Cell Biology of Astrocytes in Optic Nerve Head

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Project summary The characteristic feature of glaucoma is a progressive loss of retinal ganglion cells. Drugs that lower the intraocular pressure are the mainstay of pharmaceutical therapy of glaucoma, but they are not effective in all cases. New therapeutic approaches would therefore be welcome. Much evidence points to the optic nerve head as the point of initial insult to ganglion cell axons in glaucoma. In this anatomical location, a meshwork of astrocytes forms the direct cellular environment of the axons. Following an insult to the optic nerve, the astrocytes in the optic nerve head become reactive. We have studied the time course of astrocyte reactivity in the optic nerve head after nerve crush morphologically and on the level of gene expression. We also studied astrocyte morphology in the DBA/2J mouse line that develops glaucoma spontaneously. DBA/2J mice were crossed with a strain that expresses GFP in individual astrocytes, thus making the microscopic observation of reactive astrocytes easy. One of our findings was that, before any signs of ganglion cell degeneration become obvious in the retina, some astrocytes grow new, longitudinal processes into the retrolaminar axon bundles. We are now going to study the mechanisms that drive the growth of these processes and their function. We believe that astrocyte reactivity, at least in its early phase, is a beneficial response that aims to protect ganglion cells and their axons. A possible therapeutic approach to glaucoma would be to enhance the early astrocytic response. We therefore propose to study the regulatory mechanisms in the optic nerve that govern early astrocyte reactivity. We compared the genes that are differentially regulated in our own microarray screen (using optic nerve crush) with those that were reported in recent studies from other laboratories using DBA/2J mice or the episcleral vein injection model of ocular hypertension in rats. We identified signaling molecules and transcription factors that were up-regulated early in DBA/2J mice and after nerve crush and therefore appear to be involved in regulating astrocyte reactivity both in glaucoma and after traumatic injury. Most of these genes are also up-regulated in the episcleral vein injection model. Our candidates are the signaling molecules Bone Morphogenetic Proteins 1 and 2, Leukemia Inhibitory Factor), Secreted Phosphoprotein 1 (osteopontin), Lipocalin 2, and Transforming Growth Factor beta 1; and the transcription factors Tcf19, TGFβ-Induced Factor Homeobox 1, Runt- Related Transcription factors 1 and 2, and E2f8. We will study their involvement in three models of glaucoma, and after optic nerve crush. For this purpose, we will make use of methodological advances during the first grant period, namely the efficient transfection of optic nerve head astrocytes by AAV2/9, the ability to analyze dissociated astrocytes with well-preserved morphology from the optic nerve head, and a mouse strain that expresses GFP in astrocytes in the manner of a “live Golgi stain”.