RBFOX1 Dysfunction Unlocks APOE4-Associated Microglial Genesis and Exacerbates Alzheimer's Pathology in Human Cerebral Organoids

Abstract

Alzheimer's disease (AD) pathogenesis is strongly influenced by APOE4, though how cooperative genetic factors modulate this relationship remains unclear. While genomic studies have tentatively linked RBFOX1 to AD susceptibility, its functional synergy with APOE4 has never been experimentally defined. We engineered APOE3 or APOE4 isogenic human cerebral organoids with CRISPR/Cas9-mediated RBFOX1 knockout. Remarkably, RBFOX1 depletion selectively triggered robust microglial generation exclusively in APOE4 organoids. Time-course gene expression revealed that this APOE4-specific effect correlated with prolonged mesodermal priming during early embryoid body differentiation, creating a permissive niche for microglial lineage specification. The emergent microglia exhibited pronounced neurotoxic phenotypes, including pro-inflammatory factor secretion, synaptic architecture remodeling, and lipid droplet accumulation in organoids. These changes coincided with aggravated Tau hyperphosphorylation and electrophysiological abnormalities, collectively mirroring multifaceted AD pathology. Our findings establish RBFOX1 as a potential AD protective factor, a critical suppressor of APOE4-glia crosstalk, and demonstrate that its loss unleashes a microglia-mediated neurodegenerative cascade. By developing cerebral organoids with autonomous microglial networks, we present a platform capable of modeling genotype-dependent neuron-glia interactions in AD, opening new avenues for mechanistic and therapeutic exploration.

Keywords

Alzheimer's disease / APOE4 / microglia generation / organoids / RBFOX1

References

[1] D. Campion, C. Dumanchin, D. Hannequin, et al., “Early-onset Autosomal Dominant Alzheimer Disease: Prevalence, Genetic Heterogeneity, and Mutation Spectrum,” American Journal of Human Genetics 65 (1999): 664–670, https://doi.org/10.1086/302553.

[2] C. C. Liu, C. C. Liu, T. Kanekiyo, H. Xu, and G. Bu, “Apolipoprotein E and Alzheimer Disease: Risk, Mechanisms and Therapy,” Nature Reviews Neurology 9 (2013): 106–118, https://doi.org/10.1038/nrneurol.2012.263.

[3] J. M. Long and D. M. Holtzman, “Alzheimer Disease: An Update on Pathobiology and Treatment Strategies,” Cell 179 (2019): 312–339, https://doi.org/10.1016/j.cell.2019.09.001.

[4] J. Penney, W. T. Ralvenius, and L. H. Tsai, “Modeling Alzheimer's Disease With iPSC-derived Brain Cells,” Molecular Psychiatry 25 (2020): 148–167, https://doi.org/10.1038/s41380-019-0468-3.

[5] H. Sasaguri, P. Nilsson, S. Hashimoto, et al., “APP Mouse Models for Alzheimer's Disease Preclinical Studies,” EMBO Journal 36 (2017): 2473–2487, https://doi.org/10.15252/embj.201797397.

[6] B. W. Kunkle, M. Schmidt, H. U. Klein, et al., “Novel Alzheimer Disease Risk Loci and Pathways in African American Individuals Using the African Genome Resources Panel,” JAMA Neurology 78 (2021): 102, https://doi.org/10.1001/jamaneurol.2020.3536.

[7] N. S. Raghavan, L. Dumitrescu, E. Mormino, et al., “Association between Common Variants in RBFOX1, an RNA-Binding Protein, and Brain Amyloidosis in Early and Preclinical Alzheimer Disease,” JAMA Neurology 77 (2020): 1288, https://doi.org/10.1001/jamaneurol.2020.1760.

[8] O. Kohannim, D. P. Hibar, N. Jahanshad, et al., “Predicting Temporal Lobe Volume on MRI from Genotypes Using L(1)-L(2) Regularized Regression,” IEEE Transactions on Medical Imaging 2012 (2012): 1160–1163.

