Complement: a novel factor in basal and ischemia-induced neurogenesis

Abstract

Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3(-/-)) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase. Intriguingly, basal neurogenesis is decreased both in C3(-/-) mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3(-/-) mice had impaired ischemia-induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia-induced neurogenesis.

Figure 1

Expression of C3aR and C5aR on cultured neural stem cells. Immunostaining of clonally derived neural stem cells from adult rat hippocampus for C3aR (A) and C5aR (C) shows homogeneous distribution of both receptors in the cell membrane. Nestin immunostaining confirmed that the cells retain their stem cell phenotype. The cells were infected with retrovirus to express green fluorescent protein (GFP). Negative control immunostaining, in which the primary antibodies against C3aR, C5aR, and nestin were omitted (B, D). Scale bar equals 20 μm.

Figure 2

Expression of C3aR and C5aR on transit-amplifying precursors and migrating neuroblasts in vivo. Immunostaining of paraffin-embedded brain sections from adult mice with antibodies against C3aR (A) and C5aR (B). Both receptors are expressed by cells in the SVZ and rostral migratory stream of WT mice. The specificity of the anti-C3aR and anti-C5aR antibodies, respectively, was confirmed by the absence of immunostaining on brain sections from C3aR^−/− and C5aR^−/−mice. In the negative control immunostainings of WT brain sections, the primary antibodies were omitted. Olig2-positive transit-amplifying precursor cells in the SVZ are colabeled by anibodies against C3aR (C) and C5aR (D). Dcx^pos migrating neuroblasts in the rostral migratory stream express C3aR (E) and C5aR (F). Arrows indicate double-labeled cells in the insert. Scale bars equal 50 μm (A, B) 10 μm (C, D) and 60 μm (E, F).

Figure 3

Proliferating migrating neuroblast in the dentate gyrus SGZ visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). The number of Dcx^posBrdU^pos cells in SGZ, SVZ, and OB in control mice (n=12), C3^−/− mice (n=6), and mice treated with C3aR antagonist (n=10) (B). Representative low-power images of Dcx^posBrdU^pos cells in SGZ, SVZ, and OB in the three groups of mice; Dcx, green, BrdU, red (C). Values are the number of cells per region. ^*P<0.05, ^**P<0.0005. Scale bars equal 10 μm (A) and 200 μm (C).

Figure 4

A newly formed neuron in the dentate gyrus GCL visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). The number of NeuN^posBrdU^pos cells in the SGZ/GCL and OB in control mice (n=12), C3^−/− mice (n=6) and mice treated with C3aR antagonist (n=10) (B). Values are the number of cells per region. ^*P<0.05, ^**P<0.005. Scale bar equals 10 μm.

Figure 5

Basal neurogenesis is reduced in C3aR^−/− mice. Compared to control (WT) mice (n=6), the C3aR^−/− mice (n=6) have lower number of newly formed migrating neuroblasts in the SGZ and OB (A) and lower number of newly formed neurons in SGZ/the dentate gyrus GCL and OB (B). Values are the number of cells per region. ^*P<0.05. The higher number of NeuN^posBrdU^pos cells in control mice in this figure compared to Figure 4 is likely due to a difference in survival time after the last BrdU injection (14 versus 3 days).

Figure 6

Number of Dcx^pos migrating neuroblasts in the SVZ in the ipsilateral (I) and contralateral (C) hemispheres of WT (n=7, 8, 8) and C3^−/− (n=9, 7, 11) mice. The mice were subjected to focal cerebral ischemia by MCAO or MCAT. Dcx^pos cells in the SVZ were counted in three sections per mouse 7 days after MCAO (A) and MCAT (B) and 21 days after MCAT (C). Values are the number of cells per section. ^*P<0.05, ^**P<0.01, ^***P<0.005.

Figure 7

Neural progenitor cells in the infarct area and penumbra of WT (n=7, 8, 8) and C3^−/− (n=9, 7, 11) mice after focal cerebral ischemia. Immunostaining for nestin and GFAP was used to visualize nestin^pos GFAP^neg neural progenitor cells (A). These cells were counted in 2–3 sections/mouse in the infarct area and penumbra 7 days after MCAO and in the penumbra 7 days after MCAT (B). Values are the number of cells per section (MCAO) and the number of cells/10 mm² (MCAT). Representative low-power images of the penumbra in the two groups of mice; nestin, red, GFAP, green. Arrows indicate nestin^pos GFAP^neg cells. Broken line depicts the infarct border. i, infarct (C). ^*P<0.05, ^***P<0.005, ^*****P<0.00001. Scale bars equal 15 μm (A) and 200 μm (C).

Figure 8

Ischemia-induced generation of new neurons. A newly formed NeuN^posBrdU^pos neuron in the penumbra visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). Number of NeuN^posBrdU^pos cells in the penumbra of WT (n=10, 11) and C3^−/− (n=9, 11) mice 7 and 21 days after MCAT (B). The number of NeuN^posBrdU^pos cells/10 mm² was counted in the penumbra on 2–3 sections/mouse. ^***P<0.005, ^****P<0.001. Scale bar equals 15 μm.

Figure 9

The NeuN^posBrdU^pos cells are not undergoing ischemia-induced apoptosis. Immunostaining for BrdU, NeuN and caspase-3 on day 7 (A) and day 21 (B) after MCAT. Broken line depicts the infarct border. p, penumbra; i, infarct. Higher magnification images of boxed areas in (A) showing penumbra (P) and infarct (I). Scale bar equals 120 μm (A, B) and 60 μm (P, I).

Figure 10

Representative low-power images of the density of neurons (NeuN^pos), reactive astrocytes (GFAP^pos), and microglia (isolectin^pos) at the infarct border in C3^−/− (A) and WT (B) mice 7 days after cerebral ischemia. The sections were stained with antibodies against NeuN and GFAP, and with isolectin. Broken line depicts the infarct border. i, infarct. Scale bars equal 150 μm.