Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation
2011; Springer Nature; Volume: 30; Issue: 3 Linguagem: Inglês
10.1038/emboj.2010.336
ISSN1460-2075
AutoresAndreas M. Grabrucker, Mary Jane Knight, Christian Proepper, Juergen Bockmann, Marisa K. Joubert, Magali Rowan, G. Ulrich Nienhaus, Craig C. Garner, Jim U Bowie, Michael R. Kreutz, Eckart D. Gundelfinger, Tobias M. Boeckers,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle7 January 2011Open Access Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation Andreas M Grabrucker Andreas M Grabrucker Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author Mary J Knight Mary J Knight Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Christian Proepper Christian Proepper Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Juergen Bockmann Juergen Bockmann Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Marisa Joubert Marisa Joubert Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Magali Rowan Magali Rowan Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author G UIrich Nienhaus G UIrich Nienhaus Institute of Applied Physics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany Search for more papers by this author Craig C Garner Craig C Garner Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author Jim U Bowie Jim U Bowie Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Michael R Kreutz Michael R Kreutz PG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Tobias M Boeckers Corresponding Author Tobias M Boeckers Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Andreas M Grabrucker Andreas M Grabrucker Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author Mary J Knight Mary J Knight Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Christian Proepper Christian Proepper Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Juergen Bockmann Juergen Bockmann Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Marisa Joubert Marisa Joubert Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Magali Rowan Magali Rowan Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author G UIrich Nienhaus G UIrich Nienhaus Institute of Applied Physics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany Search for more papers by this author Craig C Garner Craig C Garner Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA Search for more papers by this author Jim U Bowie Jim U Bowie Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA Search for more papers by this author Michael R Kreutz Michael R Kreutz PG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Tobias M Boeckers Corresponding Author Tobias M Boeckers Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany Search for more papers by this author Author Information Andreas M Grabrucker1,2, Mary J Knight3, Christian Proepper1, Juergen Bockmann1, Marisa Joubert3, Magali Rowan2, G UIrich Nienhaus4, Craig C Garner2, Jim U Bowie3, Michael R Kreutz5, Eckart D Gundelfinger6 and Tobias M Boeckers 1 1Institute of Anatomy and Cell Biology, University of Ulm, Ulm, Germany 2Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA 3Department of Chemistry and Biochemistry, UC Los Angeles, CA, USA 4Institute of Applied Physics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany 5PG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany 6Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany *Corresponding author. Institute of Anatomy and Cell Biology, University of Ulm, Albert Einstein Allee 11, Ulm 89081, Germany. Tel.: +49 731 502 2331; Fax: +49 731 502 2317; E-mail: [email protected] The EMBO Journal (2011)30:569-581https://doi.org/10.1038/emboj.2010.336 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity-dependent remodelling of excitatory synapses. Here, we report that Zn2+ ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member-specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA-mediated knockdown, we could demonstrate that only the overexpression of zinc-sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn2+ along with the knockdown of zinc-insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD-95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn2+ is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity. Introduction During wiring of the central nervous system, synaptic contacts are constantly formed, stabilized or eliminated. For the establishment of new functional excitatory synapses, preformed scaffold protein complexes serve as predetermined postsynaptic sites (Gerrow et al, 2006). ProSAP/Shank