1. 1. Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration Matthew K Zabel, PhD Unit On Neuron-Glial Interactions in Retinal Disease National Eye Institute, NIH Disclosures: None
2. 2. Introduction Retinitis pigmentosa- set of blinding hereditary diseases. Typically rod and then cone photoreceptor degeneration- utility of gene therapy. The significant number and diversity of causative genes underscores an advantage of finding broadly-shared mechanisms. Clinical and laboratory findings indicate a role for non-cell autonomous mechanisms such as inflammation. Microglial infiltration is found in both animal models and human specimens. Suppression of pro-inflammatory and pro-oxidative properties alleviates degeneration.
3. 3. Microglia in RP Model Rd10 mouse model. Well-characterized model recapitulating features of RP. Recessive mutation in rod Pde6b. Infiltration of microglial processes at peak of photoreceptor apoptosis. TUNEL Transformation into amoeboid morphology Development of multiple phagosomes. Re-ramification
4. 4. Phagocytic Role of Microglia in RP Expression of phagocytic marker CD68 by infiltrating microglia. Expression and secretion of MFG-E8 by microglia and labeling of ONL photoreceptors. Expression of the eat-me signal phosphatidylserine on the outer leaflet of rods, but not cones.
5. 5. Microglial Phagocytosis of Live Rods Phagocytosed photoreceptors are positive for Rhodopsin. Negative for Cone Arrestin. Peak phagocytosis coincides with peak of microglial infiltration and photoreceptor apoptosis.
6. 6. Microglial Phagocytosis of Live Rods Microglia phagocytosis is primarily directed at non- apoptotic rods Phagocytosed rods are: Negative for TUNEL labeling Negative for early markers of apoptosis: activated caspase-3, cleaved PARP
7. 7. Alternate mouse models of RP and human RP Mouse models Rd1 Rd16 RPGRIP Human RP Autosomal Recessive Autosomal Dominant x2 X-linked
8. 8. Live cell imaging of microglial behavior in CX3CR1GFP/+, rd10 retinal explants Labeling with: GFP/Hoechst/PI Interaction of infiltrating microglia with viable photoreceptor nuclei Dynamic probing of photoreceptor somata via motile phagocytic cups Engulfment and phagocytosis of photoreceptor soma and translocation to microglial cell body Migration of microglia within ONL Dynamic microglial phagocytosis of non-apoptotic rods
9. 9. Genetic model of microglia depletion CX3CR1CreER x Rosa26- flox-STOP-flox DTA mice in rd10 background Administration of tamoxifen at P21 to deplete microglia Sustained microglial depletion delayed rod degeneration Contribution of Microglia to Photoreceptor Degeneration: Depletion of Microglia P28-29 P37-39 P50
10. 10. Inhibition of Microglial Phagocytosis in rd10 retina Inhibition of phagocytic vitronectin receptor with cRGD peptide Ex vivo effects on rd10 retinal explants microglial ramification # of microglial phagosomes In vivo effects on rd10 retina at P20 # of infiltrating microglia # of microglial phagosomes photoreceptor rescue
11. 11. Contribution of Infiltrating Microglia to Photoreceptor Degeneration Microglia potentiate degeneration by: Phagocytosis of stressed but living rod photoreceptors in non-cell autonomous manner.
12. 12. Conclusion Microglia play an active role in retinal degeneration through: 1. Infiltration of the outer retina during photoreceptor degeneration. 2. Expression of phagocytic machinery. 3. Dynamic interactions with viable photoreceptors. These microglial functions culminate in the phagocytosis of stressed, but live rod photoreceptors, augmenting disease progression. Targeting microglial phagocytosis will provide an opportunity to slow progression of degeneration across the broad spectrum of genetic etiologies.
13. 13. Acknowledgements UNGIRD Wai T. Wong Lian Zhao Xu Wang Wenxin Ma Parth Shah Biological Imaging Core Robert Fariss Functional Imaging Core Hao-hua Qian National Eye Institute, IRTA funding New York University Christopher Parkhurst Wen-Biao Gan Foundation for Fighting Blindness Joe Hollyfield Vera Bonilha Retinal Cell Biology & Degeneration Tiansen Li