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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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Increased formation of MISs by the NO donor DETA NONOate and the cell-permeable cGMP analogue 8-Br-cGMP. (A) EM sections illustrating the presence of MISs (asterisks) in slice cultures treated for 2 d with 150 μM DETA NONOate (left) or 5 mM 8-Br-cGMP (right). (B) 3D reconstruction of the two MISs illustrated in A, the left and right spines making synaptic contacts with five and four different axons, respectively. (C) Proportion of MISs observed in randomly selected volume samples of CA1 stratum radiatum in the control situation (ctrl; three slices and 615 spines analyzed), in DETA NONOate–treated cultures (DETA; three slices and 583 spines analyzed), and in 8-Br-cGMP–treated slices (cGMP; three slices and 963 spines analyzed) compared with the analysis of 234 spines in PSD-95–transfected cells (seven slices). Data are mean ± SEM (error bars; *, P < 0.01). (D) Size of individual PSDs on spines from control slices and on MISs of PSD-95–transfected cells and of DETA NONOate– or of 8-Br-cGMP–treated cultures. Data are mean ± SEM (error bars) of 114–269 reconstructed PSDs (*, P < 0.01). Bars, 0.5 μm.
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fig7: Increased formation of MISs by the NO donor DETA NONOate and the cell-permeable cGMP analogue 8-Br-cGMP. (A) EM sections illustrating the presence of MISs (asterisks) in slice cultures treated for 2 d with 150 μM DETA NONOate (left) or 5 mM 8-Br-cGMP (right). (B) 3D reconstruction of the two MISs illustrated in A, the left and right spines making synaptic contacts with five and four different axons, respectively. (C) Proportion of MISs observed in randomly selected volume samples of CA1 stratum radiatum in the control situation (ctrl; three slices and 615 spines analyzed), in DETA NONOate–treated cultures (DETA; three slices and 583 spines analyzed), and in 8-Br-cGMP–treated slices (cGMP; three slices and 963 spines analyzed) compared with the analysis of 234 spines in PSD-95–transfected cells (seven slices). Data are mean ± SEM (error bars; *, P < 0.01). (D) Size of individual PSDs on spines from control slices and on MISs of PSD-95–transfected cells and of DETA NONOate– or of 8-Br-cGMP–treated cultures. Data are mean ± SEM (error bars) of 114–269 reconstructed PSDs (*, P < 0.01). Bars, 0.5 μm.

Mentions: If NO is indeed responsible for MIS formation, their frequency should increase by exogenous application of NO or by activating signaling cascades associated with NO such as cyclic guanosine monophosphate (cGMP) formation. To test this, we incubated slice cultures for 2 d with 150 μM of the NO donor diethylenetriamine (DETA) NONOate or with the cell-permeable cGMP analogue 8-Br-cGMP (8-bromo-cGMP sodium salt). As illustrated in the EM pictures and 3D reconstructions of Fig. 7 A, these two conditions resulted in MIS formation in the entire tissue. Using serial sections and 3D reconstructions, we analyzed 2,161 spines in randomly chosen volumes in stratum radiatum taken from nine different slice cultures and determined the proportion of MISs. This proportion increased from 2.5 ± 0.3% under control condition to 23.7 ± 4.7% after DETA NONOate treatment and, thus, is very close to the values obtained in PSD-95–transfected cells (n = 3 cultures for each condition; 615 and 583 spines analyzed; P < 0.01; Fig. 7 C). Furthermore, DETA NONOate treatment increased MISs not only on all cells of treated cultures but also on cells overexpressing the PDZ2 mutant of PSD-95 (32.9 ± 8.6% of MISs; n = 4; P < 0.05; Table S1), indicating that the effect of NO is indeed downstream of PSD-95. Similarly, the application of 5 mM 8-Br-cGMP increased the proportion of MISs in the entire tissue to 12.0 ± 1.8% (n = 3 cultures; 963 spines analyzed; P < 0.01). Conversely, the blockade of cGMP by 25 μM of the inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-α]quinoxalin-1-one) prevented formation of MISs on PSD-95–transfected neurons (7.2 ± 0.5%; n = 3; Table S1). Interestingly, although both the NO donor and cGMP analogue promoted MIS formation, the PSDs expressed on these MISs did not differ in size from those measured under control conditions (Fig. 7 D). Thus, NO production is sufficient for the generation of MISs and does not require overexpression of PSD-95.


