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PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

Nikonenko I, Boda B, Steen S, Knott G, Welker E, Muller D - J. Cell Biol. (2008)

Bottom Line: Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs.NOS blockade also reduced spine and synapse density in developing hippocampal cultures.These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Neuroscience, Geneva Neuroscience Center, University of Geneva School of Medicine, CH-1211 Geneva, Switzerland.

ABSTRACT
Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

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PSD-95 overexpression correlates with increased nNOS expression at synapses. (A) Immunostaining against nNOS (red) and PSD-95 (blue) and their colocalization (white) obtained in the same field of stratum radiatum of a control hippocampal slice culture. Note the existence of PSD-95 puncta devoid of nNOS staining as well as nNOS puncta negative for PSD-95. (B) Same as in A but in a field containing a dendritic segment from a PSD-95–EGFP-transfected neuron (green). Colocalization between nNOS and PSD-95 immunostainings or nNOS and PSD-95–EGFP fluorescence are shown in white. Note the enlargement of the PSD-95 puncta and the presence of large nNOS-positive spots associated with the PSD-95 puncta. (C) Same as in A but in a field containing a dendritic segment from a PDZ2 mutant PSD-95–EGFP-transfected neuron. Note the reduced size of nNOS puncta associated with PSD-95–EGFP staining. (D) Same as in A but in a field containing a dendritic segment from a cell cotransfected with PSD-95–EGFP and nNOS siRNA. Note again the reduced nNOS staining associated with PSD-95 puncta. (E) Analysis of the size of nNOS puncta measured as the number of stained pixels under the different conditions analyzed. Data are mean ± SEM (error bars) of the analysis of four different slice cultures (75–105 puncta analyzed per condition; *, P < 0.05; Mann-Whitney test). (F) Colocalization between nNOS and PSD-95 immunostaining (or PSD-95–EGFP fluorescence for the PDZ2 condition) measured in 9–21 different fields (30 × 30 μm) obtained from four different slice cultures per condition (*, P < 0.05; Mann-Whitney test). Data are mean ± SEM (error bars). Bars, 2 μm.
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fig6: PSD-95 overexpression correlates with increased nNOS expression at synapses. (A) Immunostaining against nNOS (red) and PSD-95 (blue) and their colocalization (white) obtained in the same field of stratum radiatum of a control hippocampal slice culture. Note the existence of PSD-95 puncta devoid of nNOS staining as well as nNOS puncta negative for PSD-95. (B) Same as in A but in a field containing a dendritic segment from a PSD-95–EGFP-transfected neuron (green). Colocalization between nNOS and PSD-95 immunostainings or nNOS and PSD-95–EGFP fluorescence are shown in white. Note the enlargement of the PSD-95 puncta and the presence of large nNOS-positive spots associated with the PSD-95 puncta. (C) Same as in A but in a field containing a dendritic segment from a PDZ2 mutant PSD-95–EGFP-transfected neuron. Note the reduced size of nNOS puncta associated with PSD-95–EGFP staining. (D) Same as in A but in a field containing a dendritic segment from a cell cotransfected with PSD-95–EGFP and nNOS siRNA. Note again the reduced nNOS staining associated with PSD-95 puncta. (E) Analysis of the size of nNOS puncta measured as the number of stained pixels under the different conditions analyzed. Data are mean ± SEM (error bars) of the analysis of four different slice cultures (75–105 puncta analyzed per condition; *, P < 0.05; Mann-Whitney test). (F) Colocalization between nNOS and PSD-95 immunostaining (or PSD-95–EGFP fluorescence for the PDZ2 condition) measured in 9–21 different fields (30 × 30 μm) obtained from four different slice cultures per condition (*, P < 0.05; Mann-Whitney test). Data are mean ± SEM (error bars). Bars, 2 μm.

Mentions: As the PDZ2 domain of PSD-95 is involved in interactions with different partners and because we previously found that NO could affect axonal varicosity remodeling (Nikonenko et al., 2003), we tested the role of nNOS and NO in the formation of MISs. For this, we performed two groups of experiments to examine whether suppression of NOS activity in cells overexpressing PSD-95 prevented the formation of MISs. First, we used an NOS siRNA, which markedly inhibited nNOS expression in fibroblasts (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1) and also interfered with NOS expression at synapses (see Fig. 6). Second, we transfected pyramidal neurons with PSD-95–EGFP and treated the slice cultures for 2 d after transfection with 200 μM of the NOS inhibitor l-NG-nitroarginine methyl ester (L-NAME) in the culture medium. This treatment did not induce any toxicity, as indicated by propidium iodide staining (unpublished data). Slice cultures were then processed for EM analyses. As illustrated in Fig. 5, both down-regulation of NOS by the siRNA approach and treatment of slice cultures with the NOS inhibitor prevented the formation of MISs. The decrease was partial but significant with nNOS siRNA transfection (7.8 ± 5.1%; n = 5 cells), whereas the blockade of MIS formation was complete with L-NAME treatment of PSD-95–transfected neurons (4.4 ± 1.9%, n = 4 cells vs. 2.0 ± 1.3%, n = 8 cells for control; 29.1 ± 2.9%, n = 7 cells for PSD-95; Fig. 5 C). Interestingly, the increase in spine volume and PSD area associated with PSD-95 overexpression was not blocked by these manipulations (spine volume, 0.131 ± 0.012 μm3 and 0.125 ± 0.025 μm3; PSD area, 0.129 ± 0.014 μm2 and 0.118 ± 0.016 μm2 for siNOS [silencing RNA for NOS] and L-NAME, respectively; Fig. 5, D and E).


PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling.