proteins are observed at postsynaptic specializations early during synaptogenesis (Boeckers et al, 1999a; Petralia et al, 2005). The family of ProSAP/Shank proteins (also referred to as Synamon, CortBP, Spank and SSTRIP) consists of three members in mammals. They are efficiently targeted to synaptic sites (Sala et al, 2001; Boeckers et al, 2005) and attach receptor complexes to the local actin-based cytoskeleton within dendritic spines (Boeckers et al, 2002; Kim and Sheng, 2004; Rostaing et al, 2006). All ProSAP/Shank family members are expressed in the brain and are present at the postsynaptic density (PSD) of excitatory synapses (Boeckers et al, 1999b; Naisbitt et al, 1999); however, only Shank1 seems to be brain specific (Lim et al, 1999). The appearance of ProSAP/Shank proteins at the PSD during neuronal development has been studied in the developing rat brain and in hippocampal cell culture (Boeckers et al, 1999a; Lim et al, 1999; Naisbitt et al, 1999). Localization data demonstrate that ProSAP1/Shank2 is one of the first protein components present in developing PSDs. It also becomes anchored to the subsynaptic cytoskeleton earlier than other known components of the PSD protein fraction, including SAP90/PSD-95 and NMDA receptors (Boeckers et al, 1999a). Expression of GFP-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons reveals that the postsynaptic targeting of ProSAP1/Shank2 and ProSAP2/Shank3 is dependent on their SAM domains and the recruitment of ProSAP1/Shank2 and ProSAP2/Shank3 to the synapse is independent of their interaction with Homer (Boeckers et al, 2005). In contrast, the PDZ domain of Shank1 is mandatory for proper targeting to the synapse (Sala et al, 2001) and its localization at the PSD follows the formation of the SAP90/PSD-95-GKAP complexes (Naisbitt et al, 1999; Sheng and Kim, 2000). Overexpression of Shank1 alters spine morphology in transfected primary hippocampal neurons, leading to the enlargement of spine heads, an effect that depends on the PDZ domain and the Homer-binding site (Sala et al, 2001). Homer and ProSAP/Shank are among the most abundant scaffolding proteins in the PSD, working synergistically during the maturation of dendritic spines (Hayashi et al, 2009). The potency of ProSAP/Shank proteins to alter the morphology of the postsynaptic compartment has also been shown by Roussignol et al (2005), who demonstrated that ProSAP2/Shank3 is essential for the maintenance of spines and synapses in hippocampal cultures. Moreover, overexpression of ProSAP2/Shank3 can induces spine formation in aspiny cerebellar neurons (Roussignol et al, 2005). ProSAP/Shanks exhibit the capacity to form two-dimensional arrays by self-association via the SAM domain that are predicted to form a platform-like structure within dendritic spines (Baron et al, 2006). This association is regulated by Zn2+ ions, which might be released from the presynaptic terminal or from within dendritic spines leading to rapid changes within the PSD compartment (Gundelfinger et al, 2006). In the brain, free chelatable Zn2+ has been detected in presynaptic vesicles of glutamatergic terminals and Zn2+ released from the presynapse directly binds to and modulates glutamate receptors, thereby exerting significant effects on both acute synaptic transmission and long-term potentiation at hippocampal synapses (Li et al, 2001; Huang et al, 2008). Evidence has also been provided that Zn2+ ions are recruited into the molecular assemblies mediated by the SAM domains of ProSAP2/Shank3 and potentially ProSAP1/Shank2 (Baron et al, 2006; Gundelfinger et al, 2006) though how and if Zn2+ ions affect the molecular assembly of these proteins within dendritic spines remains unresolved. With >4 nmol/mg protein, the Zn2+ content of purified PSDs is surprisingly high (Jan et al, 2002). Given the effect of Zn2+ on the packaging density of the ProSAP2/Shank3 2D arrays (Baron et al, 2006), we have proposed that chelatable Zn2+ might act to regulate postsynaptic dynamics and structural plasticity of excitatory synapses, (Gundelfinger et al, 2006). Here, we explore the interplay between ProSAP/Shank family members and Zn2+ ions in living cells. Our results demonstrate that the zinc-sensitive isoforms, ProSAP1/Shank2 and ProSAP2/Shank3, are recruited to synapses prior to the zinc-insensitive isoform, Shank1. Moreover, changing the concentration of extracellular zinc was found to not only affect the synaptic levels of ProSAP1/Shank2 and ProSAP2/Shank3, but to dramatically influence the thickness of