Abstract
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Objectives: Phosphodiesterase type 4 (PDE4), a key enzyme that metabolizes second messenger cAMP, is regulated by protein kinase A (PKA) and by disrupted-in-schizophrenia 1 (DISC1). In vitro studies showed that phosphorylation of PDE4 by PKA activates PDE4, while presence of DISC1 inhibits the activity of PDE4. Previous studies with 11C-(R)-rolipram used PET to visualize the interaction between PDE4 and PKA in vivo (Itoh, et al. Synapse 2010). The in vivo interaction of PDE4 and DISC1 has however not yet been confirmed. To investigate this, we developed a bolus-infusion protocol plus one blood sampling using 11C-(R)-rolipram to quantify changes of 11C-(R)-rolipram binding in the brain of a Disc1 gene locus impaired mouse model. We also investigated whether the PET findings are consistent with PDE4 enzyme activity measured in vitro.
Methods: A bolus-infusion protocol was investigated to quantify 11C-(R)-rolipram binding in C57BL/6 mice. Several ratios of bolus to infusion (Kbol, min) were tested to achieve stable levels in brain. We also examined time stability of the activity in the heart chambers as an indirect measure plasma activity. The optimized protocol was then used to quantify 11C-(R)-rolipram binding in age matched (3 months) Disc1 KO (n = 11) and WT (n = 9) C57BL/6 mice. Plasma levels of 11C-(R)-rolipram were measured in heart blood at the end of scan. 11C-(R)-rolipram binding in the whole brain was measured as total distribution volume, VT/ fP, which is ratio of brain radioactivity in PET images to free 11C-(R)-rolipram concentration in plasma at equilibrium. VT/fP equals Bmax/KD (binding site density / dissociation constant) plus nonspecific binding. PDE4 enzyme activity was measured using in vitro samples of cerebral cortices from separate groups of KO (-/-) (n = 4), heterozygotes (+/-) (n = 4) and WT (n = 4) mice.
Results: Optimal Kbol was 135 min. Changes in brain radioactivity per hour after 50 min scan time were –5 ± 6% and -1 ± 9% in brain and heart, respectively, for both mouse groups indicating equilibrium was achieved within the scan time. SD/mean of VT/fP was ~30% indicating that the measurement was precise and did not have unphysiological variability caused by measurement errors. Disc1 KO mice showed a 73% significant increase in VT/fP (90 ± 31 vs. 52 ± 15 mL/cm3, P = 0.004) in Disc1 KO compared to WT mice. PDE4 enzyme activity showed significant group difference with the activity levels of KO > hetero > WT.
Conclusion: Bolus plus constant infusion and one blood sampling provides precise measurement of 11C-(R)-rolipram in mouse brain. The increased rolipram binding to PDE4 found in PET for Disc1 KO mice indicated greater PDE4 enzyme activity caused by the lack of DISC1 inhibition to PDE4. The PET findings were supported by the results of the enzyme assay. This study is the first to demonstrate that PDE4 is regulated by DISC1 in living animals.