Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons

Abstract

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur that maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or it is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this study, we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3Ј untranslated region (UTR) of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3ЈUTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions.

MicroRNA‐432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes

“…Hence, we used two reagents DC and RA that are known to induce dopaminergic and cholinergic neurons respectively [37][38][39][40]. MiRNAs that are altered in presence of two reagents may have some therapeutic roles for treatment of neurodegenerative diseases.…”

cAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

“…Using polysome distribution assays, our results suggest that mechanisms regulating TH mRNA translation may be rate-limiting for TH protein synthesis and that the lack of induction of adrenal TH protein after short-term stress is due to inadequate activation or inhibition of TH mRNA translation. More recently, we have shown regulation of TH mRNA translation by cAMP in rat midbrain slice explant cultures and mouse MN9D cells (Chen et al, 2008). In this latter report, we have shown that treatment with the cAMP analog, 8-CPT-cAMP, or forskolin leads to induction of TH protein without concomitant induction of TH mRNA.…”

“…Firefly luciferase activity was expressed as relative light units for firefly luciferase normalized to relative light units for R. reniformis luciferase for each well of cells. TH enzyme assays and immunoblotting for TH protein were performed as described previously (Chen et al, 2008). For detection of PCBP isoforms, pellets of MN9D cells were homogenized in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) with freshly added protease inhibitor cocktail (Sigma, St. Louis, MO) and incubated on ice for 30 min.…”

“…These cells express high levels of TH mRNA and TH protein under basal culture conditions. MN9D and PC12 cells were cultured as described previously (Nagamoto-Combs et al, 1997;Chen et al, 2008). For transfections, MN9D cells were cultured in 6-or 12-well dishes and transfected with 1 to 2 g of TH (3ЈU)-Luc plasmid DNA and 0.033 to 0.066 g of Renilla reniformis luciferase expression vector RL-SV40 (Promega, Madison, WI) using Lipofectamine (Invitrogen, Carlsbad, CA) as described by the manufacturer.…”

“…The TH (3ЈU)-Luc and modified GL3 plasmids were constructed as described previously (Chen et al, 2008). The deletion mutant constructs TH (3ЈU⌬AS)-Luc, TH (3ЈU⌬KA)-Luc, and TH (3ЈU⌬KS)-Luc were generated by excising the TH mRNA 3Ј-UTR sequences from ApaI to SphI, KpnI to ApaI, or KpnI to SphI from TH (3ЈU)-Luc, respectively, followed by bluntending and religation.…”