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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2017 January 27; 292(4): 1160–1177.
Published online 2016 December 9. doi:  10.1074/jbc.M116.771592
PMCID: PMC5270463

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses*An external file that holds a picture, illustration, etc.
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Abstract

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

Keywords: GABA receptor, inositol phospholipid, phosphatidylinositol phosphatase, synapse, vesicles

Introduction

Phosphoinositides, phosphorylated derivatives of phosphatidylinositol, are critical regulators of intracellular signaling, membrane traffic, and cell compartmentalization (1,3). The functional roles of phosphoinositide metabolism have been studied in great detail at the presynaptic terminal, where phosphoinositide turnover is of critical importance for synaptic vesicle (SV)3 recycling and synapse function (4). Little, however, is known about specific roles of phosphoinositides at postsynapses.

Core components of most inhibitory GABAergic postsynapses are GABAA receptors (GABAARs), the cell adhesion protein neuroligin 2 (NL2), the scaffolding protein gephyrin, and the guanine nucleotide exchange factor collybistin (Cb) (5, 6). During the formation of many GABAergic synapses, most notably those at neuronal somata, NL2 is thought to activate Cb, which promotes Cb membrane association and the subsequent recruitment of gephyrin and GABAARs (7,9). In vitro binding studies indicate that the pleckstrin homology (PH) domain of Cb specifically binds phosphatidylinositol 3-phosphate (PI3P) (9,13), and this interaction is thought to be essential for the anchoring of NL2-gephyrin-Cb complexes at the postsynaptic plasma membrane of synapses that depend on Cb (7, 9). The notion that PI3P binding by the PH domain is required for proper Cb function is supported by the observation that deletion of the PH domain (14), or the substitution therein of two arginine residues that are essential for PI3P binding (9, 11), causes a marked reduction in the density of postsynaptic gephyrin clusters in hippocampal neurons. Furthermore, an emerging feature of Cb missense mutations in patients with epilepsy and intellectual disability appears to be impaired PI3P binding (13, 15).

PI3P is enriched on early/sorting endosomes (16) and has important roles in membrane trafficking (17, 18). In addition, some studies implicate class II phosphatidylinositol 3-kinase (PI3K) isoforms (PI3K-C2α/β/γ) in the receptor-triggered accumulation of PI3P at the plasma membrane of various cell types (19, 20); however, unequivocal evidence for the presence of PI3P at the neuronal plasma membrane is presently lacking. Thus, it remains unclear whether and how PI3P might contribute to the Cb-mediated anchoring of gephyrin scaffolds beneath the postsynaptic membrane and whether the affinity and specificity of the Cb/PI3P interaction as determined in vitro correlate with a defined role of PI3P in the formation of inhibitory postsynapses.

In this study, we investigated the role of PI3P at developing GABAergic postsynapses in cultured hippocampal neurons by using a membrane-permeant PI3P derivative (21), time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results indicate that a PI3P pool associated with early/sorting endosomes is important for the formation of Cb-dependent inhibitory postsynapses. We thereby provide the first demonstration that PI3P is a critical regulator of postsynaptic gephyrin and GABAAR clustering and that it is involved in the regulation of inhibitory postsynaptic strength in neurons.

Results

PI3P Promotes Postsynaptic GFP Gephyring Clustering

To assess the specific role of PI3P in the Cb-dependent clustering of gephyrin at inhibitory postsynapses, we focused on hippocampal neurons in culture, which receive glutamatergic and GABAergic synaptic inputs and show defects in GABAergic postsynaptic composition and function upon Cb deletion (22). We first aimed to experimentally increase the intracellular PI3P concentration in cultured neurons by applying a membrane-permeable PI3P-acetoxymethyl (AM) ester derivative, its photoactivatable “caged” coumarin-PI3P-AM variant, and, as a negative control, its regioisomer PI4P-AM, all of which had previously been validated in HeLa cells (21) and were recently used for studying vesicular trafficking in muscle cells of X-linked centronuclear myopathy patients (23). Previous studies indicate a distribution of both coumarin-caged and -uncaged PIP-AM probes into most cellular membranes (21, 24). However, despite their wide subcellular distribution, these probes demonstrated high structural specificity, presumably through their specific interaction with endogenous PIP-binding proteins. For example, it has been shown that PI3P-AM induces early endosome fusion in living cells and that the resulting fused endosomes were positive for the endogenous early endosome antigen 1 (EEA1) (21), a known marker of early endosomes that interacts specifically with PI3P via its FYVE finger domain (25). In contrast, compounds of identical chemical composition as PI3P-AM but structurally slightly different, such as PI4P-AM and the enantiomer of PI3P-AM, were unable to induce early endosome fusion (21). In cultured hippocampal neurons, the AM-modified phosphoinositide variants were efficiently accumulated, as demonstrated by adding 50 μm coumarin-PI3P-AM to the culture medium. Neurons incubated with coumarin-PI3P-AM for ~10 min displayed diffuse coumarin fluorescence (Fig. 1A), indicating efficient uptake and widespread distribution of the PI3P derivative into intracellular membranes.

FIGURE 1.
PI3P-AM induces formation of GFP-gephyrin clusters in dissociated hippocampal neurons. A, images show cultured hippocampal neurons transfected from DIV 9–10 with GFP-gephyrin and treated with either DMSO-only or 50 μm caged PI3P-AM, for ...

