This study looked specifically at inhibitory synapses - "The signal it transmits hinders activation of the postsynaptic neuron, making it less likely for it to become activated and emit its action potential,” according to Paola Zacchi, a SISSA [International School for Advanced Studies (Italian: Scuola Internazionale Superiore di Studi Avanzati, SISSA) researcher who coordinated the study.
“When Pin1 is absent from the synapse, signal transmission occurs “at full strength”, but also without control. Instead, when it is present, it regulates signal strength, making it weaker. We observed that Pin1 is able to modify the number of postsynaptic receptors”. The larger the number of receptors capable of binding to the neurotransmitter, the stronger the signal that reaches the postsynaptic membrane. “This also means that Pin1 plays a role in plasticity” explains Zacchi.The whole article is open access, via Nature Communications, but it's pretty geeky, so we start with a summary of the press release from Neuroscience News, and then below that is most of the original article (minus the methods section which is not relevant to anyone outside the field of neuroscience and molecular biology).
October 10, 2014
Study reveals novel role for the Pin1 molecule.
A small, “empty” space teeming with activity: a synapse is a complex structure where the neural signal from the presynaptic neuron, as it travels towards its target turns into a chemical signal capable of crossing the synaptic space before becoming electrical again once on the other side. A synapse is a “dynamic” space not only because of the endless work that goes on there, but also for its ability to change its action over time (synaptic plasticity) as a result of either normal physiological processes (e.g., during learning) or because of disorders due to pathological conditions. A study, mainly carried out by SISSA researchers (which also involved the University of Zurich, LNCIB in Trieste, and EBRI in Rome), showed that a small enzyme (Pin1, peptidyl prolyl isomerase) that plays a mediating role in signal transmission has an effect on synaptic plasticity.
“The synapse we studied is of the inhibitory kind. The signal it transmits hinders activation of the postsynaptic neuron, making it less likely for it to become activated and emit its action potential”, explains Paola Zacchi, a SISSA researcher who coordinated the study. “When Pin1 is absent from the synapse, signal transmission occurs “at full strength”, but also without control. Instead, when it is present, it regulates signal strength, making it weaker. We observed that Pin1 is able to modify the number of postsynaptic receptors”. The larger the number of receptors capable of binding to the neurotransmitter, the stronger the signal that reaches the postsynaptic membrane. “This also means that Pin1 plays a role in plasticity” explains Zacchi.
This image depicts a neuron. Credit SISSA.
More in Detail
How does a synapse work? “A chemical synapse, the most common in vertebrates, is a small gap between nerve cells where the passage of a neural signal occurs”, explains Zacchi. In chemical synapses the two neurons are not in contact but they are separated by a distance of about 20 nanometres. For this reason, the electrical signal travelling along the presynaptic nerve ending is interrupted before resuming on the neuron on the other side of the gap. In between the two nerve cells the electrical signal is translated into a chemical signal (which then becomes electrical again).
“Arrival of the action potential on the presynaptic button causes release, into the interneural space, of molecules of neurotransmitter, which are picked up by receptors on the postsynaptic membrane”, says Zacchi. “If the synapse is excitatory, this leads to postsynaptic activation which, if sufficiently intense, triggers another action potential. If the synapse is inhibitory, as in our studies, the signal suppresses postsynaptic activation and inhibits firing of the electrical potential. In the process of neurotransmitter release and binding, other molecules come into play, such as scaffold proteins, which assemble receptors at the right place on the membrane in front of the neurotransmitter release sites, and neuroligins which act as bridges between the two ends of the synapse as well as interacting with the scaffold proteins. Pin1, the enzyme in the study, interacts with both neuroligins and scaffold proteins.
The Pin1 enzyme has long been known for its role in cancer and the development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s (whereas neuroligins seem to be involved in autism). “Studies like this enhance our understanding of the biochemical mechanisms of synaptic plasticity, extending our knowledge of healthy mechanisms, but also helping those who are trying to understand what can be done in a wide range of pathological conditions”.
Notes about this neuroscience research
Contact: Federica Sgorbissa – SISSA
Source: SISSA press release
Image Source: The image is credited to SISSA and is adapted from the press release
Original Research: Full open access research for “Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction” by Roberta Antonelli, Rocco Pizzarelli, Andrea Pedroni, Jean-Marc Fritschy, Giannino Del Sal, Enrico Cherubini and Paola Zacchi in Nature Communications; 5 (article #5066). Published online October 9 2014. doi:10.1038/ncomms6066
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Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction
Roberta Antonelli, Rocco Pizzarelli, Andrea Pedroni, Jean-Marc Fritschy, Giannino Del Sal, Enrico Cherubini & Paola Zacchi
- Nature Communications 5, Article number (2014, Oct 9): 5066; doi:10.1038/ncomms6066
AbstractThe cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis–trans isomerase Pin1. This signalling cascade negatively regulates NL2’s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1−/−) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction.
