Funding

Federal Funding

TITLE: A novel βA3/A1-crystallin gene mutation results in persistent fetal vasculature.

AGENCY: NATIONAL EYE INSTITUTE, NIH

STATUS: Active

ABSTRACT:Persistent fetal vasculature (PFV) is a human disease that results from a failure of the fetal vasculature to regress. It is a common congenital developmental disorder of the eye found in an otherwise normal child. The underlying cause of PFV disease is not well understood. We previously described a naturally occurring mutation (Nuc1) in the Sprague-Dawley rat with a novel eye phenotype involving cataract, retention of fetal vasculature, and developmental abnormalities in the retina. In Nuc1 there is failure of regression of the entire fetal intraocular vasculature and not just part of it, as reported in several other mouse models of PFV. We recently reported that the mutation causing Nuc1 is a 27 base pair insertion in exon 6 of the βA3/A1-crystallin gene on rat chromosome 10. The 27 base pair insertion is composed of near-perfect tandem repeats of a 7 base pair sequence, TGACTAT. This in-frame insertion results in the loss of a universally conserved glycine residue in exon 6 and its replacement with 10 new amino acids. We demonstrated that in the neural retina, βA3/A1-crystallin is expressed only in the astrocytes. In the Nuc1 rat, astrocytes surround the retained vessels as is found in other animal models of PFV. However, astrocytes are not associated with the hyaloid vasculature in the normal eye. While βA3/A1-crystallin is entirely cytoplasmic in lens, the protein is largely nuclear in astrocytes. Both lens fibers and astrocytes from Nuc1 homozygotes show striking structural abnormalities with profound effects on the expression and organization of intermediate filaments (IFs). We have demonstrated that both the astrocytes associated with the fetal vasculature and the lenses in Nuc1 rats and PFV patients express increased amounts of VEGF. Based on these findings, we hypothesize that mutation of βA3/A1- crystallin causes abnormal association of astrocytes with the hyaloid artery, which inhibits regression of the fetal vasculature. To test this hypothesis, the following specific aims are proposed: AIM1: To characterize and compare βA3/A1-crystallin expression in wildtype and in Nuc1 homozygous rats during lens fiber cell and astrocyte development. AIM2: To investigate if altered motility of Nuc1 homozygous astrocytes during development contributes to the abnormal association between astrocytes and the hyaloid vasculature. AIM3: To determine if VEGF produced by astrocytes expressing mutant βA3/A1-crystallin mediates the survival and stabilization of the hyaloid vasculature in the Nuc1 rat. We believe that the proposed studies should provide new insights into the cellular and molecular interactions that regulate hyaloid vascular regression. The possibility that βA3/A1-crystallin may have a role in hyaloid vascular regression is important; it may help elucidate mechanisms underlying PFV that would have potential clinical implications.

COMPETETIVE SUPPLEMENTAL REVISION: Active

ABSTRACT: We previously described a naturally occurring mutation (Nuc1) in the Sprague-Dawley rat with a novel eye phenotype involving cataract, retention of fetal vasculature, and developmental abnormalities in the retina. We have recently reported that the Nuc1 phenotype in which the development of both the lens and retina is abnormal results from mutation of the βA3/A1-crystallin gene. βA3/A1-crystallin is expressed in lens fiber cells; our recent studies show that in neural retina it is expressed only in astrocytes. Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress. It is a common congenital developmental disorder of the eye found in an otherwise normal child. The underlying cause of PFV disease is not well understood. The Nuc1 spontaneous mutant rat is the only model that accurately resembles all the clinical symptoms of human PFV disease. In the present competitive supplement, we propose to further characterize our recently established βA3/A1-crystallin transgenic mouse lines and to generate conditional knockout mice where βA3/A1- crystallin will be selectively deleted in astrocytes, or the lens. These genetically engineered mouse lines will provide additional tools for gain and loss-of-function studies and will generate more definitive data on the effects of eliminating or modulating the expression of βA3/A1-crystallin, in the etiology of PFV disease. The studies proposed in this revision application are based on progress made to date under the parent grant and are specifically designed to augment the experiments outlined in the Specific Aims of that grant. Our working hypothesis is that “mutation of βA3/A1- crystallin causes abnormal association of astrocytes with the hyaloid artery, which inhibits regression of the fetal vasculature”. To test this hypothesis, the following specific aims were proposed in the parent grant: SPECIFIC AIM 1: To characterize and compare βA3/A1-crystallin expression in wildtype and in Nuc1 homozygous rats during lens fiber cell and astrocyte development. SPECIFIC AIM 2: To investigate if altered motility of Nuc1 homozygous astrocytes during development contributes to the abnormal association between astrocytes and the hyaloid vasculature. SPECIFIC AIM 3: To determine if VEGF produced by astrocytes expressing mutant βA3/A1-crystallin mediates the survival and stabilization of the hyaloid vasculature in the Nuc1 rat. We believe that the proposed studies should provide new insights into the cellular and molecular interactions that regulate hyaloid vascular regression. The possibility that βA3/A1-crystallin may have a role in hyaloid vascular regression is important; it may help elucidate mechanisms underlying PFV that would have potential clinical implications.

