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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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NO promotes axonal varicosities differentiation and regulates spine and synapse formation under control conditions. (A) 3D reconstruction of an MIS and adjacent axons observed upon PSD-95 transfection. Seven axons were present in the vicinity, four of which made a synaptic contact. (B) 3D reconstruction of an enlarged spine observed upon PSD-95 transfection but with L-NAME treatment illustrating seven axons also present in the vicinity, of which only one made a synaptic contact. (C) 3D analysis of the environment of 12 MISs observed upon PSD-95 transfection and 12 large spines observed upon L-NAME treatment of PSD-95–transfected cells. Selection was based on similar spine head volumes. (D) The number of axons directly adjacent to the enlarged spines was similar in both conditions (7.8 ± 0.3 vs. 7.6 ± 0.5 axons; n = 12 spines). (E) The number of synaptic contacts made by adjacent axons is decreased by L-NAME treatment of PSD-95–transfected cells (*, P < 0.05). (F) The ratio of axonal shafts (black bars) versus axonal varicosities (gray bars; defined by the presence of vesicles + enlargement) is modified by L-NAME treatment (n = 12 spines; *, P < 0.05). Data are mean ± SEM (error bars). Bars, 1 μm.
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fig8: NO promotes axonal varicosities differentiation and regulates spine and synapse formation under control conditions. (A) 3D reconstruction of an MIS and adjacent axons observed upon PSD-95 transfection. Seven axons were present in the vicinity, four of which made a synaptic contact. (B) 3D reconstruction of an enlarged spine observed upon PSD-95 transfection but with L-NAME treatment illustrating seven axons also present in the vicinity, of which only one made a synaptic contact. (C) 3D analysis of the environment of 12 MISs observed upon PSD-95 transfection and 12 large spines observed upon L-NAME treatment of PSD-95–transfected cells. Selection was based on similar spine head volumes. (D) The number of axons directly adjacent to the enlarged spines was similar in both conditions (7.8 ± 0.3 vs. 7.6 ± 0.5 axons; n = 12 spines). (E) The number of synaptic contacts made by adjacent axons is decreased by L-NAME treatment of PSD-95–transfected cells (*, P < 0.05). (F) The ratio of axonal shafts (black bars) versus axonal varicosities (gray bars; defined by the presence of vesicles + enlargement) is modified by L-NAME treatment (n = 12 spines; *, P < 0.05). Data are mean ± SEM (error bars). Bars, 1 μm.

Mentions: We then investigated whether NO promoted multiple contact formation by attracting axons toward PSD-95–overexpressing spines or whether it rather favored axonal varicosity differentiation and synaptic contact formation with axons already present in the vicinity of the spine. For this, we analyzed the number of axons in contact with 12 reconstructed MISs from PSD-95–transfected cells and 12 spines from cells overexpressing PSD-95 that were treated with L-NAME. Reconstructed spines were selected so as to exhibit exactly the same size (Fig. 8, A–C). However, the mean number of synapses established with each spine was much higher in PSD-95–transfected cells (3.33 ± 0.32 vs. 1.17 ± 0.17 in PSD-95– and L-NAME–treated cells, respectively; P < 0.05; Fig. 8 E). Interestingly, the number of axons found in close proximity to the spine membrane was actually similar (7.8 ± 0.3 for PSD-95 vs. 7.6 ± 0.5 for L-NAME; Fig. 8 D). However, these axons mainly exhibited features of differentiated terminals in the case of PSD-95–transfected cells (enlargement and presence of vesicles, including docked vesicles; 4.7 ± 0.2 axonal varicosities and 3.1 ± 0.2 axonal shafts), whereas they mainly corresponded to axonal shafts in the case of L-NAME treatment (2 ± 0.3 varicosities and 5.6 ± 0.4 axonal shafts; P < 0.05; Fig. 8 F). Therefore, the main effect of NO was to promote presynaptic terminal differentiation and formation of a synaptic contact by the axons already present in the direct vicinity of the spine.