For quantitative experiments, we transfected cultured hippocampal neurons at DIV 9 with GFP-gephyrin, incubated the transfected neurons 1 day later for 2 h with 50 μm PI3P-AM (or PI4P-AM) or with the vehicle dimethyl sulfoxide (DMSO) only, and analyzed the resulting effects on GFP-gephyrin distribution (Fig. 1, B and C). This protocol allowed us to visualize newly synthesized intracellular gephyrin that is en route to the plasma membrane at early stages of inhibitory postsynapse formation (from DIV 9 to DIV 10). Under these conditions, only few dendritic GFP-gephyrin clusters were formed in the transfected neurons prior to treatment, and most GFP-gephyrin was diffusely distributed in the cells and accumulated in larger somatic structures (Fig. 1B, left). The subsequent addition of PI3P-AM for 2 h (Fig. 1, B, left and C), but not of PI4P-AM or DMSO only (Fig. 1C), led to an increase in the number of dendritic GFP-gephyrin clusters, to a concomitant reduction of the diffuse cytoplasmic GFP-gephyrin fluorescence, and to an apparent redistribution of GFP-gephyrin from the large cytoplasmic structures to numerous smaller clusters (Fig. 1B, left). Three-dimensional reconstruction of confocal image stacks and examination of internal structures in the somata of transfected neurons disclosed an enhanced accumulation of GFP-gephyrin fluorescence within both somatic (Fig. 1B, YZ plane views) and perisomatic clusters. Quantification of the total numbers of GFP-gephyrin clusters in identified neurons before and after the 2-h treatment indicated an increase by 76.6% in the neurons treated with PI3P-AM but not in those treated with DMSO or PI4P-AM (Fig. 1C; DMSO, 111.8 ± 7.2%, n = 10; PI3P-AM, 176.6 ± 15.6%, n = 10; PI4P-AM, 97.9 ± 1.6%, n = 10). Qualitatively similar effects were also observed with the coumarin-PI3P-AM derivative upon UV-light photoactivation and a 1-h incubation (Fig. 1A).

To test for PI3P effects on GFP-gephyrin recruitment to developing dendritic and perisomatic inhibitory postsynapses, we performed confocal live-imaging of GFP-gephyrin-transfected neurons prior to compound treatment (Fig. 2, A–C, left), live-imaging of the same neurons upon a 2-h treatment with DMSO, PI3P-AM, or PI4P-AM, respectively (Fig. 2, A–C, center), and confocal imaging of these neurons after fixation and post hoc staining for the vesicular inhibitory amino acid transporter (VIAAT), a marker of GABAergic presynapses (Fig. 2, A–C, right). This revealed an ~1.5-fold increase in the average fluorescence intensity of postsynaptic GFP-gephyrin clusters apposed to presynaptic VIAAT puncta in PI3P-AM-treated neurons but not in cells treated with DMSO only or PI4P-AM (Fig. 2D; DMSO, 95.9 ± 3.8%, n = 70 postsynaptic clusters; PI3P-AM, 150.4 ± 17.9%, n = 70 postsynaptic clusters; PI4P-AM, 98.9 ± 5.1%, n = 35 postsynaptic clusters). We therefore conclude that membrane-permeant PI3P promotes gephyrin clustering at inhibitory postsynaptic sites. Notably, a considerable fraction of the GFP-gephyrin clusters seen after PI3P-AM treatment were not apposed to VIAAT but appeared intracellularly localized (Fig. 2B, right). Their rounded structures suggest that these clusters represented vesicle-associated GFP-gephyrin assemblies rather than amorphous intracellular aggregates, as seen upon overexpression in heterologous cells (26, 27) or under particular in vivo conditions (28).

FIGURE 2.
PI3P-AM induces postsynaptic clustering of GFP-gephyrin in dissociated hippocampal neurons. A–C, cultures were transfected at DIV 9 with a cDNA encoding GFP-gephyrin. At DIV 10, the medium was replaced by imaging medium (see under “Experimental ...

PI3P Increases the Strength of GABAergic Postsynapses

Previous studies have demonstrated a strong interdependence between gephyrin and major GABAAR subunits in postsynaptic clustering (29, 30). To determine the physiological consequences of the enhanced postsynaptic GFP-gephyrin clustering found after PI3P-AM treatment, we recorded GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and evoked inhibitory postsynaptic currents (eIPSCs) in autaptic cultures of DIV 9–10 GABAergic neurons from striata. In these cultures, in which isolated single neurons form synapses with themselves, pre- and postsynaptic effects of PI3P-AM on GABAergic neurotransmission can be distinguished with relative ease (31). Mean mIPSC amplitudes were significantly (by 83.4%) increased in PI3P-AM-treated neurons (61.6 ± 4.7 pA, n = 30) as compared with cells treated with DMSO only (33.6 ± 2.3 pA, n = 26; Fig. 3, A and B). In contrast, mIPSC frequencies were not significantly different between the two groups (PI3P-AM, 1.3 ± 0.2 Hz, n = 30; DMSO, 1.2 ± 0.4 Hz, n = 26; Fig. 3B). The larger mIPSC amplitudes and the unchanged mIPSC frequencies in PI3P-AM-treated neurons indicate an increased number of functional postsynaptic GABAARs, which is in line with the increase in postsynaptic GFP-gephyrin clustering observed upon PI3P-AM treatment (Fig. 2D). Furthermore, exogenous application of GABA, which activates both synaptic and extrasynaptic GABAARs, led to a significant increase of the mean current amplitude (by 82.4%) in PI3P-AM-treated neurons (1.82 ± 0.24 nA; n = 33) as compared and normalized to cells treated with DMSO only (1.00 ± 0.1 nA; n = 24; Fig. 3C).

FIGURE 3.
Post- and presynaptic effects of PI3P-AM in autaptic GABAergic striatal neurons. A, representative mIPSC traces recorded at a holding potential of −70 mV in the presence of 300 nm TTX from autaptic striatal neurons treated with either PI3P-AM ...

