Tatjana C Jakobs - US grants

DESCRIPTION (provided by applicant): The ganglion cell degeneration observed in glaucoma (in humans and mice) is not spread diffusely over the whole retina but occurs in sectors that point towards the optic nerve head. The obvious localization for a first insult to the axons that would cause this geometry is the portion of the optic nerve head that lies directly behind the sclera, because there the axons are organized into bundles with a topographic relationship to the retina. It is possible that individual axon bundles are separately damaged or spared, leading to sectors of degenerated or normal ganglion cells in the same retina. In humans, the optic nerve head contains a rigid, collagenous lamina cribrosa, lined by astrocytes. In contrast, the murine optic nerve contains only a meshwork of astrocyes (the "glial lamina"), which is disorganized in glaucomatous nerves. Because astrocytes are not rigid, this suggests that the damage to axon bundles may not be mechanical. Instead it raises the possibility that astrocytes of the glial lamina may be directly involved in the pathophysiology of glaucoma. We propose four studies on the single-cell level of the cell biology of glial lamina astrocytes using a mouse that expresses GFP in sporadic, individual astrocytes. (1) We will describe the normal astrocytic architecture of the glial lamina. (2) We will introduce global optic nerve lesions (optic nerve crush) or focal lesions of ganglion cell axons on the retinal surface (by laser) to study the behavior of individual astrocytes under pathological conditions. (3) We will observe the consequences of these axonal lesions in transgenic mice whose astrocytes are compromised by knock-out of intermediate filaments or connexin43. (4) We will observe the behavior of GFP-labeled astrocyes in a new strain of GFP-DBA/2J glaucomatous mice as a realistic, slow model of glaucoma. PUBLIC HEALTH RELEVANCE: Glaucoma leads to a progressive and irreversible loss of retinal ganglion cells, whose axons form the optic nerve, and thereby severs the connection of an otherwise functional retina with the brain. Recent experimental evidence suggests that a non-neuronal cell type (astrocytes) in the optic nerve might play an active role in the disease. Our goal is to study the reaction of individual astrocytes to injury and glaucomatous degeneration in more detail than has been possible before.

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, TGF?-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?.

Project Summary Glaucoma leads to a progressive loss of retinal ganglion cells by mechanisms that are not fully understood. At present, lowering the intraocular pressure (IOP) is the only treatment, and new therapeutic approaches that prevent ganglion cell degeneration in a manner independent of IOP would be welcome. There is evidence that the first signs of ganglion cell degeneration occur in the optic nerve head where the ganglion cell axons exit the globe through a hole in the sclera to form the optic nerve. In this region, the axons are unmyelinated and come into direct contact with astrocytes. Optic nerve astrocytes react to injury ? such as an increase in IOP - with changes in their morphology and gene expression pattern. We showed that, at least in the early stages of the disease, astrocyte reactivity is a protective response and preventing it leads to a worse outcome for ganglion cells and visual function. This result suggests that astrocytes, or astrocyte-derived factors, can be harnessed for a neuroprotective glaucoma therapy that could be added to IOP-lowering drugs that are already in use. In our search for astrocyte-derived factors that mediate the protective response, we identified secreted phosphoprotein 1 (SPP1, also called osteopontin). This protein is expressed only at low levels in the normal optic nerve, but it is robustly up-regulated in all rodent glaucoma models studied so far. In addition, SPP1 is constitutively expressed in a sparse population of retinal ganglion cells. Using an SPP1 knock out mouse, we showed that SPP1 deficiency leads to morphological signs of astrocyte and axon damage in the optic nerve head even in the absence of elevated IOP. In the microbead occlusion model of glaucoma, SPP1 deficient mice lose more ganglion cells and have worse visual function than wild-type controls. Most importantly, virus- mediated overexpression of SPP1 in the retina is highly protective of ganglion cell function and prevents ganglion cell loss without affecting IOP. Based on these findings, we believe that SPP1 may be a promising candidate for a neuroprotective therapy in glaucoma. However, at present we do not know whether the SPP1 expression in reactive astrocytes or in retinal ganglion cells or both are needed to achieve optimal protection. To address this question, we have designed a transgenic mouse strain that will allow for cell-type specific deletion of SPP1 in astrocytes, retinal ganglion cells (or other cell types) separately. The mice will also express different fluorescent marker proteins in targeted cells before and after the deletion of SPP1. We will use this new tool to address our hypothesis that astrocyte-derived SPP1 in the optic nerve and ganglion cell-derived SPP1 in the retina are both necessary for optimal ganglion cell protection in glaucoma (Aims 1 and 2). In our translational Aim 3, we will test whether overexpression of SPP1 is protective of retinal ganglion cell function in the long term and assess ocular tissues for adverse effects that may result from chronic SPP1 overexpression.

SUMMARY Vascular dysfunction, with or without elevated intraocular pressure (IOP), is believed to be an important risk factor in glaucoma and other neuropathies. However, the link between vascular dysfunction and the mechanisms leading to the characteristic visual field defects in glaucoma is not fully understood. This is partly due to the lack of a solid quantitative understanding of the 3D architecture of the vasculature of the optic nerve head (ONH), its anatomical relationship with the load-bearing connective tissues, and how it is affected by IOP. Our overarching hypothesis is that features of the vasculature and its relationship with the connective tissues predispose certain ONH regions to compromised perfusion and that this susceptibility is amplified by elevated IOP. To test this hypothesis, we will sequentially collect in vivo, ex vivo, and histological 3D morphological and biomechanical data on vascular and connective tissues of the ONH in normal eyes and in eyes with experimental glaucoma (EG). We will focus on the critical lamina cribrosa (LC) region in three species: human, monkey (closest model to human, collagenous LC), and mouse (most used model, no collagenous LC). In Aim 1, we will map in 3D the vasculature and connective tissues of the ONHs of humans, monkeys, and mice, and analyze these maps quantitatively including by watershed analysis. We predict that zones of visual loss in early glaucoma will correspond to regions with the most vulnerable vascular supply, e.g., sparse capillaries with low connectivity and low perfusion redundancy. We postulate that, in primates, not all LC beams have a capillary, and conversely, that some capillaries are not within a robust collagen-rich beam. We will also address the clinically important question to which extent in vivo OCT angiography visualizes the smaller or deeper vessels inside the ONH. In Aim 2, we will perform ex vivo inflation tests on monkey and mouse eyes to quantify the effects of acute IOP elevation on vessel perfusion and biomechanics, and the LC beams support. Our preliminary data suggests that ?unprotected? vessels may be particularly vulnerable to mechanical distortion, which could, in turn, affect blood flow. In Aim 3, we will characterize the effects of chronic IOP elevation on vessels and beams. Specifically, we will compare eyes before and after chronic IOP elevation (EG), and with the contralateral control. This will allow us to discern characteristics that pre-dispose an eye to glaucoma from those that are the result of the disease. We will test the hypothesis that the patterns of vessel sensitivity to elevated IOP in mice (that have only a glial lamina) are different from those in primates. Combining multiple imaging modalities across the same ONHs in three species will provide cross-verification of the techniques, and deeper insights into the role of LC collagenous beams supporting the ONH vasculature under normal and elevated IOP. These experiments will help identifying ONH features that predict susceptibility to neural injury and vision loss.