Arylalkylamine N-acetyltransferase (AANAT): ?Nuclear regulation by blue light and its potential role in the neuron viability of vertebrate retina?

MENDOZA

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Arylalkylamine N-acetyltransferase (AANAT), is the key regulatory enzyme of melatonin (Mel) synthesis within the vertebrate retina and pineal gland, the rhythmic synthesis of melatonin is controlled by the enzyme Arylalkylamine N-acetyltransferase (AANAT) which converts serotonin to N-acetyl serotonin (NAS). In the retina, MEL is mainly synthesized by the photoreceptor cells (PRCs), with high levels at night and lower levels during the day as well in the pineal gland. In a previous report, we found that AANAT activity in RGCs displayed circadian rhythmicity with highest levels during the subjective day in constant dark (DD), light (LL), or in the light phase of a LD cycle on the contrary, AANAT in PRCs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL whereas a brief nocturnal light pulse significantly decreased AANAT activity in these cells, but had no inhibitory effect on RGCs at any time. Strikingly AA-NAT activity in RGCs of chickens undergoes a completely different regulation by light than in PRCs. The AANAT activity is controlled by a cAMP dependent phosphorylation, this post-translational modification in AANAT (pAANAT) activates the enzyme to form a protein complex with 14-3-3 proteins, leading to an increase in its activity and protecting the enzyme against the proteasome degradation. Previous results, in primary cultures of retinal cells from chicken embryos, showed that the exposure to blue light (BL) at low intensity for 15 min to 1h induces a change in the AANAT subcellular localization from the cytoplasm to nuclei, and the levels of AANAT remains higher in dark after the BL exposure as well the pAANAT levels. Here we studied the effect of BL exposure at low intensity in primary cultures of retinal neurons cells from chicken embryos, and the effect of the silencing of AANAT using specific Sh-RNA, by electroporation, in the cellular viability by MTT assay and flow cytometry using Calcein red- AM and the reactive species of oxygen (ROS) levels usuing 2′,7′-Dichlorofluorescein diacetate (DHCDA). Our results showed that the Sh-AANAT decreases the AANAT-like protein and its transcripts levels in cultures compared with the control. In addition, the silencing of AANAT also affected the cellular viability, however the BL exposure did not affect the cellular viability in the different experimental conditions. Moreover, the ROS levels showed an increase in the cultures treated with the ShAANAT compared with the control. Taken together, these results suggest an important role of AANAT in the retina, playing a protective role controlling the balance of ROS, and this function could be associated with the antioxidant products NAS and Mel. Further experiments will be necessary to investigate the nuclear role of AANAT by BL in the local production of NAS and Mel as a new mechanism of protection in retinal neuron cells.