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. 2019 Sep;150(6):666-677.
doi: 10.1111/jnc.14821. Epub 2019 Jul 31.

Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons

Armaz Aschrafi  1 , Adama Berndt  1 , Jeffrey A Kowalak  2 , Jenna R Gale  1 , Anthony E Gioio  1 , Barry B Kaplan  1
Affiliations

Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons

Armaz Aschrafi et al. J Neurochem. 2019 Sep.

Abstract

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.

Keywords: axon; catecholamine; mRNA trafficking; superior cervical ganglia neurons; sympathetic nervous system; tyrosine hydroxylase.

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Conflict of interest statement

Conflicts of Interest

The authors declare no competing financial interests

Figures

Fig. 1.
Fig. 1.. Ang II Elevates the Axonal Levels of TH and DBH mRNA in SCG Neurons.
Quantification of TH mRNA levels in the distal axons (A) and soma (B), DBH mRNA levels in the distal axons (C) and soma (D) of SCG neurons treated with either DMSO (control), L-162,313, or Eprosartan. The y-axis of Fig. 1D starts at 0.2 relative levels of DBH mRNA. TH and DBH mRNA levels were determined by qRT-PCR 2 days after treatment, using total RNA samples prepared from SCG axons and soma and gene-specific primers for TH and DBH. The relative levels of TH and DBH transcripts were normalized to β-actin mRNA to provide an internal control for reverse transcription and axonal density. Data are the mean ± SEM (n=3 independent cell culture preparations). One-way Anova, ***, p ≤ 0.0001.
Fig. 2.
Fig. 2.. Ang II Elevates the Axonal Levels of TH and DBH mRNA containing puncta in SCG Neurons.
In situ hybridization analysis of single axons from dissociated primary SCG neurons grown in monolayer cell culture. In axons hybridized with TH-specific (A) or DBH-specific (B) riboprobes, TH and DBH mRNAs appear as discrete puncta, whereas L-162,313 treatment of neurons for 2 days enhances both TH and DBH puncta in the distal axons of primary SCG neurons. Pre-treatment of SCG neurons with the Ang II antagonist, Eprosartan, followed by L-162,313 treatment reduced the number of axonal TH and DBH puncta to control levels. Arrows, denote TH and DBH mRNA puncta in the axon. C, D. The number of TH or DBH mRNA containing granules is increased in L-162,313 treated SCG neurons as compared to control and Eprosartan-treated neurons. Data are mean ± SEM from the measurement of 30–35 axons from 4 independent preparations. TH mRNA containing puncta are measured as a function of axon length. The experiment was repeated three times with similar results. One-way Anova, ***, p ≤ 0.0001.
Fig. 3.
Fig. 3.. Ang II enhances the Axonal Levels of TH Protein in SCG Neurons.
A. TH protein levels were measured using immunocytochemistry in axons and soma of SCG neurons treated either with DMSO (Control), L-162,313, or Eprosartan. Increased TH levels are detected in axons of L-162,313-treated neurons grown in Campenot chambers. Arrows, denote axonal and somal TH. B, C. Fluorescence intensity as a measure of TH levels in axons and soma of SCG neurons were quantified using ImageJ, and fluorescence levels are provided as relative fluorescence intensity. Data are mean ± SEM from the measurement of 8–10 axons and 8–10 SCG ganglia from 3 independent preparations. One-way Anova, *, p ≤ 0.05. The y-axis of Fig. 3C starts at 400,000 relative fluorescent intensity. D. Western-blot analysis of axonal protein lysates from SCG neurons treated either with DMSO (Control), L-162,313, or Eprosartan. GAPDH was used as a loading control. E. TH Immunoblots of axonal protein lysates of SCG neurons were quantified using ImageJ. Quantification showed significant elevation of TH protein levels in axons 2 days after L-162,313 treatment of SCG neurons grown in Campenot chambers, whereas TH levels in Eprosartan treated neurons remained unchanged. TH band intensities were normalized to the protein levels detected for GAPDH. One-way Anova, *, p ≤ 0.05.
