Ezh2 Regulates Early Astrocyte Morphogenesis and Influences the Coverage of Astrocytic Endfeet on the Vasculature

Abstract

Astrocytes are crucial for central nervous system (CNS) development and function, with their differentiation being stringently controlled by epigenetic mechanisms, such as histone modifications. Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase, is essential for the suppression of gene expression. However, the role of EZH2 in astrocyte early morphogenesis has remained unclear. Using an astrocyte-specific Ezh2 knockout (cKO) mouse model, we examined the effects of EZH2 deletion on astrocyte morphogenesis, blood–brain barrier (BBB) integrity and neurodevelopment. Loss of EZH2 led to increased glial fibrillary acidic protein (GFAP) expression, altered astrocyte morphology and reduced coverage of astrocytic endfeet on blood vessels, compromising BBB integrity. Vascular abnormalities, characterised by increased vascular density and smaller vessel diameter, mirrored compensatory changes seen in moyamoya disease. RNA-sequencing and ChIP-seq identified Ddn as a key upregulated gene in Ezh2^cKO astrocytes, influencing cytoskeletal changes via the MAPK/ERK pathway. Behavioural analysis revealed autism-like traits, such as reduced vocalisations, without significant anxiety-like behaviour. These findings highlight EZH2 as a critical regulator of astrocyte function, with its disruption contributing to neurodevelopmental disorders. This study provides novel insights into the molecular pathways governing astrocyte differentiation and suggests EZH2 as a promising therapeutic target for gliomas and other CNS disorders.

Keywords

astrocyte / autism / BBB / EZH2 / morphogenesis

References

[1] M. Pekny, M. Pekna, A. Messing, et al., “Astrocytes: A Central Element in Neurological Diseases,” Acta Neuropathologica 131, no. 3 (2016): 323-345.

[2] A. Verkhratsky and M. Nedergaard, “Physiology of Astroglia,” Physiological Reviews 98, no. 1 (2018): 239-389.

[3] C. Escartin, E. Galea, A. Lakatos, et al., “Reactive Astrocyte Nomenclature, Definitions, and Future Directions,” Nature Neuroscience 24, no. 3 (2021): 312-325.

[4] P. Hasel and S. A. Liddelow, “Astrocytes,” Current Biology 31, no. 7 (2021): R326-r7.

[5] J. Nagai, X. Yu, T. Papouin, et al., “Behaviorally Consequential Astrocytic Regulation of Neural Circuits,” Neuron 109, no. 4 (2021): 576-596.

[6] H. G. Lee, M. A. Wheeler, and F. J. Quintana, “Function and Therapeutic Value of Astrocytes in Neurological Diseases,” Nature Reviews. Drug Discovery 21, no. 5 (2022): 339-358.

[7] A. N. Brandebura, A. Paumier, T. S. Onur, and N. J. Allen, “Astrocyte Contribution to Dysfunction, Risk and Progression in Neurodegenerative Disorders,” Nature Reviews. Neuroscience 24, no. 1 (2023): 23-39.

[8] J. Takouda, S. Katada, and K. Nakashima, “Emerging Mechanisms Underlying Astrogenesis in the Developing Mammalian Brain,” Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 93, no. 6 (2017): 386-398.

[9] S. Kunoh, H. Nakashima, and K. Nakashima, “Epigenetic Regulation of Neural Stem Cells in Developmental and Adult Stages,” Epigenomes 8, no. 2 (2024): 22, https://doi.org/10.3390/epigenomes8020022.

[10] S. Ma, L. Wang, J. Zhang, L. Geng, and J. Yang, “The Role of Transcriptional and Epigenetic Modifications in Astrogliogenesis,” PeerJ 12 (2024): e18151.

[11] T. T. San, J. Kim, and H. J. Kim, “Histone Lysine Demethylase KDM5 Inhibitor CPI-455 Induces Astrocytogenesis in Neural Stem Cells,” ACS Chemical Neuroscience 15, no. 7 (2024): 1570-1580.

[12] Z. Zhao, A. R. Nelson, C. Betsholtz, and B. V. Zlokovic, “Establishment and Dysfunction of the Blood-Brain Barrier,” Cell 163, no. 5 (2015): 1064-1078.

[13] K. T. Baldwin, K. K. Murai, and B. S. Khakh, “Astrocyte Morphology,” Trends in Cell Biology 34, no. 7 (2024): 547-565.

[14] M. L. Suvà, E. Rheinbay, S. M. Gillespie, et al., “Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-Like Cells,” Cell 157, no. 3 (2014): 580-594.

[15] J. Park, K. Lee, K. Kim, and S. J. Yi, “The Role of Histone Modifications: From Neurodevelopment to Neurodiseases,” Signal Transduction and Targeted Therapy 7, no. 1 (2022): 217.

[16] E. Kim, M. Kim, D. H. Woo, et al., “Phosphorylation of EZH2 Activates STAT3 Signaling via STAT3 Methylation and Promotes Tumorigenicity of Glioblastoma Stem-Like Cells,” Cancer Cell 23, no. 6 (2013): 839-852.

[17] R. Cao, L. Wang, H. Wang, et al., “Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing,” Science 298, no. 5595 (2002): 1039-1043.

[18] F. Mohammad, S. Weissmann, B. Leblanc, et al., “EZH2 Is a Potential Therapeutic Target for H3K27M-Mutant Pediatric Gliomas,” Nature Medicine 23, no. 4 (2017): 483-492.

