{"doi":"10.1101/2025.06.04.657874","title":"Human microglia in brain assembloids display region- specific diversity and respond to hyperexcitable neurons carrying <i>SCN2A</i> mutation: Microglial diversity and response in assembloids","abstract":"ABSTRACT Microglia critically shape neuronal circuit development and function, yet their region-specific properties and roles in distinct circuits of the human brain remain poorly understood. In this study, we generated region-specific brain organoids (cortical, striatal, and midbrain), each integrated with human microglia, to fill this critical gap. Single-cell RNA sequencing uncovered six distinct microglial subtypes exhibiting unique regional signatures, including a subtype highly enriched for the GABA B receptor gene within striatal organoids. To investigate the contributions of microglia to neural circuitry, we created microglia-incorporated midbrain-striatal assembloids, modeling a core circuit node for many neuropsychiatric disorders including autism. Using chemogenetics to activate this midbrain-striatal circuit, we observed increased calcium signaling in microglia involving GABA B receptors. Leveraging this model, we examined microglial responses within neural circuits harboring an SCN2A nonsense (C959X) mutation associated with profound autism. Remarkably, microglia displayed heightened calcium responses to SCN2A mutation-mediated neuronal hyperactivity, and engaged in excessive synaptic pruning. These pathological effects were reversed by pharmacological inhibition of microglial GABA B receptors. Collectively, our findings establish an advanced platform to dissect human neuroimmune interactions in sub-cortical regions, highlighting the important role of microglia in shaping critical circuitry related to neuropsychiatric disorders. Graphical abstract Teaser Modeling regional microglial diversity in sub-cortical regions is challenging. We generated human organoid and assembloid models containing microglia that acquire region-specific heterogeneity. Our work shows dynamic responses of microglia when exposed to hyperexcitable midbrain-striatal circuits, providing an exciting platform to study neuroimmune interactions in human brain development and neuropsychiatric disorders, including SCN2A mutation-mediated monogenic autism. Highlights • Single-cell RNA sequencing analyses reveal six distinct microglial subtypes that spontaneously attain unique specialization in human cortical, striatal, and midbrain organoids. • Microglia facilitate axonal projections across regional organoids, promoting assembloid formation. • Microglia respond to hyperexcitable neurons via calcium signaling and exhibit excessive pruning of neuronal synapses. • Blocking microglial GABA B receptors normalizes calcium activity and reduces synaptic pruning, suggesting a potential targeting strategy for synaptic deficits.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555213,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.8992,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":337746,"name":"Xiaoling Chen","orcid":"0000-0002-2271-1140","position":1,"is_corresponding":false},{"id":337743,"name":"Jingliang Zhang","orcid":"0000-0002-2903-1287","position":2,"is_corresponding":false},{"id":663764,"name":"Kyle Wettschurack","orcid":"0000-0002-7268-8188","position":3,"is_corresponding":false},{"id":1229622,"name":"Morgan Robinson","orcid":"0000-0003-4719-196X","position":4,"is_corresponding":false},{"id":1453978,"name":"Weihao Li","orcid":null,"position":5,"is_corresponding":false},{"id":1161850,"name":"Yuanrui Zhao","orcid":null,"position":6,"is_corresponding":false},{"id":1453979,"name":"Ye-Eun Yoo","orcid":null,"position":7,"is_corresponding":false},{"id":1453515,"name":"Brody A. Deming","orcid":"0000-0002-1404-5694","position":8,"is_corresponding":false},{"id":1453980,"name":"Akila D. Abeyaratna","orcid":null,"position":9,"is_corresponding":false},{"id":337748,"name":"Zhefu Que","orcid":"0000-0002-1358-0660","position":10,"is_corresponding":false},{"id":1161258,"name":"Dongshu Du","orcid":"0000-0003-1198-1089","position":11,"is_corresponding":false},{"id":74332,"name":"Matthew Tegtmeyer","orcid":"0000-0002-9032-8207","position":12,"is_corresponding":false},{"id":631786,"name":"Chongli Yuan","orcid":"0000-0003-3765-0931","position":13,"is_corresponding":false},{"id":337751,"name":"William C. Skarnes","orcid":"0000-0002-8334-3329","position":14,"is_corresponding":false},{"id":320969,"name":"Jean‐Christophe Rochet","orcid":"0000-0002-6484-1541","position":15,"is_corresponding":false},{"id":258025,"name":"Long‐Jun Wu","orcid":"0000-0001-8019-3380","position":16,"is_corresponding":false},{"id":337753,"name":"Yang Yang","orcid":"0000-0001-6417-3654","position":17,"is_corresponding":false},{"id":337749,"name":"Jiaxiang Wu","orcid":"0000-0002-1474-3820","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T02:54:59.329539Z","pmid":"40501840","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}