TITLE: A CRYSTALLIN MUTATION WITH ABNORMAL ASTROCYTES AND RETINAL VESSELS.

AGENCY: NATIONAL EYE INSTITUTE, NIH

STATUS: Active

ABSTRACT: The goal of this grant is to investigate the role of astrocytes in retinal vascular remodeling, in general, and the regulation of the remodeling process by βA3/A1-crystallin, in particular. This is an important area of research because a clear gap exists in the literature regarding our knowledge of cellular and molecular interactions among neurons, astrocytes and endothelial cells. We have discovered a spontaneous mutation in the Sprague Dawley rat with an unusual eye phenotype that we have named Nuc1. The mutation causing Nuc1 is a 27 base pair insertion in exon 6 of the βA3/A1-crystallin gene on rat chromosome 10. We have found that in the retina, βA3/A1-crystallin is expressed only in the astrocytes and that in the Nuc1 homozygotes, the astrocytes are structurally and morphologically abnormal. During early post-natal development these rats also have a much thicker retina than normal with an excess number of vessels. As the Nuc1 homozygote rat matures, we find evidence of microaneurysm formation, intravascular deposits and leakage of blood vessels. Our studies will utilize the Nuc1 rat and βA3/A1-crystallin loss-of-function and gain-of-function genetically engineered mice to determine the function of βA3/A1-crystallin in astrocytes and to understand how loss of normal βA3/A1-crystallin leads to failure of the retinal vasculature to form properly. It is now accepted that astrocytes play a major role in the establishment of a functional retinal vasculature, however, the cellular and molecular mechanisms involved in this process remain elusive. To test our hypothesis, that “astrocytes require βA3/A1-crystallin to properly direct the development and remodeling of the retinal vasculature”, the following specific aims are proposed: AIM1: To characterize the structure, sub-cellular localization, and protein interactions of βA3/A1-crystallin protein in developing astrocytes. AIM2: To investigate the role of βA3/A1-crystallin in the proliferation and migration of astrocytes. AIM3: To demonstrate the effect of astrocytes expressing mutant βA3/A1-crystallin on retinal vasculature. Despite rapid progress made in understanding the development of the retinal vasculature, many questions remain to be answered about the mechanisms and signaling pathways regulating this complex process. We believe that the proposed studies should provide new insights into this process. Our studies on the functions of the βA3/A1-crystallin protein are highly likely to provide mechanistic insights into cellular regulatory activities associated with vascular development in the eye. In addition, the findings should also provide clues to understanding vascular diseases of the retina and central nervous system.

TITLE: Genetic analysis of a spontaneous mutation in a rat with a novel hind limb defect.

AGENCY: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT, NIH.

