ASL Metabolically Regulates Tyrosine Hydroxylase in the Nucleus Locus Coeruleus

Abstract

Patients with germline mutations in the urea-cycle enzyme argininosuccinate lyase (ASL) are at risk for developing neurobehavioral and cognitive deficits. We find that ASL is prominently expressed in the nucleus locus coeruleus (LC), the central source of norepinephrine. Using natural history data, we show that individuals with ASL deficiency are at risk for developing attention deficits. By generating LC-ASL-conditional knockout (cKO) mice, we further demonstrate altered response to stressful stimuli with increased seizure reactivity in LC-ASL-cKO mice. Depletion of ASL in LC neurons leads to reduced amount and activity of tyrosine hydroxylase (TH) and to decreased catecholamines synthesis, due to decreased nitric oxide (NO) signaling. NO donors normalize catecholamine levels in the LC, seizure sensitivity, and the stress response in LC-ASL-cKO mice. Our data emphasize ASL importance for the metabolic regulation of LC function with translational relevance for ASL deficiency (ASLD) patients as well as for LC-related pathologies.

Keywords: ASL; locus coeruleus; nitric oxide; stress response; tyrosine hydroxylase; urea cycle disorders.

Graphical abstract

Figure 1

ASL Is Highly Expressed in the LC and Regulates TH Levels (A) Left: in situ hybridization with Asl anti-sense mRNA probe showing in purple Asl prominent expression in the LC. Right: scheme of brain stem coronal section is shown. LC region is highlighted in purple (image adapted from The Mouse Brain Atlas). (B) Immunostaining of mouse brainstem for Asl (left, red), TH (center, green), and their merged co-localization (right). (C) Immunostaining of human brainstem for ASL (left, red), TH (center, green), and their merged co-localization (right). (D and E) Quantification of Asl mRNA (D) and TH mRNA (E) isolated by laser microdissection from the LC of Asl^f/f;TH Cre^+/− and from Asl^f/f control mice as measured by RT-PCR with specific TaqMan probes (n = 100 cells from 7 animals). (F) Immunohistochemistry quantification of TH protein normalized to cells number (n = 4 in each group). (G) Quantification of western blots for TH levels in LC regions taken by punch biopsies (n = 4 in each group). The bottom panels of (F) and (G) show representative images for each detection method, respectively. Data represent mean ± SEM (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001; ns, not significant). See Tables S1, S2, and S3 for RNA sequencing, ingenuity pathway analysis, and proteomic analysis, respectively.

Figure 2

ASL Regulates TH Levels by Regulating NO Availability (A) NO levels measurement using DAF-FM probe demonstrates significantly reduced NO levels in the LC of Asl^f/f,Ai9^f/f;TH Cre^+/− mice as compared to Ai9^f/f;TH Cre^+/− control mice (each experiment was performed with pair of control and Asl^f/f,Ai9^f/f;TH Cre^+/− mice; n = 3). (B) Knockdown of ASL in SH-SY5Y neurons using three different lentiviral ASL-shRNA clones associates with downregulation in TH mRNA levels. (C) shASL SH-SY5Y neurons have significantly reduced nitrosylation of GAPDH (left) and of TH (right; measurement was performed ≥3 times). (D) NO donors’ supplementation to SH-SY5Y neurons rescues the differences in GAPDH (left) and TH (right) nitrosylation between shASL and shGFP control neurons. (E) Treatment of shASL neurons with NO donors normalizes TH mRNA levels to that of shGFP control neurons (n ≥ 3; one-way ANOVA with Bonferroni). Data represent mean ± SEM (^∗p < 0.05; ^∗∗p < 0.01). See also Figure S3.

Figure 3

ASL Deficiency Associates with Decreased Catecholamine Synthesis (A) Measurement of LC catecholamine levels show alterations in quantity and in turnover rates in both dopamine (left) and in norepinephrine (right), in Asl^f/f;TH Cre^+/− as compared to Asl^f/f control mice (n ≥ 10 mice in each group). (B) Measurements of norepinephrine levels in the hippocampus following stress show decreased elevation in Asl^f/f;TH Cre^+/− as compared to Asl^f/f control mice (n ≥ 9 mice in each group). (C) Measurements of LC norepinephrine levels of mice treated with NaNO₂ show no difference between Asl^f/f;TH Cre^+/− and Asl^f/f control mice (n ≥ 5 mice in each group). Data represent mean ± SEM (non-repeated-measures two-way ANOVA with Bonferroni post hoc t tests; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001).