As PI3P is present in endosomes at presynaptic terminals (32), we tested whether evoked neurotransmitter release is affected in PI3P-AM-treated striatal autaptic neurons. Although mean mIPSC amplitudes were strongly increased (Fig. 3, A and B), mean eIPSC amplitudes were reduced (by 53%) in PI3P-AM-treated as compared with DMSO-treated cells (PI3P-AM, 1.8 ± 0.33 nA, n = 30; DMSO, 3.82 ± 0.5 nA, n = 26; Fig. 3D, left and center). Accordingly, the number of SVs released per action potential (AP) (eIPSC charge/mIPSC charge, see under “Experimental Procedures”) was strongly reduced after PI3P-AM treatment (PI3P-AM, 42.7 ± 9.6, n = 21; DMSO, 242.1 ± 58.58, n = 18; Fig. 3D, right). In contrast, postsynaptic responses triggered by hypertonic sucrose solution, which causes the release of the readily releasable pool (RRP) of SVs (33, 34), were similar between both groups (PI3P-AM, 1.62 ± 0.27 nC, n = 30; DMSO, 1.09 ± 0.17 nC, n = 26; Fig. 3, E, left and center). Likewise, the numbers of primed synaptic vesicles (PSVs; RRP charge/mIPSC charge) were not significantly different between PI3P-AM- and DMSO-treated neurons (PI3P-AM, 1245 ± 216, n = 21; DMSO, 969 ± 207, n = 18; Fig. 3E, right). Based on these results, the SV release probability (Pvr) in the PI3P-AM-treated neurons, calculated either by dividing the charge transfer during AP-evoked IPSCs by the RRP charge (Fig. 3F, left) or by dividing the number of SVs released per AP by the number of PSVs (Fig. 3F, right), was strongly reduced in the presence of PI3P-AM-treated as compared with DMSO-treated neurons. Together, our electrophysiological analyses of autaptic GABAergic neurons indicate that mIPSCs and eIPSCs are regulated differentially upon an increase of PI3P in endomembranes, likely due to an increased GABAAR density at the postsynapse and an impairment of SV recycling at the presynaptic terminal.

PI3P Increases the Size and Percentage of Endogenous Gephyrin and GABAARs at Inhibitory Postsynaptic Sites

To study the effects of PI3P-AM during the early stages of inhibitory synapse formation, we immunostained the autaptic striatal neurons for VIAAT as a marker of GABAergic presynapses, and for gephyrin and the α2 subunit of GABAARs, which are co-enriched in GABAergic postsynapses (35). After treating the striatal autaptic cultures at DIV 9 for 2 h with DMSO only (Fig. 4A, left) or 50 μm PI3P-AM (Fig. 4A, right), immunocytochemistry was performed as indicated. At this developmental stage, gephyrin and GABAAR-α2 immunoreactive clusters were distributed at both extrasynaptic and postsynaptic sites, as indicated by their only partial apposition to VIAAT-positive puncta (Fig. 4B). PI3P-AM treatment did not significantly change the total number of dendritic VIAAT, gephyrin, or GABAAR-α2 clusters, as compared with DMSO-only treated neurons (Fig. 4C, left; VIAAT: DMSO, 13.25 ± 1.01 puncta/40 μm, n = 20; PI3P-AM, 10.9 ± 0.63 puncta/40 μm, n = 20; gephyrin: DMSO, 14.3 ± 1.88 clusters/40 μm, n = 20; PI3P-AM, 13.35 ± 1.07 clusters/40 μm, n = 20; GABAAR-α2: DMSO, 9.7 ± 1.06 clusters/40 μm, n = 20; PI3P-AM, 8.8 ± 0.79 clusters/40 μm, n = 20). However, the percentages of synaptic gephyrin and GABAAR-α2 clusters colocalizing with VIAAT immunoreactivity were significantly increased upon PI3P-AM treatment, as compared with DMSO-only treated cells (Fig. 4C, center; synaptic gephyrin: DMSO, 70.71 ± 3.48%, n = 20; PI3P-AM, 83.47 ± 2.37%, n = 20; synaptic GABAAR-α2: DMSO, 61.15 ± 3.16%, n = 20; PI3P-AM, 82.52 ± 2.78%, n = 20). Furthermore, the mean sizes of these synaptic gephyrin and GABAAR-α2 clusters were significantly larger in PI3P-AM-treated neurons, as compared with cells treated with DMSO only (Fig. 4C, right; gephyrin: DMSO, 0.18 μm2 ± 0.014 μm2, n = 20; PI3P-AM, 0.26 μm2 ± 0.021 μm2, n = 20; GABAAR-α2: DMSO, 0.17 μm2 ± 0.01 μm2, n = 20; PI3P-AM, 0.22 μm2 ± 0.016 μm2, n = 20). Together, these immunocytochemical results are consistent with the observed increases in mIPSC mean amplitudes and GABA-induced responses seen in PI3P-AM-treated striatal autaptic neurons (Fig. 3, B and C) and indicate that PI3P-AM increases the densities and sizes of synaptic gephyrin and GABAAR clusters.

FIGURE 4.
PI3P-AM enhances postsynaptic clustering of gephyrin and GABAARs in autaptic GABAergic striatal neurons. A and B, representative images of autaptic striatal neurons treated at DIV 9 for 2 h with either DMSO only (left) or 50 μm PI3P-AM (right ...