the PSD and the assembly of immature synapses. Furthermore, the zinc insensitivity of mature synapses was found to dependent on the synaptic expression of Shank1. These results lead to a new model, in which zinc through its actions on specific ProSAP/Shank family members regulate the initial formation and maintenance of nascent synapses as well as the stabilization, maturation and plasticity of mature excitatory synapses. Results and discussion Results Previous studies have shown that ProSAP/Shank family members are localized to the PSDs of excitatory synapses (Boeckers et al, 1999a, 1999b, 2002; Sala et al, 2001; Grabrucker et al, 2009a), yet their contribution to synapse formation and maturation are not well understood. In an initial set of experiments, we examined whether gain (overexpression of GFP-tagged ProSAP/Shank) or loss of function (shRNA-mediated knockdown) influenced synapse number in hippocampal neuron grown for 9 days in vitro (DIV). When neurons were transfected at DIV 6 and examined at DIV 9 GFP-tagged ProSAP1/Shank2 and ProSAP2/Shank3 efficiently colocalize with the synaptic markers Bassoon and Homer (Figure 1A), while GFP-tagged Shank1 exhibited a limited synaptic localization (Figure 1A). An analysis of synaptic density/unit length of dendrite revealed that GFP-tagged ProSAP1/Shank2 and ProSAP2/Shank3 overexpression caused a significant increase in the number of synapses, while GFP-Shank1 overexpression had little effect (Figure 1B). Consistent with a role of these PSD proteins in synapse formation, knockdown of ProSAP/Shank proteins causes a significant reduction in synapse density (Figure 1B; Supplementary Figure S1). Figure 1.In young neurons, overexpressed ProSAP1/Shank2 and ProSAP2/Shank3, but not Shank1, increase synapse density and localize to synaptic contact sites. (A) Hippocampal neurons were transfected at 6 DIV with full-length and C-terminal ProSAP/Shank constructs and cells were fixed at 9 DIV. ProSAP1/Shank2 and ProSAP2/Shank3 localize at synapses in contrast to Shank1. The percentage of punctate GFP signals colocalizing with Homer/Bassoon signals was assessed. Shank1 shows significantly less localization to synapses. Although the C-terminal Shank1 construct shows a punctate distribution in contrast to full-length Shank1, the Shank1 C-terminal clusters are mostly non-synaptic. (B) The number of synapses defined by Homer/Bassoon colocalizing signals was measured per 10 μm dendrite length in ProSAP/Shank full-length and C-terminal constructs overexpressing hippocampal neurons. ProSAP1/Shank2 and ProSAP2/Shank3 overexpression from DIV 6 to DIV 9 significantly increases the number of synapses. Shank1 overexpression shows no increase. ProSAP/Shank C-terminal constructs did not show any significant alteration. The knockdown of each ProSAP/Shank member causes a significant reduction in synapse density. Images show hippocampal neurons transfected with GFP expressing constructs and stained for Homer-Alexa 647 (coloured blue in ‘Merged’ pictures) and Bassoon-Alexa-568 (coloured red in ‘Merged’ pictures). *P=0.5; **P=0.01; ***P=0.001. Download figure Download PowerPoint In a previous study, we identified the C-terminal SAM domain as a region critical for the postsynaptic localization of ProSAP1/Shank2 and ProSAP2/Shank3 (Boeckers et al, 2005; Grabrucker et al, 2009b). Intriguingly, Shank1, though it has a SAM domain, utilizes its PDZ domain for its synaptic localization (Sala et al, 2001). In the case of ProSAP1/Shank2 and ProSAP2/Shank3, the SAM domain is predicted to facilitate the oligomerization of these isoforms in a Zn2+-dependent manner and possibly the assembly of these ProSAP/Shank isoforms into a two-dimensional lattice within dendritic spines (Baron et al, 2006). We were thus interested to explore whether their overexpression and/or zinc binding affects their synaptic localization and/or synapse assembly. In initial experiments, the GFP-tagged SAM domains of ProSAP1/Shank2 and ProSAP2/Shank3 were found to reliably become synaptically localized when overexpressed in DIV 9 neurons, yet had no significant effect on synapse number (Figure 1B). Similar to full-length GFP-Shank1, the SAM domain of Shank1 only poorly localized to synapses at 9 DIV and had no effect on synapse number (Figure 1A and B). Interestingly, although full-length Shank1 exhibits a microtubular-like pattern, in immature neurons 9 DIV (Figure 1A), it becomes synaptic in older cultures (Supplementary Figure S1B and C), indicating that the postsynaptic localization of