Nikonenko I, Boda B, Steen S, Knott G, Welker E, Muller D - J. Cell Biol. (2008)

PSD-95 overexpression correlates with increased nNOS expression at synapses. (A) Immunostaining against nNOS (red) and PSD-95 (blue) and their colocalization (white) obtained in the same field of stratum radiatum of a control hippocampal slice culture. Note the existence of PSD-95 puncta devoid of nNOS staining as well as nNOS puncta negative for PSD-95. (B) Same as in A but in a field containing a dendritic segment from a PSD-95–EGFP-transfected neuron (green). Colocalization between nNOS and PSD-95 immunostainings or nNOS and PSD-95–EGFP fluorescence are shown in white. Note the enlargement of the PSD-95 puncta and the presence of large nNOS-positive spots associated with the PSD-95 puncta. (C) Same as in A but in a field containing a dendritic segment from a PDZ2 mutant PSD-95–EGFP-transfected neuron. Note the reduced size of nNOS puncta associated with PSD-95–EGFP staining. (D) Same as in A but in a field containing a dendritic segment from a cell cotransfected with PSD-95–EGFP and nNOS siRNA. Note again the reduced nNOS staining associated with PSD-95 puncta. (E) Analysis of the size of nNOS puncta measured as the number of stained pixels under the different conditions analyzed. Data are mean ± SEM (error bars) of the analysis of four different slice cultures (75–105 puncta analyzed per condition; *, P < 0.05; Mann-Whitney test). (F) Colocalization between nNOS and PSD-95 immunostaining (or PSD-95–EGFP fluorescence for the PDZ2 condition) measured in 9–21 different fields (30 × 30 μm) obtained from four different slice cultures per condition (*, P < 0.05; Mann-Whitney test). Data are mean ± SEM (error bars). Bars, 2 μm.
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Related In: Results  -  Collection

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fig6: PSD-95 overexpression correlates with increased nNOS expression at synapses. (A) Immunostaining against nNOS (red) and PSD-95 (blue) and their colocalization (white) obtained in the same field of stratum radiatum of a control hippocampal slice culture. Note the existence of PSD-95 puncta devoid of nNOS staining as well as nNOS puncta negative for PSD-95. (B) Same as in A but in a field containing a dendritic segment from a PSD-95–EGFP-transfected neuron (green). Colocalization between nNOS and PSD-95 immunostainings or nNOS and PSD-95–EGFP fluorescence are shown in white. Note the enlargement of the PSD-95 puncta and the presence of large nNOS-positive spots associated with the PSD-95 puncta. (C) Same as in A but in a field containing a dendritic segment from a PDZ2 mutant PSD-95–EGFP-transfected neuron. Note the reduced size of nNOS puncta associated with PSD-95–EGFP staining. (D) Same as in A but in a field containing a dendritic segment from a cell cotransfected with PSD-95–EGFP and nNOS siRNA. Note again the reduced nNOS staining associated with PSD-95 puncta. (E) Analysis of the size of nNOS puncta measured as the number of stained pixels under the different conditions analyzed. Data are mean ± SEM (error bars) of the analysis of four different slice cultures (75–105 puncta analyzed per condition; *, P < 0.05; Mann-Whitney test). (F) Colocalization between nNOS and PSD-95 immunostaining (or PSD-95–EGFP fluorescence for the PDZ2 condition) measured in 9–21 different fields (30 × 30 μm) obtained from four different slice cultures per condition (*, P < 0.05; Mann-Whitney test). Data are mean ± SEM (error bars). Bars, 2 μm.
Mentions: As the PDZ2 domain of PSD-95 is involved in interactions with different partners and because we previously found that NO could affect axonal varicosity remodeling (Nikonenko et al., 2003), we tested the role of nNOS and NO in the formation of MISs. For this, we performed two groups of experiments to examine whether suppression of NOS activity in cells overexpressing PSD-95 prevented the formation of MISs. First, we used an NOS siRNA, which markedly inhibited nNOS expression in fibroblasts (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1) and also interfered with NOS expression at synapses (see Fig. 6). Second, we transfected pyramidal neurons with PSD-95–EGFP and treated the slice cultures for 2 d after transfection with 200 μM of the NOS inhibitor l-NG-nitroarginine methyl ester (L-NAME) in the culture medium. This treatment did not induce any toxicity, as indicated by propidium iodide staining (unpublished data). Slice cultures were then processed for EM analyses. As illustrated in Fig. 5, both down-regulation of NOS by the siRNA approach and treatment of slice cultures with the NOS inhibitor prevented the formation of MISs. The decrease was partial but significant with nNOS siRNA transfection (7.8 ± 5.1%; n = 5 cells), whereas the blockade of MIS formation was complete with L-NAME treatment of PSD-95–transfected neurons (4.4 ± 1.9%, n = 4 cells vs. 2.0 ± 1.3%, n = 8 cells for control; 29.1 ± 2.9%, n = 7 cells for PSD-95; Fig. 5 C). Interestingly, the increase in spine volume and PSD area associated with PSD-95 overexpression was not blocked by these manipulations (spine volume, 0.131 ± 0.012 μm3 and 0.125 ± 0.025 μm3; PSD area, 0.129 ± 0.014 μm2 and 0.118 ± 0.016 μm2 for siNOS [silencing RNA for NOS] and L-NAME, respectively; Fig. 5, D and E).

Bottom Line: Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs.NOS blockade also reduced spine and synapse density in developing hippocampal cultures.These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Neuroscience, Geneva Neuroscience Center, University of Geneva School of Medicine, CH-1211 Geneva, Switzerland.

ABSTRACT
Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Show MeSH
Related in: MedlinePlus