[9] M. Vounou, E. Janousova, R. Wolz, et al., “Sparse Reduced-Rank Regression Detects Genetic Associations With Voxel-Wise Longitudinal Phenotypes in Alzheimer's Disease,” NeuroImage 60 (2012): 700–716, https://doi.org/10.1016/j.neuroimage.2011.12.029.

[10] B. L. Fogel, E. Wexler, A. Wahnich, et al., “RBFOX1 Regulates Both Splicing and Transcriptional Networks in Human Neuronal Development,” Human Molecular Genetics 21 (2012): 4171–4186, https://doi.org/10.1093/hmg/dds240.

[11] L. T. Gehman, P. Stoilov, J. Maguire, et al., “The Splicing Regulator Rbfox1 (A2BP1) Controls Neuronal Excitation in the Mammalian Brain,” Nature Genetics 43 (2011): 706–711, https://doi.org/10.1038/ng.841.

[12] X. Zhang, M. H. Chen, X. Wu, et al., “Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex,” Cell 166 (2016): 1147–1162.e15, e1115. https://doi.org/10.1016/j.cell.2016.07.025.

[13] D. Lal, H. Trucks, R. S. Møller, et al., “Rare Exonic Deletions of the RBFOX1 Gene Increase Risk of Idiopathic Generalized Epilepsy,” Epilepsia 54 (2013): 265–271, https://doi.org/10.1111/epi.12084.

[14] B. R. Bill, J. K. Lowe, C. T. Dybuncio, and B. L. Fogel, “Orchestration of Neurodevelopmental Programs by RBFOX1: Implications for Autism Spectrum Disorder,” International Review of Neurobiology 113 (2013): 251–267.

[15] N. Fernàndez-Castillo, G. Gan, M. M. J. van Donkelaar, et al., “RBFOX1, Encoding a Splicing Regulator, Is a Candidate Gene for Aggressive Behavior,” European Neuropsychopharmacology 30 (2020): 44–55, https://doi.org/10.1016/j.euroneuro.2017.11.012.

[16] J. Zhao, Y. Fu, Y. Yamazaki, et al., “APOE4 exacerbates Synapse Loss and Neurodegeneration in Alzheimer's Disease Patient iPSC-derived Cerebral Organoids,” Nature Communications 11 (2020): 5540, https://doi.org/10.1038/s41467-020-19264-0.

[17] E. H. Corder, A. M. Saunders, W. J. Strittmatter, et al., “Gene Dose of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer's Disease in Late Onset Families,” Science 261 (1993): 921–923, https://doi.org/10.1126/science.8346443.

[18] E. Kok, S. Haikonen, T. Luoto, et al., “Apolipoprotein E–dependent Accumulation of Alzheimer Disease–related Lesions Begins in Middle Age,” Annals of Neurology 65 (2009): 650–657, https://doi.org/10.1002/ana.21696.

[19] C. C. Liu, N. Zhao, Y. Fu, et al., “ApoE4 Accelerates Early Seeding of Amyloid Pathology,” Neuron 96 (2017): 1024–1032.e3, https://doi.org/10.1016/j.neuron.2017.11.013, e1023.

[20] M. Tachibana, M. L. Holm, C. C. Liu, et al., “APOE4-mediated Amyloid-β Pathology Depends on Its Neuronal Receptor LRP1,” Journal of Clinical Investigation 129 (2019): 1272–1277, https://doi.org/10.1172/JCI124853.

[21] C. Wang, M. Xiong, M. Gratuze, et al., “Selective Removal of Astrocytic APOE4 Strongly Protects Against Tau-mediated Neurodegeneration and Decreases Synaptic Phagocytosis by Microglia,” Neuron 109 (2021): 1657–1674.e7, https://doi.org/10.1016/j.neuron.2021.03.024, e1657.

[22] M. A. Lancaster and J. A. Knoblich, “Generation of Cerebral Organoids From Human Pluripotent Stem Cells,” Nature Protocols 9 (2014): 2329–2340, https://doi.org/10.1038/nprot.2014.158.

