Research Project
10317323
Diurnal RPE phagocytosis is an essential aspect of lifelong continuous renewal of photoreceptor outer segments. Phagocytosis of distal photoreceptor outer segment tips requires activity of RPE surface receptors of the TAM (Tyro3/Axl/MerTK) receptor tyrosine kinase family. In human patients, mutations in the mertk gene cause an unusually severe form of retinitis pigmentosa with blindness in teenage years. The retina in mice and rats lacking MerTK develops normally but rapidly degenerates postnatally, with onset of photoreceptor death before 4 weeks of age. Neuroinflammation and specifically activation and redistribution of retinal microglia has been reported in adult MerTK-deficient animals and has been assumed to be a consequence of photoreceptor distress and debris accumulation caused by lack of daily clearance phagocytosis of spent outer segment tips by the RPE. However, we find that, surprisingly, robust increase in pro-inflammatory cytokines and microglia activation in MerTK-deficient rat and mouse retina precede postnatal onset of photoreceptor outer segment renewal and daily RPE phagocytosis. Moreover, our results show that anti-inflammatory treatment is effective in preventing microglia activation and extending photoreceptor survival and function only if applied at early postnatal age several days prior to the onset of diurnal outer segment renewal and RPE phagocytosis. In wild- type retina, MerTK is expressed early postnatally and mainly by the RPE (and not detected in microglia). This implies that loss of MerTK affects the RPE specifically, directly and before outer segment renewal begins. Thus, all available evidence supports microglia stimulation by MerTK-deficient RPE independently of RPE phagocytic activity and outer segment renewal. Here, we propose highly focused, key proof-of-principle pilot studies to rigorously scrutinize our completely novel concept that lack of MerTK in itself and unrelated to RPE phagocytosis causes RPE cells to release a pro-inflammatory cytokine, which elicits early onset, harmful neuroinflammation that in turn accelerates photoreceptor dysfunction and demise. We will test our hypothesis in 3 independent specific aims. Aim 1 will explore double mutant mice we just generated to test if cytokine is required for neuroinflammation in mertk-/- retina. Aim 2 will explore if inducing cytokine loss in the RPE alone is sufficient to eliminate early postnatal harmful neuroinflammation and whether cytokine mediator must be expressed early postnatally to induce neuroinflammation in mertk-/- retina. Aim 3 will explore polarized, differentiated RPE cell models (primary, unpassaged wild-type and MerTK-deficient RPE and passage 1 human RPE cells) to determine whether MerTK expression and its kinase signaling activity are directly linked mechanistically to cytokine transcription, translation, and/or release by RPE cells. Upon completion, this pilot study will have yielded unambiguous confirmation or disproof of our novel hypothesis and generated in vivo and culture models for large scope mechanistic and pre-translational follow-up.
