Abstract
Purpose :
cGMP-dependent cytotoxicity is part of a common photoreceptor cell death pathway in multiple models of inherited retinal degenerations, including the prototypical rd10 model of retinitis pigmentosa. Mycophenolate mofetil (MMF) is a reference inhibitor of IMPDH, a key enzyme for the biosynthesis of guanine nucleotide precursors to cGMP. We previously showed that MMF confers potent neuroprotection in rd10 mice. This study aims to elucidate the neuroprotective mechanisms of MMF by quantifying retinal guanine nucleotides longitudinally.
Methods :
rd10 mice received intraperitoneal injections of MMF 100 mg/kg daily postnatal day (P)12-22. After dark-adaptation, whole retina was harvested and flash frozen in liquid nitrogen under dim red light from c57 wildtype, naïve rd10, and MMF-treated rd10 mice every 1-2 days, P10-22. Supernatant from homogenized (0.1 M HCl on ice) and centrifuged tissue samples were used to quantify GMP, GDP, GTP, and cGMP using a 4000 QTRAP hybrid/triple quadrupole linear ion trap mass spectrometer (AB Sciex) with electrospray ionization (ESI) in positive mode, interfaced to a SIL-20AC XR auto-sampler followed by 2LC-20AD XR LC pumps (Shimadzu).
Results :
The levels of all guanine nucleotides from rd10 mice were significantly abnormal at multiple time points P10-22. Prior to the onset of structural degeneration (P16), rd10 retina exhibited 1) significantly elevated levels of GMP and GDP at P10-15 with an additional relative GMP spike at P13 (p<0.05 for all), 2) loss of a normal GTP spike at P13 that was observed in c57 mice (p<0.05), and 3) significantly depressed levels of cGMP at P12-15 (P<0.05 for all) except at P14 where there was a relative cGMP spike. After the onset of degeneration, GMP and GDP levels continued to be elevated until P17-19, and cGMP levels were continually depressed to P22 (p<0.05 for all). Treatment with MMF suppressed the relative spikes in GMP at P13 (p<0.05) and cGMP at P14 (p<0.05), and normalized levels of GDP at P13-19 (p<0.05 for all).
Conclusions :
Our data support a new concept that dysregulation of all guanine nucleotides is associated with the photoreceptor cell death pathway in rd10 mice. Moreover, MMF normalized GDP levels and mitigated abnormal spikes in GMP and cGMP levels, supporting our hypothesis that the neuroprotective effect of MMF is via the inhibition of IMPDH and guanine nucleotide biosynthesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.