Structural and functional changes of post-synaptic density (PSD) components contribute to regulate synapse formation and plasticity. These remodelling events can affect trafficking, lateral mobility and turnover of several classes of structural and signalling molecules. They often involve interactions among specific proteins regulated by post-translational modifications, such as phosphorylation. At GABAergic synapses, the impact of phosphorylation on the gating properties, surface mobility and trafficking of the gamma-aminobutyric acid A receptors (GABAARs) has been extensively studied1, 2. Much less is known about the effects of phosphorylation of other post-synaptic proteins functionally linked to GABAARs.
An important class of molecules involved in synapse formation, maturation and stabilization comprizes the cell adhesion molecules of the neuroligin (NLs) family3. These post-synaptic proteins functionally coordinate pre and post-synaptic rearrangements by binding, via their extracellular domain, the presynaptically localized neurexins (NRXs) and via specific intracellular motifs, synapse-specific scaffolding molecules4, 5, 6. Neuroligin2 (NL2) isoform is the only known adhesion molecule constitutively present at GABAergic PSDs7, where it drives the recruitment of inhibitory neurotransmitter receptors as well as the scaffolding molecule gephyrin6. Gephyrin, initially identified as a constituent of purified glycine receptor preparations (GlyR)8, 9, was soon recognized a key player in α2 and γ2 subunit-containing GABAARs clustering10, 11 and to be a central component of the GABAergic (and glycinergic) PSD8, 12. On the basis of its auto-oligomerization properties, gephyrin builds a bidimensional lattice underneath the synaptic membrane, which exposes a high number of binding sites to accumulate GlyR and GABAARs in front of the presynaptic releasing sites13, 14, 15, 16, 17.
NL2 interacts with gephyrin through a conserved stretch of amino acid residues highly conserved among all family members6. Site-directed mutagenesis within this binding module identified a specific tyrosine residue (Y770A) whose alanine substitution impairs NL2 ability to recruit recombinant and endogenous gephyrin to post-synaptic sites6. Notably, the corresponding tyrosine residue on NL1, the isoform enriched at excitatory synapses, was found to be phosphorylated in vivo, preventing NL1–gephyrin interaction while favouring PSD95 recruitment at excitatory synapses18. Altogether, these findings point to the existence of intracellular signalling mechanisms able to modulate NL-scaffolding protein interactions by modifying specifically NL properties, leading to alteration in excitatory and inhibitory synaptic transmission.
In the present study, we have investigated whether post-phosphorylation prolyl-isomerization may affect GABAergic transmission in a similar manner. This signalling cascade targets serine and threonine residues preceding a proline residue to promote conformational changes on its substrate19. This effect is achieved by a unique enzyme, peptidyl-prolyl isomerase Pin1, whose catalytic activity facilitates the cis–trans isomerization of the peptide bond20, 21. Notably, Pin1 was found to interact with gephyrin and to alter its overall conformation, thus enhancing its ability to bind the GlyR22.
Here, we provide evidence that endogenous NL2 can be phosphorylated at its unique Pin1 consensus motif thus rendering it able to physically recruit the phospho-specific effector Pin1. We show that post-phosphorylation prolyl-isomerization can regulate NL2’s ability to complex with gephyrin. Specifically, Pin1-mediated propyl-isomerization of phosphorylated serine 714 negatively modulates NL2–gephyrin complex formation, down-regulating GABAergic synaptic transmission.
Endogenous NL2 undergoes proline-directed phosphorylation
The cytoplasmic domain (CD) of NL2 possesses a unique consensus motif for proline-directed phosphorylation, S714-P, located 15 amino acids apart from the transmembrane domain (Fig. 1a). To assess whether this site can undergo phosphorylation in vivo we used the mitotic phosphoprotein monoclonal 2 (MPM2) antibody that specifically recognizes phosphorylated S/T-P motifs (Davis et al.23). Endogenous NL2 was therefore immunoprecipitated from mouse brain homogenates using an affinity-purified polyclonal antibody raised against its CD or normal mouse IgG as negative control. Western blotting using the MPM2 antibody revealed a band at around 120 kDa that corresponds to the upper band of the doublet recognized by the NL2 antibody in parallel immunoprecipitation experiments (Fig. 1b), suggesting that at least a fraction of NL2 can be phosphorylated at its unique Pin1 consensus motif. To demonstrate that phosphorylation at serine 714 is the event responsible for NL2 detection by the MPM2 antibody, we generated the phospho-defective point mutant NL2HA-S714A. This mutation was introduced into a NL2HA hampered in gephyrin binding (NL2HA-S714A-Δgephyrin-binding domain, GBD) (see Supplementary Fig. 1), to exclude the possibility that the MPM2 antibody would immune-react with phosphorylated Pin1 consensus motifs on endogenous gephyrin, which is, at the same time, a Pin1 target22 and an interacting partner of NL2 (ref. 6). Under these conditions, the MPM2 antibody efficiently immunoprecipitated only NL2HA-ΔGBD but not the corresponding point mutant, as indicated by the anti-HA immunoblot (Fig. 1c), thus demonstrating that S714 can be found phosphorylated on NL2.
