NeuroD1 protein in the rat pineal gland.

A.E. CASTRO; S.G. BENITEZ; L.E. SAVASTANO; M.J. OCHOCINSKA; J.L. WELLER; D.C. KLEIN; E.M. MUÑOZ

Galveston, TX

Congreso; Gordon Research Conference on Pineal Cell Biology; 2012

Gordon Research Conference

NeuroD1/BETA2 is a bHLH transcription factor that functions in neuronal and endocrine tissues, including the pineal gland and retina. NeuroD1 orchestrates complex regulatory programs through heterodimerization with promiscuous members of the E protein family and subsequent binding to E-box-like DNA consensus sequences in target genes. The fact that NeuroD1 has been identified in areas with strong circadian phenotypes (e.g. retina and pineal gland) makes NeuroD1 a potential molecular player in circadian biology. Studies using two murine NeuroD1 knockout (KO) models (Muñoz et al., 2007; Ochocinska et al., 2009; 2011) indicate that the pineal gland is present in both total KO and Cre-LoxP conditional KO mice, with subtle morphological changes. Pineal transcriptome analyses at neonatal and adult stages identified a defined number of affected genes linked to transcription and signal transduction, in addition to other processes. Interestingly, loss of NeuroD1 had more dramatic morphological effects in the retina than in the pineal gland in both models, suggesting that NeuroD1 function might be more modulatory than essential in the pineal gland, as compared to the retina. To further characterize the role of NeuroD1 in pineal biology, we focused our study on NeuroD1 protein dynamics. To achieve this goal a polyclonal anti-NeuroD1 serum was developed and NeuroD1 protein abundance was characterized throughout pineal development and at multiple times during a 24-hour period via immunohistochemistry, confocal microscopy, and WB analysis. Cells were characterized using antisera against S-antigen, vimentin, GFAP, protein Go and glutamic acid decarboxylase 1 (GAD1). NeuroD1-like immunoreactivity was detected in S-antigen-positive pinealocytes and vimentin-positive/GAD1-negative interstitial cells. A few vimentin- and GAD1-positive astrocyte-like cells were nuclear NeuroD1-negative. In adult rats, NeuroD1 protein levels were 2- to 3-fold higher at night (ZT14; two hours after lights off), as compared with daytime values. Analysis of the subcellular location of NeuroD1 in pinealocytes revealed a rhythmic pattern with higher nuclear presence at the beginning of the night, coincident with the nocturnal activation of the pinealocytes by noradrenaline released from sympathetic nerves. NeuroD1 nuclear-cytoplasmic trafficking was disrupted by surgical elimination of these nerves, indicating that these changes are under neural control. NeuroD1 protein is known to be phosphorylated; here we found via WB analysis that pSer(336)-NeuroD1 exhibited a nuclear-cytoplasmic trafficking pattern similar to that of NeuroD1 total protein. Phosphorylation of this specific amino acid residue might not be influencing the nuclear import of NeuroD1 but it might be stabilizing the protein and facilitating heterodimer formation.