[23] M. A. Lancaster, M. Renner, C. A. Martin, et al., “Cerebral Organoids Model Human Brain Development and Microcephaly,” Nature 501 (2013): 373–379, https://doi.org/10.1038/nature12517.

[24] A. M. Paşca, S. A. Sloan, L. E. Clarke, et al., “Functional Cortical Neurons and Astrocytes From Human Pluripotent Stem Cells in 3D Culture,” Nature Methods 12 (2015): 671–678.

[25] X. Qian, H. N. Nguyen, M. M. Song, et al., “Brain-Region-Specific Organoids Using Mini-Bioreactors for Modeling ZIKV Exposure,” Cell 165 (2016): 1238–1254, https://doi.org/10.1016/j.cell.2016.04.032.

[26] G. Sun, F. Chiuppesi, X. Chen, et al., “Modeling Human Cytomegalovirus-Induced Microcephaly in Human iPSC-Derived Brain Organoids,” Cell Reports Medicine 1 (2020): 100002, https://doi.org/10.1016/j.xcrm.2020.100002.

[27] S. J. Yoon, L. S. Elahi, A. M. Pașca, et al., “Reliability of human Cortical Organoid Generation,” Nature Methods 16 (2019): 75–78, https://doi.org/10.1038/s41592-018-0255-0.

[28] D. Dutta, I. Heo, and H. Clevers, “Disease Modeling in Stem Cell-Derived 3D Organoid Systems,” Trends in Molecular Medicine 23 (2017): 393–410, https://doi.org/10.1016/j.molmed.2017.02.007.

[29] A. Fatehullah, S. H. Tan, and N. Barker, “Organoids as an in Vitro Model of Human Development and Disease,” Nature Cell Biology 18 (2016): 246–254, https://doi.org/10.1038/ncb3312.

[30] E. M. Abud, R. N. Ramirez, E. S. Martinez, et al., “iPSC-Derived Human Microglia-Like Cells to Study Neurological Diseases,” Neuron 94 (2017): 278–293.e9, e279, https://doi.org/10.1016/j.neuron.2017.03.042.

[31] G. Popova, S. S. Soliman, C. N. Kim, et al., “Human Microglia States Are Conserved Across Experimental Models and Regulate Neural Stem Cell Responses in Chimeric Organoids,” Cell Stem Cell 28 (2021): 2153–2166.e6, e2156. https://doi.org/10.1016/j.stem.2021.08.015.

[32] P. Wörsdörfer, N. Dalda, A. Kern, et al., “Generation of Complex human Organoid Models Including Vascular Networks by Incorporation of Mesodermal Progenitor Cells,” Scientific Reports 9 (2019): 15663.

[33] S. Sabate-Soler, S. L. Nickels, C. Saraiva, et al., “Microglia Integration Into Human Midbrain Organoids Leads to Increased Neuronal Maturation and Functionality,” Glia 70 (2022): 1267–1288, https://doi.org/10.1002/glia.24167.

[34] F. L. Yeh, Y. Wang, I. Tom, L. C. Gonzalez, and M. Sheng, “TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia,” Neuron 91 (2016): 328–340, https://doi.org/10.1016/j.neuron.2016.06.015.

[35] R. Jansen, J. J. Hottenga, M. G. Nivard, et al., “Conditional eQTL Analysis Reveals Allelic Heterogeneity of Gene Expression,” Human Molecular Genetics 26 (2017): 1444–1451, https://doi.org/10.1093/hmg/ddx043.

[36] N. Maphis, G. Xu, O. N. Kokiko-Cochran, et al., “Reactive Microglia Drive Tau Pathology and Contribute to the Spreading of Pathological Tau in the Brain,” Brain 138 (2015): 1738–1755, https://doi.org/10.1093/brain/awv081.

[37] M. T. Heneka, D. T. Golenbock, and E. Latz, “Innate Immunity in Alzheimer's Disease,” Nature Immunology 16 (2015): 229–236, https://doi.org/10.1038/ni.3102.

