Abstract
Light induced retinal degeneration (LIRD) models have been extensively used as disease models. However, the widespread areas of retinal damage do not closely match disease conditions in humans, where areas of healthy and areas of damaged cells exist next to each other. In this project, we aim to develop a mouse model of focal light induced retinal degeneration, which results in a targeted area of damage surrounded by areas of healthy cells. We characterise the retinal cellular remodelling events in both areas for up to 120 days after exposure.The model developed was used to study the effects of targeting the translocator protein 18kDa (TSPO) with its ligand XBD173. TSPO is an outer mitochondrial transmembrane protein associated with inflammatory modulation, energy metabolism, cell death, steroidogenesis and mitochondrial health. In recent years, the widely accepted functions of TSPO and its ligands, such as cholesterol transport and mitochondrial permeability transition regulation, have been challenged. For this reason, we focused mainly on characterising the effects of XBD173 supplementation on cell death, proliferation and energy metabolism.
Reactive microglial cells are commonly observed in degenerative diseases and can exacerbate neuronal cell death. TSPO has been reported as a biomarker of microglial activation and supplementation with XBD173 dampens microglial activation in vitro. We investigated the effects of targeting TSPO with XBD173 on photoreceptor damage in vivo. RPE cells were also observed to express constitutive levels of TSPO. We thus evaluated the effects of supplementation of RPE cells on cell death.
The focal light exposed area of the LIRD model showed early photoreceptor, horizontal cell, and OPL connectivity loss by day 23 after exposure. This was accompanied by increased number and activation of microglial cells and macrogliosis. Rod- and cone- bipolar cells did not exhibit any loss in cell numbers but showed significant loss and remodelling of their OPL dendrites. Third order neurons remained unaffected by the light exposure. Vascular leakage restricted to the subretinal space and photoreceptor layer was observed in the model. The non-exposed areas showed a milder, but significant decrease in photoreceptor and horizontal cells. Microglial activation through CD68 expression was cell gliosis, observed in those areas. There was no Muller but a sustained increased expression of GFAP was noted in astrocytes.
Supplementation of LIRD mice with the TSPO agonist, XBD173, showed lower number and more ramified microglia in both damaged and non-damaged areas. This event was accompanied by an increase in photoreceptor survival within the damaged and non-damaged areas. TSPO expression was confirmed in a mouse cone-like cell line: 661W. Supplementation with XBD173 rescued 661W from antimycin A (AMA), an electron transport chain complex III inhibitor, induced ATP depletion and cell death. This was also accompanied by a decrease in cell proliferation.
Light exposure did not result in RPE damage or change in TSPO expression, while aged RPE cells exhibited lower TSPO levels. The mouse RPE cell line, B6-RPE07, expressed TSPO in a confluency-dependent manner. B6-RPE07 showed TSPO levels changes according to the insult the cells were exposed to. Supplementation of XBD173 to RPE cells prevented AMA-induced ATP depletion and necrosis. The treatments also induced cell cycle arrest but did not change apoptotic markers, oxidant levels and mitochondrial membrane potential.
These research findings highlight the importance of developing animal models that closely represent human diseases. Second order neurons are very susceptible to changes when the photoreceptor layer is affected. Microglial cell activation likely exacerbates disease conditions by phagocytosis of living neurons. Dampening microglial activation should be considered in the future as a therapeutic target in degenerative diseases. The neuroprotection seen after XBD173 supplementation is possibly due to a decrease in microglial activation and a direct biological effect on photoreceptor cells. XBD173 is also a potent drug for targeting RPE cell death. These results altogether suggest that XBD173 is a promising drug that warrants further investigation as a potential new therapy in a multitude of ocular diseases.
| Date of Award | Jul 2019 |
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| Original language | English |
| Awarding Institution |
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| Sponsors | Fight for Sight (Inc British Eye Research Foundation) |
| Supervisor | Heping Xu (Supervisor) & Mei Chen (Supervisor) |