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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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MISs induced by PSD-95 overexpression. (A) EM picture of a section through an MIS showing three axonal terminals (arrowheads) making synaptic contacts with a transfected spine. (B) 3D reconstruction of the same MIS making synaptic contacts with six different presynaptic terminals. Axons have been omitted, and the black regions represent PSDs. (C) Proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines), in cells transfected with EGFP (n = 4 cells; 164 spines), and PSD-95 (n = 7 cells; 234 spines; *, P < 0.05). Data are mean ± SEM (error bars). (D) Same as in B but including the presynaptic terminals and axons. See the animated versions in Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). (E) Contour representation of a dendritic segment from a PSD-95–transfected cell showing the distribution and complexity of MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (F) Graph showing the distribution of total PSD areas (sum of all PSDs on a given spine) as a function of spine volume under control conditions (closed circles), in PSD-95–transfected cells (open circles), and for MISs of PSD-95–transfected cells (triangles). The two lines indicate the linear regression obtained for control spines and for spines of PSD-95–transfected cells (r = 0.7832, P < 0.0001; and r = 0.9091, P < 0.0001, respectively). Bars: (A) 0.5 μm; (B and C) 1 μm; (E) 5 μm.
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fig3: MISs induced by PSD-95 overexpression. (A) EM picture of a section through an MIS showing three axonal terminals (arrowheads) making synaptic contacts with a transfected spine. (B) 3D reconstruction of the same MIS making synaptic contacts with six different presynaptic terminals. Axons have been omitted, and the black regions represent PSDs. (C) Proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines), in cells transfected with EGFP (n = 4 cells; 164 spines), and PSD-95 (n = 7 cells; 234 spines; *, P < 0.05). Data are mean ± SEM (error bars). (D) Same as in B but including the presynaptic terminals and axons. See the animated versions in Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). (E) Contour representation of a dendritic segment from a PSD-95–transfected cell showing the distribution and complexity of MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (F) Graph showing the distribution of total PSD areas (sum of all PSDs on a given spine) as a function of spine volume under control conditions (closed circles), in PSD-95–transfected cells (open circles), and for MISs of PSD-95–transfected cells (triangles). The two lines indicate the linear regression obtained for control spines and for spines of PSD-95–transfected cells (r = 0.7832, P < 0.0001; and r = 0.9091, P < 0.0001, respectively). Bars: (A) 0.5 μm; (B and C) 1 μm; (E) 5 μm.

Mentions: EM analysis of PSD-95–transfected neurons revealed the presence of numerous MISs characterized by several presynaptic terminals contacting the same postsynaptic spine through independent PSDs (Fig. 3, A, B, and D; Fig. S1, and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). MISs contacted by two terminals can be observed in the hippocampus in vivo and in vitro and account for a small fraction of all spines, although during the early postnatal development, dendritic protrusions exhibiting multiple synaptic contacts are rather prominent (Fiala et al., 1998). PSD-95 transfection resulted in a significant increase in the number of MISs (mean number: EGFP control, 2.0 ± 1.3%; nontransfected, 1.8 ± 1.1%; PSD-95, 29.1 ± 2.9%; P < 0.01; Fig. 3 C) and in their complexity. On some dendritic segments, their occurrence increased markedly, as illustrated in Fig. 3 E in which 16 out of 28 spines exhibited synaptic contacts with multiple presynaptic terminals. The increased complexity of these structures is indicated by the number of presynaptic terminals making contact with a single spine (Fig. 3 E). This number varied between two and seven in PSD-95–transfected cells, whereas the maximum observed under control conditions was only two (see Fig. S2 for a distribution of the number of contacts). Furthermore, in the case of MISs, the individual PSDs could show a complex organization with perforated or segmented PSDs in the synaptic contacts made by several axons on the same spine (Videos 1 and 2). A final characteristic of MISs is their unusually large size (0.29 ± 0.028 μm3 compared with 0.064 ± 0.011 μm3 in EGFP-transfected cells) and the fact that they exhibit large PSD areas both at individual contacts (0.164 ± 0.012 μm2 compared with 0.033 ± 0.004 μm2 in EGFP-transfected cells; n = 190 and 175 PSDs, respectively; P < 0.01) and on the entire spine (sum of all PSDs on a given spine, 0.466 ± 0.047 μm2; n = 68 MISs; Fig. 3 F).


