Abstract
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-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).
Highlights
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
Sequence analysis revealed that the patient is a compound heterozygote for a novel, previously unidentified missense mutation and the same splicing mutation in intron 1 that was identified in other patients (Fig. 1A) [12]
Expression and Characterization of Wild-type and Mutant DBH in Chinese hamster ovary (CHO) Cells—We investigated the potential functional effects of the missense mutations identified in NE-deficient patients
Summary
Genomic DNA Analysis—The region containing both the 12 exons and intron-exon boundaries of the DBH gene was PCRamplified. Cells were washed with PBS, and the chase was performed by adding 1 ml/well DMEM supplemented with 5% dialyzed FBS, containing a 10-fold excess of methionine and a 5-fold excess of cysteine. Cells were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 2% NDS. Immunogold Electron Microscopy—PC12 cells infected by retrovirus expressing WT or A348E mutant DBH were fixed for 1 h at 4 °C in PBS containing 0.1% glutaraldehyde, 4% formaldehyde, and 3.5% sucrose. Grids were incubated for 2 h at room temperature in a humidified chamber on 50-l droplets of anti-rabbit ␣-Myc antibody appropriately diluted in solution B (solution A but with 1% normal goat serum), followed by rinses in solution B. Equal sample volumes were loaded onto SDS-PAGE, and secretion was determined by Western blotting analysis with anti-Myc antibody. DBH enzyme activity was determined by the photometric method of Nagatsu and Udenfriend [15] using tyramine as substrate
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
