Browsing by Author "Fallatah, Azdah Hamed"

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Investigation of the role of Kv1.3 and Kv1.6 potassium channels in retinal ganglion cell degeneration in vitro.
(Eberhard-Karls-University Tübingen, 2023-12-11) Fallatah, Azdah Hamed; Paquet-Durand, François
1.0. Abstract 1.1. Introduction Organotypic retinal explants are an ideal system for identifying and studying potential treatments for retinal diseases, such as inherited retinal degeneration (IRD). In the organotypic retinal explant culture procedure, retinal ganglion cells (RGCs) usually progressively die in response to optic nerve axotomy. Previous studies connected RGC death to excessive NO/cGMP/PKG signalling. Another study showed that PKG targets Kv1.3 and Kv1.6 potassium channels, which are located in the ganglion cell layer (GCL). Others showed that Kv1.3/Kv1.6 potassium channels contribute to RGC degeneration after optic nerve crush in vivo. Interestingly, recent studies showed that PKG inhibition in retinal explants restored the viability and function of RGCs in vitro. Here, we investigated whether the direct inhibition of Kv1.3 and Kv1.6 channels with 50 nM Margatoxin (MrgX) might rescue RGCs in vitro to find a possible relationship between RGCs survival, PKG inhibition, and Kv1.3/Kv1.6 channels. 1.2. Methods For this study, cultures of organotypic retinal explants derived from post-natal day (P) 12 wild type (WT) mice were treated with the Kv1.3/Kv1.6 inhibitor of MrgX until P13 (24h) or P14 (48h). The PKG inhibitor CN238 was used to treat retinal explants at P13 (24h), P14 (48h), and P24. The rate of RGC degeneration was evaluated using both TUNEL-assay, which can label dead cells, and RNA-binding protein with multiple splicing (RBPMS) staining, which is a marker for RGCs. Furthermore, Amacrine cell (AC) staining was used to determine the protective effect of MrgX and CN238. 1.3. Results RGCs survived in vitro after 48h of culture with MrgX, RBPMS-positive cells showed a 70 % increase per mm2. On the other hand, the number of TUNEL-positive cells remained below 1,000 per mm2 in both MrgX-treated and non-treated groups. The overall percent difference in cell death between the two groups was 2%. In cultures treated with CN238, the amount of TUNEL-positive cell/mm2 dropped significantly after 24h and 48h. Cultures treated with CN238 for 10 days showed a significant increase in RBPMS-positive cells/mm2 and a significant decrease in TUNEL-positive cells/mm2. 1.4. Conclusion The inhibition of the Kv1.3 and Kv1.6 channels with 50 nM MrgX protected RGCs from the induced degeneration due to optic nerve transection in WT retina in vitro. This suggests a relationship between Kv1 channels and RGC death. In addition, the CN238-mediated PKG inhibition rescued RGCs. Together these results suggest that RGC degeneration in retinal explants following the severing of the optic nerve could be triggered by a potential new putative cell death pathway, which involves the signalling along the cGMP/PKG/Kv1 pathway.
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