The essential feature of proline-directed phosphorylation as a signalling mechanism relies on the ability of phosphorylated S/T-P motifs to recruit the prolyl isomerase Pin1 (refs 19, 24). To test whether this unique phospho-epitope is able to recruit the effector molecule of the signalling cascade, we performed co-immunoprecipitation experiments from Pin1+/+ and Pin1−/− brain lysates. This approach unveiled that Pin1 can be detected in NL2, but not in control, immunoprecipitates or in the absence of Pin1 expression (Fig. 1d). To exclude the possibility that Pin1 co-precipitated by NL2 is bound to endogenous gephyrin, these assays were performed on co-expression of NL2HA-ΔGBD and Pin1-FLAG in HEK293 cells. Cell lysates were immunoprecipitated with the anti-FLAG antibody and bound protein complexes analysed by western blotting using anti-HA and anti-FLAG antibodies for NL2 and Pin1 detection, respectively. As shown in Fig. 1e, while NL2HA-ΔGBD was still able to be immunoprecipitated from cells expressing Pin1-FLAG, S714 to alanine mutagenesis completely abolished such interaction, indicating that S714 represents a newly identified Pin1 target.
Pin1 modulates gephyrin–NL2 interaction
The observation that two fundamental components of the GABAergic PSD are both targets of proline-directed phosphorylation prompted us to investigate whether such signalling cascade would modulate their interaction. To this end, we initially co-expressed gephyrin-FLAG and NL2HA in HEK293 cells and examined the amount of NL2HA that complex with gephyrin-FLAG at 48 h after treating the cells with the selective and reversible inhibitor of Pin1 isomerase activity PiB (IC50 of approximately 1.5 μM) (ref. 25). As shown in Fig. 2a, even though the anti-FLAG antibody immunoprecipitated comparable amounts of gephyrin-FLAG, a significant increase (64%) in the amount of co-precipitated NL2HA was observed on PiB treatment as compared with mock-treated cells (dimethylsulfoxide, DMSO). Interestingly, a marked increase (140%) was detected on gephyrin-FLAG co-precipitation by NL2HA-S714A as compared with NL2HA, indicating that Pin1 exerts a negative control on NL2–gephyrin complex formation, at least in part, through NL2 prolyl-isomerization (Fig. 2b).
This issue was then investigated using a source of native NL2–gephyrin complexes mouse brain homogenates from both genotypes. For these experiments, endogenous NL2 was immunoprecipitated using a rabbit polyclonal anti-NL2 antibody and the co-precipitated gephyrin fraction was visualized by the monoclonal 3B11 antibody (Fig. 2c). In the absence of Pin1 expression, the amount of gephyrin co-precipitated by NL2 was increased by 40% as compared with Pin1 expressing neurons. This approach was also applied on hippocampal tissues isolated from both mouse genotypes. Here, the enrichment of gephyrin co-precipitated by NL2 in the absence of Pin1 expression was even more dramatic as compared with the amount detected from whole brain (130% increase; Fig. 2d), suggesting a strong impact of such signalling pathway on GABAergic synapses of the hippocampus.