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)

Increased formation of MISs by the NO donor DETA NONOate and the cell-permeable cGMP analogue 8-Br-cGMP. (A) EM sections illustrating the presence of MISs (asterisks) in slice cultures treated for 2 d with 150 μM DETA NONOate (left) or 5 mM 8-Br-cGMP (right). (B) 3D reconstruction of the two MISs illustrated in A, the left and right spines making synaptic contacts with five and four different axons, respectively. (C) Proportion of MISs observed in randomly selected volume samples of CA1 stratum radiatum in the control situation (ctrl; three slices and 615 spines analyzed), in DETA NONOate–treated cultures (DETA; three slices and 583 spines analyzed), and in 8-Br-cGMP–treated slices (cGMP; three slices and 963 spines analyzed) compared with the analysis of 234 spines in PSD-95–transfected cells (seven slices). Data are mean ± SEM (error bars; *, P < 0.01). (D) Size of individual PSDs on spines from control slices and on MISs of PSD-95–transfected cells and of DETA NONOate– or of 8-Br-cGMP–treated cultures. Data are mean ± SEM (error bars) of 114–269 reconstructed PSDs (*, P < 0.01). Bars, 0.5 μm.
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fig7: Increased formation of MISs by the NO donor DETA NONOate and the cell-permeable cGMP analogue 8-Br-cGMP. (A) EM sections illustrating the presence of MISs (asterisks) in slice cultures treated for 2 d with 150 μM DETA NONOate (left) or 5 mM 8-Br-cGMP (right). (B) 3D reconstruction of the two MISs illustrated in A, the left and right spines making synaptic contacts with five and four different axons, respectively. (C) Proportion of MISs observed in randomly selected volume samples of CA1 stratum radiatum in the control situation (ctrl; three slices and 615 spines analyzed), in DETA NONOate–treated cultures (DETA; three slices and 583 spines analyzed), and in 8-Br-cGMP–treated slices (cGMP; three slices and 963 spines analyzed) compared with the analysis of 234 spines in PSD-95–transfected cells (seven slices). Data are mean ± SEM (error bars; *, P < 0.01). (D) Size of individual PSDs on spines from control slices and on MISs of PSD-95–transfected cells and of DETA NONOate– or of 8-Br-cGMP–treated cultures. Data are mean ± SEM (error bars) of 114–269 reconstructed PSDs (*, P < 0.01). Bars, 0.5 μm.
Mentions: If NO is indeed responsible for MIS formation, their frequency should increase by exogenous application of NO or by activating signaling cascades associated with NO such as cyclic guanosine monophosphate (cGMP) formation. To test this, we incubated slice cultures for 2 d with 150 μM of the NO donor diethylenetriamine (DETA) NONOate or with the cell-permeable cGMP analogue 8-Br-cGMP (8-bromo-cGMP sodium salt). As illustrated in the EM pictures and 3D reconstructions of Fig. 7 A, these two conditions resulted in MIS formation in the entire tissue. Using serial sections and 3D reconstructions, we analyzed 2,161 spines in randomly chosen volumes in stratum radiatum taken from nine different slice cultures and determined the proportion of MISs. This proportion increased from 2.5 ± 0.3% under control condition to 23.7 ± 4.7% after DETA NONOate treatment and, thus, is very close to the values obtained in PSD-95–transfected cells (n = 3 cultures for each condition; 615 and 583 spines analyzed; P < 0.01; Fig. 7 C). Furthermore, DETA NONOate treatment increased MISs not only on all cells of treated cultures but also on cells overexpressing the PDZ2 mutant of PSD-95 (32.9 ± 8.6% of MISs; n = 4; P < 0.05; Table S1), indicating that the effect of NO is indeed downstream of PSD-95. Similarly, the application of 5 mM 8-Br-cGMP increased the proportion of MISs in the entire tissue to 12.0 ± 1.8% (n = 3 cultures; 963 spines analyzed; P < 0.01). Conversely, the blockade of cGMP by 25 μM of the inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-α]quinoxalin-1-one) prevented formation of MISs on PSD-95–transfected neurons (7.2 ± 0.5%; n = 3; Table S1). Interestingly, although both the NO donor and cGMP analogue promoted MIS formation, the PSDs expressed on these MISs did not differ in size from those measured under control conditions (Fig. 7 D). Thus, NO production is sufficient for the generation of MISs and does not require overexpression of PSD-95.

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