PI3P-AM Treatment Substantially Reduces Evoked Excitatory Transmission but Has No Effect on Glutamatergic Postsynapses

To further test whether the presynaptic changes produced by PI3P-AM treatment of autaptic GABAergic neurons contribute to the observed increase of their postsynaptic strength, we recorded mEPSCs and eEPSCs from glutamatergic neurons in autaptic DIV 9–11 hippocampal cultures after a 2-h incubation with either DMSO only or 50 μm PI3P-AM. Notably, here the amplitudes and frequencies of mEPSCs were not significantly different between these treatments (Fig. 5, A and B; mEPSC amplitudes: DMSO, 22.42 pA ± 1.38 pA, n = 15; PI3P-AM, 25.43 pA ± 1.57 pA, n = 21; mEPSC frequencies: DMSO, 1.11 Hz ± 0.24 Hz, n = 15; PI3P-AM, 1.33 ± 0.18, n = 21), indicating that the density of synaptic excitatory glutamate receptors is unchanged in PI3P-AM-treated neurons, as compared with DMSO-only treated cells. Furthermore, exogenous application of glutamate led to similar responses in both groups (Fig. 5C; DMSO normalized, 1.00 nA ± 0.11 nA, n = 19; PI3P-AM, 1.02 nA ± 0.10 nA, n = 24); thus the total number of surface glutamate receptors (synaptic + extrasynaptic) was not altered upon PI3P-AM treatment. In contrast, exogenous application of GABA resulted in a significantly larger (by 62%) mean current amplitude in PI3P-AM-treated neurons (1.62 ± 0.18 nA; n = 24) as compared and normalized to cells treated with DMSO only (1.00 ± 0.07 nA; n = 19; Fig. 3C). Thus, PI3P specifically increases the density of surface GABAARs, but not glutamate receptors, in autaptic hippocampal neurons.

FIGURE 5.
Post- and presynaptic effects of PI3P-AM in autaptic glutamatergic hippocampal neurons. A, representative mEPSC traces recorded at a holding potential of −70 mV in the presence of 300 nm TTX from autaptic hippocampal neurons treated with either ...

In contrast to its lack of an effect on mEPSCs, PI3P-AM treatment abolished eEPSCs recorded from autaptic hippocampal neurons almost completely (Fig. 5E). Notably, 65% of the PI3P-AM-treated cells showed no detectable eEPSCs, whereas the remaining cells (9 of 26 tested) displayed markedly reduced mean eEPSC amplitudes, as compared with DMSO controls (Fig. 5E, center; DMSO, 1.95 nA ± 0.38 nA, n = 20; PI3P-AM, 0.28 nA ± 0.04, n = 9/26). Analysis of the postsynaptic responses triggered by hypertonic sucrose solution indicated that the RRPs of SVs were similar between both groups (Fig. 5F; DMSO, 0.16 ± 0.03 nC, n = 17; PI3P-AM, 0.18 ± 0.02 nC, n = 25). Accordingly, Pvr in the PI3P-AM-treated neurons, calculated by dividing the charge transfer during AP evoked EPSCs by the RRP charge, was strongly reduced as compared with DMSO-treated neurons (Fig. 5G; DMSO, 6.14 ± 0.81%, n = 17; PI3P-AM, 0.54 ± 0.13%, n = 16/25). Together, these data indicate that the impairment of SV exocytosis seen after PI3P-AM treatment is not caused by or associated with changes in the density of glutamate receptors in the postsynaptic neuron. Together, these electrophysiological analyses of both GABAergic and glutamatergic autaptic neurons demonstrate a specific increase of surface and synaptic GABAARs, but not glutamate receptors, upon PI3P-AM treatment.

Phosphatidylinositol 3-Kinases and Phosphatases Involved in the Regulation of PI3P Levels during GABAergic Synapse Formation

Earlier reports have shown that individual PI3K inhibitors differentially affect the PI3P pools in endosomal and plasma membrane compartments, which indicate that multiple different enzymes are involved in the generation of PI3P (36, 37). Current consensus is that PI3K-C3 is responsible for the production of the constitutive endosomal pool of PI3P at early/sorting endosomes (17), whereas class II PI3K isoforms (PI3K-C2α/β/γ) are implicated in both the synthesis of PI(3,4)P2 and the agonist-mediated accumulation of PI3P at the plasma membrane (18,20). Specifically, the α-isoform has been demonstrated to preferentially synthesize PI(3,4)P2 both in vitro and at plasma membrane endocytotic pits in vivo, a site where PI(3,4)P2 is required for membrane constriction prior to endocytic vesicle fission (18, 38). Of the three known class II PI3K isoforms, PI3K-C2α and PI3K-C2β are expressed in the mammalian brain, whereas PI3K-C2γ is not (39, 40).

To determine which PI3K enzymes might be relevant for the generation of the PI3P pool involved in postsynaptic gephyrin clustering, we conducted miRNA-based knockdown experiments targeting PI3K-C3, PI3K-C2α, and PI3K-C2β in dissociated rat hippocampal neurons. Knockdown efficiencies of the different miRNAs were determined by Western blotting of lysates of Rat2 fibroblasts (ATCC CRL-1764; a fibroblast cell line of rat origin) stably expressing the corresponding miRNAs or control miRNAs. This led to the identification of miRNAs, which reduced the expression levels of PI3K-C3, PI3K-C2α, and PI3K-C2β by about 79, 83, and 83%, respectively (Fig. 6, A and B). To focus on postsynaptic defects resulting from down-regulating the PI3P-specific kinases and to minimize confounding effects of parallel presynaptic changes, we performed Ca2PO4 transfections with vectors expressing GFP and a given miRNA at low transfection efficiencies (1–2%) and concentrated our immunocytochemical analysis on the sparse morphologically identifiable pyramidal neurons coexpressing GFP and the respective miRNAs. Cultured rat hippocampal neurons were transfected at DIV 4, and the effects of down-regulating the corresponding kinases on gephyrin clustering were analyzed at DIV 14. We detected a significant reduction by ~33% in the density of dendritic gephyrin immunoreactive puncta in neurons expressing the miRNA directed against PI3K-C3 but not in neurons expressing the miRNAs directed against PI3K-C2α or PI3K-C2β, as compared with neurons transfected with the control miRNA (Fig. 7, A and B; control miRNA, 24.2 ± 1.2 puncta/40-μm dendritic length, n = 21; PI3K-C3 miRNA, 16.1 ± 0.8 puncta/40-μm dendritic length, n = 22; PI3K-C2α miRNA, 22.2 ± 1.1 puncta/40-μm dendritic length, n = 14; PI3K-C2β miRNA, 21.6 ± 1.4 puncta/40-μm dendritic length, n = 14). Notably, expression of the PI3K-C3 miRNA had no effect on the density of dendritic VIAAT-immunoreactive puncta (Fig. 7C; control miRNA, 17.9 ± 0.7 puncta/40-μm dendritic length, n = 13; PI3K-C3 miRNA, 18.0 ± 1.3 puncta/40-μm dendritic length, n = 12), indicating that the number of contacting GABAergic presynapses had not changed. EEA1 is a protein containing a FYVE finger domain that binds to PI3P and targets EEA1 to early/sorting endosomes (25). In agreement with the involvement of PI3K-C3 in generating the PI3P pool localized on early/sorting endosomes, down-regulation of PI3K-C3 by miRNA reduced the density of EEA1 immunoreactive puncta in the dendrites (Fig. 7F; control miRNA, 22.6 ± 1.8 puncta/40-μm dendritic length, n = 10; PI3K-C3 miRNA, 11.8 ± 1.7 puncta/40-μm dendritic length, n = 10) but not the somata (Fig. 7E; control miRNA, 18.6 ± 1.2 puncta/100 μm2, n = 10; PI3K-C3 miRNA, 16.3 ± 2.7 puncta/100 μm2, n = 10) of transfected neurons (Fig. 7, D–F).