Shank1 is tightly controlled limiting its influence to mature synapses. The overexpression in young neurons might lead to an accumulation of protein outside synaptic compartments, resulting in a shift of Shank1 localization to other interaction partners. In the next set of experiments, we were keen to investigate the role of Zn2+ ions on SAM domain function and a possible influence on synapse formation and maturation. For these experiments, we took advantage of a dye (Zinquin) that fluoresces when it binds zinc (Coyle et al, 1994; Fahrni and O'Halloran, 1999) (Supplementary Figure S2A). To test the hypothesis that ProSAP/Shank SAM domains bind Zn2+ in vivo, transfected HeLa cells cultured in media supplemented with Zn2+ (30 μM) were stained with Zinquin. In untransfected HeLa cells, Zinquin staining labels small naturally occurring zinc-positive clusters. The application of ZnCl2 to the medium strongly enhances the intracellular Zinquin signals (Supplementary Figure S2B). Zn2+ binds to Zinquin by complexing with either one or two nitrogen atoms to form complexes in a ratio of 1:1 (Zn2+:Zinquin) or 1:2. However, a wide range of free Zn2+ concentrations have been estimated in studies with Zinquin, ranging from femtomolar to micromolar concentrations, thereby raising the question of what Zinquin is measuring. Strikingly, Coyle et al (1994) showed in hepatocyte homogenates that Zinquin fluoresced with protein-bound Zn2+ across a broad range of molecular weights. There was no evidence that Zinquin removed Zn2+ from high molecular weight proteins. Taken together, these data indicate that Zinquin sequesters zinc that is labile or loosely bound to proteins, using ill-defined ligand-exchange mechanisms (Coyle et al, 1994; Fahrni and O'Halloran, 1999). In Hela cells transfected with GFP-tagged ProSAP1/Shank2 or ProSAP2/Shank3, we observed the appearance of large intracellular clusters, which nicely colocalize with Zinquin fluorescent puncta (Figure 2A and C). A high degree of colocalization with Zinquin fluorescent puncta was also seen in cells expressing the C-terminal SAM domain of ProSAP1/Shank2 or ProSAP2/Shank3 tagged with GFP (Figure 2A and C). No colocalization of zinc dye with GFP was observed upon transfection of HeLa cells with other cluster-forming PSD molecules, such as Abi-1 (Supplementary Figure S2C). Similarly, expression of the Shank1-SAM domain, constructs lacking the ProSAP1/Shank2- or ProSAP2/Shank3-SAM domains, or constructs harbouring a mutation in the Zn2+-binding site within the ProSAP2/Shank3-SAM domain do not display any overlap with Zinquin fluorescent puncta (Figure 2A). Intriguingly, the addition of 300 μM ZnCl2 leads to an increase in cluster size of GFP-ProSAP2/Shank3 in HeLa cells (Supplementary Figure S2D). In contrast, the localization pattern of Shank1 is not affected by the addition of 300 μM ZnCl2 (Supplementary Figure S2D). Together, these data suggest that zinc levels may control the oligomerization state of specific ProSAP/Shank family members via the self-association of isoform-specific SAM domains. To more directly test this hypothesis, we created and purified fusion proteins between maltose-binding protein (MBP) and the SAM domains of ProSAP2/Shank3 or Shank1. When MBP-ProSAP2/Shank3(SAM) was incubated with an equimolar amount of zinc, the majority of the protein precipitates (data not shown). In contrast, when MBP-Shank1(SAM) protein is incubated with zinc, only a minor fraction of the protein precipitates (data not shown). Note, in previous work, we found that the Zn2+-induced precipitate of ProSAP2/Shank3(SAM) contains a large fraction of SAM-organized sheets (Baron et al, 2006). To confirm that Zn2+ was changing the oligomerization state, the supernatant of zinc-treated samples of MBP-ProSAP2/Shank3(SAM) and MBP-Shank1(SAM) were loaded onto a gel-filtration column and individual fractions assayed with Zinquin. A significant Zinquin fluorescence signal was detected in fractions that contain both MBP-ProSAP2/Shank3(SAM) monomers and polymers, but no signal was detected in MBP-Shank1(SAM) fractions (Figure 2B). As a further measure that the SAM domain of ProSAP2/Shank3 binds Zn2+, we assessed Zinquin fluorescence in the zinc-induced precipitates of ProSAP/Shank proteins. While the purified MBP-ProSAP2/Shank3(SAM) samples produced a strong fluorescence signal, the MBP-Shank1(SAM) samples only yielded a Zinquin signal of roughly 8% of the ProSAP2/Shank3 signal (Figure 2B). These results demonstrate that ProSAP2/Shank3-SAM domains are able