The Effects Of Metabolic Regulation Of Retinoic Acid On Spatial Learning And Memory In Rats And The Expression Pattern Of RALDHs And CYP26s During The Neural Differentiation

Part1Synthetic regulator (citral) and metabolic enzymes inducer (triadimefon) of retinoic acid on spatial learning and memory in ratsRetinoic acid (RA) plays a critical role in embryonic development and function maintenance of vital organs in adult. And RA signal is reported to be critical in the development and maintenance of spatial learning and memory. Subtle regulation of RA signaling is relied on spatio-temporal control of RA synthesis and catabolism.I Biphasic effect of citral, a synthetic regulator of retinoic acid, on spatial learning and memory in ratsAim:To investigate the effects of changes in RA synthesis on the spatial learning and memory, we used a RA synthetic regulator, citral, to alter the synthetic rate of RA to oberve the consequent changes in RA concentration in hippocampus and spatial learning and memory and the relationship between them.Methods:In this study, we administrated citral via intraperitoneal injection to SD rats, and then applied Morris water maze to test the effect of citral on animals'spatial learning and memory. To elucidate the mechanism of this effect, we also measured the retinoic acid concentration in rats'hippocampus by high performance liquid chromatography (HPLC).Results:Our data implied biphasic effects of citral. The low dose (0.1mg/kg) of citral improved the spatial learning capability, and enhanced the spatial reference memory of rats, whereas the high dose (1.0mg/kg) was like to produce the opposite effects. Meanwhile, the low dose of citral increased the hippocampal retinoic acid concentration, while the high dose decreased it.Conclusions:The changes in hippocampal retinoic acid concentration induced by different doses of citral might be responsible for the biphasic effect of citral on spatial learning and memory.II Suppressive effect of triadimefon, an inducer of metabolic enzymes of retinoic acid, on spatial learning and memory in ratsAim:We have discovered that changes of RA concentration in hippocampus induced by administration of citral, a synthetic regulator of RA, is associated with alteration of spatial learning and memory in rats. Therefore, we applied an inducer of metabolic enzymes of RA, triadimefon, to induce changes in metabolic rate of RA, and observed the subsequent changes in RA concentration in hippocampus and spatial learning and memory in rat and the relationship between them.Methods:We administrated triadimefon via intraperitoneal injection to SD rats, and then applied Morris water maze to test the effects of triadimefon on animals'spatial learning and memory. To elucidate the mechanism of this effect, we also measured the retinoic acid concentration in ratsshippocampus by high performance liquid chromatography (HPLC).Results:Our data implied that triadimefon dose-dependently inhibited the spatial learning capability, and impaired the spatial reference memory in rats. Meanwhile, triadimefon induced a reduction of hippocampal retinoic acid concentration in a dose-dependent manner.Conclusions:The reduction in hippocampal retinoic acid concentration induced by triadimefon might be responsible for the suppressive effect of triadimefon on spatial learning and reference memory. Retinoic acid level in hippocampus plays an important role in spatial learning and reference memory, and the interference with synthesis and metabolism of retinoic acid may be responsible for the change in spatial learning capability and reference memory. Part2Expression of RALDHs and CYP26s in human tissues and during the neural differentiation of P19embryonal carcinoma stem cellAim:To further investigate the physiological functions of RA and mechanism of the regulation of RA signal, we measured the expression patterns of RA synthases RALDHs and metabolic enzymes CYP26s, which play critical role in the regulation of RA signaling, in multiple human tissues and during the neuronal differentiation of P19embryonal carcinoma stem cell.Methods:To measure the expression patterns of RALDHs and CYP26s in human tissues, normalized first-strand cDNAs from multiple human fetal, adult, and tumor tissues and cell lines were analyzed by PCR. To investigate the dynamic expression of RALDH isoforms and CYP26isoforms during neural differentiation, we applied a P19embryonal carcinoma stem cell neural differentiation model. Undifferentiated P19cells were treated under three different conditions:(1) suspension culture without ATRA induction;(2) ATRA induction without cell suspension;(3) suspension culture and ATRA induction. Total RNA was extracted at various time points, and one-step RT-PCR was applied to measure the expression of RALDHs, CYP26s and differentiation marker genes.Results:Each isoform of these two enzyme families revealed unique expression pattern in human tissues. And it was worth noting that there were evident differences between fetal brain and adult brain in the expression of RALDHs and CYP26s. During P19cell neural differentiation induced by ATRA and cell aggregate formation, RALDH1, RALDH2, CYP26A1, and CYP26B1could be notably up-regulated by ATRA, and maintenance of the high-level expression of RALDH1and RALDH2was dependent on the further neural differentiation, but not continuous ATRA induction. RALDH3transcription could not be directly induced by ATRA, and obvious up-regulation of its transcription initiated at the late stage of ATRA induction or after the removal of ATRA. This implied its neural differentiation-dependent expression pattern. CYP26C1transcription was significantly repressed by ATRA, and this down-regulation also showed a neural differentiation-dependent pattern, in respect that CYP26C1expression was kept in low-level even after the withdrawal of ATRA.Conclusions:Different isoforms of RALDHs and CYP26s play distinct role in the regulation of RA signal, and their proper spatio-temporal expression and the synergism or antagonism among these isoforms might be crucial for appropriate RA signal.