STATUS: Active

ABSTRACT: We describe here a novel rat mutant that arose spontaneously from a mating of two Sprague Dawley rats in a colony that we maintain to study another mutation, Nuc1. Nuc1 is an eye-specific phenotype with developmental abnormalities that we have been investigating for several years. Hind limbs of the new mutant animals are extended and abducted so severely that they do not effectively support the animal's weight. Because of this unusual appearance and gait anomaly, we have named the mutant strain frogleg. The brain of a frogleg rat is smaller in size and weight relative to normal littermates; however body weight, which is typically lower in the affected newborn rats, does catch up to normal after several months. Histological analysis of frogleg sciatic nerve revealed some abnormal changes in Schwann cells. Neurometric studies also showed that sciatic compound muscle action potential is significantly reduced in frogleg rats. Attempts to breed frogleg animals with each other or with phenotypically normal males and females were unsuccessful. Carrying the line was complicated by the absence of a heterozygote phenotype. We were able to perpetuate the frogleg line by breeding unaffected littermates of frogleg rats in random combination until specific matings produced frogleg progeny, thereby identifying the parents as heterozygotes. We have established that the condition is inherited in a Mendelian fashion as a single autosomal recessive trait, and have used genetic linkage analysis to map the gene to rat chromosome 1q36-37. This disease interval has been reduced to a chromosomal region of 2.7 Mbp. Based on the Reference Sequence annotation database for the most current rat genome assembly, there are 8 potential frogleg gene candidates. However, augmentation of this number by alignment with syntenic portions of the human and mouse genomes increases the total number of genes in the interval to approximately 19. In the current application, we seek to identify the gene and the specific mutation responsible for the frogleg phenotype. In addition we will better characterize the neurological abnormalities of frogleg rats. Knowing the gene's identity and the nature of the mutation will help us to further understand the molecular and cellular mechanisms responsible for this interesting phenotype. Our working hypothesis is that identification of the mutation and the gene responsible for the frogleg phenotype will provide a novel model for studying cellular and molecular mechanisms of neurodevelopmental disorders. Therefore, we propose the following aims: SPECIFIC AIM 1: To refine the linkage map of the frogleg locus. SPECIFIC AIM 2: To identify the frogleg gene and mutation. SPECIFIC AIM 3: To characterize the pathology that underlies the phenotype of the frogleg rat. The identification of the gene responsible for frogleg would be a major step toward defining the potential value of the model for the study of mechanisms involved in neurodevelopmental disorders.

TITLE: Modulation of CIITA by Cannabinoids in Human Microglia.

AGENCY: NATIONAL INSTITUTE OF DRUG ABUSE, NIH.

STATUS: Completed.

ABSTRACT: Cannabinoids are established as being immunomodulatory, however, the involvement of the cannabinoid receptors in specific aspects of immune function has been demonstrated in few studies. Many effects of cannabinoids are mediated by two G-protein coupled receptors, designated CB1 & CB2. Most modern studies on immunomodulatory effects of cannabinoids have been focused on CB2 receptor, since it is primarily expressed on immune cells. We have shown that CB1 receptor is expressed in microglia that also express MHC class II antigen. The regulation of class II MHC genes occur primarily at the transcriptional level, and a non-DNA-binding protein, class II transactivator (CIITA), has been shown to be the master activator for class II transcription. Our recent data indicate that cannabinoids affect MHC class II expression through actions on CIITA at the transcriptional level. The CIITA gene is known to be controlled by multiple promoters. Interferon (IFN)- γ -inducible expression of CIITA is primarily regulated by promoter IV which contains three binding sites, an IFN-γ activation sequence, an E Box and an IFN regulatory factor site that bind the transcription factors STAT-lα, USF-1 and IRF-1 & IRF-2 respectively. Synergistic activation of the promoter by STAT-1, USF-1 and IRF-1 and IRF-2 leads to expression of CIITA, which then activates transcription of class II MHC genes. The present proposal will elucidate the mechanism by which the cannabinoids mediate the expression of CIITA in human microglia. To address our goal, we will isolate and study primary cultures of microglial cells from human brain. Cells activated with IFN-γ will be exposed to cannabinoid agonists and antagonists to determine the effect of cannabinoid receptor activation on stimulations of CIITA mRNA and protein levels. The specific aims of the proposal are 1) Determine whether cannabinoid effects on CIITA are receptor mediated and 2) Determine whether the binding of specific transcription factors to CIITA promoter is affected by cannabinoids. Data obtained from our proposed pilot study will provide valuable information for our long-range plan to define the functional significance of cannabinoid receptors in microglia. Microglia forms the first line of defense during infection in the CNS and is involved in various immune diseases including AIDS.

Non-Federal Funding

  1. RESEARCH TO PREVENT BLINDNESS (the unrestricted grant to the Wilmer Eye Institute)
  2. POOLED PROFESSORS FUND, The Wilmer Eye Institute
  3. THE KNIGHTS TEMPLAR EYE FOUNDATION
  4. HELENA RUBINSTEIN FOUNDATION
  5. NATIONAL FISHERIES INSTITUTE
  6. WILLIAM AND ELLA OWENS MEDICAL RESEARCH FOUNDATION
  7. MORTON F. GOLDBERG, M.D. DIECTOR’S DISCOVERY FUND
  8. THE RAYMOND KWOK RESEARCH FUND