[38] A. Olmos-Alonso, S. T. Schetters, S. Sri, et al., “Pharmacological Targeting of CSF1R Inhibits Microglial Proliferation and Prevents the Progression of Alzheimer's-Like Pathology,” Brain 139 (2016): 891–907, https://doi.org/10.1093/brain/awv379.

[39] Y. T. Lin, J. Seo, F. Gao, et al., “APOE4 Causes Widespread Molecular and Cellular Alterations Associated With Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types,” Neuron 98 (2018): 1141–1154.e7, https://doi.org/10.1016/j.neuron.2018.05.008, e1147.

[40] Y. Qi, X. J. Zhang, N. Renier, et al., “Combined Small-molecule Inhibition Accelerates the Derivation of Functional Cortical Neurons From Human Pluripotent Stem Cells,” Nature Biotechnology 35 (2017): 154–163, https://doi.org/10.1038/nbt.3777.

[41] C. A. Trujillo, E. S. Rice, N. K. Schaefer, et al., “Reintroduction of the Archaic Variant of NOVA1 in Cortical Organoids Alters Neurodevelopment,” Science 371 (2021): eaax2537, https://doi.org/10.1126/science.aax2537.

[42] P. R. Ormel, R. Vieira de Sá, E. J. van Bodegraven, et al., “Microglia Innately Develop Within Cerebral Organoids,” Nature Communications 9 (2018): 4167, https://doi.org/10.1038/s41467-018-06684-2.

[43] R. Xu, X. Li, A. J. Boreland, et al., “Human iPSC-derived Mature Microglia Retain Their Identity and Functionally Integrate in the Chimeric Mouse Brain,” Nature Communications 11 (2020): 1577, https://doi.org/10.1038/s41467-020-15411-9.

[44] Y. Li, Z. Li, M. Yang, et al., “Decoding the Temporal and Regional Specification of Microglia in the Developing Human Brain,” Cell Stem Cell 29 (2022): 620–634.e6, e626, https://doi.org/10.1016/j.stem.2022.02.004.

[45] S. Ma, M. Skarica, Q. Li, et al., “Molecular and Cellular Evolution of the Primate Dorsolateral Prefrontal Cortex,” Science 377 (2022): eabo7257, https://doi.org/10.1126/science.abo7257.

[46] M. L. Bennett and F. C. Bennett, “The Influence of Environment and Origin on Brain Resident Macrophages and Implications for Therapy,” Nature Neuroscience 23 (2020): 157–166, https://doi.org/10.1038/s41593-019-0545-6.

[47] E. G. Perdiguero and F. Geissmann, “The Development and Maintenance of Resident Macrophages,” Nature Immunology 17 (2016): 2–8, https://doi.org/10.1038/ni.3341.

[48] C. Schulz, E. Gomez Perdiguero, L. Chorro, et al., “A Lineage of Myeloid Cells Independent of Myb and Hematopoietic Stem Cells,” Science 336 (2012): 86–90, https://doi.org/10.1126/science.1219179.

[49] B. Cakir, Y. Tanaka, F. R. Kiral, et al., “Expression of the Transcription Factor PU.1 Induces the Generation of Microglia-Like Cells in Human Cortical Organoids,” Nature Communications 13 (2022): 430, https://doi.org/10.1038/s41467-022-28043-y.

[50] A. P. Tsai, C. Dong, P. B. Lin, et al., “Genetic Variants of Phospholipase C-γ2 Alter the Phenotype and Function of Microglia and Confer Differential Risk for Alzheimer's Disease,” Immunity 56 (2023): 2121–2136.e6, e2126, https://doi.org/10.1016/j.immuni.2023.08.008.

[51] R. Martins-Ferreira, J. Calafell-Segura, B. Leal, et al., “The Human Microglia Atlas (HuMicA) Unravels Changes in Disease-associated Microglia Subsets Across Neurodegenerative Conditions,” Nature Communications 16 (2025): 739, https://doi.org/10.1038/s41467-025-56124-1.

