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. 2017 May 2;8:15096.
doi: 10.1038/ncomms15096.

Developmental Activities of the Complement Pathway in Migrating Neurons

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Free PMC article

Developmental Activities of the Complement Pathway in Migrating Neurons

Anna Gorelik et al. Nat Commun. .
Free PMC article

Abstract

In recent years the notion that malfunctioning of the immune system may result in developmental brain diseases has emerged. However, the role of immune molecules in the developing brain has not been well explored. The complement pathway converges to cleave C3. Here we show that key proteins in the lectin arm of this pathway, MASP1, MASP2 and C3, are expressed in the developing cortex and that neuronal migration is impaired in knockout and knockdown mice. Molecular mimics of C3 cleavage products rescue the migration defects that have been seen following knockdown of C3 or Masp2. Pharmacological activation of the downstream receptors rescue Masp2 and C3 knockdown as well as C3 knockout. Therefore, we propose that the complement pathway is functionally important in migrating neurons of the developing cortex.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Complement components are expressed in the developing brain and affect neuronal migration.
(a) Schematic presentation of the complement pathway. (bg) Immunostaining of cortices (E16) with anti-C3, anti-MASP1 and anti-MASP2 antibodies (bd, respectively) demonstrate wide expression of these proteins. Immunostaining with anti-TBR2, anti-TUJ1 and anti-TBR1 antibodies (eg, respectively) show the borders of the ventricular zone, intermediate zone and cortical plate, respectively. (h) Scheme showing the route of radial migration in the cerebral cortex. Neurons born at E14 in the VZ (green cells) migrate and on E18 are located at superficial extremity of the cortex. (ik) Extracellular localization of the tested complement proteins. Brains were in utero electroporated (E14–E16) with Lifeact-GFP to mark the cell periphery. Sections were immunostained with anti-C3, anti-MASP1 and anti-MASP2 antibodies (ik, respectively). High-magnification 3D reconstructions of individual neurons from the IZ were performed using confocal microscopy and IMARIS software. The asterisks in bd demonstrate the approximate position of the chosen neurons. (lq) C3, Masp1 and Masp2 KO affect neuronal migration. (l,m) WT or C3 KO embryos were in utero electroporated with GFP (E14–E18). The GFP positive neurons in the C3 KO (n=5) resided in deeper layers than in the WT (n=3).The position of the GFP+ cells was quantified in five bins (from the VZ to the CP) (m). (nq) Birth-dating by IdU at E14 and analysis at E18 show that in WT (n=3) sections (n,p), labelled cells are located in more superficial layers than in Masp1 KO (n=3) sections (n) or in the Masp2 KO (n=3) sections (p). The distribution of IdU-positive neurons (o,q). C3 KO brain (n=8) sections (r), Masp1 KO brain (n=5) sections (u) and Masp2 KO brain (n=5) sections (x) and WT brain (n=8, n=5, n=5 respectively) sections immunostained with anti-CUX1 and anti-TBR1 antibodies. The relative proportion of the CUX1-positive and TBR1-positive domains relative to the total cortical width are displayed (Student t-test, s,v,y and t,w,z, respectively). (ik), scale bars are 5 μm, (bg,l,n,p,r,u,x, scale bars are 50 μm). *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001, histograms present means±s.e.m.
Figure 2
Figure 2. C3, Masp1 and Masp2 knockdown affect neuronal migration.
(ae) Brains were electroporated in utero (E14–E18) with control shRNA (a, n=4), C3 shRNA (b, n=5), Masp1 shRNA (c, n=5) or Masp2 shRNA (d, n=4). The scale bars are 50 μm. (e) The position of GFP+ neurons across the width of the cortex was analysed and is shown in five bins (from the VZ to the CP). All shRNA treatments were compared to control shRNA. (fn) Postnatal positioning and the identity of C3, Masp1 and Masp2 knockdown cells. Brains electroporated in utero on E14 with control shRNA (f,j), C3 shRNA (g,k), Masp1 shRNA (h,l) or Masp2 shRNA (i,m) were immunostained at postnatal day 8 (P8) with anti-CUX1 or anti-TBR1 antibodies (fi,jm, respectively). Black and red boxes show the position of the enlargements underneath each slice. The immunostainings for the enlarged areas are shown together with GFP or with the outlines of GFP-positive cells. (n) The position of GFP+ neurons across the width of the P8 cortex was analysed and is shown in five bins (from the VZ to the CP). All shRNA treatments were compared to control shRNA. The scale bars are 50 μm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, histograms present means±s.e.m.
Figure 3
Figure 3. C3 cleavage products can rescue upstream migration deficits.
(a) Schematic presentation of C3 cleavage (top) and the two peptides used in this study, C3a with an attached signal peptide (SP) and C3b beta. (bg) C3 knockdown impairs neuronal migration and can be rescued in part by addition of either C3res, or C3a, or C3b beta. Brains were electroporated in utero (E14–E18) with control shRNA (b, n=4), C3 shRNA alone (c, n=5), or in combination with C3 resistant to the shRNA (d, n=3), C3a (e, n=3) or C3b beta (f, n=4). The distribution of neurons along the cortex is shown in five bins (from the VZ to the CP) for all the treatments (g). All the treatments were compared to control shRNA (the statistical significance is shown in violet) and to C3 shRNA (the statistical significance is shown in orange). (hm) Masp2 knockdown impairs neuronal migration via the complement pathway. Brains were electroporated in utero (E14–E18) with control shRNA (h, n=4), Masp2 shRNA alone (i, n=4), or in combination with Masp2 resistant to the shRNA (j, n=3), C3a (k, n=4) or C3b beta (l, n=4). Quantification of the distribution of neurons across the cortex is shown for all the treatments (m). All the treatments were compared to control shRNA (the statistical significance is shown in violet) and to C3 shRNA (the statistical significance is shown in orange). The scale bars are 50 μm. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001, histograms present means±s.e.m.
Figure 4
Figure 4. Neuronal migration impairment by either knockdown of Masp2 or C3, or knockout of C3 is ameliorated by activation of the C5a and/or the C3a receptors.
(ae) Treatment by Masp2 shRNA (b, n=4) electroporated in utero (E14–E18) is partially rescued by addition of C3aR agonist (c, n=5) or the dual receptor agonist (d, n=3). Quantification of the distribution of neurons across the cortex is shown for all the treatments (e). All the treatments were compared to control shRNA (a, n=4, the statistical significance is shown in violet) or Masp2 shRNA (the statistical significance is shown in orange). (fj) Knockdown of C3 (g, n=4) electroporated in utero (E14–E18) is partially rescued by either addition of C3aR agonist (h, n=4) or the dual receptor agonist (i, n=4). Quantification of the distribution of neurons across the cortex is shown for all the treatments (j). All the treatments were compared to control shRNA (f, n=4, the statistical significance is shown in violet) or C3 shRNA (the statistical significance is shown in orange). (kn) Knockout of C3 is rescued by addition of the dual agonist. C3 KO embryos were electroporated in utero (E14–E18) with a GFP expression construct (l, n=6), or with a GFP expression construct together with the C3a/C5a receptors agonist (m, n=4). None of the agonist treated brains showed any neuronal migration impairment. Comparison to the WT (k, n=3) is shown in violet. Comparison to C3 KO is shown in orange, histograms present means±s.e.m.

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