Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study researchers from the University of Leeds investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. The researchers report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function.
Toxoplasma-induced neuronal extracellular vesicles (TINEV)
induce transcriptional regulation in neuronal cells
(A) Scheme summarising sequential ultracentrifugation steps undertaken to produce purified extracellular vesicles. Sucrose gradient densities range from 0.104 to 1.43 g/ml. The density between 1.15 and 1.19 g/ml was collected as this represents extracellular vesicles. (B) Transmission electron microscopy of extracellular vesicles isolated from uninfected (top) and infected (bottom) PC12 cell cultures. Arrows indicate extracellular vesicles; scale bar represents 100 nm. (C) Plot of extracellular vesicle diameter from uninfected (EV) and T. gondii infected PC12 cultures (iEV); p = 0.81 (D) DBH mRNA expression following treatment with purified TINEVs. PC12 cells were treated with EVs from uninfected (grey) and T. gondii-infected (black) cultures for 24 h; n = 3, **p = 0.006. Graphs show ± SEM with p values for Student’s t tests. (E) A schematic representation of experimental design illustrating the site of injection for delivery of EVs directly into the brain’s noradrenergic center, the locus coeruleus (LC), of rats. Brain regions (prefrontal cortex, midbrain, and the pons/LC as illustrated by the dotted lines) were harvested two days following 3 days of twice daily treatment. RNA was purified from tissues and RT-qPCR performed to quantitate DBH expression. Stereotaxic coordinates from Paxinos and Watson. (F) DBH mRNA levels in brain tissues of rats receiving TINEVs (iEVs) and uninfected cell EVs, relative to the neuronal marker MAP2. The pons containing locus coeruleus (pons/LC) contained significantly down-regulated DBH. ± SEM shown, n = 5, Mann–Whitney unpaired t test, **p = 0.0079.