UC Riverside

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell, however, cyst formation in the central nervous system (CNS) is only found in neurons. Previously our lab discovered that primary cortical neurons infected with T. gondii resulted in a downregulation of astrocytic glutamate transporter, GLT-1. This transporter is responsible for ~90% of extracellular glutamate uptake, the primary excitatory neurotransmitter in the brain. In vivo astrocytes are one CNS cell type that is able to evade cyst formation, but their presence is fundamental to neuronal health. Although previous work has demonstrated defects in neuronal physiology, it is not yet known how infection disrupts the crucial communication between astrocytes and neurons. Extracellular vesicles (EVs) function in intracellular communication and contain proteins, lipids, DNA, miRNA, and other RNAs subtypes. Normal expression of GLT-1 is driven and regulated by post-translational modifications and neuronal release of EVs. Toxoplasma infection of neurons leads to many changes in neuronal function. It is therefore possible that changes in EV production by neurons is responsible for the downregulation of astrocytic GLT-1 observed during infection. To evaluate if EVs from T. gondii infected neurons will alter astrocyte and neuron communication, primary neuronal cultures were infected and EVs were harvested and analyzed for morphology, size, content, and concentration. Neuronal production of EVs was significantly reduced following infection, as measured by NTA (nanoparticle tracking analysis) and CD63 ELISA. Neuronal EV content was also altered following infection, as determined by liquid chromatography (LC)-mass spectrometry (MS)/MS. To determine if EV-dependent neuronal-astrocyte communication was altered by infection, EV uptake assays and bulk RNA Sequencing were conducted on astrocytes incubated with EVs from uninfected or infected neurons. Results support EV uptake by astrocytes along with EV regulation of astrocytic GLT-1, in addition to pinpointing other genes regulated by this process. Furthermore, protein expression analysis of astrocytes incubated with infected neuronal-derived EVs demonstrate a significant decrease in GLT-1 protein concentration. These results support the concept that Toxoplasma infection causes changes in the production and content of neuronal derived EVs and helps better understand how a parasitic infection in the brain alters neuronal-astrocyte communication.