Figure 4

ASL Deficiency in the LC Associates with Increased Firing from LC Neurons (A) Representative recordings of spontaneous action potentials of LC neurons. Upper panel —Asl^f/f control, showing typical inter-spike interval variability; lower panel—Asl^f/f;TH Cre^+/− (frequency 7.9 ± 0.6 Hz in Asl^f/f control; 13.8 ± 2.0 Hz in Asl^f/f;TH Cre^+/−; p < 0.01). (B) Averaged normalized inter-spike interval (ISI) histograms. (Left) Asl^f/f control n = 14 is shown; (right) Asl^f/f;TH Cre^+/− n = 15 cells are shown; red, gamma distribution probability density function fit with shape parameters 26 and 60 and scale parameters 5.9 and 1.7, respectively. ISI distributions from single recordings were also fit with gamma distribution (data not shown) with shape parameters 31 ± 6 and 122 ± 37 (p < 0.03) and scale parameters 8.4 ± 2.7 and 2.4 ± 0.8 (p < 0.04) in Asl^f/f control and Asl^f/f;TH Cre^+/−, respectively. (C) Spiking frequency presented as mean ISI (left; 144 ± 17 ms in Asl^f/f control [n = 14 cells from 3 animals] and 93 ± 13 ms in Asl^f/f;TH Cre^+/− [n = 15 cells from 3 animals] p < 0.03) and SEM. ISI as a measure of ISI variability (right; SEM ISI 33 ± 6 ms in Asl^f/f control and 14 ± 3 ms in Asl^f/f;TH Cre^+/−; p < 0.02). (D) Examples for averaged spike (first 10 spikes from a recording per cell), showing the “landmarks” used for analyzing the spike shape (red asterisks: spike threshold; spike peak; and after-hyperpolarization). Red line, AHP recovery slope. (E) Firing frequency and after-hyperpolarization (AHP) recovery slope scatterplot and per-cell linear regression plot: Ct ● y = 15x + 5; r2 = 0.25; dashed line; cKO ♦ y = 61x − 2; r2 = 0.79; solid line. (F) After hyperpolarization current properties; left to right, amplitude (ahpAmpl −19.5 ± 0.7 mV n = 13 and −19.0 ± 1.0 mV n = 14; n.s.) and recovery slope (ahpSlope 0.18 ± 0.01 mV/ms n = 13 and 0.23 ± 0.02 mV/ms n = 14; p < 0.05). In Asl^f/f;TH Cre^+/− group, this slope is correlated with firing frequency (data not shown; correlation coefficient 0.79; p < 0.001; Pearson correlation), whereas in the Asl^f/f control cells, this correlation was not observed (0.25; p = 0.41). Other passive and active properties of LC neurons were not significantly different between the groups (data not shown; resting membrane potential −48.9 ± 0.7 mV n = 12 and −48.6 ± 0.9 mV n = 15; input resistance 113 ± 7 MOhm n = 13 and 121 ± 11 MOhm n = 15; spike threshold −39.8 ± 0.9 mV n = 12 and −38.6 ± 1.0 mV n = 15; spike amplitude 83.1 ± 2.2 mV n = 14 and 86.1 ± 2.5 mV n = 15; spike width 2.09 ± 0.08 ms n = 14 and 2.04 ± 0.06 ms n = 15 in Asl^f/f control and Asl^f/f;TH Cre^+/−, respectively).

Figure 5

Phenotypic Consequences of ASL Deficiency in LC (A) Measured mean arterial pressure (MAP) levels following induction of acute stress by exposure to intruder mice. The increase in blood pressure levels following stress is higher in Asl^f/f;TH Cre^+/− mice and requires a longer recovery time as compared to Asl^f/f control and to endothelial-ASL cKO mice (n ≥ 8 in each group; repeated-measures two-way ANOVA with Holm-Sidak’s post hoc t tests). (B) Continuous blood pressure measurement during the first 3 h after stress induction demonstrates a significantly higher blood pressure in Asl^f/f;TH Cre^+/− mice as compared to Asl^f/f control mice (n ≥ 8 in each group). (C and D) Open-field test locomotor evaluation. Following stressful stimuli, Asl^f/f;TH Cre^+/− mice present increase locomotor activity as indicated in distance (C) and speed (D), in comparison to Asl^f/f control mice. Following treatment with NO donors, Asl^f/f;TH Cre^+/− mice response to stressful stimuli is similar to that of Asl^f/f control mice (n ≥ 11 in each group; non-repeated-measures two-way ANOVA with Bonferroni post hoc t tests). (E) Seizure score after injection of 50 mg/kg PTZ is higher in Asl^f/f;TH Cre^+/− mice as compared to Asl^f/f control mice. Following treatment with NO donor, there was no significant difference in the seizure reaction to injection between the two genotypes (n = 6 in each group). (F) Kaplan Meier estimator presenting the probability of mice to develop myoclonic jerks from the time of PTZ injection. Asl^f/f;TH Cre^+/− mice have more than 4-fold chance of developing myoclonic jerks as compared to Asl^f/f control mice. Data represent mean ± SEM (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). See also Figure S4.