Early Involvement of Immune/Inflammatory Response Genes in Retinal Degeneration in DBA/2J Mice.

PURPOSE: The DBA/2J (D2) mouse carries mutations in two of its genes, Tyrp1 and Gpnmb. These alterations result in the development of an immune response in the iris, leading to iris atrophy and pigment dispersion. The development of elevated intraocular pressure (IOP) in this model of glaucoma is considered to be a significant factor leading to the death of retinal ganglion cells (RGCs). Changes in gene expression in the retina have already been correlated with the appearance of elevated IOP in the D2 mouse. The purpose of the present study was to determine if any changes in gene expression occur prior to the development of IOP. METHODS: The IOP was measured monthly using a rebound tonometer in D2 and age-matched C57/BL6 (B6) mice (normal controls). D2 animals with normal IOP at 2 and 4 M were used. In addition, mice at the age of 6-7 M were included to look for any trends in gene expression that might develop during the progression of the disease. Separate RNA samples were prepared from each of three individual retinas for each age, and gene expression profiles were determined with the aid of mouse oligonucleotide arrays (Agilent). A subset of genes was examined with the aid of real-time PCR. Immunocytochemistry was used to visualize changes in the retina for some of the gene-products. RESULTS: Four hundred and thirteen oligonucleotide probes were differentially expressed in the retinas of 4 M versus 2 M old D2 mice. The most significantly up-regulated genes (181) were associated with immune responses including interferon signaling, the complement system and the antigen presentation pathway, whereas the down-regulated genes (232) were linked to pathways related to cell death and known neurological diseases/disorders. These particular changes were not revealed in the age-matched B6 mice. By 6 M, when IOP started to increase in many of the D2 mice, more robust changes of these same genes were observed. Changes in the levels of selected genes, representative of different functions/pathways, were validated with RT-PCR, and changes in glial responses were visualized in the retina with immunocytochemistry. CONCLUSIONS: The results showed that the expression of genes related to the immune response and acute stress were altered independently of the development of elevated IOP, and indicated early involvement of the immune system in the onset of the disease. The later development of elevated IOP, observed in this animal model, was coincident with continued changes in expression of genes observed at earlier time points. Further studies are warranted to identify the roles of specific genes identified here with respect to the death of the RGCs.

Differential distribution of exchange proteins directly activated by cyclic AMP within the adult rat retina.

The recently discovered exchange protein directly activated by cAMP (Epac), a guanine exchange factor for the G-protein RAP-1, is directly activated by cAMP independently of protein kinase A (PKA). While cAMP is known to be an important second messenger in the retina, the presence of Epac has not been investigated in this tissue. The goal of the present study was to determine if the Epac1 and Epac2 genes are present and to characterize their location within the retina. Western blot analysis revealed that Epac1 and Epac2 proteins are expressed within the retina, and the presence of mRNA was demonstrated with the aid of reverse transcriptase polymerase chain reaction (RT-PCR). Additionally, we used immunofluorescence and confocal microscopy to demonstrate that Epac1 and Epac2 have overlapping as well as unique distributions within the retina. Both are present within horizontal cells, rod and cone bipolar cells, cholinergic amacrine cells, retrograde labeled retinal ganglion cells, and Müller cells. Uniquely, Epac2 was expressed by cone photoreceptor inner and outer segments, cell bodies, and synaptic terminals. In contrast, Epac1 was expressed in vesicular glutamate transporter 1 (VGlut1) and C-terminal binding protein 2 (CtBP2) positive photoreceptor synaptic terminals. Together, these results provide evidence that Epac1 and Epac2 are differentially expressed within the retina and provide the framework for further functional studies of cAMP pathways within the retina.

The novel distribution of phosphodiesterase-4 subtypes within the rat retina.

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. While much is known about the function of cyclic GMP-specific PDEs in the retina, much less is known about the closely related, cyclic AMP-specific PDEs. The purpose of the present study is to characterize and localize PDE4 within the adult rat retina. We have used Western blotting, RT-PCR, and immunohistochemistry together with retrograde labeling to determine the presence and location of each PDE4 subtype. Western blot analysis revealed that multiple isoforms of PDE4A, B, and D subtypes are present within the retina, whereas the PDE4C subtype was absent. These data were confirmed by RT-PCR. Using immunohistochemistry we show that all three PDE4s are abundantly expressed within the retina where they all colocalize with retrograde-labeled retinal ganglion cells, as well as bipolar cells, horizontal cells, and cholinergic amacrine cells, whereas Müller cells lack PDE4 expression. Uniquely, PDE4B was expressed by the inner and outer segments of rod photoreceptors as well as their terminals within the outer plexiform layer. Collectively, our results demonstrate that PDE4s are abundantly expressed throughout the rodent retina and this study provides the framework for further functional studies.

The relationship between neurotrophic factors and CaMKII in the death and survival of retinal ganglion cells.

The scientific discourse relating to the causes and treatments for glaucoma are becoming reflective of the need to protect and preserve retinal neurons from degenerative changes, which result from the injurious environment associated with this disease. Knowledge, in particular, of the signal transduction pathways which affect death and survival of the retinal ganglion cells is critical to this discourse and to the development of a suitable neurotherapeutic strategy for this disease. The goal of this chapter is to review what is known of the chief suspects involved in initiating the cell death/survival pathways in these cells, and what still remains to be uncovered. The least controversial aspect of the subject relates to the potential role of neurotrophic factors in the protection of the retinal ganglion cells. On the other hand, the postulated triggers for signaling cell death in glaucoma remain controversial. Certainly, the restricted flow of neurotrophic factors has been cited as one possible trigger. However, the connections between glaucoma and other factors present in the retina, such as glutamate, long held to be a prospective culprit in retinal ganglion cell death are still being questioned. Whatever the outcome of this particular debate, it is clear that the downstream intersections between the cell death and survival pathways should provide important foci for future studies whose goal is to protect retinal neurons, situated as they are, in the stressful environment of a cell destroying disease. The evidence for CaMKII being one of these intersecting points is discussed.