FIGURE 6.
Down-regulation of kinases involved in PI3P synthesis. A, Western blotting analysis of lysates from Rat2 cells stably expressing negative control (neg. Cntrl.-miRNA) or miRNAs specific for the rat isoforms of PI3K-C3, PI3K-C2α, or PI3K-C2β, ...
FIGURE 7.
PI3K-C3 knockdown impairs postsynaptic gephyrin clustering. A, cultured hippocampal neurons were transfected at DIV 4 with a cDNA encoding a GFP-tagged negative control (neg. Cntrl.)-miRNA (left) or an miRNA specific for rat PI3K-C3 (right). At DIV 14, ...

To evaluate whether the effects of PI3K-C3 miRNA down-regulation can be rescued by exogenously added PI3P-AM, we transfected hippocampal cultures at DIV 4, e.g. prior to the onset of inhibitory synapse formation, with the PI3K-C3 miRNA or the control miRNA, and subsequently treated them at DIV 8 with 50 μm PI3P-AM or DMSO for 2 h prior to fixation and immunocytochemical analysis. Again, the PI3K-C3 miRNA-expressing neurons showed a significant reduction by ~54% in the density of dendritic gephyrin cluster neurons, as compared with cells expressing the control miRNA, when treated with DMSO only (Fig. 7, G and H, top/left; control miRNA, 6.06 ± 0.71 puncta/40-μm dendritic length, n = 16; PI3K-C3 miRNA, 2.76 ± 0.32 puncta/40-μm dendritic length, n = 17). Furthermore, we detected an ~37% reduction in the size of dendritic gephyrin clusters in the PI3K-C3 miRNA-expressing neurons, as compared with control miRNA expressing cells (Fig. 7, G and H, bottom/left; control miRNA, 0.217 ± 0.014 μm2, n = 16; PI3K-C3 miRNA, 0.137 ± 0.011 μm2, n = 17). In neurons treated for 2 h with PI3P-AM, the difference in endogenous gephyrin cluster densities between PI3K-C3 and control miRNA-expressing cells was similar to that observed with DMSO only-treated cells (Fig. 7, G, and H, top/right; control miRNA, 6.82 ± 0.42 puncta/40-μm dendritic length, n = 17; PI3K-C3 miRNA, 3.35 ± 0.61 puncta/40-μm dendritic length, n = 17). Similarly, upon PI3P-AM treatment, the mean cluster sizes of the gephyrin immunoreactive puncta did not differ significantly between the PI3K-C3 miRNA and control miRNA-expressing neurons but in both cases were larger than upon DMSO treatment (Fig. 7H, bottom/right; control miRNA, 0.286 ± 0.022 μm2, n = 17; PI3K-C3 miRNA, 0.231 ± 0.019 μm2, n = 17). Thus, consistent with the results obtained from striatal autaptic neurons (Fig. 4), hippocampal neurons also show a significant change in the mean size, but not density, of endogenous gephyrin clusters after a 2-h treatment with PI3P-AM. Furthermore, these findings demonstrate that the comparatively short incubation with PI3P-AM rescues the reduction in mean gephyrin cluster size, but not density, observed upon prolonged PI3K-C3 miRNA expression.

We next wondered whether raising intracellular PI3P levels by overexpression of recombinant PI3K-C3 similarly would result in enhanced gephyrin clustering, as observed upon PI3P-AM treatment. Hence, we expressed the GFP-tagged human orthologue of PI3K-C3, GFP-hVps34, or as a control GFP from DIV 7 to 14 in cultured hippocampal neurons and then analyzed the resulting effects on the clustering of endogenous gephyrin by immunostaining. In contrast to the results obtained with a 2-h PI3P-AM treatment, prolonged overexpression of GFP-hVps34 led to significant increases in the density (GFP, 10.9 ± 1.1 puncta/100 μm2, n = 15; GFP-hVps34, 17.1 ± 1.5 puncta/100 μm2, n = 15) and size (GFP, 0.30 ± 0.03 μm2, n = 562 clusters; GFP-hVps34, 0.54 ± 0.08 μm2, n = 851 clusters) of perisomatic, but not dendritic, gephyrin clusters (Fig. 8, A and B). The reasons for the opposing effects on gephyrin perisomatic and dendritic clusters observed upon prolonged hVps34 expression are currently unknown but might reflect different functions of PI3K-C3 within the cell or differences in the compartmental contents of PI3P-related kinases and phosphatases (see under “Discussion”). In either case, these results further confirm that increases in intracellular PI3P levels lead to enhanced gephyrin clustering.