Fig. 4.
Fig. 4.. Ang II Enhances the Axonal Levels of DBH Protein in SCG Neurons.
A. DBH protein levels were measured using immunocytochemistry in axons and soma of SCG neurons treated with either DMSO (Control), L-162,313, or Eprosartan. Increased DBH levels are detected in axons of ANGII agonist-treated neurons grown in Campenot chambers. Arrows, denote axonal DBH. B, C. The y-axis of Fig. 4C starts at 450,000 relative fluorescent intensity. Fluorescence intensity as a measure of DBH levels in axons and soma of SCG neurons were quantified using ImageJ, and fluorescence levels are provided as relative fluorescence intensity. Data are mean ± SEM from the measurement of 8–10 axons and 8–10 SCG ganglia from 3 independent preparations. One-way Anova, ***, p ≤ 0.0001.
Fig. 5.
Fig. 5.. Enhanced axonal transport of TH and DBH mRNA increased axonal catecholamine levels in SCG axons.
A. DA and NE levels were measured using immunocytochemistry in axons and soma of SCG neurons treated with either DMSO (Control), L-162,313, or Eprosartan. Arrows, denote axonal and soma DA and NE. B-E. Fluorescence intensity as a measure of DA (B, D) and NE (C, E) levels was quantified in the soma and distal axons of SCG neurons, respectively, using ImageJ, and fluorescence levels are indicated as relative fluorescence intensity. Enhanced DA and NE levels are detected in distal axons of ANGII-treated neurons, whereas the parental soma DA and NE levels remained at control levels. Data are the mean ± SEM from the measurement of neurons cultured in 6 Campenot chambers from three independent experiments. One-way Anova, **, p ≤ 0.001. F. Distal axons located in the lateral compartment of Campenot chambers were treated for 10 min with 100 mM KCl. NE release into culture media was subsequently measured using an ELISA immunoassay, and NE concentration in the culture media was calculated using linear regression analysis. Elevated NE levels are detected in distal axons of L-162,313 neurons, as compared to NE levels measured in the axons of control and Eprosartan neurons. Values are mean ± SEM of four different experiments each from the measurement of 8–10 axons and 8–10 SCG ganglia. One-Way ANOVA analysis ***, P ≤ 0.0003
Fig. 6.
Fig. 6.. A. DBH mRNA co-purifies with TH mRNA in the axons of SCG neurons using a TH zipcode-affinity immune-precipitation assay.
RT-PCR analyses were conducted on TH zipcode affinity purified RNA fractions derived from the axonal lysates to detect the presence of TH mRNA. DBH mRNA co-purified with TH mRNA using the TH zipcode as bait, whereas the sodium channel mRNA SCN1A was not co-purified using the TH or scrambled oligomers used as a negative control (A). Treatment with L-162,313 enhanced the association of TH and DBH mRNAs as shown by enhanced PCR band intensity on the agarose gel. This experiment was repeated at least 4 times with highly reproducible outcomes. B. RT-PCR analyses of the presence of SCN1A mRNA in the axons and soma of SCG neurons indicates that this message is present in both neuronal compartments and can be detected in the RT-PCR assay. C., D. TH and DBH cDNA bands as shown in Fig. 6.A. were quantified using ImageJ. Quantification showed significant elevation of axonal TH mRNA (C) and DBH mRNA (D) levels after L-162,313 treatment of SCG neurons grown in Campenot chamber. TH and DBH band intensities were normalized to the corresponding levels of axonal mRNA levels detected in control cells. Student’s t-test, *, p ≤ 0.0004.
Fig. 7.
Fig. 7.. A Venn diagram showing the overlap between the TH zipcode affinity purified proteins immuno-purified from the axons and soma of SCG neurons.
The number of proteins shown represent those associated with TH zipcode in the axon and soma of SCG neurons. The proteins associated with both axons and soma immuno-precipitated RNPs are shown in the intersection of the Venn diagram.
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