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)

MISs induced by PSD-95 overexpression. (A) EM picture of a section through an MIS showing three axonal terminals (arrowheads) making synaptic contacts with a transfected spine. (B) 3D reconstruction of the same MIS making synaptic contacts with six different presynaptic terminals. Axons have been omitted, and the black regions represent PSDs. (C) Proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines), in cells transfected with EGFP (n = 4 cells; 164 spines), and PSD-95 (n = 7 cells; 234 spines; *, P < 0.05). Data are mean ± SEM (error bars). (D) Same as in B but including the presynaptic terminals and axons. See the animated versions in Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). (E) Contour representation of a dendritic segment from a PSD-95–transfected cell showing the distribution and complexity of MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (F) Graph showing the distribution of total PSD areas (sum of all PSDs on a given spine) as a function of spine volume under control conditions (closed circles), in PSD-95–transfected cells (open circles), and for MISs of PSD-95–transfected cells (triangles). The two lines indicate the linear regression obtained for control spines and for spines of PSD-95–transfected cells (r = 0.7832, P < 0.0001; and r = 0.9091, P < 0.0001, respectively). Bars: (A) 0.5 μm; (B and C) 1 μm; (E) 5 μm.
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fig3: MISs induced by PSD-95 overexpression. (A) EM picture of a section through an MIS showing three axonal terminals (arrowheads) making synaptic contacts with a transfected spine. (B) 3D reconstruction of the same MIS making synaptic contacts with six different presynaptic terminals. Axons have been omitted, and the black regions represent PSDs. (C) Proportion of MISs observed under control conditions (ctrl; n = 8 cells; 145 spines), in cells transfected with EGFP (n = 4 cells; 164 spines), and PSD-95 (n = 7 cells; 234 spines; *, P < 0.05). Data are mean ± SEM (error bars). (D) Same as in B but including the presynaptic terminals and axons. See the animated versions in Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). (E) Contour representation of a dendritic segment from a PSD-95–transfected cell showing the distribution and complexity of MISs (numbers refer to presynaptic terminals [*] making synaptic contacts on the spine). (F) Graph showing the distribution of total PSD areas (sum of all PSDs on a given spine) as a function of spine volume under control conditions (closed circles), in PSD-95–transfected cells (open circles), and for MISs of PSD-95–transfected cells (triangles). The two lines indicate the linear regression obtained for control spines and for spines of PSD-95–transfected cells (r = 0.7832, P < 0.0001; and r = 0.9091, P < 0.0001, respectively). Bars: (A) 0.5 μm; (B and C) 1 μm; (E) 5 μm.
Mentions: EM analysis of PSD-95–transfected neurons revealed the presence of numerous MISs characterized by several presynaptic terminals contacting the same postsynaptic spine through independent PSDs (Fig. 3, A, B, and D; Fig. S1, and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200805132/DC1). MISs contacted by two terminals can be observed in the hippocampus in vivo and in vitro and account for a small fraction of all spines, although during the early postnatal development, dendritic protrusions exhibiting multiple synaptic contacts are rather prominent (Fiala et al., 1998). PSD-95 transfection resulted in a significant increase in the number of MISs (mean number: EGFP control, 2.0 ± 1.3%; nontransfected, 1.8 ± 1.1%; PSD-95, 29.1 ± 2.9%; P < 0.01; Fig. 3 C) and in their complexity. On some dendritic segments, their occurrence increased markedly, as illustrated in Fig. 3 E in which 16 out of 28 spines exhibited synaptic contacts with multiple presynaptic terminals. The increased complexity of these structures is indicated by the number of presynaptic terminals making contact with a single spine (Fig. 3 E). This number varied between two and seven in PSD-95–transfected cells, whereas the maximum observed under control conditions was only two (see Fig. S2 for a distribution of the number of contacts). Furthermore, in the case of MISs, the individual PSDs could show a complex organization with perforated or segmented PSDs in the synaptic contacts made by several axons on the same spine (Videos 1 and 2). A final characteristic of MISs is their unusually large size (0.29 ± 0.028 μm3 compared with 0.064 ± 0.011 μm3 in EGFP-transfected cells) and the fact that they exhibit large PSD areas both at individual contacts (0.164 ± 0.012 μm2 compared with 0.033 ± 0.004 μm2 in EGFP-transfected cells; n = 190 and 175 PSDs, respectively; P < 0.01) and on the entire spine (sum of all PSDs on a given spine, 0.466 ± 0.047 μm2; n = 68 MISs; Fig. 3 F).

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