FIGURE 8.
hVPS34 (PI3K-C3) overexpression leads to an increase in the density and size of perisomatic gephyrin clusters in cultured neurons. A, cultured hippocampal neurons were transfected at DIV 7 with cDNAs encoding either GFP alone (left) or GFP-hVps34 (right ...

The ability of PI3P to serve dynamic functions in membrane trafficking and protein sorting relies on the tight spatial and temporal control of the kinases and phosphatases involved in its generation and degradation. Members of the myotubularin phosphatase family, MTMs, are the principal PI3P and PI(3,5)P2 3-phosphatases. Notably, a pool of membrane-associated PI3K-C3 is bound to MTM1, which competes with GTPases for PI3K-C3 binding (2). This indicates a complex interplay of small GTPases, MTMs, and PI3K-C3 in the tuning of endosomal PI3P levels. Furthermore, the activity of certain inositol polyphosphate 5-phosphatases can lead to an enrichment of PI3P in endosomes or at the plasma membrane. For example, ectopic expression of the 72-kDa 5-phosphatase (72-5ptase; also called pharbin) leads to the depletion of PI(3,5)P2 and the accumulation of PI3P at the plasma membrane, and thereby it promotes the plasma membrane translocation of the glucose transporter 4 in adipocytes (41). This 72-5ptase contains a C-terminal CAAX motif for membrane targeting.

To investigate the effects of a PI3P/PI(3,5)P2-specific 3-phosphatase and a PI(3,5)P2-specific 5-phosphatase in neurons, we overexpressed HA-tagged 72-5ptase and mCherry-tagged MTM1 fused to a C-terminal CAAX-box prenylation sequence for membrane targeting (38) in dissociated rat hippocampal cultures at early stages of inhibitory postsynapse formation for 2 days (DIV 8–10) and analyzed their effects on endogenous gephyrin clustering. MTM1-CAAX expression led to significant reductions in both perisomatic and dendritic gephyrin cluster densities, as compared with the corresponding values in neurons expressing mCherry alone (Fig. 9, A–E; mCherry perisomatic, 7.2 ± 0.9 per 100 μm2, n = 19; mCherry-MTM1-CAAX perisomatic, 3.5 ± 0.5 per 100 μm2, n = 16; mCherry dendritic, 10.4 ± 0.8 per 40 μm, n = 32; mCherry-MTM1-CAAX dendritic, 6.8 ± 1.1, n = 29). In contrast, overexpression of 72-5ptase did not have any effects on the density of perisomatic and dendritic gephyrin puncta (Fig. 9, B and C) but led to a significant increase in the size of perisomatic clusters (Fig. 9D; mCherry, 0.13 ± 0.01 μm2, n = 370 clusters from 19 neurons; HA-72-5ptase, 0.18 ± 0.01 μm2, n = 647 clusters from 30 neurons) but not dendritic (Fig. 9E), gephyrin clusters. Together, these results indicate that changes in the total amount of PI3P in neurons caused by overexpression of kinases and phosphatases that control PI3P generation and degradation regulate the extent of gephyrin clustering.

FIGURE 9.
Overexpression of MTM1-CAAX or 72-5ptase affects gephyrin clustering in cultured neurons. A, cultured hippocampal neurons were transfected at DIV 8 with cDNAs encoding either mCherry alone (left), mCherry-MTM1-CAAX (center), or HA-72-5ptase (right). At ...

Characteristics of Synaptic PI3P Pools

Our finding that the down-regulation of PI3K-C3, but not of PI3K-C2α or PI3K-C2β, causes a significant reduction in gephyrin cluster density on the dendrites of rat hippocampal neurons indicates that the PI3P pool required for gephyrin clustering is located on early/sorting endosomes, because PI3K-C3 is the kinase responsible for the generation of the constitutive PI3P pool on these endosomal compartments (17). To investigate whether gephyrin associates with PI3P-containing membranes during the initial stages of cluster formation, we transfected rat hippocampal neurons for 1 day (DIV 9–10) with vectors expressing monomeric red fluorescent protein (mRFP)-gephyrin and GFP-2×FYVE, a probe consisting of two tandem PI3P-binding FYVE finger domains that binds PI3P specifically and with high affinity in vitro and in vivo (16). Newly clustered mRFP-gephyrin partially colocalized with GFP-2×FYVE at both extrasynaptic and inhibitory postsynaptic sites (Fig. 10A), as indicated by a 25% coapposition of the two proteins to presynaptic VIAAT puncta (Fig. 10, B and C).

FIGURE 10.
Coapposition of GFP-2×FYVE and mRFP-gephyrin at inhibitory postsynapses. A, cultured hippocampal neurons were transfected at DIV 9 with cDNAs encoding GFP-2×FYVE and mRFP-gephyrin. At DIV 10, the cultures were fixed and stained with a ...

