Phox2a and Phox2b are two homeodomain transcription factors playing a pivotal role in the development of noradrenergic neurons during the embryonic period. However, their expression and function in adulthood remain to be elucidated. Using human postmortem brain tissues, rat stress models and cultured cells, this study aimed to examine the alteration of Phox2a and Phox2b expression. The results show that Phox2a and Phox2b are normally expressed in the human locus coeruleus (LC) in adulthood. Furthermore, the levels of Phox2a protein and mRNA and protein levels of Phox2b were significantly elevated in the LC of brain donors that suffered from the major depressive disorder, as compared to age-matched and psychiatrically normal control donors. Fischer 344 rats subjected to chronic social defeat showed higher mRNA and protein levels of Phox2a and Phox2b in the LC, as compared to non-stressed control rats. In rats chronically administered oral corticosterone, mRNA and protein levels of Phox2b, but not Phox2a, in the LC were significantly increased. In addition, the corticosterone-induced increase in Phox2b protein was reversed by simultaneous treatment with either mifepristone or spironolactone. Exposing SH-SY5Y cells to corticosterone significantly increased expression of Phox2a and Phox2b, which was blocked by corticosteroid receptor antagonists. Taken together, these experiments reveal that Phox2 genes are expressed throughout the lifetime in the LC of humans and Fischer 344 rats. Alterations in their expression may play a role in major depressive disorder and possibly other stress-related disorders through their modulatory effects on the noradrenergic phenotype.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.