Retinitis Pigmentosa (RP) are a group of inherited retinal degenerative diseases for which more than forty-five have been identified. Patients began to experience night blindness, followed by a sight in tunnel vision due to the lost of their peripheral photoreceptors, eventually leading to total blindness about the age of thirty-five or forty years. In this study, we focus of one of the most severe form of RP (RP38) due to a mutation in the Mertk gene. Mertk is a receptor found in the retinal-pigmented epithelium (RPE) involved in the very important process of daily phagocytosis of the photoreceptors. When this function is impaired the retina degenerate leading to lost of vision. It is still unclear why this particular form of the disease is such severe. Recent studies have shown that inflammation could exacerbate the degeneration of the retina. Usually inflammatory cells can be detected in degenerating retinas of mice, but if they could be detected in young mice, when the retina is still intact, that could explain part of the severity of the disease. A recent study has been able to detect inflammatory cells in aged Mertk knock-out (KO) mice by immunostaining of cryosection of the retina, when the retina is already degenerated. Here, we wanted to develop a molecular technique to detect inflammatory in the retina as a complementary tool to immunostaining, that would help detect the presence or not of inflammatory cells in younger mice, in order to answer the question of the severity.
Figure 1-Detection of inflammatory cells by RT-PCR in three-month-old mice. A. Nox2 macrophage specific primer. A high level of the macrophage specific transcript Nox2 is only found in the KO mice. B. IBA1 macrophages specific primer. Increase of IBA1 transcript level is only found in the KO mice. C. Retinal specific marker Brn3a. Same level of Brn3a transcript is found in WT and KO mice. D. Housekeeping gene. Rplp0 used as a loading control show same amount of transcripts both in WT and KO mice; * shows expected size. WT: Wild-type mouse; KO: Mertk Knock-out mice.
Figure 2-Detection of inflammatory cells by RT-PCR in five-week-old mice. A. Nox2 macrophage specific primer. A high level of the macrophage specific transcript Nox2 is only found in the KO mice. B. IBA1 macrophages specific primer. Increase of IBA1 transcript level is only found in the KO mice. C. Retinal specific marker Brn3a. Same level of Brn3a transcript is found in WT and KO mice. D. Housekeeping gene. Rplp0 used as a loading control show same amount of transcripts both in WT and KO mice. WT: Wild-type mouse; KO: Mertk Knock-out mice
Figure 3-Fig.1S: Validation of the primers. A. Macrophage specific primers. Two pairs of primers for Nox2 and IBA1 were tested in duplicata on WT macrophages B. Housekeeping gene. Rplp0 primers tested in duplicata in WT macrophages. C. Retinal specific marker Brn3a. Three pairs of primers were tested in duplicata in WT macrophages and WT retinas. WT: Wild-type mouse; M: Macrophages.
Retinitis Pigmentosa (RP) are a group of inherited retinal degenerative diseases, that lead to a progressive vision loss starting from the periphery of the retina. Researchers have identified more than 45 genes involved in RP. Here we focus on one of the most severe forms of RP: RP 38, which is caused by a mutation in the Mertk receptor gene. The Mertk receptor is required for the daily phagocytosis of the photoreceptor outer segment extremities. Without daily phagocytosis, photoreceptors accumulate oxydative reactives and degenerate. Recent studies have used immunostaining to show positive inflammatory cells in the retinas of five-week-old Mertk KO mice. Finding inflammatory cells in younger mice could help explain the severity of the disease. Here we sought to develop molecular tools to detect inflammatory cells in the retina of mice. We selected three markers: two markers that are known to be macrophage specific (Nox2 and IBA1), and one marker that is a retinal ganglion cell specific gene (Brn3a). Rplp0, a ribosomic protein, has been used as housekeeping gene. Because of the immune privilege in the eye we did not expect detection of macrophage specific markers (Nox2 or IBA1) in the WT retinas but did expect detection of the macrophage specific markers in Mertk KO mice. Confirming our prediction we observed no or very few Nox2 or IBA1 transcripts in WT mice respectfully, but a high level of these transcripts in the one-month-old and three-month-old Mertk KO mice. Additionally, we determined that Brn3a is a good retina specific marker. Taken together these results suggest that Nox2 and IBA1 can be used to detect inflammatory cells in retinas by RT-PCR, and Brn3a can be used as a retinal specific molecular. These methods will be useful for investigating the particular severity of Retinitis Pigmentosa 38.
I would like to thank Dr. Finnemann for providing the mice. I would also like to greatly thank Catharina Grubaugh and Tony Evans for their amazing dedication and support. Finally I would like to thank Dr. Rubin for his guidance and for letting me do at the end of each meeting a “one more last RT”.
|This document was last modified 05/18/2016.|
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