Project Summary/Abstract Microglia-induced neuroinflammation (MiN) plays a critical role in many neurological disorders, contributing to disease progression and symptomology. In Temporal Lobe Epilepsy (TLE), hippocampal sclerosis (HS) forms a nidus for persistent MiN that is associated with chronic epileptic seizures. MiN presents a novel therapeutic target and represents a complementary therapeutic approach to current anti-seizure medications. TLE is the most common form of drug resistant epilepsy, impacting 200,000 Americans, and resulting in significant morbidity and mortality. Discovery of compounds active against MiN have been based almost exclusively on screening in an immortalized murine BV2 cell model stimulated with pathogenic LPS to induce neuroinflammation. However, this is a poor surrogate for human TLE-related neuroinflammation and models with greater fidelity to TLE are critically needed for successful translation. Stem Pharm’s proprietary human stem cell derived neural organoids allow for incorporation of microglia in a reproducible, 96-well plate format amenable to drug screening applications. We have demonstrated that microglia incorporated into our organoids demonstrate a gene signature that strongly correlates with human in vivo microglia, respond appropriately to inflammatory stimuli, and model features of neuroinflammation related to TLE. While LPS stimulation can produce a form of MiN in vitro, it is based on pathogen signaling largely through the TLR4 receptor. In contrast, we will stimulate Stem Pharm’s organoids with Damage Associated Molecular Pattern (DAMP) molecules that interact with a broad set of microglial receptors and cell signaling pathways. This is expected to produce MiN more closely resembling that found in TLE and result in a more effective and translatable drug discovery model. Specific Aims will 1) develop and characterize DAMP cocktails or DAMP-enriched conditioned media (CM) that optimally stimulate MiN, 2) apply optimized DAMP cocktails/CM that elicit neuroinflammatory responses in organoids and assess transcriptional changes with comparison to TLE patient data, and 3) validate the organoid model by evaluating the ability of known anti-inflammatory compounds to prevent DAMP-induced MiN. Completion of these aims will result in an in vitro DAMP-based model of MiN with demonstrable relevance to TLE. This will represent a significant advancement in the ability to evaluate anti-inflammatory activity of candidate therapeutics in a physiologically relevant model. A Phase 2 proposal will focus on screening drug candidates capable of regulating microglia activation in TLE and performing preclinical activities.