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Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

ABSTRACT: Human norepinephrine (NE) deficiency (or dopamine ?-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T?C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T?C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

SUBMITTER: Kim CH

PROVIDER: S-EPMC3059068 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Kim Chun-Hyung CH Leung Amanda A Huh Yang Hoon YH Yang Eungi E Kim Deog-Joong DJ Leblanc Pierre P Ryu Hoon H Kim Kyungjin K Kim Dong-Wook DW Garland Emily M EM Raj Satish R SR Biaggioni Italo I Robertson David D Kim Kwang-Soo KS

The Journal of biological chemistry 20110105 11

Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-de ...[more]

PMID: 21209083

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Project description:In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine β-hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.

Project description:Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. unc13d-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including Atg9b. Downregulation of TFEB decreased Atg9b levels and decreased macroautophagy in unc13d-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders. Abbreviations ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type.

Dataset Information

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Publications

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

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