To exclude the possibility that the observed colocalization of mRFP-gephyrin with GFP-2×FYVE results from a random signal-match due to overexpression of the two proteins in dendritic shafts, we performed time-lapse confocal microscopy to monitor the movement of mRFP- and GFP-positive particles over a period of 30 min. This revealed that mRFP-gephyrin clusters remained stable over the observation period, whereas the GFP-2×FYVE fluorescence associated with early/sorting endosomes showed extensive movement, particularly in the dendrites of transfected neurons (Fig. 7D and supplemental Movie 1). However, the GFP-2×FYVE puncta that colocalized with mRFP-gephyrin and were apposed to VIAAT-positive presynapses, as indicated by post hoc staining for VIAAT upon time-lapse imaging and fluorescence intensity plot analysis (Fig. 10, D, bottom, and E), showed less movement over time as compared with those that were mRFP-gephyrin negative and not apposed to VIAAT (Fig. 10D and supplemental Movie 1). Notably, prolonged expression of GFP-2×FYVE in cultured neurons (DIV 4–11) led to a significant reduction of endogenous gephyrin clusters in the dendrites, as compared with neurons expressing GFP alone (Fig. 11, A and B; untransfected, 11.4 ± 0.5 puncta/40-μm dendritic length, n = 26; GFP, 10.3 ± 0.5 puncta/40-μm dendritic length, n = 26; GFP-2×FYVE, 7.8 ± 0.6 puncta/40-μm dendritic length, n = 26). We attribute this to a competitive masking of membrane-associated PI3P by high levels of GFP-2×FYVE (16, 42). Together, the results presented above support the view that PI3P is present within endosomal membrane domains that are closely associated with inhibitory postsynapses and hence are restricted to lateral diffusion.

FIGURE 11.
Prolonged expression of GFP-2×FYVE inhibits clustering of endogenous gephyrin in cultured hippocampal neurons. A, cultures were transfected at DIV 4 with cDNAs encoding either GFP alone (top) or GFP-2×FYVE (bottom). At DIV 11, neurons ...

PI3P-AM-induced Redistribution of Gephyrin Depends on Cb

Different lines of evidence suggest that the stimulation of neuronal gephyrin clustering by PI3P-AM involves Cb. First, Cb is the only component of inhibitory postsynapses known to specifically bind this phosphoinositide (10,12, 15). Second, Cb deletions and mutations impairing PI3P binding have been demonstrated to impair Cb-dependent gephyrin clustering in cultured neurons (9,11). Third, several collybistin mutations in humans causing X-linked intellectual disability are associated with a loss of, or reduced, PI3P binding capacity (10, 13, 15). We therefore examined whether the observed effect of PI3P-AM on gephyrin clustering depends on Cb by using HEK 293 cells expressing both GFP-gephyrin and Cb. This heterologous coexpression system was chosen because of the following: (i) it has proven highly reliable for the identification of proteins that are implicated in gephyrin clustering, including Cb (43), NL2 (7), and its homolog NL4 (44) as well as the Cb-interacting GTPase TC10 (8); and (ii), most importantly, it displays a stringent dependence on Cb of gephyrin cluster formation (14, 43).

To allow for proper control of GFP-gephyrin expression levels, we generated a HEK 293 cell line (Flp-In T-Rex HEK 293), which inducibly expresses GFP-gephyrin upon addition of tetracycline (TET) to the culture medium (see “Experimental Procedures”). Fig. 12A, left (Tet-On), shows that in this cell line TET induction led to the formation of large intracellular GFP-gephyrin aggregates, as described previously (43). Coexpression of the constitutively active CbII-splice variant lacking the N-terminal SH3 domain (ΔSH3CbII) redistributed gephyrin into membrane-associated microclusters (Fig. 12A, right). This redistribution is due to the ability of ΔSH3CbII to simultaneously bind gephyrin and membrane lipids (e.g. PI3P (6, 9)). Notably, the majority of the mammalian Cb isoforms detected in vivo contain an inhibitory SH3 domain that renders the protein inactive in this assay, resulting in the preferential accumulation of gephyrin in cytoplasmic aggregates (14, 43). To examine the effects of PI3P-AM on gephyrin distribution in the absence or presence of the SH3 domain containing Cb, we transfected Flp-In T-Rex-GFP-gephyrin HEK 293 cells with Myc-SH3(+)CbII for 10 h prior to a 4-h TET induction of GFP-gephyrin expression. Subsequently, the cells were treated for an additional 2 h with either DMSO only (Fig. 12B, top) or 50 μm PI3P-AM (Fig. 12B, bottom), followed by immunocytochemical analysis. In the absence of SH3(+)CbII (“untransfected”), PI3P-AM treatment had no effect on both the density (Fig. 12D, left; DMSO, 1.38 ± 0.18 clusters/cell, n = 21; PI3P-AM, 1.43 ± 0.14 clusters/cell, n = 21) and mean size (Fig. 12D, right; DMSO, 0.32 ± 0.04 μm2, n = 21; PI3P-AM, 0.37 ± 0.04 μm2, n = 21) of GFP-gephyrin clusters, as compared with DMSO-only treated cells. In contrast, in the presence of Myc-SH3(+)CbII (“transfected”), PI3P-AM increased significantly the density (Fig. 12D, left; DMSO, 3.76 ± 0.89 clusters/cell, n = 21; PI3P-AM, 9.38 ± 1.27 clusters/cell, n = 21), and correspondingly reduced the mean size (Fig. 12D, right; DMSO, 0.25 ± 0.04 μm2, n = 21; PI3P-AM, 0.12 ± 0.023 μm2, n = 21), of GFP-gephyrin clusters, as compared with controls. These results indicate that in HEK 293 cells (i) PI3P-AM alone has no effect on the distribution of gephyrin and (ii) Cb is required for the stimulation of gephyrin redistribution into submembranous clusters by this phosphoinositide.

FIGURE 12.
In HEK 293 cells, PI3P-AM stimulation of GFP-gephyrin clustering requires Cb. A, Flp-In T-Rex-GFP-gephyrin HEK 293 cells inducibly express GFP-gephyrin upon TET-induction (compare Tet-Off with Tet-On state in the 1st two panels). Transfection of the Myc-ΔSH3CbII ...

