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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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Deletion of the PDZ2 but not the PDZ3 domain of PSD-95 prevents MIS formation. (A, top) Contour representation of a dendritic segment reconstructed from a PDZ3 mutant PSD-95–overexpressing cell illustrating the preservation of numerous MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (bottom) Three-dimensionally reconstructed segment expressing PDZ3 mutant PSD-95 illustrating large, complex spines. (B, top) Same as in A (top) but for a PDZ2 mutant PSD-95. Note the absence of MISs. (bottom) Same as in A (bottom) but for a cell expressing PDZ2 mutant PSD-95. MISs are absent, but large spines are still present. (C) Quantitative analysis of the proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines) and in cells transfected with EGFP (n = 4 cells; 164 spines), PSD-95 (n = 7 cells; 234 spines), PDZ3 mutant PSD-95 (n = 3 cells; 86 spines), and PDZ2 mutant PSD-95 (n = 3 cells; 56 spines; *, P < 0.05 vs. control and PDZ2). (D) Same as in C but for the spine volume. Expression of PDZ mutants of PSD-95 does not prevent PSD-95–induced spine enlargement (*, P < 0.05 vs. control). (E) Same as in C but for the total PSD area. Expression of PDZ mutants of PSD-95 does not reduce or only partially reduces the increase in PSD area (*, P < 0.05 vs. control). Data are mean ± SEM (error bars). Bars: (A and B, top) 5 μm; (A and B, bottom) 1 μm.
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fig4: Deletion of the PDZ2 but not the PDZ3 domain of PSD-95 prevents MIS formation. (A, top) Contour representation of a dendritic segment reconstructed from a PDZ3 mutant PSD-95–overexpressing cell illustrating the preservation of numerous MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (bottom) Three-dimensionally reconstructed segment expressing PDZ3 mutant PSD-95 illustrating large, complex spines. (B, top) Same as in A (top) but for a PDZ2 mutant PSD-95. Note the absence of MISs. (bottom) Same as in A (bottom) but for a cell expressing PDZ2 mutant PSD-95. MISs are absent, but large spines are still present. (C) Quantitative analysis of the proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines) and in cells transfected with EGFP (n = 4 cells; 164 spines), PSD-95 (n = 7 cells; 234 spines), PDZ3 mutant PSD-95 (n = 3 cells; 86 spines), and PDZ2 mutant PSD-95 (n = 3 cells; 56 spines; *, P < 0.05 vs. control and PDZ2). (D) Same as in C but for the spine volume. Expression of PDZ mutants of PSD-95 does not prevent PSD-95–induced spine enlargement (*, P < 0.05 vs. control). (E) Same as in C but for the total PSD area. Expression of PDZ mutants of PSD-95 does not reduce or only partially reduces the increase in PSD area (*, P < 0.05 vs. control). Data are mean ± SEM (error bars). Bars: (A and B, top) 5 μm; (A and B, bottom) 1 μm.

Mentions: Fig. 4 (A and B) shows three-dimensionally reconstructed dendritic segments from cells transfected with each of those constructs. Interestingly, expression of PDZ3 mutant PSD-95 in neurons still resulted in numerous MISs (Fig. 4, A and C). Their proportion did not change, nor did the mean spine volume or PSD area of three-dimensionally reconstructed spines as compared with PSD-95–transfected neurons (MISs, 22.1 ± 4.5% vs. 29.1 ± 2.9%; spine volume, 0.168 ± 0.012 μm3 vs. 0.144 ± 0.023 μm3; PSD area, 0.169 ± 0.03 μm2 vs. 0.189 ± 0.043 μm2; n = 3 and 7 cells for PDZ3 mutant and PSD-95–transfected cells, respectively; Fig. 4, C–E). In contrast, the number of MISs markedly decreased in cells expressing the PDZ2 mutant PSD-95 (PDZ2 mutant, 1.7 ± 1.7% vs. PSD-95, 29.1 ± 2.9%; n = 3 and 7 cells, respectively; P < 0.01; Fig. 4, B and C), reaching values similar to those found in control cells (neighboring, nontransfected neurons, 2.0 ± 1.3%; n = 8 cells). The mean spine volume, however, remained significantly enlarged compared with control cells (0.131 ± 0.021 μm3; P < 0.05) as well as the mean PSD area (0.094 ± 0.019 μm2; P < 0.05; Fig. 4, D and E). Thus, expression of a PDZ2 mutant PSD-95 resulted in the enlargement of spines with increased PSD areas but specifically prevented formation of multiinnervated structures.


