Effects Of MTOR Pathway In Cone Photoreceptors Of Retinitis Pigmentosa & Wild Type Mice

ObjectivePhotoreceptors(PR) are metabolically highly active cells requiring phosphoinositide 3-kinase(PI3K) activity for long-term survival. Downstream of PI3 K is the kinase mechanistic target of rapamycin(m TOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3 K and m TOR are part of the insulin/m TOR signaling pathway, however if m TOR is required for long-term photoreceptor survival remains unknown. Here we try to explore the effects of m TOR pathway in both Retinitis Pigmentosa(RP) and wild type cones through cone specific knockout of important regulator Pten, Tsc1, Raptor or Rictor.Methods1. Animals: To dissect the role of the insulin/mTOR pathway on cone survival, Pten which is an upstream suppressive regulator of both m TORC1 and m TORC2, Tsc1 which has suppress effect on m TORC1, Raptor which encodes m TORC1 unique accessory protein RAPTOR, as well as Rictor which encodes m TORC2 unique accessory protein RICTOR, were chosen for creating conditional allele. The conditional allele was first crossed to the cone-specific Middle-wave opsin Cre-driver line and then to the rd1 line to generate Cre– and Cre+ rd1-conditional allele lines. In some cases double conditional alleles were also generated. The Cre positive and negative lines were crossed to each other(e.g. rd1, PtenC/C × rd1_PtenC/C, M-Cre+) to generate Cre+ and Cre– littermates that were used for analyses. 2. PCR of tail tissue DNA extract is used to identify mice genotype. 3. The Ai9 Cre reporter line was used to validate uniform expression of Cre recombinase across the retina 4. Retina flat mount and/or cryosection combined with immunochemical fluorescent staining was used to evaluate cone cell morphology, cone survival rate, cone specific protein, as well as AKT/m TOR pathway related protein expression level. 5. Whole retina Western blot was used to evaluate expression level changes of key regulators in m TOR pathway. 6. q RT-PCR was used to monitor c DNA level of key regulator genes in m TOR pathway. 7. Whole retina NADPH assay was used to test the NADPH level of rd1, Tsc1c/c, MCre+ mice.8. Analyse Casp2-/-, rd1 retina to determine the importance of NADPH level in cones under disease stress. 9. Photopic electroretinogram(photopic ERG) was used to track cone function changes. 10. Funduscopy is used to check the overall retina fundus morphology. 11. Transmission Electron Microscopy(TEM) was performed to evaluate the ultra structure of cone photoreceptors inner and outer segments.ResultsI. Target gene knockout mice were successfully established; Cre recombinase uniformly expressed across the retina. II. Retinitis pigmentosa mice 1. Retina flat mount combined with immunochemical fluorescent staining for cone counting results: ① Cone survival is significantly improved in rd1_Ptenc KO retinas for up to eight months of age when compared to Cre– littermate control retinas. Loss of RAPTOR in cones alone or concurrent loss of PTEN and RAPTOR accelerated cone death, while loss of RICTOR in cones had no effect on cone survival in rd1(p<0.05). ② Comparing to Cre– littermate control retinas, survival is significantly improved in rd1_Tsc1c KO retinas for up to eight months of age. The protection effect was abolished when concurrent loss of both TSC1 and RAPTOR(p<0.05). 2. Photopic ERG results:Cone function was profoundly preserved in rd1_Tsc1c KO at the onset of cone loss(postnatal day 21), as well as at 2 months when compared to Cre- littermates(p<0.05). 3. Immunofluorescence on rd1_Ptenc KO retina flat mounts at P21 revealed an increase in the number of cones positive for phosphorylated m TORC1 target phosphorylated ribosomal protein S6(p-S6), while the number of cones phosphorylated m TORC2 target serum/glucocorticoid regulated kinase 1(p-SGK-1) was reduced. In rd1_Tsc1c KO retinas S6 was uniformly phosphorylated in cones when compared to loss of PTEN. 4. Loss of TSC1 in rd1 cones did not induce cone proliferation as assessed by the proliferation maker Ki-67. 5. At 2 months of age, we found by immunofluorescence analysis an increase in the expression of metabolic genes: glucose transporter 1(GLUT1), hexokinase II(HKII), glucose-6-phosphate dehydrogenase(G6PD), pyruvate kinase muscle isoform 2(PKM2), malic enzyme 1(ME1), and the transcription factor hypoxia inducible factor-1 alpha(HIF-1a) in cones of rd1_Tsc1c KO mice. The increase was not seen upon concurrent loss of TSC1 and RAPTOR. 6. Higher levels of NADPH in whole retinal extracts of rd1_Tsc1c KO mice at postnatal day 21(P21) was observed by whole retina NADPH assay(p<0.05). 7. q RT-PCR analysis showed that most metabolic genes started to display a modest at P21, but statistically significant difference between Cre– and Cre+ retinas was observed by P24(p<0.05). 8. Systematically deletion of CASP2 prlonged cone survival in rd1 mice. 9. Similar to the observations made in the rd1 mouse model, loss of TSC1 in cones of Rho-/- mice was able to significantly prolong cone survival at 30 weeks of age. III. Wild type mice 1. Photopic ERG showed that neither loss of RAPTOR nor RICTOR alone affected function of wild type cones up to one year of age, however concurrent loss of RAPTOR and RICTOR caused a significant decline of cone function as early as 2-month of age, and cone function continued to decline over time(p<0.05). 2. Funduscopy showed no significant different between Cre+ and Cre- mice in Raptor or Rictor single as well as double knockout mice up to one year. 3. Retina cryosection combined with immunoflorescent staining showed: ①The expression level of cone specific proteins MIDDLE-WAVE OPSIN, BLUE OPSIN, cone ARRESTIN, cone TRANSDUCIN showed slight decrease in Raptor and Rictor double knockout retinas, however the density of cones was not affected. ②Ventral retina MIDDLE-WAVE OPSIN transiently decreased in the gene knockout retinas. ③When compare with wild type retina, the outer segment(OS) of Raptorc KO, and Raptorc KO,Rictorc KO photoreceptors seemed less defined. 4. Transmission Electron Microscopy(TEM) revealed thickened cone inner segments(IS) upon loss of RAPTOR and concurrent loss of RAPTOR and RICTOR. Higher magnification TEM showed that upon loss of Raptor many cones with a thickened IS had strongly reduced OSs, both in length and thickness. The morphology changes in Rictorc KO retinas was less sever.Conclusions1. mTORC1 activity is required and sufficient to promote cone survival in RP. m TORC2 and AKT-mediated pro-survival mechanisms do not contribute to cone survival upon loss of PTEN. 2. m TORC1 promotes cone survival in RP mainly through improving glucose uptake,retention, and divergence into the Pentose Phosphate Pathway. 3. Normal cone function needs both m TORCs to be activated, but single loss of either m TORC has no effect on cone function. 4. m TOR activity is not required for wild type cone survival, suggesting that PI3 K pro survival mechanism maybe independent of m TOR signaling...