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)

NO promotes axonal varicosities differentiation and regulates spine and synapse formation under control conditions. (A) 3D reconstruction of an MIS and adjacent axons observed upon PSD-95 transfection. Seven axons were present in the vicinity, four of which made a synaptic contact. (B) 3D reconstruction of an enlarged spine observed upon PSD-95 transfection but with L-NAME treatment illustrating seven axons also present in the vicinity, of which only one made a synaptic contact. (C) 3D analysis of the environment of 12 MISs observed upon PSD-95 transfection and 12 large spines observed upon L-NAME treatment of PSD-95–transfected cells. Selection was based on similar spine head volumes. (D) The number of axons directly adjacent to the enlarged spines was similar in both conditions (7.8 ± 0.3 vs. 7.6 ± 0.5 axons; n = 12 spines). (E) The number of synaptic contacts made by adjacent axons is decreased by L-NAME treatment of PSD-95–transfected cells (*, P < 0.05). (F) The ratio of axonal shafts (black bars) versus axonal varicosities (gray bars; defined by the presence of vesicles + enlargement) is modified by L-NAME treatment (n = 12 spines; *, P < 0.05). Data are mean ± SEM (error bars). Bars, 1 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2600742&req=5

fig8: NO promotes axonal varicosities differentiation and regulates spine and synapse formation under control conditions. (A) 3D reconstruction of an MIS and adjacent axons observed upon PSD-95 transfection. Seven axons were present in the vicinity, four of which made a synaptic contact. (B) 3D reconstruction of an enlarged spine observed upon PSD-95 transfection but with L-NAME treatment illustrating seven axons also present in the vicinity, of which only one made a synaptic contact. (C) 3D analysis of the environment of 12 MISs observed upon PSD-95 transfection and 12 large spines observed upon L-NAME treatment of PSD-95–transfected cells. Selection was based on similar spine head volumes. (D) The number of axons directly adjacent to the enlarged spines was similar in both conditions (7.8 ± 0.3 vs. 7.6 ± 0.5 axons; n = 12 spines). (E) The number of synaptic contacts made by adjacent axons is decreased by L-NAME treatment of PSD-95–transfected cells (*, P < 0.05). (F) The ratio of axonal shafts (black bars) versus axonal varicosities (gray bars; defined by the presence of vesicles + enlargement) is modified by L-NAME treatment (n = 12 spines; *, P < 0.05). Data are mean ± SEM (error bars). Bars, 1 μm.
Mentions: We then investigated whether NO promoted multiple contact formation by attracting axons toward PSD-95–overexpressing spines or whether it rather favored axonal varicosity differentiation and synaptic contact formation with axons already present in the vicinity of the spine. For this, we analyzed the number of axons in contact with 12 reconstructed MISs from PSD-95–transfected cells and 12 spines from cells overexpressing PSD-95 that were treated with L-NAME. Reconstructed spines were selected so as to exhibit exactly the same size (Fig. 8, A–C). However, the mean number of synapses established with each spine was much higher in PSD-95–transfected cells (3.33 ± 0.32 vs. 1.17 ± 0.17 in PSD-95– and L-NAME–treated cells, respectively; P < 0.05; Fig. 8 E). Interestingly, the number of axons found in close proximity to the spine membrane was actually similar (7.8 ± 0.3 for PSD-95 vs. 7.6 ± 0.5 for L-NAME; Fig. 8 D). However, these axons mainly exhibited features of differentiated terminals in the case of PSD-95–transfected cells (enlargement and presence of vesicles, including docked vesicles; 4.7 ± 0.2 axonal varicosities and 3.1 ± 0.2 axonal shafts), whereas they mainly corresponded to axonal shafts in the case of L-NAME treatment (2 ± 0.3 varicosities and 5.6 ± 0.4 axonal shafts; P < 0.05; Fig. 8 F). Therefore, the main effect of NO was to promote presynaptic terminal differentiation and formation of a synaptic contact by the axons already present in the direct vicinity of the spine.

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