Novel Findings about the Role of Glial Cells in Retinal Function, Disease, and Therapy

Abstract

The retina is highly specialized and, due to its unique physiology, at the same time a very vulnerable neural tissue. Numerous mutations are known today that lead to photoreceptor degeneration, and the exact mechanisms causing cell death are still not completely understood. However, photoreceptor cell death is not a stand-alone event, but triggers secondary reactive changes in other neurons as well as non-neuronal cells. The key goal of this thesis was to elucidate some of the dynamics of reactive changes and how these affect the progression of photoreceptor cell loss in several models of human retinal degeneration. Up-regulation of the intermediate filament proteins, glial fibrilary acidic protein (GFAP) and vimentin, in astrocytes and Müller cells is a feature of gliotic stress responses in the retina. The inability to produce these filament proteins has been shown to alter the reactivity of glial cells to numerous insults. For the present study, mice deficient for GFAP and vimentin (GFAP-/-Vim-/- mice) were characterized with regards to their retinal function. The electroretinography measurements (ERG, experiments performed by N. Tanimoto) revealed alterations of the scotopic responses of post-photoreceptor neurons. While immunohistochemical and western blot analysis could show that the number of neurons and glia was comparable, the expression of glutamine synthetase (GS) and inwardly rectifying potassium (Kir) channels was markedly reduced in GFAP-/-Vim-/- and Vim-/-, but not GFAP-/- mice. The Kir4.1 channel was also observed to mislocalize along the Müller cells, which could possibly underlie the electrophysiological phenotype. Similar results were obtained from GFAP-/-Vim-/- mice on the rd1 (retinal degeneration 1) background. Levels of GS, Kir2.1, Kir4.1, and the water channel aquaporin 4 were lower in rd1 mice lacking GFAP and vimentin than in rd1 mice expressing these proteins. According to immunohistochemical results, no alteration of the cell death progression could be detected although the ERG analysis revealed a small improvement of retinal function. Müller cells up-regulate also neuroprotective substances upon activation. In chapter 4.3, the spatio-temporal expression changes of the putative neuroprotective metal-binding proteins metallothioneins were evaluated at RNA and protein levels in the rd1 and the rds (retinal degeneration slow) mouse models as well as in the RCS rat. During the course of the disease, the expression of metallothioneins was up-regulated in Müller cells and microglia. With the proximity ligation assay, a putative interaction with the endocytic receptor megalin that mediates the transport of metallothioneins into neurons could be visualized in situ in the inner and outer photoreceptor segments but was lost at later stages of the retinal degeneration. The rd1 and rds mouse models were also employed to evaluate the microglial reactivity during retinal degeneration. In association with photoreceptor cell death, microglia migrated into the outer nuclear layer and the subretinal space. The macrophage-restricted cell adhesion molecule sialoadhesin, which has been shown to support immunoregulating functions influencing T-cell behavior in a pro-inflammatory manner, had been reported to be present in activated microglia in the rds mouse retina. This fact could not be reproduced with the present study, nor were any sialoadhesin-positive cells found in the rd1 model. However, after intraocular transplantation of neonatal cells to wildtype and rd1 mice, sialoadhesinpositive cells were observed in association with the graft and in the subretinal space. These results add to the evidence that retinal glial cells are important both under physiological and pathological conditions.