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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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Spine enlargement induced by PSD-95 overexpression. (A) Three-dimensionally reconstructed dendritic segment of a PSD-95–transfected cell illustrating the presence of numerous large spines regularly exhibiting perforated PSDs. (B) Three-dimensionally reconstructed dendritic segment of a control cell. Note the smaller size of the spines. (C) Spine volume measured in control nontransfected (ctrl; n = 8 cells; 145 spines), EGFP-transfected (n = 4 cells; 164 spines), and PSD-95–transfected (n = 7 cells; 234 spines) pyramidal neurons. Data are mean ± SEM (error bars; *, P < 0.05). (D) Same as in C but for the total PSD area. (E) Same as in C but for the proportion of spines with complex PSDs. Bars, 1 μm.
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fig2: Spine enlargement induced by PSD-95 overexpression. (A) Three-dimensionally reconstructed dendritic segment of a PSD-95–transfected cell illustrating the presence of numerous large spines regularly exhibiting perforated PSDs. (B) Three-dimensionally reconstructed dendritic segment of a control cell. Note the smaller size of the spines. (C) Spine volume measured in control nontransfected (ctrl; n = 8 cells; 145 spines), EGFP-transfected (n = 4 cells; 164 spines), and PSD-95–transfected (n = 7 cells; 234 spines) pyramidal neurons. Data are mean ± SEM (error bars; *, P < 0.05). (D) Same as in C but for the total PSD area. (E) Same as in C but for the proportion of spines with complex PSDs. Bars, 1 μm.

Mentions: As shown in Fig. 2, PSD-95 overexpression modified the ultrastructural features of dendritic spines and PSDs. Analysis of 234 spines from PSD-95–transfected neurons (n = 7 cells), 164 spines from EGFP-transfected cells (n = 4 cells), and 145 spines from nontransfected neurons (n = 8 cells) showed that spines in PSD-95–transfected cells become much larger, often with complex shapes and membrane extensions that engulf presynaptic terminals. The spine volume increased by factors of 2.3 and 2.8 when compared with either EGFP neurons (0.144 ± 0.023 μm3 vs. 0.064 ± 0.011 μm3) or nontransfected neurons (0.051 ± 0.006 μm3; P < 0.05; Fig. 2 C). The increase of the PSD area was even more pronounced (5.7× and 8.2× the mean value of EGFP and control neurons; 0.189 ± 0.043 μm2 vs. 0.033 ± 0.004 μm2 and 0.023 ± 0.002 μm2, respectively; P < 0.05; Fig. 2 D). The proportion of spines with complex PSDs, characterized by a discontinuity on a single section, was also significantly increased in comparison with control cells (46.6 ± 5% vs. 8.5 ± 1% or 5.7 ± 1.4%; P < 0.05; Fig. 2 E). In addition, PSD-95 overexpression resulted in a decrease in spine density as indicated by analysis of the three-dimensionally reconstructed segments (PSD-95, 0.63 ± 0.12 spines/μm of dendritic length; EGFP, 0.96 ± 0.29 spines/μm of dendritic length; n = 7 and 4 cells, respectively; P < 0.05; Table S1), a result also confirmed by analyzing the number of fluorescent puncta observed on transfected cell dendrites (PSD-95–transfected cells, 0.74 ± 0.03 puncta/μm; spines on EGFP-transfected cells, 0.94 ± 0.9 puncta/μm; n = 14 and 8 cells, respectively; P < 0.05).


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)

Spine enlargement induced by PSD-95 overexpression. (A) Three-dimensionally reconstructed dendritic segment of a PSD-95–transfected cell illustrating the presence of numerous large spines regularly exhibiting perforated PSDs. (B) Three-dimensionally reconstructed dendritic segment of a control cell. Note the smaller size of the spines. (C) Spine volume measured in control nontransfected (ctrl; n = 8 cells; 145 spines), EGFP-transfected (n = 4 cells; 164 spines), and PSD-95–transfected (n = 7 cells; 234 spines) pyramidal neurons. Data are mean ± SEM (error bars; *, P < 0.05). (D) Same as in C but for the total PSD area. (E) Same as in C but for the proportion of spines with complex PSDs. Bars, 1 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600742&req=5

fig2: Spine enlargement induced by PSD-95 overexpression. (A) Three-dimensionally reconstructed dendritic segment of a PSD-95–transfected cell illustrating the presence of numerous large spines regularly exhibiting perforated PSDs. (B) Three-dimensionally reconstructed dendritic segment of a control cell. Note the smaller size of the spines. (C) Spine volume measured in control nontransfected (ctrl; n = 8 cells; 145 spines), EGFP-transfected (n = 4 cells; 164 spines), and PSD-95–transfected (n = 7 cells; 234 spines) pyramidal neurons. Data are mean ± SEM (error bars; *, P < 0.05). (D) Same as in C but for the total PSD area. (E) Same as in C but for the proportion of spines with complex PSDs. Bars, 1 μm.
Mentions: As shown in Fig. 2, PSD-95 overexpression modified the ultrastructural features of dendritic spines and PSDs. Analysis of 234 spines from PSD-95–transfected neurons (n = 7 cells), 164 spines from EGFP-transfected cells (n = 4 cells), and 145 spines from nontransfected neurons (n = 8 cells) showed that spines in PSD-95–transfected cells become much larger, often with complex shapes and membrane extensions that engulf presynaptic terminals. The spine volume increased by factors of 2.3 and 2.8 when compared with either EGFP neurons (0.144 ± 0.023 μm3 vs. 0.064 ± 0.011 μm3) or nontransfected neurons (0.051 ± 0.006 μm3; P < 0.05; Fig. 2 C). The increase of the PSD area was even more pronounced (5.7× and 8.2× the mean value of EGFP and control neurons; 0.189 ± 0.043 μm2 vs. 0.033 ± 0.004 μm2 and 0.023 ± 0.002 μm2, respectively; P < 0.05; Fig. 2 D). The proportion of spines with complex PSDs, characterized by a discontinuity on a single section, was also significantly increased in comparison with control cells (46.6 ± 5% vs. 8.5 ± 1% or 5.7 ± 1.4%; P < 0.05; Fig. 2 E). In addition, PSD-95 overexpression resulted in a decrease in spine density as indicated by analysis of the three-dimensionally reconstructed segments (PSD-95, 0.63 ± 0.12 spines/μm of dendritic length; EGFP, 0.96 ± 0.29 spines/μm of dendritic length; n = 7 and 4 cells, respectively; P < 0.05; Table S1), a result also confirmed by analyzing the number of fluorescent puncta observed on transfected cell dendrites (PSD-95–transfected cells, 0.74 ± 0.03 puncta/μm; spines on EGFP-transfected cells, 0.94 ± 0.9 puncta/μm; n = 14 and 8 cells, respectively; P < 0.05).

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