Reproducing Increased Dopamine with Infection To Evaluate the Role of Parasite-Encoded Tyrosine Hydroxylase Activity

LETTER

In a recent publication, Wang et al. (1) commented on their inability to reproduce findings from a prior publication. In that study, a sophisticated gene knockout was performed, disrupting one of the two copies of the Toxoplasma gondii AAH gene to study its biological roles. Further strains were engineered that contained the disrupted gene, AAH2, reintroduced and incorporated into the genome. Although the study was conducted by a laboratory well established in Toxoplasma biology and a major contributor to our knowledge of the biology of T. gondii, it is our belief that variation of the study design led to the different results of the studies of Wang et al. and Prandovszky et al. (2).

Prior work has found altered dopamine neurotransmission with T. gondii infection (2–5). In the study of Wang et al., dopaminergic PC12 cell cultures were incubated at alkaline pH to examine dopamine levels in bradyzoite-infected cells, a standard procedure used to induce parasite conversion in fibroblasts, yet the synthesis of dopamine in PC12 cells is sensitive to pH changes (6). In prior work by Prandovszky et al., numerous precautions were taken to ensure that PC12 cells were competent to express high levels of dopamine and to circumvent the problematic effect of high pH on PC12 production of dopamine. These include maintaining a low passage number (7) and preincubating liberated tachyzoites under conditions that induce bradyzoite conversion prior to infection of PC12 cells so that the culture pH remains neutral. We did not see the same precautions taken in the study by Wang et al. In brief, we incubated liberated parasites for 16 to 18 h under conditions that stress the parasites (alkaline pH medium, low serum and CO₂ concentrations) for preinduction of bradyzoite stage conversion prior to host cell infection and then assayed the dopamine content on the 5th day of infection. However, in the study by Wang et al. the infected culture was incubated for 48 h and then assayed for dopamine content. Significant differences between the protocols would likely contribute to the different outcomes. Indeed, in the study by Wang et al., the high-pH medium decreased dopamine levels by 25-fold in uninfected cells (Table 1). Hence, it is to be expected that these pH-shocked cells do not show changes in dopamine expression with infection. Also, without viability assay data, it cannot be excluded that the 25-fold dopamine decrease could be due to cell death caused by alkaline pH. This may explain why the positive control did not reproduce previously published work showing that T. gondii infection can increase levels of released and total dopamine in PC12 cells severalfold (2). Infection of PC12 cells with tachyzoites under neutral pH conditions did not elevate dopamine levels, similar to our findings (unpublished observations).

Additionally, there are parasite strain-dependent differences in the effects on dopamine and behavior (2, 8). Hence, it is essential to prove that the strain used in this study, PruΔku80Δhxg, induces an increase in dopamine prior to gene knockout studies. An increase in dopamine in brains of infected mice was also not reproduced as in previously published studies (3–5). Furthermore, Wang et al. used a different outbred mouse line for their in vivo study than that reported by Prandovszky at al. Host genetic differences can contribute to decreased susceptibility to T. gondii infection (9).

The abstract of reference 1 states that expression levels were “negligible,” yet the transcript levels for several strains in genomic analyses of ToxoDB by different research groups show T. gondii AAH expression in line with important metabolic enzymes (e.g., central pyrimidine enzyme OMPDC-OPRT) although considerably lower than transcript levels for structural proteins. Also, the authors show levels greater than those of lactate dehydrogenase 2 in Fig. 3. Hence, T. gondii AAH is expressed in the range of other metabolic enzymes.

Lastly, with two gene copies that are 97.5% identical, interpretation of the effects of disrupting one of the copies is difficult. In this case, the gene that was not disrupted (AAH1) is constitutively expressed (10; http://www.toxodb.org/toxo/ [accessed 2 April 2015]) and may compensate for the disrupted copy.

Hence, appropriate positive controls are needed for interpretation of this study, with demonstration that the parental strain increases dopamine levels in PC12 cells, similar to previously published work. Indeed, in the absence of reproduction of previously published data on changes in dopamine, it is not possible to interpret the effects of gene knockouts. Once these conditions are established, conditional knockout of both T. gondii AAH genes is needed to reassess their biological role.