Abstract
Purpose :
We previously reported that the retinal pigment epithelium (RPE) preferentially metabolizes proline into TCA cycle metabolites and non-essential amino acids (NEAAs), which are exported to be utilized by the neural retina. Proline dehydrogenase (PRODH) is a key enzyme in proline catabolism. In this study, we aim to test the hypothesis that PRODH controls proline utilization and its metabolic support to the neural retina.
Methods :
We obtained PRODH deficient (PRO/Re) mice and their parental control strain (C57BL/6J) from the Jackson Laboratory and validated the deficiency of PRODH expression in liver, RPE, and retina by Western blot. We measured the steady-state proline level in the plasma and RPE from PRO/Re mice by Gas Chromatography and Mass Spectrometry (GC MS). To study whether proline utilization was blocked in vivo in PRODH deficient mice, we delivered either 13C or 15N proline through retro-orbital injection to trace proline-derived TCA cycle metabolites and NEAAs using GC MS.
Results :
In the wildtype (WT) C57BL/6J mice, PRODH protein was abundant in the liver and RPE, but much lower in the retina. In PRO/Re mice, however, PRODH protein was undetectable in the liver, RPE, and retina. We also found the steady-state proline was substantially accumulated in both the plasma and RPE from PRO/Re mice. These results validate that PRODH is deleted in the PRO/Re mice. Tracing isotope-labeled metabolites in vivo showed proline-derived TCA cycle metabolites and NEAAs were highly enriched in the RPE and retinas from the WT mice. However, in the PRODH deficient mice, both 13C or 15N proline were accumulated in the RPE, and proline-derived metabolites were almost abolished in the retina.
Conclusions :
Proline catabolism through PRODH occurs mainly in the RPE but not the neural retina. RPE catabolizes proline into mitochondrial intermeidates and amino acids to provide substrates for retinal metabolism.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.