directly on the mechanism that cause dying ganglion cells to lose function long before committing to the cell death pathway.

CORE 1: VECTOR AND GENE DELIVERY/QUANTITATIVE MOLECULAR BIOLOGY ABSTRACT The goal of Core 1 is to provide the following services to users with qualifying grants and other vision researchers. The services include consultation on the use of gene delivery vectors, construction of plasmid and viral vectors, packaging and validating viral vectors, training individuals to construct and package viral vectors, providing consultation for quantitative PCR, providing access to qPCR equipment, carrying out qPCR reactions for users, providing qPCR data analysis, allelic characterization of normal and genetically modified mice, nucleic acid probe design, in situ hybridization, quantitative reporter gene analysis, and to facilitate use of campus resources such as the Biotechnology Center (NextGen Sequencing, data handling and analysis, proteomics, etc.).

BAX is a proapoptotic protein, which is critical for the execution of intrinsic apoptosis in retinal ganglion cells (RGCs) after optic nerve damage, suggesting that targeting BAX may be an effective therapeutic strategy for neuroprotection. It has been reported, however, that reducing BAX levels only provides a transient protection to axons in a mouse model of glaucoma. Whether or not this transient protection is sufficient to allow axons to recover after elimination of the original stressor, has not been tested. Aim 1 will test the protective effect of BAX reduction on the ability of axons to survive and/or recover in models that mimic the IOP-lowering therapy experienced by patients with ocular hypertension (OHT). Two models of inducible OHT in mice, microbead injection and steroid induced OHT, will be employed on both wild type and Bax+/- mice (where reduced BAX more likely mimics a human treatment). In both models IOP levels can be returned to normal after a desired interval. We will test if reducing BAX protein levels both reduces soma and axon pathology, and either protects axon function, or allows functional recovery. We will also examine the effects of controlled experimental IOP (CEI). The advantage of CEI is that it normalizes the IOP insult, which eliminates the confounding factors of variable IOP associated with other models of OHT. Dorsal Root Ganglion cells contain a BAX-dependent degenerative pathway that is activated early and before other more well-characterized catabolic reactions involving calcium influx. This pathway is activated by a BH3- only protein produced in the cell soma and then transported to the axon. A similar mechanism in RGC axons may explain the partial protective effect of BAX reduction. Aim 2 will evaluate the presence of this pathway in RGC axons where degeneration will be induced in optic nerves ex vivo. We will also interrogate axons for the presence and localization of key molecules that activate BAX, and molecules that act down-stream of BAX activation. Preliminary studies suggest that the BH3-only protein NOXA may be an important regulator of BAX activation in axons. We will explore this in detail using Noxa-deficient mice subjected to induced OHT. Protected RGCs silence transcription of genes required for function. New studies show that the committed process of dying in Bax-deficient cells, is shut down after 8 weeks. Thus cells can be classified into two categories, those actively dying (AD-mode) and those that have gone quiescent (Q-mode). Aim 3 will investigate if cells in Q-mode respond differently to 3 attempts to reactivate them, including using HDAC inhibitors to attenuate the transcriptional silencing of RGC-specific gene expression, the addition of Zymosan and CPT-cAMP to induce regeneration, or the up-regulation of transcription factors that can specify retinal precursor cells as RGCs. These experiments will be conducted on RGCs after acute damage to the optic nerve, which serves as a model where regeneration has a high priority for a therapeutic outcome.

PROJECT SUMMARY/ABSTRACT Technological advances are contributing to significant progress in different areas of basic research. However, such advances often exceed their clinical translation. The vision community is not exempt from this dilemma. Resolution of the gap requires an interdisciplinary approach to training vision scientists in a collaborative setting of scientists and clinicians that will ultimately advance medical care. We are re-submitting a Vision Research Training Program (VRTP) T32 grant to help achieve this objective. The VRTP is an interdisciplinary training program in the visual sciences that crosses traditional department and institute boundaries. The goal of the program is to provide research training in basic science disciplines relevant to vision research. The VRTP also supports interdisciplinary, collaborative and translational research so that trainees are prepared to enter and compete within emerging, interactive research environments. Currently, there is no vision-related T32 grant or equivalent support mechanism at UW-Madison. In fact, any training in vision research is now conducted in a disbursed fashion with trainees mentored by individual faculty and not provided a broader context and cross collaboration among faculty from multiple disciplines. If funded, the VRTP will provide exceptional scientific training as well as provide a structure for interactions with translational and basic scientists to meet our stated goal of understanding, preventing and treating diseases of the visual system. VRTP will act as a focal point for the vision community on campus in accomplishing these research and societal objectives. UW-Madison is an outstanding environment for the training of future vision scientists and provides the essential elements for pre- and post-doctoral training. These include a Department of Ophthalmology and Visual Sciences (DOVS) with equal exemplary prowess in research and clinical activities; the McPherson Eye Research Institute (MERI); a School of Medicine and Public Health (SMPH) with renown faculty and physical facilities; an equally renown Graduate School with Schools and Colleges containing numerous graduate programs and outstanding research mentors in the biological, engineering, and computational sciences; and a history of collegiality and collaborative research among faculty that allows students to develop into outstanding translational investigators. Sixteen faculty affiliated with DOVS and MERI currently comprise the VRTP. They are organized according to four areas of research emphasis: (a) Development and Diseases of the Anterior Segment, (b) Development and Diseases of the Posterior Segment; (c) Ocular Epidemiology and Genetics; (d) Higher Order Visual Processing. Support is being requested for 2 pre-doctoral trainees and 2 post-doctoral trainees. Pre-doctoral trainees will be supported by the training grant for 2-3 years and post-doctoral trainees for 1-3 years. Additional years of support will be obtained through several sources, including the Advisor's federal and non- federal funding, individual NRSA grants and SMPH and Graduate School resources. Training will consist of didactic and research components. Trainees will benefit from individual development plans as well as career planning and counseling. Significant institutional commitment to training will help ensure the success of the program, in addition to recruitment and retention plans to enhance diversity and plans for instruction in the responsible conduct of research.

CORE 1: VECTOR AND GENE DELIVERY/QUANTITATIVE MOLECULAR BIOLOGY ABSTRACT The goal of Core 1 is to provide the following services to users with qualifying grants and other vision researchers. The services include consultation on the use of gene delivery vectors, construction of plasmid and viral vectors, packaging and validating viral vectors, training individuals to construct and package viral vectors, providing consultation for quantitative PCR, providing access to qPCR equipment, carrying out qPCR reactions for users, providing qPCR data analysis, allelic characterization of normal and genetically modified mice, nucleic acid probe design, in situ hybridization, quantitative reporter gene analysis, and to facilitate use of campus resources such as the Biotechnology Center (NextGen Sequencing, data handling and analysis, proteomics, etc.).