[19] G. Zhao, Z. Deng, X. Li, et al., “Targeting EZH2 Regulates the Biological Characteristics of Glioma Stem Cells via the Notch1 Pathway,” Experimental Brain Research 241, no. 10 (2023): 2409-2418.

[20] H. Zhang, Z. Wang, X. Qiao, et al., “Unveiling the Therapeutic Potential of IHMT-337 in Glioma Treatment: Targeting the EZH2-SLC12A5 Axis,” Molecular Medicine 30, no. 1 (2024): 91.

[21] B. S. Khakh and B. Deneen, “The Emerging Nature of Astrocyte Diversity,” Annual Review of Neuroscience 42 (2019): 187-207.

[22] J. Niu, H. H. Tsai, K. K. Hoi, et al., “Aberrant Oligodendroglial-Vascular Interactions Disrupt the Blood-Brain Barrier, Triggering CNS Inflammation,” Nature Neuroscience 22, no. 5 (2019): 709-718.

[23] B. Cebolla, A. Fernández-Pérez, G. Perea, A. Araque, and M. Vallejo, “DREAM Mediates cAMP-Dependent, Ca2+−Induced Stimulation of GFAP Gene Expression and Regulates Cortical Astrogliogenesis,” Journal of Neuroscience 28, no. 26 (2008): 6703-6713.

[24] G. J. Rahme, N. M. Javed, K. L. Puorro, et al., “Modeling Epigenetic Lesions That Cause Gliomas,” Cell 186, no. 17 (2023): 3674.

[25] S. Dhar, S. Gadd, P. Patel, et al., “A Tumor Suppressor Role for EZH2 in Diffuse Midline Glioma Pathogenesis,” Acta Neuropathologica Communications 10, no. 1 (2022): 47.

[26] B. Díaz-Castro, S. Robel, and A. Mishra, “Astrocyte Endfeet in Brain Function and Pathology: Open Questions,” Annual Review of Neuroscience 46 (2023): 101-121.

[27] M. Ihara, Y. Yamamoto, Y. Hattori, et al., “Moyamoya Disease: Diagnosis and Interventions,” Lancet Neurology 21, no. 8 (2022): 747-758.

[28] X. Zhang, W. Xiao, Q. Zhang, et al., “Progression in Moyamoya Disease: Clinical Features, Neuroimaging Evaluation, and Treatment,” Current Neuropharmacology 20, no. 2 (2022): 292-308.

[29] S. Mehra, A. U. Ahsan, M. Sharma, M. Budhwar, and M. Chopra, “Gestational Fisetin Exerts Neuroprotection by Regulating Mitochondria-Directed Canonical Wnt Signaling, BBB Integrity, and Apoptosis in Prenatal VPA-Induced Rodent Model of Autism,” Molecular Neurobiology 61, no. 7 (2024): 4001-4020.

[30] W. P. Ge, A. Miyawaki, F. H. Gage, Y. N. Jan, and L. Y. Jan, “Local Generation of Glia Is a Major Astrocyte Source in Postnatal Cortex,” Nature 484, no. 7394 (2012): 376-380.

[31] S. Clavreul, L. Abdeladim, E. Hernández-Garzón, et al., “Cortical Astrocytes Develop in a Plastic Manner at Both Clonal and Cellular Levels,” Nature Communications 10, no. 1 (2019): 4884.

[32] E. A. Bushong, M. E. Martone, and M. H. Ellisman, “Maturation of Astrocyte Morphology and the Establishment of Astrocyte Domains During Postnatal Hippocampal Development,” International Journal of Developmental Neuroscience 22, no. 2 (2004): 73-86.

[33] M. Neuner-Jehle, J. P. Denizot, A. A. Borbély, and J. Mallet, “Characterization and Sleep Deprivation-Induced Expression Modulation of Dendrin, a Novel Dendritic Protein in Rat Brain Neurons,” Journal of Neuroscience Research 46, no. 2 (1996): 138-151.

[34] A. Herb, W. Wisden, M. V. Catania, D. Maréchal, A. Dresse, and P. H. Seeburg, “Prominent Dendritic Localization in Forebrain Neurons of a Novel mRNA and Its Product, Dendrin,” Molecular and Cellular Neurosciences 8, no. 5 (1997): 367-374.

[35] A. Kawata, J. Iida, M. Ikeda, et al., “CIN85 Is Localized at Synapses and Forms a Complex With S-SCAM via Dendrin,” Journal of Biochemistry 139, no. 5 (2006): 931-939.

[36] F. Dunér, J. Patrakka, Z. Xiao, et al., “Dendrin Expression in Glomerulogenesis and in Human Minimal Change Nephrotic Syndrome,” Nephrology, Dialysis, Transplantation 23, no. 8 (2008): 2504-2511.

[37] H. Zhao, Z. Tu, H. Xu, et al., “Altered Neurogenesis and Disrupted Expression of Synaptic Proteins in Prefrontal Cortex of SHANK3-Deficient Non-Human Primate,” Cell Research 27, no. 10 (2017): 1293-1297.

[38] Q. Wang, Y. Kong, D. Y. Wu, et al., “Impaired Calcium Signaling in Astrocytes Modulates Autism Spectrum Disorder-Like Behaviors in Mice,” Nature Communications 12, no. 1 (2021): 3321.

[39] Y. Li, T. Fan, X. Li, et al., “Npas3 Deficiency Impairs Cortical Astrogenesis and Induces Autistic-Like Behaviors,” Cell Reports 41, no. 10 (2022): 111767.

RIGHTS & PERMISSIONS

2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.