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)

Deletion of the PDZ2 but not the PDZ3 domain of PSD-95 prevents MIS formation. (A, top) Contour representation of a dendritic segment reconstructed from a PDZ3 mutant PSD-95–overexpressing cell illustrating the preservation of numerous MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (bottom) Three-dimensionally reconstructed segment expressing PDZ3 mutant PSD-95 illustrating large, complex spines. (B, top) Same as in A (top) but for a PDZ2 mutant PSD-95. Note the absence of MISs. (bottom) Same as in A (bottom) but for a cell expressing PDZ2 mutant PSD-95. MISs are absent, but large spines are still present. (C) Quantitative analysis of the proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines) and in cells transfected with EGFP (n = 4 cells; 164 spines), PSD-95 (n = 7 cells; 234 spines), PDZ3 mutant PSD-95 (n = 3 cells; 86 spines), and PDZ2 mutant PSD-95 (n = 3 cells; 56 spines; *, P < 0.05 vs. control and PDZ2). (D) Same as in C but for the spine volume. Expression of PDZ mutants of PSD-95 does not prevent PSD-95–induced spine enlargement (*, P < 0.05 vs. control). (E) Same as in C but for the total PSD area. Expression of PDZ mutants of PSD-95 does not reduce or only partially reduces the increase in PSD area (*, P < 0.05 vs. control). Data are mean ± SEM (error bars). Bars: (A and B, top) 5 μm; (A and B, bottom) 1 μm.
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fig4: Deletion of the PDZ2 but not the PDZ3 domain of PSD-95 prevents MIS formation. (A, top) Contour representation of a dendritic segment reconstructed from a PDZ3 mutant PSD-95–overexpressing cell illustrating the preservation of numerous MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (bottom) Three-dimensionally reconstructed segment expressing PDZ3 mutant PSD-95 illustrating large, complex spines. (B, top) Same as in A (top) but for a PDZ2 mutant PSD-95. Note the absence of MISs. (bottom) Same as in A (bottom) but for a cell expressing PDZ2 mutant PSD-95. MISs are absent, but large spines are still present. (C) Quantitative analysis of the proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines) and in cells transfected with EGFP (n = 4 cells; 164 spines), PSD-95 (n = 7 cells; 234 spines), PDZ3 mutant PSD-95 (n = 3 cells; 86 spines), and PDZ2 mutant PSD-95 (n = 3 cells; 56 spines; *, P < 0.05 vs. control and PDZ2). (D) Same as in C but for the spine volume. Expression of PDZ mutants of PSD-95 does not prevent PSD-95–induced spine enlargement (*, P < 0.05 vs. control). (E) Same as in C but for the total PSD area. Expression of PDZ mutants of PSD-95 does not reduce or only partially reduces the increase in PSD area (*, P < 0.05 vs. control). Data are mean ± SEM (error bars). Bars: (A and B, top) 5 μm; (A and B, bottom) 1 μm.
Mentions: Fig. 4 (A and B) shows three-dimensionally reconstructed dendritic segments from cells transfected with each of those constructs. Interestingly, expression of PDZ3 mutant PSD-95 in neurons still resulted in numerous MISs (Fig. 4, A and C). Their proportion did not change, nor did the mean spine volume or PSD area of three-dimensionally reconstructed spines as compared with PSD-95–transfected neurons (MISs, 22.1 ± 4.5% vs. 29.1 ± 2.9%; spine volume, 0.168 ± 0.012 μm3 vs. 0.144 ± 0.023 μm3; PSD area, 0.169 ± 0.03 μm2 vs. 0.189 ± 0.043 μm2; n = 3 and 7 cells for PDZ3 mutant and PSD-95–transfected cells, respectively; Fig. 4, C–E). In contrast, the number of MISs markedly decreased in cells expressing the PDZ2 mutant PSD-95 (PDZ2 mutant, 1.7 ± 1.7% vs. PSD-95, 29.1 ± 2.9%; n = 3 and 7 cells, respectively; P < 0.01; Fig. 4, B and C), reaching values similar to those found in control cells (neighboring, nontransfected neurons, 2.0 ± 1.3%; n = 8 cells). The mean spine volume, however, remained significantly enlarged compared with control cells (0.131 ± 0.021 μm3; P < 0.05) as well as the mean PSD area (0.094 ± 0.019 μm2; P < 0.05; Fig. 4, D and E). Thus, expression of a PDZ2 mutant PSD-95 resulted in the enlargement of spines with increased PSD areas but specifically prevented formation of multiinnervated structures.

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