A circadian NeuroD1 protein as a potential Aanat gene regulator in the adult rat pineal gland.

A.E. CASTRO; S. BENITEZ; M.R. FITT; V. ORTIZ MALDONADO; L.E. SAVASTANO; E. MUÑOZ

Huerta Grande, Córdoba.

Congreso; SAN2011; 2011

Sociedad Argentina de Investigación en Neurociencias

A circadian NeuroD1 protein as a potential Aanat gene regulator in the adult rat pineal gland Castro Analía, Benitez Sergio, Fitt Marcos, Ortiz Maldonado Valentín, Savastano Luis and Muñoz Estela. Laboratorio de Neurobiología, Sección de Cronobiología, IHEM-CONICET, FCM-UNCuyo, Mendoza, Argentina. ANPCyT. acastro@fcm.uncu.edu.ar NeuroD1 is a transcription factor responsible for the establishment and maintenance of endocrine phenotypes. In the pineal gland, the Aanat gene encodes a key enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase. E-Box motifs identified in the Aanat gene promoter have been associated with its highly tissue-specific expression. In this work, we characterized NeuroD1 protein expression in adult rat pineal glands via IHC and WB. We also determined, via EMSA, whether an E-Box motif located upstream of the CRE-inverted CCAAT cassette in the Aanat promoter might recruit NeuroD1. NeuroD1-immunoreactivity was observed in pinealocytes and in a subpopulation of vimentin-positive astrocytes. Whereas at ZT14 NeuroD1 was mainly nuclear, at ZT6 there was a weaker signal in the cytoplasm. After SCGx, a heterogeneous pattern of subcellular NeuroD1-immunostaining was observed among pinealocytes at ZT14. These data were confirmed by WB. Aanat E-Box-containing probe was able to bind proteins from pineal nuclear extracts; NeuroD1 may form part of these complexes, according to supershift assays with an anti-NeuroD1 antibody. These results suggest that NeuroD1 protein dynamics are under circadian control and that NeuroD1 modulates Aanat gene expression in the adult rat pineal gland.