Characterization of gephyrin Pin1 sites S270-P and S319-P
The scaffolding molecule gephyrin possesses 10 putative Pin1 consensus motifs, the majority of them being concentrated in the central region (C-domain)26. To determine whether specific Pin1 sites may contribute to enhance NL2/gephyrin complex formation, we decided to focus on those located close to, or within, the NL2 binding site on gephyrin. A previous yeast two-hybrid screening identified a large portion of gephyrin encompassing the E-domain and part of the C-domain as the region involved in NL2 interaction6. We re-examine this issue by generating eGFP-tagged gephyrin truncated version to be tested in GST-NL2-CD pulldown assays. HEK293 cells transfected with different eGFP-gephyrin variants were incubated with GST-NL2-CD loaded beads or with GST alone as negative controls. As shown in Fig. 3a, while gephyrin 310–736 was recruited even better than the wild-type (WT) version, the mutants gephyrin 326–736 and gephyrin 1–310 (gephyrin GC) displayed a reduced binding activity as compared to both gephyrin full-length (FL) and the truncated version 310–736 (Fig. 3a). Since the two E-domain gephyrin versions, showing such a striking difference in the binding affinity, differ only for a short stretch of amino acids, we generated the deletion mutant removing, from the FL protein, only the residues contained in this region but belonging to the E-domain itself (gephyrinΔ319–329) and assayed it for NL2 binding. Interestingly, the lack of this short sequence almost completely abolished the interaction of gephyrin with NL2 (Fig. 3b), indicating that epitope(s) contained in the C-domain together with this minimal binding module are involved in gephyrin recruitment.
On the basis of these results, two Pin1 consensus sites were further characterized, namely S319-P, located at the edge of the minimal binding module, and S270-P, positioned in its proximity, still contained, in the C-domain participating in NL2 binding. To this end, we introduced point mutations in eGFP-gephyrin to create S319A and S270A mutants and tested them for their ability to interact with NL2HA. As judged by co-immunoprecipitation experiments, no significant differences were observed in binding capacity of the mutants as compared with gephyrin WT (Fig. 3c). These constructs were also overexpressed in cultured hippocampal neurons to analyse and quantify their impact on endogenous NL2 distribution using immunofluorescence staining and confocal microscopy. As previously reported, neurons expressing the S270A mutants had an increased number, unchanged in size, of gephyrin clusters compared with eGFP-gephyrin WT27 (18.9±1.7 per 20 μm dendritic segment versus 6.5±0.6, P=0.00015). The expression of the S319A construct produced a dramatic decrease in cluster density associated with a diffuse cytoplasmic staining. This latter effect seems to correlate with the intrinsic instability of the mutant protein that undergoes a high rate of degradation on neuronal expression (data not shown), hampering its further characterization. Clusters formed by gephyrin S270A co-localized with NL2 at the same extent as the WT protein (around 78%; Fig. 3d,e). The fraction of NL2 clusters co-localizing with S270A mutant as well as their synaptic localization were increased as compared with gephyrin WT but their intensity values (calculated by normalizing cluster fluorescence intensity to cluster area and expressed in a.u.: 119 a.u.±15.2 versus 102±6.3) were unchanged (Fig. 3e). These data indicate that the increase in NL2/gephyrin S270 interaction observed by immunoprecipitation is simply due to the augmented S270A cluster density and not to an enhance affinity of the mutant for NL2.
Pin1 selectively controls NL2 synaptic enrichment
Pin1 has emerged as a negative regulator of gephyrin–NL2 interaction. Since these protein complexes are mainly localized at the plasma membrane, we tested whether Pin1 affects the amount of NL2 transported to, or maintained at, the neuronal plasma membrane. To this end, cultured hippocampal neurons derived from Pin1+/+ and Pin1−/− mice were subjected to surface biotinylation assay. Cell surface proteins were treated with the membrane-impermeant sulfo-NHS-biotin reagent, then isolated by binding to Streptavidin beads and probed with anti-NL2 antibody. To check for unspecific protein binding during surface biotinylation experiments, hippocampal neurons not labelled with biotin were processed with biotinylated samples. Western blot detecting the intracellular glycophosphatidylinositol-anchored protein Flotilin1 was included to ensure that similar amount of associated membrane proteins, biotinylated or not, where incubated with Streptavidin beads. No major differences on the total content of membrane localized NL2 were observed between Pin1+/+ and Pin1−/− (Fig. 4a).
These results allow excluding the involvement of Pin1 in NL2 transport and/or turnover at the plasma membrane. Surface biotinylation represents an experimental approach that cannot provide an accurate analysis of protein distributions among different membrane domains. Since NL2 is enriched at GABAergic synapses, but is also distributed on extrasynaptic sites28, with this approach differences in NL2 partitioning between these two compartments might have been missed.