[52] A. E. Cardona, E. P. Pioro, M. E. Sasse, et al., “Control of Microglial Neurotoxicity by the Fractalkine Receptor,” Nature Neuroscience 9 (2006): 917–924, https://doi.org/10.1038/nn1715.

[53] S. Lee, G. Xu, T. R. Jay, et al., “Opposing Effects of Membrane-anchored CX3CL1 on Amyloid and Tau Pathologies via the p38 MAPK Pathway,” Journal of Neuroscience 34 (2014): 12538–12546, https://doi.org/10.1523/JNEUROSCI.0853-14.2014.

[54] A. D. Bachstetter, J. M. Morganti, J. Jernberg, et al., “Fractalkine and CX3CR1 Regulate Hippocampal Neurogenesis in Adult and Aged Rats,” Neurobiology of Aging 32 (2011): 2030–2044, https://doi.org/10.1016/j.neurobiolaging.2009.11.022.

[55] V. Zujovic and V. Taupin, “Use of Cocultured Cell Systems to Elucidate Chemokine-Dependent Neuronal/Microglial Interactions: Control of Microglial Activation,” Methods 29 (2003): 345–350, https://doi.org/10.1016/S1046-2023(02)00358-4.

[56] H. A. Mattison, H. Nie, H. Gao, et al., “Suppressed Pro-inflammatory Response of Microglia in CX3CR1 Knockout Mice,” Journal of Neuroimmunology 257 (2013): 110–115, https://doi.org/10.1016/j.jneuroim.2013.02.008.

[57] C. Lauro, M. Catalano, F. Trettel, et al., “The Chemokine CX3CL1 Reduces Migration and Increases Adhesion of Neurons With Mechanisms Dependent on the β1 Integrin Subunit,” Journal of Immunology 177 (2006): 7599–7606, https://doi.org/10.4049/jimmunol.177.11.7599.

[58] C. Kaur, G. Rathnasamy, and E. A. Ling, “Biology of Microglia in the Developing Brain,” Journal of Neuropathology and Experimental Neurology 76 (2017): 736–753, https://doi.org/10.1093/jnen/nlx056.

[59] C. A. Mosser, S. Baptista, I. Arnoux, and E. Audinat, “Microglia in CNS Development: Shaping the Brain for the Future,” Progress in Neurobiology 149-150 (2017): 1–20, https://doi.org/10.1016/j.pneurobio.2017.01.002.

[60] M. W. Salter and B. Stevens, “Microglia Emerge as central Players in Brain Disease,” Nature Medicine 23 (2017): 1018–1027, https://doi.org/10.1038/nm.4397.

[61] J. Zhou, Y. Zhang, Q. Lin, et al., “Embryoid Bodies Formation and Differentiation From Mouse Embryonic Stem Cells in Collagen/Matrigel Scaffolds,” Journal Of Genetics And Genomics 37 (2010): 451–460, https://doi.org/10.1016/S1673-8527(09)60064-3.

[62] N. Sagy, S. Slovin, M. Allalouf, et al., “Prediction and Control of Symmetry Breaking in Embryoid Bodies by Environment and Signal Integration,” Development 146 (2019): dev181917.

[63] I. Kelava and M. A. Lancaster, “Dishing out Mini-brains: Current Progress and Future Prospects in Brain Organoid Research,” Developmental Biology 420 (2016): 199–209, https://doi.org/10.1016/j.ydbio.2016.06.037.

[64] S. J. Arnold and E. J. Robertson, “Making a Commitment: Cell Lineage Allocation and Axis Patterning in the Early Mouse Embryo,” Nature Reviews Molecular Cell Biology 10 (2009): 91–103, https://doi.org/10.1038/nrm2618.

[65] C. Ross and T. E. Boroviak, “Origin and Function of the Yolk Sac in Primate Embryogenesis,” Nature Communications 11 (2020): 3760, https://doi.org/10.1038/s41467-020-17575-w.

