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

Toxoplasma gondii (T. gondii), a prolific protozoan parasite, forms cysts within neurons of the central nervous system that maintain infection 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 and play an important role in neuronal-astrocyte interactions, including the regulation of glutamate receptors on astrocytes. Previous work has demonstrated that 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), ELISA, western blot, 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. In addition to changes in host protein content, EVs from infected neurons contained the Toxoplasma proteins GRA1, GRA2, GRA7, MAG1 and MAG2. Following incubation of neuronal EVs with primary astrocytes, GRA7 protein could be observed within intracellular EVs and the nuclei of GRA7+EV-containing cells. EVs from infected neurons altered gene expression of astrocytes resulting in an increase in pro-inflammatory transcriptional signatures, along with a downregulation of GLT-1 protein expression with similar transcriptional changes found in astrocytes in vivo. These results demonstrate the ability of a parasitic infection in the brain to alter EV production and the fundamental communication between neurons and astrocytes. To determine if infection alters astrocyte and neuron communication EVs from T. gondii infected primary cortical murine neurons were isolated and added onto primary murine astrocyte cultures for 24 hours prior to bulk RNA-Sequencing. The addition of LPS/IFNy was used as a positive control, media without Dexamethasone (Dex) as a negative control, and media as an experimental control. Primary murine cortical astrocytes were grown and treated with 100nM of Dexamethasone one day prior to the addition of the four different conditions. Samples were incubated for 24 hours prior to RNA extraction and bulk RNA Sequencing. Each sample was done in triplicate. For the negative control Dexamethasone was never added.