To this aim, immunocytochemical experiments were performed in dissociated Pin1+/+ and Pin1−/− hippocampal neurons co-labelled for NL2, gephyrin and VGAT, a specific marker of GABAergic innervations29 (Fig. 4b). In the absence of Pin1 expression a significant increase in NL2 cluster size (2.4 μm2±0.2 versus 1.7 μm2±0.2, P=0.00044) and intensity (92 a.u.±4.0 versus 58 a.u.±2, P<0.00048) was observed as compared with WT neurons, while no major changes in NL2 cluster density were detected (Fig. 4c). The fraction of NL2-positive clusters co-localized with endogenous gephyrin puncta was also enhanced in Pin1−/− cells (80±3.0% versus 60±5%, P=0.00013) and found enriched at post-synaptic sites, as demonstrated by the higher percentage of NL2/gephyrin co-stained puncta overlapping with the presynaptic marker VGAT (48±4% versus 33±4%, P=0.0008; Fig. 4d). Gephyrin puncta appeared slightly, but significantly, increased in size while their density and intensity values were unchanged as compared with Pin1+/+ (Fig. 4e). These observations suggest that the absence of Pin1 promotes the formation and/or stabilization of NL2/gephyrin complexes at GABAergic post-synaptic sites.
NL2/gephyrin complex modulates synaptic abundance of GABAARs
The recruitment of GABAARs at synaptic sites is functionally coupled to NLs expression levels as well as to the gephyrin scaffold6. To assess whether the enhanced NL2/gephyrin complex formation detected at GABAergic synapses similarly affects the distribution of synaptic γ2 subunit-containing GABAARs, we performed a quantitative evaluation of the γ2 subunit present in synaptosome suspensions isolated from the hippocampus of Pin1+/+ and Pin1−/− mice. Quantitative immunoblot analysis was also extended to NL2 and gephyrin to further verify their synaptic enrichment. As shown in Fig. 5a, the amount of all three markers investigated was significant increased in Pin1−/− mice as compared with Pin1+/+. The synaptic enrichment (synaptic fraction versus homogenate) was 35±5% for the NL2, 30±6% for the γ2 subunit and 20±4% for gephyrin.
We also examined the number of puncta labelled for gephyrin and γ2 subunit-specific antibodies, as well as their levels of colocalization with the presynaptic marker VGAT, in the CA1 region of the hippocampus of both genotypes. The staining pattern of gephyrin in Pin1−/− demonstrated a slight increase in the number of clusters both in the stratum oriens (SO) and stratum radiatum (SR) as compared with Pin1+/+ (SO 16±3 clusters per 100 μm2 and SR 28±3 clusters per 100 μm2 versus SO 10±2 clusters per 100 μm2 and SR 19±3 clusters per 100 μm2; P<0.05; Fig. 5b,c). This increase was paralleled by a small increase (around 6–8%) in gephyrin puncta co-localized with presynaptic VGAT (SO 30±2% and SR 39±1.4% versus SO 24±2% and SR 31±2%; P<0.05; Fig. 5b,c). The average cluster size and intensity were similar in both genotypes (3.6 μm2±0.2 versus 3.5 μm2±0.3 and 61±7 versus 65±4 a.u. for cluster size and intensity in Pin1−/− versus Pin1+/+, respectively).
The γ2 subunit staining pattern exhibited a similar cluster density in the two strata analysed in both genotypes (SO 8±2 and SR 18±2 versus SO 8±1 and SR 17±1.2; P>0.05; Fig. 5d,e). A small, although significant, increase in their intensity was evident (120±3 RFU versus 106±2 RFU in Pin1−/− versus Pin1+/+; P<0.05) but they were similar in size (4.3 μm2±0.5 versus 3.7 μm2±0.5). VGAT colocalization was increased by 10–15% in tissue from knockout animals (SO 38.9±2.7% and SR 52±3% versus SO 29±2% and SR 36±3%; P<0.05; Fig. 5d,e). The changes in gephyrin and γ2 subunit synaptic fraction are not due to an increase in synapses numbers, the density of inhibitory terminals being unaltered between the two genotypes, as assessed by quantification of VGAT immunolabeling (SO 14±2% and SR 22±3% versus SO 13±2% and SR 21±3%; P>0.05).
Altogether, these data indicate that the enhanced interaction between gephyrin and NL2 observed in the absence of Pin1 is associated with a concomitant increase in the synaptic recruitment of γ2 subunit-containing GABAARs.
