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, 6 (9), e23866

The Neurotropic Parasite Toxoplasma Gondii Increases Dopamine Metabolism

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The Neurotropic Parasite Toxoplasma Gondii Increases Dopamine Metabolism

Emese Prandovszky et al. PLoS One.

Abstract

The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Dopamine in tissue cysts of T. gondii in brain tissue sections.
(A) Dopamine was detected in brain tissue sections of chronically infected Swiss Webster mice by immunohistochemical staining with anti-dopamine antibody and horseradish peroxidase. Tissue cysts containing hundreds of bradyzoites are visible as brown circular structures (arrowheads) in infected brains. The bottom right panel is a control lacking anti-dopamine antibody. All black bars are 10 µm long. (B) Localization by indirect immunofluorescence of brain sections stained with anti-dopamine antibody (green), DAPI (blue), and TRITC-lectin (red). Three sections are shown from different regions of the brain in the top, middle and bottom rows of panels with the negative control (no primary antibody) in the bottom row. In each series all three channels are illuminated (left), the anti-dopamine and lectin channels are illuminated (center), and only the anti-dopamine channel is illuminated (right). The DAPI identifies neural cells and the individual bradyzoites within the tissue cyst and the lectin stains the surface of the cyst. The dopamine staining appeared specific (also see Fig. 2) as the antibody stained neurons in the striatum, amygdala and hippocampus. (C) A 3D projection of a Z-stack reconstruction of serial images of a tissue cyst within a brain section stained with anti-dopamine antibody and lectin as described in B. Control without the primary anti-dopamine antibody is shown in the right panel.
Figure 2
Figure 2. Specificity of dopamine staining T. gondii tissue cysts.
(A) Histochemical (glyoxylic acid) staining of dopamine in brain sections from chronically-infected mice detected by fluorescence. Glyoxylate reacts with dopamine to fluoresce blue-white . Cells containing T. gondii cysts in brain tissue exhibited blue-white fluorescence. The tissue cysts stained darkly, similar to mouse cell nuclei, presumably due the high density of bradyzoites. (B) Brain tissue sections from chronically-infected mice were stained with indirect fluorescein staining as in Fig. 1 except the anti-dopamine primary antibody was incubated in the presence of 50 µg/ml dopamine (top) and 50 µg/ml serotonin (botto). From left to right: bright field, fluorescein only channel (green), fluorescein and lectin-TRITC (green and red channels, respectively), and both channels plus bright field. Serotonin did not compete for dopamine staining.
Figure 3
Figure 3. Elevated dopamine from T. gondii infected dopaminergic cells.
(A) Overlay of HPLC-ED chromatograms derived from PC12 cells DA release assay, where cells were infected with increasing numbers of induced tachyzoites. PC12 cells are the classic dopaminergic neuron model since they contain all the machinery for dopamine synthesis, packaging and release. Equivalent numbers of cells were infected with T. gondii (brown, 7.5×105; yellow, 5×105; blue, 2.5×105; and black, control) and incubated for 5 days followed by assaying DA release in high K+buffer. Increased dopamine was released from infected cultures. The amount of dopamine released is correlated with number of parasites in the culture. The experiment was repeated several times (n = 4) with a representative experiment shown. (B) Graph of dopamine released from the K+ induced cultures (squares) described in A. The total dopamine measured in each of the cultures is shown (circles). The dopamine measured in the low KCl wash buffer for each culture is also plotted (triangles).
Figure 4
Figure 4. Dopamine enzyme tyrosine hydroxylase in intracellular T. gondii.
(A) Immunohistochemical localization of tyrosine hydroxylase (TH) in brain sections of chronically-infected mice with commercial antibody and horseradish peroxidase labelling. Tissue cysts are visible as brown circular structures (left, four cysts, and right, single cyst, highlighted with arrowheads). (B) TH in intracellular parasites in vitro. Alkaline-induce parasite cultures were probed with anti-tyrosine hydroxylase antibody (green), RFP-GRASP (red), and DAPI (blue) shown separately and as a composite image. Scale bars on all images are 10 µM.
Figure 5
Figure 5. Expression of a parasite-encoded tyrosine hydroxylase in brain tissue cysts.
(A) 3D projections of serial images of T. gondii tissue cysts within brain sections were triple stained with T. gondii encoded tyrosine hydroxylase (TgTH) antibody (green), DAPI (blue), and lectin (red). The panels (from left to right) show all three channels, the lectin and antibody, and TgTH antibody alone (green). Staining was not apparent in control sections that received only secondary antibody (data not shown). DAPI identified neuronal cells and the individual bradyzoites within the tissue cyst and lectin stained the surface of the cyst. (B) Western analysis for specificity of the custom antibody for TgTH. Recombinant protein from Δ29TgAaaH2 and mouse brain were probed with TgTH antibody. No bands were detected in uninfected mouse brain. β-actin was used as a loading control.

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