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First published online 14 February 2007
doi: 10.1242/dev.02808

Development 134, 1181-1190 (2007)
Published by The Company of Biologists 2007
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PSA-NCAM in postnatally generated immature neurons of the olfactory bulb: a crucial role in regulating p75 expression and cell survival

Eduardo Gascon1, Laszlo Vutskits1,2, Benoit Jenny1, Pascale Durbec3 and Jozsef Zoltan Kiss1,*

1 Department of Neuroscience, University of Geneva Medical School, CH-1211 Geneva, Switzerland.
2 Department of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva, Geneva, Switzerland.
3 Laboratoire de Génétique et Physiologie du Développement, CNRS 9943, Parc Scientifique de Luminy, Case 907, F-13288 Marseille Cedex 9, France.


Figure 1
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  		Fig. 1. Enhanced cell death in the SVZ-RMS of NCAM-deficient animals. (A) Photomicrographs of HE-stained sagittal sections illustrating the enlarged SVZ-RMS in NCAM-deficient (NCAM KO) as compared with WT mice. (B) TUNEL assay (lower panels, green fluorescence) reveals that apoptosis is increased in the SVZ-RMS of mice lacking NCAM (right) as compared with WT (left). Hoechst counterstaining on the same sections (upper panels) shows dying cells located in the SVZ-RMS but not in neighboring areas. These pictures correspond to the boxed regions in A. (C) Quantification of the density of TUNEL+ cells in WT and NCAM-/- brains demonstrates a significant increase in cell death in the SVZ and RMS of NCAM mutant animals. *, P<0.05 (two-tailed unpaired t-test). CC, corpus callosum; V, lateral ventricle; St, striatum. The limit between SVZ and St is represented by the dashed line. Scale bar: 1 mm in A; 100 µm in B.

 

Figure 2
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  		Fig. 2. Characterization of SVZ-derived cultures from mice. In culture, SVZ-derived cells prepared from WT mice (top panels) display immunocytochemical markers of immature neurons (strongly positive for ßIII-tubulin, GABA and PSA-NCAM). Neither genetic deletion of NCAM (NCAM-/-, middle panels) nor the enzymatic removal of PSA (WT+EndoN, bottom panels) affects the morphology or the pattern of markers expressed by these cells. Scale bar: 20 µm.

 

Figure 3
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  		Fig. 3 . Loss of PSA-NCAM reduces survival of cultured SVZ-derived neurons under basal conditions and in response to neurotrophins. (A) Epifluorescence images illustrating apoptotic cells in SVZ-derived cultures co-labeled for activated caspase 3 (red) and TUNEL (green). ßIII-tubulin staining (blue) was used to confirm the neuronal phenotype of cultured cells. (B) Quantification of cell death rate in the presence or absence of PSA-NCAM. When WT cultures are treated with Endo-N (PSA- NCAM+) for 20 hours, or with a PSA-blocking antibody (mAb 735), a significant increase in death rate is found. *, P<0.05 (ANOVA, Holm-Sidak test). (C) Survival of WT cells is promoted by exogenous application of BDNF (100 ng/ml), but not of NGF. By contrast, cells lacking either PSA (WT+EndoN) or PSA and NCAM (NCAM-/-) exhibit an enhanced cell death rate in response to NGF (50 ng/ml) and BDNF (100 ng/ml), as compared with WT neurons. *, P<0.05 (ANOVA, Holm-Sidak test).

 