[66] S. G. Utz, P. See, W. Mildenberger, et al., “Early Fate Defines Microglia and Non-Parenchymal Brain Macrophage Development,” Cell 181 (2020): 557–573.e18, e518. https://doi.org/10.1016/j.cell.2020.03.021.

[67] Y. Shi, K. Yamada, S. A. Liddelow, et al., “ApoE4 Markedly Exacerbates Tau-mediated Neurodegeneration in a Mouse Model of Tauopathy,” Nature 549 (2017): 523–527, https://doi.org/10.1038/nature24016.

[68] M. Valdearcos, J. D. Douglass, M. M. Robblee, et al., “Microglial Inflammatory Signaling Orchestrates the Hypothalamic Immune Response to Dietary Excess and Mediates Obesity Susceptibility,” Cell Metabolism 27 (2018): 1356, https://doi.org/10.1016/j.cmet.2018.04.019.

[69] J. Marschallinger, T. Iram, M. Zardeneta, et al., “Lipid-droplet-accumulating Microglia Represent a Dysfunctional and Proinflammatory State in the Aging Brain,” Nature Neuroscience 23 (2020): 194–208, https://doi.org/10.1038/s41593-019-0566-1.

[70] M. S. Haney, R. Pálovics, C. N. Munson, et al., “APOE4/4 is Linked to Damaging Lipid Droplets in Alzheimer's Disease Microglia,” Nature 628 (2024): 154–161, https://doi.org/10.1038/s41586-024-07185-7.

[71] M. B. Victor, N. Leary, X. Luna, et al., “Lipid Accumulation Induced by APOE4 Impairs Microglial Surveillance of Neuronal-network Activity,” Cell Stem Cell 29 (2022): 1197–1212.e8, e1198, https://doi.org/10.1016/j.stem.2022.07.005.

[72] A. Ghalali, F. Alhamdan, S. Upadhyay, et al., “Contrasting Effects of Intracellular and Extracellular human PCSK9 on Inflammation, Lipid Alteration and Cell Death,” Communications Biology 7 (2024): 985, https://doi.org/10.1038/s42003-024-06674-9.

[73] H. Zhang, A. Lin, P. Gong, et al., “The Chemokine-Like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding,” Journal of Neuroscience 40 (2020): 6991–7007, https://doi.org/10.1523/JNEUROSCI.0455-20.2020.

[74] M. Mai, X. Guo, Y. Huang, et al., “DHCR24 Knockdown Induces Tau Hyperphosphorylation at Thr181, Ser199, Ser262, and Ser396 Sites via Activation of the Lipid Raft-Dependent Ras/MEK/ERK Signaling Pathway in C8D1A Astrocytes,” Molecular Neurobiology 59 (2022): 5856–5873, https://doi.org/10.1007/s12035-022-02945-w.

[75] H. Sakane, H. Yamamoto, and A. Kikuchi, “LRP6 is Internalized by Dkk1 to Suppress Its Phosphorylation in the Lipid Raft and is Recycled for Reuse,” Journal of Cell Science 123 (2010): 360–368, https://doi.org/10.1242/jcs.058008.

[76] L. Wu, C. Liu, D. Y. Chang, et al., “The Attenuation of Diabetic Nephropathy by Annexin A1 via Regulation of Lipid Metabolism through the AMPK/PPARα/CPT1b Pathway,” Diabetes 70 (2021): 2192–2203, https://doi.org/10.2337/db21-0050.

[77] S. E. Flaherty, O. Bezy, W. Zheng, et al., “Chronic UCN2 Treatment Desensitizes CRHR2 and Improves Insulin Sensitivity,” Nature Communications 14 (2023): 3953, https://doi.org/10.1038/s41467-023-39597-w.

[78] E. D. Hamlett, E. Hjorth, A. Ledreux, et al., “RvE1 Treatment Prevents Memory Loss and Neuroinflammation in the Ts65Dn Mouse Model of Down Syndrome,” Glia 68 (2020): 1347–1360, https://doi.org/10.1002/glia.23779.

