Microglial GRB2 is essential for brain ventriculogenesis and cerebrospinal fluid homeostasis
Phan Q. Duy, Benjamin C. Reeves, Huanxing Sun, Xueyan Peng, Pazhanichamy Kalailingam, Garrett Allington, Evan Dennis, Le Thi Hao, Lei Wang, David Rufino-Ramos, Shujuan Zhao, Qiang Li, Neel H. Mehta, William C. Davalan, Mason Blacker, Anthony J. Piscopo, Shozeb Haider, Baojian Fan, Kedous Y. Mekbib, Shuai Shao, Carol Nelson-Williams, Tukiet T. Lam, Benjamin P. Kleinstiver, Patricia L. Musolino, Seth L. Alper, Sheng Chih Jin, Erica L. Herzog, Kristopher T. Kahle
Phan Q. Duy, Benjamin C. Reeves, Huanxing Sun, Xueyan Peng, Pazhanichamy Kalailingam, Garrett Allington, Evan Dennis, Le Thi Hao, Lei Wang, David Rufino-Ramos, Shujuan Zhao, Qiang Li, Neel H. Mehta, William C. Davalan, Mason Blacker, Anthony J. Piscopo, Shozeb Haider, Baojian Fan, Kedous Y. Mekbib, Shuai Shao, Carol Nelson-Williams, Tukiet T. Lam, Benjamin P. Kleinstiver, Patricia L. Musolino, Seth L. Alper, Sheng Chih Jin, Erica L. Herzog, Kristopher T. Kahle
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Research Article Genetics Neuroscience

Microglial GRB2 is essential for brain ventriculogenesis and cerebrospinal fluid homeostasis

Abstract

Microglia play essential yet poorly understood roles in brain development, including axon guidance, regulation of neurogenesis, and pruning of neuronal projections. Congenital hydrocephalus (CH), characterized by enlarged ventricles filled with cerebrospinal fluid (CSF), is a leading cause of pediatric brain surgery, but its molecular mechanisms remain unclear. We have identified what we believe to be novel, recurrent, damaging missense variants in the SH3-binding domain of the adaptor protein growth factor receptor-bound protein 2 (GRB2) in unrelated patients with CH. GRB2 is significantly coexpressed with 1 of its known upstream RTK partners, CSF1R, in the developing human brain, particularly in a microglial subtype associated with regulation of neural stem cells. IP validated GRB2-CSF1R binding in mouse microglial cells and a human monocyte cell line. Cx3cr1-Grb2fl/fl mice engineered with conditional deletion of Grb2 in microglia exhibit congenital absence of microglia and early postnatal severe communicating (nonobstructive) hydrocephalus, mimicking the condition of patients with the GRB2 mutation. The severe ventriculomegaly of Cx3cr1-Grb2fl/fl mice is associated with both depletion of cerebral cortical neurons and impairment of glia-lymphatic-mediated CSF flow. Together, these findings implicate a role of GRB2 in microglia that could be essential for brain development and CSF homeostasis.

Authors

Phan Q. Duy, Benjamin C. Reeves, Huanxing Sun, Xueyan Peng, Pazhanichamy Kalailingam, Garrett Allington, Evan Dennis, Le Thi Hao, Lei Wang, David Rufino-Ramos, Shujuan Zhao, Qiang Li, Neel H. Mehta, William C. Davalan, Mason Blacker, Anthony J. Piscopo, Shozeb Haider, Baojian Fan, Kedous Y. Mekbib, Shuai Shao, Carol Nelson-Williams, Tukiet T. Lam, Benjamin P. Kleinstiver, Patricia L. Musolino, Seth L. Alper, Sheng Chih Jin, Erica L. Herzog, Kristopher T. Kahle

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Figure 1

Recurrent, damaging variants in the GRB2 c-terminal SH3 domain in patients with CH.

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Recurrent, damaging variants in the GRB2 c-terminal SH3 domain in patien...
(A) Coronal T1-weighted fetal MRI of proband KCHYD52-1 and axial head CT scan of proband KCHYD65-1 (white dot indicates ventricular catheter of a ventriculo-peritoneal shunt used to treat hydrocephalus). (B) Schematic of variant locations in GRB2 protein domains: red variants are from our CH cohort, blue variants are from the GMKF database. The axis depicts protein length (aa, in gray). (C) Final AF3 model of the CSF1R-GRB2-SHP2 multimeric complex, with sites of GRB2 missense substitutions highlighted. Shown are CSF1R aa 521–926, GRB2 aa 1–217 and SHP2 aa 1–573. AlphaFold models of deleterious missense variant p.R178C (ΔΔG 0.93) (D) and p.R179Q (ΔΔG 0.40) (E), both predicted to be detrimental to GRB2 structure and function due to loss of hydrogen-bonding interactions between GRB2-mutant side chains and adjacent SHP2 backbone atoms. (F) Schematic of the CSF1R/GRB2/SHP2 signaling pathway with all variants summarized. LoF, loss of function.