Pin1 signalling affects the number of synaptic GABAARs
To functionally explore whether the enrichment of γ2 subunit-containing GABAARs in Pin1−/− mice affects GABAergic transmission, whole-cell recordings in voltage clamp configuration were performed from CA1 principal cells in hippocampal slices obtained from Pin1+/+ and Pin1−/− mice at postnatal (P) day P10–P13. These neurons presented similar resting membrane potential (Vrest) and input resistance (Rin) values (data not shown), thus indicating that Pin1 does not affect the passive membrane properties of principal cells. Spontaneous GABAA-mediated inhibitory post-synaptic currents (sIPSCs) were then recorded from both genotypes in the presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX; 20 μM) to block AMPA-mediated excitatory post-synaptic currents (sEPSCs). As shown in Fig. 6a, recordings from Pin1−/− mice exhibited sIPSCs of higher amplitude values compared with control littermates (106±12 pA versus 62±8 pA; P<0.05), in the absence of any significant change in frequency (4.2±0.5 Hz versus 3.6±0.6 Hz; P>0.05; Fig. 6b). The amplitude distribution histogram of sIPSCs recorded in Pin1−/− unveiled a clear peak at ~200 pA (Fig. 6c). The observed effects were selective for sIPSCs since no significant differences in amplitude (22±2 pAin Pin1−/− mice and 27±4 pA in Pin1+/+; n=6 for both genotypes; P>0.05) or frequency (1.7±0.3 Hz in Pin1−/− mice and 1.3±0.4 Hz in Pin1+/+ mice; P>0.05) of sEPSCs (recorded in the presence of picrotoxin, PTX, 100 μM) were detected between the two genotypes (Supplementary Fig. 2a,b).
Spontaneous inhibitory events from hippocampal neurons in culture overexpressing the NL2HA-S714A mutation exhibited, compared with NL2HA-transfected cells, a significant increase in amplitude (but not frequency), which in part mimicked the phenotype observe in Pin1−/− mice, suggesting that the interaction of Pin1 with NL2 is critical for this effect (Fig. 7a). As shown in the cumulative amplitude plot (Fig. 7b), the curve obtained from NL2HA-S714A transfected cells was shifted to the right as compared with cells expressing NL2HA (P<0.05).
The selective increase in amplitude of sIPSCs detected in Pin1−/− mice suggest a post-synaptic site of action. This may involve an increase in the number of active GABAARs or changes in single-receptor channel conductance. To distinguish between these two possibilities, peak-scaled non-stationary fluctuations analysis of sIPSCs was performed only on stable recordings with no time-dependent changes in either peak amplitude, 10–90% rise time and decay time (Fig. 8a) (electrotonic filtering was excluded on the basis of no correlation between 10–90% rise time and decay time30). Plotting the mean current amplitude versus variance and fitting individual points with the parabolic equation (equation (2) in the methods; Fig. 8b), allowed estimating single-channel conductance and the number of channels open at the peak of spontaneous IPSCs. The single-channel conductance was calculated according to equation (3), assuming a reversal potential for chloride equal to 0. Interestingly, while the values of single-channel conductance were similar in both genotypes (Fig. 8c), the average number of active channels open at the peak of sIPSCs (Np) was significantly increased in Pin1−/− mice compared with controls (53±11 versus 26±5; P=0.03; Fig. 8c).
To further evaluate the possibility that higher amplitude inhibitory events recorded in Pin1−/− mice may originate from GABAARs containing different subunits, we measured in both genotypes the decay time constants of small and large amplitude events. Spontaneous IPSCs were plotted against their decay half-widths and arbitrarily divided in two main classes whose amplitude was <or>150 pA (Fig. 9a, in green and blue, respectively). Notably, larger amplitude events (>150 pA) prevailed in Pin1−/− mice. No differences in decay of sIPSCs <or>150 pA were observed between Pin1+/+ and Pin1−/− mice, thus excluding the involvement of multiple receptor subtypes with different kinetics (the 90–10% decay (τ) of sIPSCs <150 pA was 9±1 ms in Pin−/− mice and 11±2 ms in Pin1+/+; P>0.05; τ of sIPSCs >150 pA was 11±2 ms in Pin−/− mice and 10±2 ms in Pin1+/+; P>0.05. The 90–10% decay time (τ90–10%) of all sIPSCs was 11±2 ms and 10±2 ms in Pin1+/+ and Pin1−/− mice, respectively; Fig. 9b,c, P>0.05). These data altogether suggest that the observed increase in amplitude of sIPSCs in Pin1−/− mice is exclusively due a genuine increase in number of GABAARs composed of the same subunits.
GABA release and tonic inhibition are unaltered in Pin1−/−
In a previous study, we demonstrated that the functional knockdown of NL2 was accompanied by a reduction in the probability of GABA release31, thus underlying the role of NLs as retrograde regulators of presynaptic function. Therefore, we evaluated here whether Pin1-dependent modulation of NL2–gephyrin interaction could also affect GABA release from presynaptic nerve terminals. To this end, we used 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a low affinity competitive GABAAR antagonist32. This approach allowed to compare differences in presynaptic GABA transients between Pin1+/+ and Pin1−/− mice. Similar reduction of sIPSCs amplitude in both genotypes (51±6% versus 54±8%, P>0.05, Supplementary Fig. 3a,b) was detected on bath application of TPMPA (200 μM), thus excluding a transsynaptic action of Pin1 on GABA release.