[79] J. L. Cash, R. Hart, A. Russ, et al., “Synthetic Chemerin-derived Peptides Suppress Inflammation Through ChemR23,” Journal of Experimental Medicine 205 (2008): 767–775, https://doi.org/10.1084/jem.20071601.

[80] C. Ricci, M. Ruscica, M. Camera, et al., “PCSK9 Induces a Pro-inflammatory Response in Macrophages,” Scientific Reports 8 (2018): 2267, https://doi.org/10.1038/s41598-018-20425-x.

[81] S. Cohen-Cory, “The Developing Synapse: Construction and Modulation of Synaptic Structures and Circuits,” Science 298 (2002): 770–776, https://doi.org/10.1126/science.1075510.

[82] J. Zhou, W. Du, K. Zhou, et al., “Critical Role of TRPC6 Channels in the Formation of Excitatory Synapses,” Nature Neuroscience 11 (2008): 741–743, https://doi.org/10.1038/nn.2127.

[83] J. Meng, X. Ma, H. Tao, et al., “Myrf ER-Bound Transcription Factors Drive C. elegans Synaptic Plasticity via Cleavage-Dependent Nuclear Translocation,” Developmental Cell 41 (2017): 180–194.e7, e187, https://doi.org/10.1016/j.devcel.2017.03.022,.

[84] Z. Luo, “Synapse Formation and Remodeling,” Science China Life Sciences 53 (2010): 315–321, https://doi.org/10.1007/s11427-010-0069-5.

[85] A. Dityatev, R. Frischknecht, and C. I. Seidenbecher, “Extracellular Matrix and Synaptic Functions,” Results and Problems in Cell Differentiation 43 (2006): 69–97, https://doi.org/10.1007/11372318.

[86] C. Cangalaya, W. Sun, S. Stoyanov, I. R. Dunay, and A. Dityatev, “Integrity of Neural Extracellular Matrix Is Required for Microglia-Mediated Synaptic Remodeling,” Glia 72 (2024): 1874–1892, https://doi.org/10.1002/glia.24588.

[87] O. Senkov, P. Andjus, L. Radenovic, E. Soriano, and A. Dityatev, “Neural ECM Molecules in Synaptic Plasticity, Learning, and Memory,” Progress in Brain Research 214 (2014): 53–80.

[88] A. Dityatev and M. Schachner, “Extracellular Matrix Molecules and Synaptic Plasticity,” Nature Reviews Neuroscience 4 (2003): 456–468, https://doi.org/10.1038/nrn1115.

[89] A. Dityatev, M. Schachner, and P. Sonderegger, “The Dual Role of the Extracellular Matrix in Synaptic Plasticity and Homeostasis,” Nature Reviews Neuroscience 11 (2010): 735–746, https://doi.org/10.1038/nrn2898.

[90] T. E. Sutherland, D. P. Dyer, and J. E. Allen, “The Extracellular Matrix and the Immune System: A Mutually Dependent Relationship,” Science 379 (2023): eabp8964, https://doi.org/10.1126/science.abp8964.

[91] A. J. L. Ridley, Y. Ou, R. Karlsson, et al., “Chemokines Form Complex Signals During Inflammation and Disease That Can be Decoded by Extracellular Matrix Proteoglycans,” Science Signaling 16 (2023): eadf2537, https://doi.org/10.1126/scisignal.adf2537.

[92] P. T. Nguyen, L. C. Dorman, S. Pan, et al., “Microglial Remodeling of the Extracellular Matrix Promotes Synapse Plasticity,” Cell 182 (2020): 388–403.e15, e315, https://doi.org/10.1016/j.cell.2020.05.050.

[93] J. B. Kwok, M. Hallupp, C. T. Loy, et al., “GSK3B Polymorphisms Alter Transcription and Splicing in Parkinson's Disease,” Annals of Neurology 58 (2005): 829–839, https://doi.org/10.1002/ana.20691.

