Downregulation of the Central Noradrenergic System by Toxoplasma gondii Infection

Abstract

Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro The mechanism responsible for the NE suppression was found to be downregulation of dopamine β-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

Keywords: apicomplexan parasites; behavior; bradyzoite; host-pathogen interactions; intracellular parasites; norepinephrine; protozoa.

FIG 1

Catecholamine levels with T. gondii infection in the brain and catecholaminergic cells. (A) A graph of the norepinephrine concentration in the brains of uninfected and infected rats, with each point representing one animal and bars showing the means ± SEM (P = 0.0019, Student’s t test; n = 6 infected and 6 mock-infected animals). (B) Dopamine levels in the brains of the same uninfected and infected rats shown graphically (P = 0.12, Student’s t test). (C) Norepinephrine levels in uninfected and infected catecholaminergic PC12 cells at day 5 of infection (P = 0.0024, n = 3 biological replicates). (D) Levels of dopamine in the same infected PC12 cells, plotted as described for panel B. P = 0.0043, n = 3 biological replicates with triplicate readings. (E) Overlay of chromatograms from HPLC-ED of uninfected and infected PC12 cells.

FIG 2

Norepinephrine biosynthesis in catecholaminergic cells with T. gondii infection. (A) Dopamine and norepinephrine biosynthetic pathway showing synthesis from tyrosine. DBH, dopamine β-hydroxylase; AADC, aromatic amino acid decarboxylase (also DDC); TH, tyrosine hydroxylase. Reactions in which dopamine and/or norepinephrine are bound (e.g., dopamine receptor D1 [DRD1], dopamine receptor D2 [DRD2]) or degraded (e.g., monoamine oxidase A [MaoA]) are not included in this schematic. (B) Expression of the set of catecholaminergic genes during infection (black) or without infection (gray). Only DBH gene expression was significantly altered by infection (n = 3 biological replicates with triplicate readings; ***, P = 0.008). PAH, phenylalanine hydroxylase. Error bars are ±SEM. (C) Dopamine β-hydroxylase mRNA levels during a time course of infection (black) relative to levels for uninfected (gray) PC12 catecholaminergic cells and a rat housekeeping gene. ***, P = 0.0046, n = 3 biological replicates. (D) Plot of the level of DBH mRNA in human BE(2)-M17 neuronal cells over a time course of infection relative to human GAPDH, showing that T. gondii induces DBH downregulation in rat and human neuronal cells. **, P = 0.0010; ***, P = 0.00032; n = 3 biological replicates.

FIG 3

Infection downregulates dopamine β-hydroxylase gene expression in the brain. (A) DBH gene expression in the brains of uninfected (gray) and chronically infected (black) male rats plotted relative to GAPDH (P = 0.0023; n = 4 uninfected and 5 infected animals). (B) Brain region-specific DBH gene expression in uninfected and infected rats in the prefrontal cortex (PFC) (P = 0.0034), midbrain, and pons/locus coeruleus (LC) (P = 0.012). Error bars are ±SEM. n = 4 uninfected and infected animals. (C) Plot showing expression of the neuronal MAP2 gene (as a percentage of GAPDH) in uninfected (gray) and chronically infected (black) brains for the animals shown in panel A (P = 0.57).

FIG 4

Dopamine β-hydroxylase expression was not suppressed in infected females. (A) Plot of DBH mRNA in the brains of uninfected (gray) and chronically infected (black) female rats (±SEM; n = 3 uninfected and 5 infected animals; P = 0.45). (B) Expression of estradiol receptor 1 (ESR1) gene in brains of the same female rats shown graphically (±SEM; P = 0.40). (C) Correlation of DBH versus ESR1 gene expression in the brains (Pearson’s correlation coefficient, 0.86).

FIG 5

Dopamine β-hydroxylase suppression is pathogen specific. (A) Plot of DBH gene expression over a time course of 48 h. Uninfected (gray) and human cytomegalovirus-infected (black) human BE(2)-M17 neuronal cell line, shown as a percentage of the housekeeping gene; n = 2 biological repeats of triplicate measures. (B) Accumulation of HCMV UL123 IE as percent gene expression (normalized to GAPDH) over a time course. (C) Plot shows DBH expression over a similar time course for uninfected (gray) and T. gondii-infected (black) human neuronal cells as a percentage of the housekeeping gene. **, P = 0.0015; ***, P = 0.0012; n = 3 biological repeats with triplicate measures; error bars indicate SEM. (D) The intensity of T. gondii infection over the time course based on levels of T. gondii actin plotted as a percentage of host GAPDH.

FIG 6

Locomotion and anxiety-related behaviors are altered in infected animals. (A) Mean ambulation of uninfected (gray) and infected (black) mice in the open field at single-minute time points. **, P = 0.0015; ***, P = 0.000097; n = 24 uninfected and 27 infected mice. (B) Graph of distance moved for each mouse over the 15-min time course of the experiment plotted as a box plot, with whiskers representing minimum and maximum at single-minute time points followed by 5-min time points. **, P = 0.0015; ***, P = 0.000097. (C) Tracking in the open field for representative uninfected (top) and infected (bottom) mice from 0 to 180 s in the trial.