Figure 4
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  		Fig. 4 . Decreased PSA-NCAM expression in SVZ-derived neurons correlates with enhanced cell death in response to exogenous NGF. (A) Confocal photomicrographs of PSA-immunostained neurons demonstrate the downregulation of PSA expression between 3 and 9 DIV. A color scale, where bright regions correspond to PSA-rich areas, was used to better illustrate these differences. (B) Surface intensity of PSA immunoreactivity was measured by confocal microscopy and plotted (arbitrary units) against the time in culture. This reveals that the levels of PSA decrease during maturation of neurons in vitro. (C) Time-dependent response of SVZ-derived cultures to exogenous NGF. In WT cultures, NGF induces a significant increase in cell death at late stages (9 DIV), but not before (3 and 6 DIV). Results are expressed as the percentage of cell death increase as compared with sister cultures not treated with NGF (basal conditions). *, P<0.05, as compared with basal conditions (ANOVA, unpaired t-test). Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5 . Enhanced cell death after PSA removal involves the activation of p75 pathways. (A) K252a effectively blocks phosphorylation of Trk receptors. At 5 DIV, SVZ-derived neurons were exposed to BDNF (100 ng/ml) in the presence or absence of the Trk-inhibitor K252a (400 nM) and the phosphorylation state of Trk receptors was assessed by western blotting. (B) The presence of the Trk receptor blocker K252a (400 nM) does not modify the increased cell death observed in response to exogenously applied NGF (50 ng/nl) or BDNF (100 ng/ml) after PSA removal. *, P<0.05; **, P<0.001 (ANOVA, Holm-Sidak test). (C) Blocking p75-dependent pathways by inhibitors of ceramide production (10 µM fumonisin B1 and 50 nM myriocin) or c-Jun N-terminal kinase (SP600125, 20 µM) was able to prevent cell death induced by PSA removal. *, P<0.05 (ANOVA, Holm-Sidak test).

 

Figure 6
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  		Fig. 6. Increased expression of p75 after PSA removal. (A) Real-time RT-PCR analysis of p75, TrkB and TrkC mRNA after Endo-N treatment. RNA extracted from SVZ-derived neurons treated either with Endo-N (black bars) or with vehicle (white bars) were used to quantify the expression of p75, TrkB and TrkC in three independent experiments. Compared with vehicle-treated cultures, removal of PSA specifically affected p75 levels. *, P<0.05 (two-tailed unpaired t-test). (B) Confocal photomicrographs of ßIII-tubulin (green) and p75 (red) double-labeling in neurons treated with vehicle or with Endo-N demonstrate the upregulation of p75 expression in cells lacking PSA. (C) Quantification of p75 immunoreactivity using confocal microscopy. Cells treated with Endo-N for 20 hours showed a significant increase in p75 immunoreactivity compared with controls. Scale bar: 20 µm.

 

Figure 7
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  		Fig. 7 . p75 upregulation in the SVZ-RMS of NCAM-/- animals is accompanied by the presence of abnormally mature neurons. (A) Western blot analysis of p75 expression in WT and NCAM-/- brain. p75 is expressed at higher levels in the RMS-OB of NCAM mutant mice during early postnatal life. *, P<0.05 (two-tailed unpaired t-test). (B) Confocal image of sagittal brain slices stained for p75 and doublecortin (DCX) demonstrating the presence of small round p75+ DCX- cells in the WT SVZ-RMS (arrows). p75+ cells occasionally form small clusters (insert). (C) Immunolabeling for p75 in NCAM-/- brains revealed an increased number of p75 cells in the SVZ-RMS. These cells often exhibited a branched morphology (arrowheads). A higher magnification image of the area depicted in the left-hand panel illustrates that most of these p75+ cells colocalized with the neuronal marker doublecortin. Scale bars: 20 µm.

 

Figure 8
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  		Fig. 8 . Abnormal neuronal maturation in the SVZ-RMS of NCAM-/- animals is associated with apoptosis and p75 expression. (A) Morphological analysis of cells in SVZ-RMS 4 days after a GFP lentiviral vector injection in the lateral ventricles. In WT animals, GFP+ cells display a unipolar morphology typical of migrating cells, whereas in NCAM mutant mice many GFP+ cells exhibit a complex branched morphology suggestive of maturating neurons. GFP+ cells colocalized with the neuronal marker doublecortin (DCX). (B) NeuN staining in the SVZ-RMS revealed numerous NeuN-positive nuclei in the NCAM mutant mice (arrows). These nuclei were smaller than those found on the cortex (arrowheads). The dashed line indicates the limit between the SVZ-RMS and the overlying corpus callosum and cortex. (C) Colocalization of TUNEL labeling and p75 immunostaining in the NCAM-deficient SVZ-RMS. The upper panel depicts an example of a TUNEL+ nucleus that colocalizes with p75, whereas the lower panels illustrate a strongly TUNEL+ nucleus that does not colocalize with p75 staining. Note that this latter nucleus is highly condensed, as illustrated by the Hoechst staining. Scale bar: 20 µm in A,B; 10 µm in C.

 

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© The Company of Biologists Ltd 2007