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First published online 16 September 2008
doi: 10.1242/jcs.031872

Journal of Cell Science 121, 3357-3365 (2008)
Published by The Company of Biologists 2008
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Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin

Nathalie Lebeurrier1,2,3,*, Séverine Launay1,2,3,*, Richard Macrez1,2,3, Eric Maubert1,2,3, Hélène Legros4, Arnaud Leclerc5,6, Soazik P. Jamin5,6, Jean-Yves Picard5,6, Stéphane Marret4, Vincent Laudenbach4, Philipp Berger7,{ddagger}, Peter Sonderegger7, Carine Ali1,2,3, Nathalie di Clemente5,6 and Denis Vivien1,2,3,§

1 INSERM, INSERM U919, Serine Proteases and Pathophysiology of the neurovascular Unit (SP2U), Cyceron, F-14074 France
2 CNRS, UMR CNRS 6232 Ci-NAPs `Center for imaging Neurosciences and Applications to PathologieS', Cyceron, F-14074 France
3 University of Caen Basse-Normandie, Caen Cedex, F-14074 France
4 INSERM-Avenir, Institute for Biomedical Research, IFRMP23, University of Rouen & Department of Neonatal Pediatrics and Intensive Care, Rouen University Hospital, Rouen, F-76183, France
5 INSERM, U782, Clamart, F-92140
6 University of Paris-Sud, UMR-S0782, Clamart, F-92140
7 Department of Biochemistry, University of Zurich, Zurich, CH-8057, Switzerland


Figure 1
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  		Fig. 1. Analysis of the putative mouse neuroserpin promoter. Regions of the putative mouse neuroserpin promoter were subcloned into pGL3-Basic vector, upstream of the firefly luciferase gene. Mv1Lu (mink lung epithelial) cells were transiently transfected with the pRL-TK control vector and pGL3-Basic vector containing the different regions of the mouse neuroserpin promoter. Firefly and Renilla luciferase activities were measured as described in the Materials and Methods. After normalization to Renilla luciferase activity, values were expressed as a percentage of the activity of construct A (valued as 100%) (mean ± s.d.; n=8). Asterisks indicates a significant difference from the A construct: *P<0.05, **P<0.01, ***P<0.001; hash marks indicate a significant difference from the E construct: #P<0.05, ##P<0.01; Kruskall-Wallis test followed by a Mann-Whitney post-hoc test.

 

Figure 2
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  		Fig. 2. AMH promotes the transcription of neuroserpin. (A,B,C) Mv1Lu cells were transiently transfected with the pRL-TK control vector and pGL3-Basic vector containing the E (–346 to +72) or A (–3941 to +72) constructs. At the same time, cells were co-transfected with pcDNA3 containing the cDNA encoding for either auto-activated versions of type-I receptors for members of the TGFβ family (Alk proteins, A,B) (n=11-12) or proteins of the Smad family (C) (n=8). Luciferase activities were measured as described in Materials and Methods. Values are indicated as a percentage of the activity in controls (–; E or A reporter vector co-transfected with the empty pcDNA3 vector). Bars represent mean values ± s.d. **P<0.01, ***P<0.001; Kruskall-Wallis test followed by Mann-Whitney posthoc test. (D) Summary of the Alk and Smad proteins involved in intracellular signalling pathways of the TGFβ family members TGFβ, BMP and AMH.

 

Figure 3
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  		Fig. 3. Regulation of neuroserpin expression in vitro. (A) mRNA levels of neuroserpin (NS), tPA, PAI-1 and protease nexin 1 (PN1) in cultured neurons (N; n=3) and astrocytes (A; n=3). (B) Evaluation of the mRNA levels for neuroserpin in cultured neurons (n=5) and astrocytes (n=3) treated over 24 hours with either recombinant BMP2, BMP4, BMP7 or AMH at a concentration of 1 or 10 ng/ml. Relative levels of mRNA expression were measured by quantitative PCR as described in the Materials and Methods. C, control. Results were computed by calculating both the 2–{Delta}Ct (white bars) and the 2–{Delta}{Delta}Ct (grey bars). Bars represent mean values ± s.d. (2–{Delta}Ct:*P<0.05, **P<0.01; 2–{Delta}{Delta}Ct: #P<0.05, ##P<0.01; Kruskall-Wallis test followed by Mann-Whitney post-hoc test).

