Toxoplasma gondii infection of neurons alters the production and content of extracellular vesicles altering astrocyte phenotype and contributing to the loss of GLT-1 in the infected brain

Toxoplasma gondii (T. gondii) cyst formation in the central nervous system only occurs in neurons allowing the parasite to remain latent for the lifetime of the host. Astrocytes are fundamental to neuronal health by providing nutrients and structural support and help regulate neurotransmitters by continuous communication with neurons. It is not yet known how infection and the presence of intracellular cysts, disrupts the crucial relationship between these cells. Extracellular vesicles (EVs) function in intracellular communication and can contain proteins, lipids, DNA, miRNA, and other RNA subtypes. EVs are produced by all cells including neurons and play an important role in neuronal-astrocyte interactions including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated Toxoplasma infection reduces astrocytic expression of the primary glutamate transporter, GLT-1. Here we tested if cyst infection of neurons alters the production and content of EVs. EVs were isolated from uninfected and infected primary murine cortical neurons and their size, concentration, and characterization were confirmed with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), CD63 ELISA, liquid chromatography (LC)-mass spectrometry (MS)/MS, and microRNA Sequencing. Analysis reveals that infection of neurons reduced neuronal production of EVs and altered their protein and miRNA content. EVs from infected neurons contained secreted Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2 associated with cyst formation. Following incubation of neuronal EVs with primary astrocytes, a proportion of EVs colocalize to the nucleus. EVs from infected neurons altered gene expression of astrocytes leading to a downregulation of GLT-1 protein expression and an increase in pro-inflammatory transcriptional signatures. These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes.