Abstract
Purpose: :
MFRP is a type-II transmembrane protein that is expressed on the apical membrane of the RPE. Rd6 mice have a splicing mutation in mfrp, rendering it undetectable at the protein level. Previously we identified a novel mutation in mouse mfrp, called rdx, which is likewise a null mutation at the protein level. The phenotype of both of these mutants includes progressive photoreceptor degeneration and white fundus flecks corresponding with pigmented cells in the subretinal space. Furthermore, we have previously shown evidence of geographic atrophy of the RPE in 21-month old rdx mice. We have now further characterized these mutants by examining the ultrastructure of the RPE of mutant animals before onset of RPE atrophy, in an effort to determine a key deficiency that may lead to photoreceptor degeneration. We also have evaluated mRNA and protein expression of MFRP and its binding partner, CTRP5. Finally, since CTRP5 has been shown to activate AMPK in L6 myoblasts, we tested for the presence of a similar pathway in the RPE, and examined the possibility that MFRP might mediate this signaling mechanism.
Methods: :
For ultrastructural studies of RPE, eyecups were treated with 1% hyaluronidase in HBSS for 40 minutes, and the neural retina was gently removed. Tissues were then processed for standard TEM. We used quantitative PCR and western blotting to analyze expression of MFRP and CTRP5. Activation of AMPK was measured using antibodies specific to total AMPKα and phospho-AMPKα (Thr 172).
Results: :
We found that the density of RPE microvilli is increased in both rd6 and rdx animals, while their length is unaffected. Furthermore, we noted large, lipid-rich cytoplasmic inclusions in rdx mice that were not observed in controls. MFRP protein is undetectable in mutants, but the corresponding mRNA is upregulated. CTRP5 message is likewise upregulated, and CTRP5 protein is increased as well. We also found AMPK to be activated in mutants compared to controls.
Conclusions: :
Mice with mutations in mfrp have increased RPE microvilli. Cytoplasmic inclusions in rdx mice were suggestive of lipoprotein accumulation. Mfrp mRNA is upregulated in mutant animals, suggesting that the gene may be somewhat autoregulatory. Interestingly, our evidence suggests that mfrp mRNA in rdx mice is not subject to nonsense-mediated decay. The upregulation of CTRP5 in mutant RPE, which clearly has an abnormal phenotype, is consistent with previous reports of CTRP5 upregulation and subsequent AMPK activation in L6 myoblasts during stress response. Finally, we have also shown that MFRP is not required for AMPK activation in RPE in vivo.
Keywords: retinal pigment epithelium • retinal degenerations: cell biology • microscopy: electron microscopy