The TREM2-R47H Variant Drives Alzheimer’s-Relevant Alterations in Forebrain Organoids Beyond Microglial Populations

Abstract Recent genetic studies highlight microglia as central drivers of Alzheimer’s disease (AD), yet how specific risk variants like TREM2-R47H influence broader neurocellular networks remains elusive. Here, we utilize an iPSC-derived forebrain organoid co-culture system to investigate the multi-lineage impact of the TREM2-R47H variant. High-resolution transcriptomic profiling, paired with confocal imaging, demonstrate that mutant organoids recapitulate AD-specific pathological signatures. Representative confocal imaging revealed phosphorylated-Tau (pTau) and amyloid-beta (Aβ) internalization by WT microglia, while R47H variants showed a qualitative reduction in pTau accumulation. Single-cell RNA sequencing (scRNA-seq) revealed neurodegenerative transcriptional profiles in TREM2-R47H neurons as early as day 139, occurring independently of microglia presence. By day 173, these cell-intrinsic signatures intensified, characterized by disrupted oxidative phosphorylation and impaired maturation trajectories. Interaction analysis further demonstrated that the addition of microglia exacerbated this phenotype; while WT cells adapted to the microglia niche by activating homeostatic, neuro-supportive programs, TREM2-R47H cells underwent ‘identity erosion’ and failed to transition into HLA-enriched activation states. This state was characterized by a failure to adopt brain-resident signatures and a divergent shift toward inflammatory myeloid phenotypes. These findings reveal that the TREM2-R47H mutation exerts a dual burden: it drives a baseline neurodegenerative state in neural lineages and renders them incapable of proper niche integration. Our study provides an in vitro human platform to dissect the interplay between genetic risk and multi-cellular dysfunction, establishing a scalable system for evaluating novel therapeutic interventions and drug screening aimed at restoring neuro-immune homeostasis in AD. Competing Interest Statement The authors have declared no competing interest. Footnotes This revised version includes updated analyses, figure refinements, and textual revisions made during manuscript preparation for journal submission. Several datasets were reprocessed using an improved analytical pipeline, resulting in minor updates to quantitative results and visualizations. These changes do not alter the overall conclusions of the study but strengthen the robustness and clarity of the findings.