Targeting Müller cells for therapy of diabetic retinopathy
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
To understand the pathomechanisms of diabetic retinopathy and ultimately to successfully treat respective patients, there is the urgent need to identify underlying cellular pathways involved in the tissue response. Approximately a third of people with diabetes are diagnosed with diabetic retinopathy (DR) and a tenth develop vision threatening effects, making DR the leading cause of blindness among working-age adults. Here, we investigated how Müller cells, the major macroglia of the retina, respond to stress factors found in a type II diabetes mouse model. We used unbiased RNA sequencing and mass spectrometric approaches to characterize their signature of gene expression changes at mRNA and protein levels. These experiments clearly showed major gene expression changes in Müller cells that very likely lead to their observed reduced efficiency to maintain the retinal immune and ion homeostasis. Importantly, those alterations in Müller cell gene expression clearly proceeded neuronal cell loss and major vascular reorganization indicating that they play a central role in disease onset and do not only seem to act as bystanders of neurodegeneration. For that reason, we tried to identify a genetic master regulator that would account for a broad spectrum of these Müller cell-specific expression changes. To do so, we used bioinformatics tools to narrow done the candidate list from our big omics data sets primarily focusing on transcription factors since they hold the potential to act as a master regulator. Performing this analysis the glucocorticoid receptor (GR) caught our attention for several reasons: (1) GR expression is almost exclusive to Müller glia, in a highly conserved manner amongst warm-blooded vertebrates (i.e. retinae of chicks, mice, guinea pigs, dogs and, importantly, humans), (2) we found that many transcription factors downstream or co-active with GR are specifically expressed in Müller glia in the mouse retina, (3) the GR expression is significantly downregulated in Müller cells of the diabetic mice, and, (4) a transcription factor binding site cluster analysis based on our RNAseq data identified GR as one of few strong candidates to explain the diabetes-associated expression changes in Müller cells. Of note, treatment with GR agonists (e.g. triamcinolone) has proven to be effective in inflammatory retinal diseases, DR included, as it counterbalances typical changes associated with DR such as breakdown of the blood retinal barrier or onset of neuroinflammation. However, this treatment regimen requires repetitive intravitreal injection of the compound with all the possible complications with this route of application. Accordingly, the second part of the project was dedicated to establish a gene addition approach to overexpress GR in Müller cells of diabetic mice via an adeno associated (AAV) virus-gene shuttle. Even though first very promising results could be obtained that the AAV approach leads to successful overexpression in the target cells, we had to tackle methodological problems. For that reason, last experiments are currently under way to proof our hypothesis that stimulating GR signaling in Müller cells via gene therapeutic tools holds potential to maintain the neuron-supportive Müller cell functions thereby fostering neuronal survival in multifactorial retinal degeneration such as DR for which no long-term efficient treatment exists to date. The main work awaits publication as soon as experiments regarding the effect of GR overexpression in the diabetic mice are finalized.
Publications
- The role of Müller cell glucocorticoid signaling in diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 2020 Feb; 258(2):221-230
Ghaseminejad F, Kaplan L, Pfaller AM, Hauck SM, Grosche A
(See online at https://doi.org/10.1007/s00417-019-04521-w)
