ArylalkylamineN‐acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells

Abstract

AbstractIn vertebrates, arylalkylamineN‐acetyltransferase (AANAT; EC 2.3.1.87) is the time‐keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin toN‐acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy‐o‐methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub‐cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7–10 (E7‐10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1–2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh‐RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh‐AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh‐control. Our results support the idea that AANAT is a BL‐sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.