Project summary: Scientists and clinicians have studied the underlying mechanisms of HIV-associated neurocognitive disorder (HAND) for decades, spanning clinical trials and preclinical studies in animal models and cellular systems. Although this research has undoubtedly pushed the field forward, there is still no approved adjuvant therapy to relieve the cognitive impairment affecting a significant portion of ART-treated patients. This could be partially due to the preclinical models themselves, which may incompletely model human brain’s cellular heterogeneity, organization, and properties. We propose to bridge this gap by developing a human brain slice culture model to study HIV neuropathogenesis. Human tissues are provided from brain surgeries via a collaboration with a neurosurgeon colleague. These include adult normal human brain tissues, cerebrospinal fluid, and peripheral blood. We will isolate monocyte-derived macrophages (MDM) from a donor’s blood sample, infect them with a fluorescent HIV-1, and expose the infected MDMs to patient- matched HIV-negative brain cultures. After the brain slice infection is established, HIV replication will be controlled with relevant concentrations of standard-of-care ART. This human model may better recapitulate pathological features of HAND thought to be present in treated patients, including infection of appropriate CNS cell types, subtle brain inflammation, and impaired neurotransmission, and serve as a highly relevant model system for future studies on neuroHIV and preclinical testing of neuroprotective strategies. Aim 1 will optimize brain slice culture conditions and examine neuronal structure and function (and neuroinflammation) over the life of the culture. Cell viability will be assessed in slice cultures with different culture media and slice thickness by longitudinal vital staining approaches and flow cytometry. Neuronal activity will also be longitudinally measured with multielectrode arrays or calcium imaging, and dendritic branching and spine changes will be monitored at multiple timepoints using DiI staining and Neurolucida 360 software analysis. Glial function will also be studied using calcium signaling and glutamate probes. Aim 2 will examine optimized slice cultures infected with patient-matched HIV+ MDMs and treated with ART. Infection progress will be tracked by confocal imaging of the fluorescent HIV, flow cytometry, and HIV p24 Alphalisa. The inflammatory environment of HIV- infected/uninfected slices pre- and post-ART will be assessed using flow cytometry and Alphalisa for select inflammatory mediators and trophic factors. Neuronal status in ART-treated HIV+ brain slices will be determined with the same techniques listed in Aim 1. Completion of the project will lay the foundation for a human tissue-based model system that largely maintains the composition and local connectivity of the adult brain, reflects the virally suppressed pathology in today’s patients, and can help validate imp...