to bind bivalent Zn2+ ions in vitro while Shank1 binds much lower levels of Zn2+. This is consistent with our prediction that amino-acid exchanges in the sheet interface of the Shank1 SAM domain allows it to form zinc-independent sheets (Baron et al, 2006; Gundelfinger et al, 2006). Intriguingly, the Zn2+-binding sites of ProSAP1/Shank2 and ProSAP2/Shank3, however, are completely conserved (Boeckers et al, 2005) and binding of other divalent metal ions is nearly absent (Supplementary Figure S2E). Figure 2.The SAM domains of ProSAP1/Shank2 and of ProSAP2/Shank3 specifically bind zinc ions. (A) Full-length GFP-ProSAP1/Shank2, ProSAP2/Shank3 or their C-terminal SAM domains colocalize with Zn2+ stained with Zinquin (arrowheads) in HeLa cells. GFP-Shank1 or its C-terminal SAM domain do not colocalize with Zinquin. Mutations of the ProSAP2/Shank3 SAM domain within the zinc-binding site (H22A) or the site of oligomerization (M56E) (Baron et al, 2006) abolish Zinquin colocalization. (B) Gel filtration of purified maltose-binding protein (MBP)-Shank1 (upper panel) and MBP-ProSAP2/Shank3 (middle panel) after zinc preincubation (solid black line). The plots are overlayed with Zinquin fluorescence profile (red lines, open circles). Quantification of the Zinquin fluorescence signals per nmole of zinc-precipitated MBP-AH-Shank1 and MBP-AH-ProSAP2/Shank3 protein (lower panel). (C) Transfection of ProSAP2/Shank3-GFP shows that EGFP signals cocluster with the fluorescence zinc dye Zinquin in hippocampal neurons in culture. Similarly, Zinquin signals colocalize with endogenous ProSAP2/Shank3 staining. (D) Immunolabelling of excitatory synapses of cultured hippocampal neurons (14 DIV). The zinc signal (Newport Green) colocalizes with the PSD protein ProSAP1/Shank2 and is juxtaposed to the presynaptic marker Bassoon. (E) The postsynaptic enrichment of zinc can be visualized in living cells, using the FM dye 4–64 that labels recycling presynaptic vesicles and Zinquin. Download figure Download PowerPoint One prediction of these biochemical studies is that ProSAP/Shank-positive dendritic spines should also contain Zn2+. To test this concept, we overexpressed GFP-ProSAP2/Shank3 in cultured hippocampal neurons and stained these neurons with Zinquin. Figure 2C reveals a striking colocalziation of dendritic GFP-ProSAP2/Shank3 clusters and Zinquin fluorescent puncta. Cultures stained with antibodies against ProSAP1/Shank2 and ProSAP2/Shank3 revealed that Zinquin or Newport Green puncta also colocalized with endogenous ProSAP2/Shank3 and ProSAP1/Shank2 postsynaptic clusters, respectively (Figure 2C and D). Previous studies on zinc in neurons have found that it is particularly enriched in presynaptic nerve terminal especially those expressing the zinc transporter ZnT3 (Palmiter et al, 1996; Wenzel et al, 1997). To assess whether the detected synaptic zinc was presynaptic or postsynaptic, we compared the spatial localization of Zinquin fluorescent puncta with endogenous ProSAP1/Shank2 and Bassoon, a presynaptic active zone protein, or presynaptic boutons loaded with the styryl dye FM4-64 (Figure 2D and E). The results show that the detected Zn2+ signal colocalizes with postsynaptic markers. Note, that Newport Green (a second zinc-binding dye) was used to confirm our data with Zinquin that postsynaptic structures contain zinc. A critical question raised by these studies is whether Zn2+ levels in neurons affect the synaptic distribution of zinc-binding isoforms of ProSAP/Shank. This question was initially addressed by acutely removing Zn2+ from cultured hippocampal neurons by the application of a more potent chelator than Zinquin such as TPEN. Here, we found that 10 μM TPEN caused a translocation of ProSAP1/Shank2 and ProSAP2/Shank3 proteins from PSDs into the dendritic compartment within a few minutes (Figure 3A and B; Supplementary Figure S3A). The localization of other molecular PSD components including Shank1 and glutamate receptors as well as the overall number of PSDs remained unchanged. Additionally, hippocampal neuronal cultures were treated with TPEN at DIV 14 and protein fractionation was performed. Consistent with the previous results, the amount of ProSAP1/Shank2 and ProSAP2/Shank3, but not Shank1 increases in the S2 protein fraction after zinc depletion compared with untreated neurons (Figure 3B). This shift is further emphasized by the fact that the ProSAP2/Shank3 protein can be washed out with Triton X-100 from the dendritic compartment to a higher degree in zinc-depleted neurons (Supplementary Figure S3A). Importantly, the effect of zinc depletion on the localization of ProSAP2/Shank3 depends upon the applied concentration of the zinc chelators, but leaves the staining of the presynaptic marker protein Bassoon unchanged. These effects of zinc depletion are reversible by the supplementation of Zn2+ ions, but not by Ca2+ or Mg2+ (Supplementary Figure S3). Figure 3.Zinc can rapidly alter the morphology of synaptic contacts and the attachment of ProSAP1/Shank2 and ProSAP2/Shank3 at PSDs. (A) A 20-min application of the zinc chelator TPEN results in the loss of defined ProSAP2/Shank3 dots (green) adjacent to presynaptic specializations as marked by Bassoon immunoreactivity (red). Measurement of the ProSAP2/Shank3-positive area above a present fluorescence limit illustrates the significant reduction within the TPEN group (control 0.17±0.055 versus 0.06±0.03) and a slight enlargement upon ZnCl2 treatment (0.2±0.05). Altogether, >500 synapses were measured for each condition and the results were corrected for inhibitory synapses, which are positive for Bassoon but a priori negative for ProSAP/Shanks. The mean number of inhibitory synapses per cell was assessed via Gepyhrin and Bassoon staining and amounted to 23.1±1.33%. (B) Western blot analysis of neuronal P2 and S2 fractions without and with short TPEN treatment shows that the zinc-binding proteins ProSAP1/Shank2 and ProSAP2/Shank3 are shifted towards the soluble fraction while the Shank1 signal stays unchanged upon TPEN application. (C) Ultrastructural analysis of PSD depth and area by electron microscopy shows a significant reduction of both parameters for TPEN and CaEDTA application and a slight enlargement in the ZnCl2 group. (D) The ultrastructural analysis of synaptic contacts according to the semiquantitative criteria ‘normal PSD', ‘strong PSD' or ‘without PSD' indicates that the percentage of synaptic contacts without a PSD is higher in the zinc chelator groups (TPEN, CaEDTA), while ZnCl2 treatment leads to the increased appearance of ‘strong’ PSDs. *P=0.5; **P=0.01; ***P=0.001. Download figure Download PowerPoint One hallmark of excitatory synapses is the presence of a pronounced PSD that is thought to arise due to a high concentration of postsynaptic scaffold proteins (Gray, 1959). Given the striking effects of TPEN and ZnCl2 on the postsynaptic levels of ProSAP/Shank proteins, we examined the ultrastructural organization of synapses following these manipulations. Intriguingly, the application of 10 μM TPEN led to a significant reduction of PSD thickness and area, while the application of 300 μM Zn2+ caused in a significant increase in PSD thickness (Figure 3C). Quantifying synapse morphology using semiquantitative criteria ‘normal PSD', ‘strong PSD' or ‘without PSD' indicates that the percentage of synaptic contacts without a PSD is higher in the zinc chelator groups (TPEN, CaEDTA), while ZnCl2 treatment leads to the increased appearance of ‘strong’ PSDs (Figure 3D). These data indicate that altering zinc levels can have dramatic effects on PSDs by modulating the postsynaptic levels of Zn2+-sensitive ProSAP/Shank isoforms. Intriguingly, the effect of Zn2+ chelators on postsynaptic morphology was particularly pronounced in young neurons (DIV 7). This was explored by staining cultured neurons with antibodies against Homer1 to mark excitatory postsynapses and Bassoon as a presynaptic marker. For evaluation, fluorescent puncta along dendrites were counted and the mean number of monofluorescent (‘Bassoon only’) and double-fluorescent (‘Bassoon and Homer’) signals were compared under control and zinc-depleted conditions. Here, we observed a complete loss of Homer1 immunoreactivity, in about 40% of synapses, within a short time after treatment, with little if any effect on Bassoon fluorescence (Figure 4A and B). Importantly, the overall number of presynaptic contact sites remains unchanged. These data suggest that zinc depletion is causing a selective disassembly of the PSD scaffold. Surprisingly, at later stages of synaptic maturation (DIV 14), the reduction of the postsynaptic Homer1 signal is not observed (Figure 4B). This latter observation suggests that PSDs undergo a developmental change in their molecular composition. To explore whether this is due to changes in the synaptic expression patterns of ProSAP/Shank family members, cultures at different stages were fixed and stained with antibodies against Bassoon and different ProSAP/Shank is
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