Photochemical restoration of visual responses in blind mice
- Author(s): Polosukhina, Aleksandra
- Advisor(s): Kramer, Richard H
- et al.
This dissertation addresses two non-genetic therapeutic approaches for treating inherited retinal diseases such as retinitis pigmentosa and age related retinal degeneration. The first approach utilizes a light sensitive molecule Azobenzene-acrylomide-quaternary-amonium (AAQ) to restore light sensitivity to retinal degenerated (rd1) mice. AAQ molecules permeate through the cell membrane and photosensitize voltage-gated K+ words channels in response to different wavelengths of light. Consequently, we achieved robust photosensitization of most retinal cells ex-vivo upon application of AAQ on rd1 mouse retinal explants. We then characterized the effect of AAQ on mouse behavior and restored a pupillary light reflex and light elicited behavior in these blind rd1 mice.
The third chapter addresses improvements to the AAQ therapeutic approach. We utilize Di-ethyl-amine-azobenzene quaternary ammonium (DeNAQ) that also confers light sensitivity on RGCs by photosensitizing voltage-gated channels in response to 480 nm light and silencing the channels in the dark (trans form). The property of DeNAQ being sensitive in the visible range is one of the major advantages of this therapeutic. The quick relaxation of DeNAQ in the dark is also an advantage, since only one wavelength is required for photosensitization. In addition to the red-shifted properties we found that DeNAQ can confer light sensitivity to retinal cells for several weeks and restores light elicited behavioral responses.
The fourth chapter addresses the question of whether the physiological and anatomical changes that accompany retinal degeneration lead to functional changes in the retinal ganglion cells (RGCs). We used a membrane impermeable Quarternary-azobenzene-quaternary molecule (QAQ) molecule that can only enter the cells and render them light sensitive through large pore openings, those seen during pore dilation of P2X and TRP channels. We characterized the path of entry of QAQ using pharmacology and found that photosensitization of RGCs could be reduced by adding P2XR antagonists. In agreement with other studies suggesting that pore dilation has been associated with neurodegenerative processes, we show that QAQ can only confer light sensitivity to RGCs from rd1 and not from wildtype (WT) mice. We also demonstrate that QAQ does not photosensitize RGCs in glaucoma mouse models, suggesting that this physiological change in permeability is specific to retinal degenerating disease.
The therapeutic approaches addressed in this thesis apply to a variety of retinal disease genotypes. The high promiscuity of AAQ delivery allows us to optically stimulate any cell type in the retina. Additionally, the red-shifted molecule DeNAQ, combined with its ability to target only diseased tissue, offers a promising clinical alternative for vision restoration. Finally, using another photosensitive molecule, QAQ, we study the physiological impact of retinal degeneration on pore-dilating ion channel activity. Understanding the sources of physiological changes could help prevent further damage caused by degenerative retinal diseases.