PROJECT SUMMARY/ABSTRACT Tuberculosis (TB) of the central nervous system (CNS) is the most dangerous form of M. tuberculosis (Mtb) infection. For these cases, the risk of mortality is extremely high; those who survive are left with an elevated risk of severe neurological damage and disability. Despite its public health importance, current understanding of the dissemination of Mtb in the brain and anti-mycobacterial immunity within the human brain parenchyma is limited. This represents a roadblock for developing a better cure for TB meningitis. This concern motivated the NIH to organize a TB Meningitis workshop to develop a roadmap for advancing CNS TB research. The workshop established that creating human in vitro platforms would facilitate research on the field and significantly contribute to the understanding of cellular and molecular insights into the pathogenesis of CNS TB. This proposal will test and optimize a new platform, Mtb infection of human brain organoids, to study CNS Mtb infection. Previously, we have found that mycobacterial infection of the murine brain leads to protective immune responses and bacterial control. While useful, murine model systems do not always reflect the many aspects of human disease, and the application of human single and multi-component neuronal tissues would be more appropriate. Mtb infects phagocytes, and most of the Mtb in the brain after CNS infection is located in recruited monocytes and local microglia. We discovered that neural progenitors could also be infected with Mtb. Under this proposal's aegis, we will test how the different infected phagocytes contribute to brain tuberculosis. The two main objectives of this proposal are to understand the mechanism and consequences of bacterial uptake by neural progenitors (Aim 1) and to apply neuronal organoids to test mechanisms of Mtb dissemination and brain cell responses to Mtb infection. We propose to compare the effects of different Mtb-infected phagocytes on human neural tissue (Aims 2 and 3). Successful completion of this work will lead to new knowledge on a novel aspect of brain TB concerning neural progenitors' infection. It raises the possibility that Mtb effects neuronal replacement at sites where neurons are constitutively generated. In addition, we develop new human model platforms to study the dissemination of Mtb into the brain and brain-specific responses to Mtb infection.