Part of GABA released during synaptic activity may escape the cleft and invade the extracellular space to activate extrasynaptic high affinity GABAARs. This feature generates a persistent GABAA-mediated conductance33 that is involved in a number of physiological processes34. To determine whether Pin1 signalling affects extrasynaptic GABAARs, we analysed the tonic GABAA-mediated conductance in both Pin1+/+ and Pin1−/−mice. The tonic conductance was assessed by the shift of the holding current induced by application of the GABAAR channel blocker PTX (100 μM) (Supplementary Fig. 4a). This drug caused a similar shift in holding current in Pin1−/− and Pin1+/+ mice (Supplementary Fig. 4b,c), indicating that extrasynaptic GABAA receptors are not influenced by Pin1-mediated signalling.
The present study shows that NL2 is a newly identified substrate of proline-directed phosphorylation. This post-translational modification, acting on its unique Pin1 consensus motif localized within the CD (S714-P), modulates the amount of NL2–gephyrin complexes at synaptic sites. This modulation impacts on GABAergic transmission, by selectively affecting the total number of synaptic GABAARs. On the basis of these findings, post-phosphorylation prolyl-isomerization can play a crucial role in remodelling the GABAergic PSD to sustain plasticity processes.
Protein phosphorylation on serine and threonine residues preceding a proline, the so-called proline-directed phosphorylation, has emerged as a mechanism regulating signalling events through conformational changes that are catalysed by the phospho-dependent recruitment of the peptidyl-prolyl isomerase Pin1. While the different roles of Pin1 in dividing cells have long been established and characterized19, its function in post-mitotic neurons in general and at synapses in particular is still poorly understood. In a previous study, we identified gephyrin, the main scaffolding protein of inhibitory PSD, as a new target of post-phosphorylation prolyl-isomerization22.
Here, by inspecting the protein sequence of NL CDs, we identified S/T-P motifs that may provide Pin1 binding sites if phosphorylated in vivo. In particular, NL2 presents a unique Pin1 consensus site in its cytoplasmic region, S714-P, which is located 15 amino acids apart from the transmembrane domain. Even though this proximity to the plasma membrane raises doubts about its accessibility by a proline-directed kinase, several lines of evidence suggest that endogenous NL2 can undergo proline-directed phosphorylation. First, this isoform was recognized by the MPM2 antibody on NL2 immunoprecipitation from mouse brain homogenates. Second, MPM2-mediated NL2 immunoprecipitation was still maintained on removal of the NL2–gephyrin-binding domain, excluding the possibility of an indirect recognition mediated by endogenous gephyrin. Third, such detection was completely lost on NL2HA-S714A mutagenesis. This phosphorylation event is then able to directly recruit the effector molecule of the signalling cascade Pin1, as shown by co-immunoprecipitation experiments with endogenous neuronal proteins. Also in this case, Pin1 binding to NL2 was still maintained on the removal of the GBD, while it was completely abolished by mutating S714 to alanine, thus suggesting that the prolyl isomerase can be directly recruited by the unique NL2 Pin1 consensus motif in a phosphorylation-dependent manner. These results altogether indicate that NL2 represents a newly identified substrate for proline-directed signalling cascade in vivo.
Our biochemical data demonstrate that NL2–gephyrin interaction is negatively regulated by proline-directed phosphorylation. Co-immunoprecipitation experiments on recombinantly expressed gephyrin-FLAG and NL2HA unveiled an enhanced complex formation on pharmacological inhibition of Pin1 catalytic activity. Similarly, endogenous NL2/gephyrin complexes pulled down from whole brain or hippocampal tissues of Pin1−/− animals were significantly augmented as compared with the corresponding WT tissues. These biochemical findings were also validated by immunocytochemistry performed on cultured hippocampal neurons, where we could detect a high number of clusters co-labelled for NL2 and gephryin as well as their increased apposition to presynaptic GABAergic inputs in the absence of Pin1 expression. Interestingly, the NL2 point mutant unable to undergo prolyl-isomerization was capable to recruit gephyrin even more efficiently as compared with the WT form, whereas gephyrin mutagenesis at two putative Pin1 consensus motifs, S270A and S319A, located within, or close to, the minimal NL2 binding domain, was completely ineffective. The fact that this post-translational modification seems to control the strength of NL2 association with gephyrin by acting mainly on NL2, and not vice versa, further reinforces the emerging idea that cell adhesion molecules are key determinant in regulating synapse function. In a recent study by Giannone et al.18, it has been demonstrated that the level of NL1 phosphorylation at a specific tyrosine residue located within the GBD dictates the strength of NL1/gephyrin interaction. In other words, NL1, the isoform enriched at excitatory synapses and therefore mostly associated with PSD95, can potentially recruit gephyrin as well as NL2, but its phosphorylation, promoted by neurexin–adhesion signalling, precludes such interaction while favouring PSD95 binding. Our experimental data indicate that proline-directed phosphorylation is acting similarly to tyrosine phosphorylation signalling. Since NL2 S714 is not positioned within the GBD, but is located just 50 amino acid upstream, it is reasonable to believe that Pin1-driven conformational changes, by affecting the overall folding of the CD, will induce gephyrin release (Fig. 10a). Alternatively, these conformational changes may promote NL2 tyrosine phosphorylation, an event shown to impede NLs/gephyrin interaction18 (Fig. 10b). Interestingly, tyrosine to alanine mutagenesis on NL2 was shown to completely abolish recombinant gephyrin recruitment by the mutant protein or to strongly reduce its interaction with endogenous gephyrin6. Whether NL2 phosphorylation occurs at tyrosine 770 and whether this event is able to hamper gephyrin binding is still unknown.