 

Figure 4
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  		Fig. 4. AMH promotes the expression of the protein neuroserpin in cultured neurons. (A) Neurons were treated during 24 hours with either recombinant BMP2 (n=9), BMP4 (n=6), BMP7 (n=3) or AMH (n=7) at the concentration of 1 ng/ml. Immunoblots were revealed with an antibody raised against mouse neuroserpin [antibody G64 (Lebeurrier et al., 2005Go)]. (B) Densitometries of immunoblots performed with ImageJ software. Bars represent mean values ± s.d. **P<0.01; Kruskall-Wallis test followed by Mann-Whitney post-hoc test.

 

Figure 5
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  		Fig. 5. Expression of neuroserpin in AMHR-II-deficient mice. Evaluation of the mRNA levels of neuroserpin (NS) in the cerebral cortex, hippocampus and striatum of C57Bl6/J mice. Mice strains are wild type (WT; n=7-12), or heterozygote (HT; n=7-11) or homozygote (KO; n=6) AMHR-II deficients. Results were computed by calculating both the 2–{Delta}Ct (open bars) and the 2–{Delta}{Delta}Ct (filled bars). Symbols indicate significant difference from wild-type mice (2–{Delta}Ct: **P<0.01; 2–{Delta}{Delta}Ct: ##P<0.01); Kruskall-Wallis test followed by Mann-Whitney post-hoc test.

 

Figure 6
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  		Fig. 6. Distribution of AMHR-II immunoreactivity in the developing forebrain of E16 mouse embryos (coronal sections). (A-D) AMHR-II immunoreactivity (TRITC) is detected in the neuroepithelium of the ventricular zone (arrows). AMHR-II immunoreactivity is also detected in the developing parenchyma (* in B). Higher magnifications of the boxed region in B and C (C and D, respectively) show that the AMHR-II immunostaining is mainly confined to the ventricular zone (VZ) and the subventricular zone (SVZ). (B-D) Nuclei are counterstained with DAPI (blue). di, diencephalon; iz, intermediate zone; mz, marginal zone. Scale bars: 500 µm (A); 200 µm (B); 50 µm (C); 20 µm (D).

 

Figure 7
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  		Fig. 7. AMHR-II immunostaining in adult mouse brain. In the cortex, immunostaining for AMHR-II (TRITC) is predominantly associated with capillaries (arrows in A-D) and the external zone of the cortical layer (arrowheads in A and B), with a weaker immunostaining in the parenchyma. In the cortex, AMHR-II immunostaining is associated with cortical nerve fibres (arrowheads in C and D), which are positive for the 160-kDa neurofilament (insert in D). In the hippocampus (E), immunostaining for AMHR-II (TRITC) is observed in the parenchyma surrounding the large vessels of the stratum lacunosum molecularis (SLM). At higher magnification (F), the perivascular AMHR-II immunoreactivity is associated with glial cells (arrows) and, more precisely, astrocytes, which are characterized by GFAP immunoreactivity (inserts in F). The nuclei are counterstained with DAPI (blue; A-F).

 

Figure 8
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  		Fig. 8. Neuroprotective effect of AMH against NMDA-induced excitotoxicity. (A) Pure cultures of neurons (14 DIV) were exposed for 24 hours to NMDA (12.5 µM) without (black bars) or with (white bars) increasing concentrations of AMH (1 or 10 ng/ml) and neuronal death was measured as described in the Materials and Methods (n=4). Asterisks indicate a significant difference from NMDA alone by one way Kruskall-Wallis test followed by Mann-Whitney post-hoc test (*P<0.05). (B) Effects of intra-striatal injection of recombinant AMH (0.45 pg) on the extent of neuronal death induced by the striatal administration of NMDA (10 nmol) in mice (n=9). Bars represent mean values ± s.e.m. **P<0.01; Mann-Whitney test. Diagram on the right illustrates on a brain section the typical lesion size obtained by the striatal injection of NMDA alone versus NMDA + AMH.

 

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© The Company of Biologists Ltd 2008