Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ

Abstract

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Keywords: C5a; C5aR1; aPKC; complement; neurogenesis; polarity.

Figure 1.

Localization of C5aR1 and ligands. A, C5aR1 (red) is expressed in the developing neocortex at the apical surface of the ventricular zone at E14.5 (top row). Shown is counterstain with Pax6 (green) and DAPI (blue). Scale bar, 50 μm. Merged images of the ventricular zone at E12.5, E16.5, and E18.5 are shown in the second row. RT-PCR demonstrates C5aR1 expression in embryonic brain tissue, neurosphere, and NE-4C culture. B, C5aR1 expression (red) within sectioned neurosphere. Neg, Secondary-only negative control. Scale bar, 50 μm. C, C5aR1 expression (red) on NE-4C cells grown in monolayer. Neg, Secondary-only negative control. Scale bar, 20 μm. D, Embryonic CSF contains mC5a at significantly greater concentrations than brain tissue or maternal CSF. E, C5aR1 is detected within NE-4C cultures by Western blot at the predicted molecular weight (50 kDa) and decreases at stage VI of differentiation. Stages indicate morphologically distinct progression of NE-4C differentiation (see Materials and Methods). F, Expression of C5aR1 mRNA decreases with differentiation of NE-4C cells. Progenitor marker Sox2 is assayed as a comparison. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 2.

Expression of C5aR1 in human embryonic stem cell-derived rosettes. A, Immunocytochemistry of human neural rosettes showing staining for neural marker TUBB3, tight junction marker ZO-1, and C5. C5aR1 localizes to the apical membrane, colocalizing with PKCζ, but not markers of cilia (Arl13b) or tight junctions (NCAD). Negative controls are shown at top right. Scale bar, 20 μm. B, Transcript expression as hES cells (day 0) are differentiated through the cortical rosette stage (day 28) to a mature neuronal lineage. C5AR1 expression is highest at the rosette stage of neuronal differentiation. C, hC5a is detected through ELISA within the lysate of rosette cultures and is not derived from the exogenous extracellular matrix (Matrigel). *p ≤ 0.05, **p ≤ 0.01.

Figure 3.

C5aR1 signals through PKCζ to maintain cell polarity in vitro. A, Mouse neurosphere cultures demonstrate C5a concentration-dependent p42/44 phosphorylation. B, The response to 100 nm C5a is prevented by PKCζ inhibition. C, Human rosette cultures demonstrate time-dependent p42/44 phosphorylation to 10 nm hC5a. The response is prevented through pretreatment with C5aR1-A or PKCζ inhibition. D–G, Mouse and human neurosphere cultures dissociated and grown over a 7 d period demonstrate an increase in number (D, F) and diameter (E, G) in response to C5a. H–K, Treatment of human rosettes with 10 nm C5a. H, C5a increases M-phase-positive cells in neural rosettes as determined by pHH3 immunocytochemical analysis. I, DAPT (gray bars) treatment induced loss of rosettes and a decrease in mRNA of NCAD, C5AR1, and C5 compared with vehicle (black bars) treatment. J, K, Maintenance of rosette architecture after single-cell dissociation (K) or DAPT treatment (J) was promoted by exogenous C5a addition. Adjacent images are representative of DAPT-treated rosettes in the presence or absence of C5a. NCAD (white) and computational outlines (green) of rosette apical lumens are shown. L, M, NE-4C cells grown on transwell membranes demonstrate reorganization of the mitotic spindle (L), as determined by acetylated tubulin staining (green), and reduction in apical surface area (M), outlined by ZO-1 (red) in response to C5a. White arrows are representative distances from mitotic spindle to calculated cell center as shown in L. Scale bar, 20 μm. Veh., Vehicle. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4.

C5aR1 signaling alters neural progenitor division planes and proliferation in vivo. A, Schema of the in utero injection process. Briefly, 1 μl of 100 nm mC5a, 1 μm PMX53, or vehicle was delivered to the embryonic ventricle in utero. After 24 h, brains were processed for immunohistochemistry. M-phase cells, as determined by pHH3 staining, were counted along the ventricular surface of the neocortex. B, In utero injection of mC5a to the embryonic ventricle increases, whereas blockade of C5aR1 signaling using PMX53 decreases, the number of M-phase apical progenitor cells. C, Cleavage plan analysis demonstrates a shift from symmetric division toward asymmetric division after treatment with C5aR1 antagonist. S, Symmetric division; A, asymmetric division; O, oblique division; Veh., vehicle. **p ≤ 0.01, ***p ≤ 0.001.

Figure 5.

Blockade of C5aR1 signaling at E12.5–E14.5 causes behavioral changes in adult mice. A, Schema of the experimental process. Briefly, 1 mg · kg⁻¹ · d⁻¹ of the C5aR1 antagonist PMX53 was delivered by intraperitoneal injection to pregnant dams at E12.5–E14.5. Resultant litters were taken through behavioral testing from 6 to 8 weeks and killed, and brains were prepared for ex vivo MRI. Data show results for vehicle (V, black bars)- and PMX53 (P, gray bars)-treated mice. B, Sox2/DCX ratio of the ventricular zone of E16.5 embryos. C, D, F, No change in postnatal growth (C), litter size (D), and snout-occiput length (F) was seen. E, A significant reduction in crown-rump length was observed. G, No difference between treatment groups was found in grip strength for both forelimb (F) and hindlimb (H). H, Time to cross balance beam was increased in male animals from PMX53-treated litters. I, Footfall errors crossing balance beam. J–L, Distance moved in center of open-field arena (J), frequency of entry to novel arm Y-maze (K), and time spent immobile during the forced swim test (L) were significantly different in PMX53-treated litters. Veh., Vehicle; Fem., female. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 6.

Blockade of C5aR1 signaling at E12.5–E14.5 results in microstructural changes in adult brains. Top row, Significance map of Jacobian warping projected onto T1-weighted template average. Middle row, Significance map of FA values projected onto template average. For both maps: blue/purple, vehicle>PMX53 (C5aR1-A) treatment; red/yellow, PMX53>vehicle. Color-coded p values are shown at the bottom. Bottom row, Anatomical areas are labeled. Mot., Motor cortex; Orb., orbital cortex; OB, olfactory bulb; SS, somatosensory cortex; Gust., gustatory cortex; Pir., piriformis; CP, caudate/putamen; NA, nucleus accumbens; P., pallidum; H., hypothalamus; Veh, vehicle.