Background: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the βGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice.

Retinal ganglion cell somas and nuclei undergo the AVD in response to optic nerve damage. Atrophy is rapid and precedes the Bax-dependent committed step of the intrinsic apoptotic pathway.

Angiogenesis contributes to pathogenesis of many diseases including exudative age-related macular degeneration (AMD). It is normally kept in check by a tightly balanced production of pro- and anti-angiogenic factors. The up-regulation of the proangiogenic factor, vascular endothelial growth factor, is intimately linked to the pathogenesis of exudative AMD, and its antagonism has been effectively targeted for treatment. However, very little is known about potential changes in expression of antiangiogenic factors and the role they play in choroidal vascular homeostasis and neovascularization associated with AMD. Here we will discuss the important role of thrombospondins and pigment epithelium derived factor, two major endogenous inhibitors of angiogenesis, in retinal and choroidal vascular homeostasis and their potential alterations during AMD and choroidal neovascularization. We will review the cell autonomous function of these proteins in retinal and choroidal vascular cells. We will also discuss the potential targeting of these molecules and use of their mimetic peptides for therapeutic development for exudative AMD.

Background: Intrinsic apoptosis of neuronal somas is one aspect of neurodegenerative diseases that can be influenced by genetic background. Genes that affect this process may act as susceptibility alleles that contribute to the complex genetic nature of these diseases. Retinal ganglion cell death is a defining feature of the chronic and genetically complex neurodegenerative disease glaucoma. Previous studies using an optic nerve crush procedure in inbred mice, showed that ganglion cell resistance to crush was affected by the Mendelian-dominant inheritance of 1-2 predicted loci. To assess this further, we bred and phenotyped a large population of F2 mice derived from a resistant inbred strain (DBA/2J) and a susceptible strain (BALB/cByJ).

ROSA3 mice were developed by retroviral insertion of the ␤Geo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of ␤Geo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for ␤Geo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the ␤Geo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1c R3/R3 ) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1c R3/R3 mice may still be able to express a putative FEM1C protein. To this extent, Fem1c R3/R3 mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of ␤Geo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the ␤Geo reporter gene a potentially useful marker to study early molecular events in damaged neurons.Promoter trap experiments using the reverse orientation splice acceptor (ROSA) ␤Geo retroviral gene trap vector (9) have been useful in identifying genes involved in mouse development (4, 5). The coding region for the trap vector contains bacterial ␤-galactosidase fused in frame to neomycin phosphotransferase (Neo) to produce a fusion protein (␤Geo) that processes substrates like X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) and neomycin. Transcription of ␤Geo is dependent on the trap vector being inserted into the vicinity of an existing gene in the mouse genome, where the presence of a 5Ј splice acceptor facilitates the splicing of the ␤Geo coding region into the nearest upstream exon. The ROSA3 line of mice was initially part of 31 lines established using retroviral insertion of the ␤Geo vector into embryonic stem cells (9). The insertion of the ␤Geo gene caused embryonic recessive lethality in some of the lines. ROSA3 mice showed no overt defects, however, and exhibited a widespread pattern of staining by embryonic day 12.Here, we report the further characterization of the ROSA3 line of mice. As with the results seen with the embryos, ROSA3 mouse adult tissues show widespread staining (including selective staining of certain populations of neurons in the central nervous system) in ␤-galactosidase assays. Analysis of the genomic insertion site shows that the ␤Geo gene has been inserted into the first intron of Fem1c, the third member of a murine gene family that shares homolo...

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.