[94] J. B. Kwok, C. T. Loy, G. Hamilton, et al., “Glycogen Synthase Kinase-3β and Tau Genes Interact in Alzheimer's Disease,” Annals of Neurology 64 (2008): 446–454, https://doi.org/10.1002/ana.21476.

[95] Z. Shen, D. Sun, A. Savastano, et al., “Multivalent Tau/PSD-95 Interactions Arrest in Vitro Condensates and Clusters Mimicking the Postsynaptic Density,” Nature Communications 14 (2023): 6839, https://doi.org/10.1038/s41467-023-42295-2.

[96] M. J. Kim, K. Futai, J. Jo, et al., “Synaptic Accumulation of PSD-95 and Synaptic Function Regulated by Phosphorylation of Serine-295 of PSD-95,” Neuron 56 (2007): 488–502, https://doi.org/10.1016/j.neuron.2007.09.007.

[97] I. Nikonenko, B. Boda, S. Steen, et al., “PSD-95 Promotes Synaptogenesis and Multiinnervated Spine Formation Through Nitric Oxide Signaling,” Journal of Cell Biology 183 (2008): 1115–1127, https://doi.org/10.1083/jcb.200805132.

[98] Z. Yentür, T. Kagermeier, K. Sarieva, et al., “Human Dorsal Forebrain Organoids Show Differentiation-state-specific Protein Secretion,” Iscience 28 (2025): 112935.

[99] I. Fagerlund, A. Dougalis, A. Shakirzyanova, et al., “Microglia-Like Cells Promote Neuronal Functions in Cerebral Organoids,” Cells 11 (2021): 124, https://doi.org/10.3390/cells11010124.

[100] B. Spittau, N. Dokalis, and M. Prinz, “The Role of TGFβ Signaling in Microglia Maturation and Activation,” Trends in Immunology 41 (2020): 836–848, https://doi.org/10.1016/j.it.2020.07.003.

[101] Y. Jiang, X. Fu, Y. Zhang, et al., “Rett Syndrome Linked to Defects in Forming the MeCP2/Rbfox/LASR Complex in Mouse Models,” Nature Communications 12 (2021): 5767, https://doi.org/10.1038/s41467-021-26084-3.

[102] H. Zhang, X. Chen, P. Xue, et al., “FN1 Promotes Chondrocyte Differentiation and Collagen Production via TGF-β/PI3K/Akt Pathway in Mice With Femoral Fracture,” Gene 769 (2021): 145253, https://doi.org/10.1016/j.gene.2020.145253.

[103] L. A. Farrer, L. A. Cupples, J. L. Haines, et al., “Effects of Age, Sex, and Ethnicity on the Association Between Apolipoprotein E Genotype and Alzheimer Disease,” JAMA 278 (1997): 1349, https://doi.org/10.1001/jama.1997.03550160069041.

[104] Y. Hou, X. Dan, M. Babbar, et al., “Ageing as a Risk Factor for Neurodegenerative Disease,” Nature Reviews Neurology 15 (2019): 565–581, https://doi.org/10.1038/s41582-019-0244-7.

[105] F. Yuan, Y. Li, R. Hu, et al., “Modeling Disrupted Synapse Formation in Wolfram Syndrome Using hESCs-derived Neural Cells and Cerebral Organoids Identifies Riluzole as a Therapeutic Molecule,” Molecular Psychiatry 28 (2023): 1557–1570, https://doi.org/10.1038/s41380-023-01987-3.

[106] G. M. Parfitt, E. Coccia, C. Goldman, et al., “Disruption of Lysosomal Proteolysis in Astrocytes Facilitates Midbrain Organoid Proteostasis Failure in an Early-Onset Parkinson's Disease Model,” Nature Communications 15 (2024): 447, https://doi.org/10.1038/s41467-024-44732-2.

[107] K. Wang, H. Zhang, D. Ma, et al., “Common Genetic Variants on 5p14.1 Associate With Autism Spectrum Disorders,” Nature 459 (2009): 528–533, https://doi.org/10.1038/nature07999.

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