Project Summary:Toxoplasma gondii is an intracellular parasite that latently infects many hosts including humans.Successful latent infection requires T. gondii to evade cytokine-induced cell intrinsic responses andswitch from a fast-growing form to a slow-growing encysted form. In humans this life-long infectionoccurs in the brain where T. gondii can reactivate in the setting of acquired immune deficiencies. InAIDS patients toxoplasmic encephalitis is the most common cause of focal brain lesions and cancause prolonged neurologic deficits even after appropriate treatment. Recent studies also suggest thateven in HIV+ patients on effective antiretroviral therapy persistent T. gondii infection may adverselyaffect cognition and global immune responses. Despite its clinical importance we lack a mechanisticunderstanding of what is actually responsible for T. gondiis pathogenesis in the CNS including whatenables long-term latent infection. Such understanding is crucial to eventually preventing symptomaticdisease in HIV/AIDs patients. Within the CNS cysts are primarily found in neurons. Based on limited invitro and in vivo studies it has been presumed that while infected astrocytes cleared intracellularparasites neurons did not and thus were the de facto host cell for persistent infection. In the lastdecade our pioneering work has questioned this model including showing that IFN--stimulatedneurons mount anti-parasitic defenses. As most of this work has occurred in murine models andneurons it will have missed human neuron-specific pathways (e.g. humans lack the terminal IFN-dependent proteins used by murine cells to kill intracellular parasites). The goal of this grant is toaddress this gap by establishing robust stem cell derived human neuron models (hPSC neurons) whichcan be used to mechanistically define human neuron-T. gondii interactions that enable immune evasionand persistence. To accomplish this goal our will leverage our synergistic expertise in CNStoxoplasmosis (Dr. Koshy) and stem cell biology (Dr. Churko) to generate Cre reporter hPSC neuronsand CRISPRi-expressing hPSC neurons (Aim 1) and to begin to define the mechanisms by whichcytokine stimulated hPSC-neurons control T. gondii (Aim 2). With the completion of these aims we willhave established and validated tools that will form the essential foundation of a long-term program tomolecularly define the neuron-T. gondii interactions that enable control and persistence of T. gondii inhuman neurons. The work proposed here represents an important first step toward developing humanneuron-specific therapies for acute and chronic toxoplasmosis. Such therapies will be of great benefitfor the HIV/AIDS population at risk for toxoplasmic encephalitis.