The other partner of the complex is represented by gephyrin, a recognized target of Pin1 (ref. 22). Gephyrin contains 10 consensus motifs mostly concentrated in its C-domain, and all of them found to be phosphorylated in vivo35, 36. This region of the protein is positioned between the amino-terminal G- and carboxyl-terminal E-domains, which are directly involved in gephyrin multimerization. Conformational changes induced by phosphorylation, possibly followed by prolyl-isomerization, are expected to alter the conformation of the gephyrin C-domain and in turn, regulate specific functional properties of gephyrin, in particular its binding to interacting proteins, including possibly NL2. However, the complexity of the system under investigation makes it very difficult to determine whether and how a specific phosphorylation event can contribute, directly or indirectly, to enhance gephyrin association to NL2. Nevertheless, it should be emphasized that gephyrin is robustly phosphorylated at several residues in vivo, thus suggesting that a specific pattern of phosphorylation, rather than a single post-translational modification, is functionally determinant. In contrast, NL2 possesses a unique target for prolyl-isomerization suggesting that it could represent the master switch of the signalling cascade.
Our electrophysiological experiments clearly demonstrate that deletion of Pin1 specifically affects GABAergic transmission, causing a dramatic increase in amplitude, but not in frequency, of sIPSCs due to an increase in the number of GABAARs at post-synaptic sites. Notably, such enhancement was detected on neuronal overexpression of the NL2 mutant unable to undergo prolyl-isomerization, suggesting a functional link between the signalling cascade strengthening NL2/gephyrin interaction and the increased synaptic recruitment of GABAARs. There is a large body of evidence underlying the key role played by NL2 in promoting clustering and/or stabilization of GABAARs at post-synaptic sites. By employing a heterologous expression system, it was shown that GABAARs are able to co-aggregate with NL2 and only the presence of this isoform can induce strong GABAergic presynaptic differentiation from co-cultured neurons and promote the establishment of fully functional hemi-synapses37. In NL2-deficient mice, the number of functional GABAARs detected in the retina was shown to be drastically reduced38. Furthermore, targeting of GABAARs and gephyrin scaffold appeared severely compromised in the pyramidal cell layer of the CA1 region of the hippocampus, a morphological phenotype accompanied by a strong deficit in synaptic inhibition6.
The increased recruitment of synaptic GABAA receptors in Pin1−/− mice may simply depend on the enhanced gephyrin targeting at synaptic sites. More scaffold deposition should offer a high number of binding sites available for the transient immobilization of GABAARs at inhibitory synapses. In addition, or alternatively, we cannot exclude the possibility that the extracellular domain of NL2 could also participate in GABAARs receptor recruitment. The unique S714-P consensus motif, located very close to the NL2 transmembrane domain, could influence the folding of the extracellular domain of NL2, rendering it incapable to interact in cis with GABAAR subunits. This type of mechanism has been shown to operate at excitatory synapses, where the abundance of NMDARs is controlled by the interaction occurring between the GluN1 subunit with NL1-specific sequences located in its extracellular domain39.
In conclusion, our findings unveil the existence of a new signalling pathway operating at GABAergic synapses to alter the efficacy of GABAergic transmission by modulating NL2/gephyrin interaction. Although a comprehensive understanding of the molecular mechanisms underlying the action of Pin1 on NL2/gephyrin interaction is still lacking, we believe that our study further emphasizes the key role played by NL2 in organizing and stabilizing GABAergic synapses.
How to cite this article: Antonelli, R. et al. Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction. Nat. Commun. 5:5066 doi: 10.1038/ncomms6066 (2014).