Characterizing the influence of Toxoplasma infection on opioid receptor expression using human neuroblastoma cells (SKNSH)

Abstract

BackgroundToxoplasma gondii is an obligate intracellular parasite that has permanently infected billions of people. The parasite can convert to a latent form that is found preferentially in the brain and heart. Recent serological studies have linked Toxoplasma infection to many brain disorders including schizophrenia, depression, substance abuse and Alzheimer’s. However, the molecular mechanism by which Toxoplasma influences these brain disorders remains unclear. Previous studies have shown that Toxoplasmaincreases dopamine synthesis, and dopamine is an important neurotransmitter in the reward pathway, where the opioid system has been shown to play a major role in modulating this pathway. We aim to further explore these mechanisms by studying the influence of Toxoplasmainfection on opioid receptor expression.ObjectivesThe goal of our project is to elucidate the influence of Toxoplasma gondiion brain physiology through studying the impact of Toxoplasma infection on opioid receptor expression. The aim of this study is to determine the effect of Toxoplasma infection on opioid receptor expression in neuroblastoma cells (SKNSH) in the presence or absence of morphine.MethodsTo evaluate the potency of morphine against Toxoplasma, a doubling assay was performed. Briefly, Human foreskin fibroblasts (HFF) were infected with Toxoplasma. Six‐hours post‐infection, the uninvaded parasites were removed and the media was replaced with media containing 10 µM morphine, 10 µM pyrimethamine, or DMSO. The flasks were fixed with ice‐cold methanol and the number of parasites per vacuole were enumerated at 24‐ and 48‐hours post‐treatment. To evaluate the expression of opioid receptors, the neuroblastoma cell SKNSH, which has been shown to express functional mu and delta opioid receptors, was infected with Toxoplasma type 1. Four hours post‐infection, uninvaded parasites were removed and the medium was replaced with media containing DMSO or 1 µM morphine or 10 µM morphine. Thirty‐two hours post‐treatment, the cells were harvested and protein was isolated. The expression of mu and delta opioid receptors was evaluated using western blot analysis.ResultsWe observed changes in the morphology of SKNSH upon infection with Toxoplasma type 1. The results from our doubling assay demonstrate the lack of potency for morphine against Toxoplasma type 1 at 24 hours and 48 hours post treatment with the highest morphine concentration we have been using for these experiments. Preliminary results suggest that the expression of the delta opioid receptor is downregulated in the presence of Toxoplasma, independent of morphine administration. In addition, the expression of delta opioid receptor is increased in the presence of morphine alone. When pyrimethamine was added to cells containing both Toxoplasma and morphine, delta receptor expression was comparable to its expression in the presence of morphine alone.ConclusionWe have confirmed the expression of muand delta opioid receptors in SKNSH, as reported previously. In addition, we were able to successfully infect this cell line with Toxoplasma. Additionally, we have found that morphine lacks potency against Toxoplasma, unlike previously reported. Preliminary data suggest that the collective the presence of Toxoplasma can negatively influence the expression of delta opioid receptors. Future experiments are being designed to elucidate the molecular mechanism of these findings, as well as observing similar findings in the animal model of Toxoplasmosis.