Discussion

This study provides the first direct demonstration of a key role of PI3P in the regulation of postsynaptic gephyrin clustering and inhibitory synaptic strength in neurons. Specifically, by using cultured hippocampal neurons transiently expressing GFP-gephyrin, we show that increasing intracellular PI3P concentrations with the membrane-permeant derivative PI3P-AM suffices to promote the formation of new gephyrin clusters and to increase the gephyrin content of inhibitory postsynapses by enhancing the membrane recruitment of gephyrin from the cytosol and intracellular vesicle-like structures. In autaptic GABAergic neurons derived from the striatum, the increase in postsynaptic gephyrin induced by PI3P-AM correlated with an increase in postsynaptic GABAARs, as illustrated by larger mIPSC amplitudes but unaltered mIPSC frequencies and by significant increases in the number and mean size of synaptic gephyrin and GABAAR clusters. Furthermore, current responses to externally applied GABA were significantly larger, showing that the number of GABAARs at the cell surface had increased. A very similar increase in GABA-induced responses was found in autaptic hippocampal glutamatergic neurons upon PI3P-AM treatment. However, here PI3P-AM had no effect on the densities of surface and postsynaptic glutamate receptors, as indicated by the unaltered sizes of glutamate-induced currents and mEPSC amplitudes. Thus, PI3P-AM does not simply enhance plasma membrane incorporation of receptor-containing endosomes at postsynaptic sites but specifically up-regulates the formation of inhibitory, but not excitatory, postsynapses.

In both GABAergic and glutamatergic autaptic neurons, PI3P-AM treatment resulted in marked decreases in eIPSC and eEPSC amplitudes without altering RRP sizes; this is consistent with a markedly reduced probability of presynaptic neurotransmitter release. Based on these findings, we conclude that PI3P negatively affects the fusion or exocytosis of SVs at both inhibitory and excitatory presynapses. Currently, the reasons for the presynaptic effects of PI3P-AM are not clear, but it is conceivable that higher local PI3P concentrations might compete with and reduce the phosphatidylinositol 4,5-bisphosphate-regulated binding of Ca2+ to synaptotagmins and thereby inhibit SNARE-dependent synaptic vesicle fusion, as reported previously (45). Further work will be required to elucidate the precise role of PI3P in presynaptic terminals.

Our knockdown experiments targeting different PI3K isoforms in hippocampal neurons indicate an involvement of PI3K-C3, the enzyme that generates the constitutive pool of PI3P on early/sorting endosomes, in postsynaptic gephyrin clustering. In contrast, PI3K-C2α and PI3K-C2β, which are involved in both the synthesis of PI(3,4)P2 (38) and the de novo biosynthesis of PI3P at the plasma membrane (19, 20, 37, 46), appear not to be involved in postsynaptic gephyrin accumulation. These findings lead to the unexpected conclusion that the pool of PI3P on endomembranes, and not PI3P generated at the plasma membrane, is of crucial relevance for postsynaptic Cb-dependent gephyrin clustering. In addition, we consistently found the following: (i) miRNA-mediated down-regulation of PI3K-C3 in cultured hippocampal neurons caused an ~35% reduction in the density of dendritic gephyrin clusters as compared with control cells; (ii) a 2-h treatment with PI3P-AM rescued the reduction in mean size, but not density, of dendritic gephyrin clusters observed in neurons overexpressing the PI3K-C3 miRNA; and (iii) overexpression of hVps34, the human homologue of PI3K-C3, had opposing effects on gephyrin perisomatic and dendritic clusters, compared with a 2-h treatment of the cells with PI3P-AM. Based on these data, one might speculate that PI3P may not be the only signaling lipid involved in the Cb-dependent accumulation of gephyrin at postsynapses. In agreement with this idea, a recent study indicates broader PIP specificities for the short Cb splice variant, which lacks the N-terminal SH3 domain, than those determined for an open-conformation mutant (W24A/E262A) of the SH3 domain-containing isoform (12). Furthermore, Vps34 has been shown to exist in two complexes in both yeast and mammalian cells, which not only have opposing effects but can potentially negatively regulate one another (18). Finally, phosphoinositide interconversion by additional kinases or phosphatases present at different subcellular compartments might partially restore the endosomal PI3P pool in the absence of PI3K-C3. In line with the latter interpretation, overexpression of the PI(3,5)P2-specific phosphatase 72-5ptase/pharbin, which is predicted to increase PI3P levels in the transfected neurons, caused a significant increase in the size of perisomatic gephyrin clusters. This result supports the notion of a complex interplay between additional kinases, phosphatases, and PI3K-C3 in the tuning of endosomal PI3P levels and gephyrin clustering.

Of note in this context, a comprehensive siRNA screen employing a functional cell-based assay for gephyrin clustering in HeLa cells as a readout identified PI3K-C3 and PI3K-C2γ as candidate kinases involved in the stabilization of GFP-gephyrin clusters (47). To examine the involvement of Cb in the enhancement of gephyrin clustering by PI3P-AM, we employed a similar cell-based assay that relied on a HEK 293 cell line, which inducibly expresses GFP-gephyrin upon TET application and thereby allows us to investigate the clustering of newly synthesized gephyrin independently of other neuronal proteins. In agreement with previous reports (9,11, 13, 15), our results obtained with this heterologous expression system clearly show that PI3P-AM increases gephyrin cluster formation only upon coexpression of Cb.

The importance of early endosomal PI3P pools for gephyrin clustering, which we demonstrate here, extends our understanding of the mechanisms underlying Cb activation and the subsequent nucleation of gephyrin scaffolds (Fig. 13). Our findings support the view that, at least at early stages of synaptogenesis, PI3P-enriched early/sorting endosomes contribute to the membrane recruitment of Cb-gephyrin complexes. Indeed, a significant fraction of 2×FYVE-labeled early/sorting endosomes was closely associated with inhibitory postsynapses and apposed to both VIAAT and gephyrin in DIV 10 cultured hippocampal neurons. Furthermore, prolonged overexpression of the 2×FYVE probe significantly reduced the density of gephyrin clusters along dendrites of cultured hippocampal neurons, presumably by masking PI3P on endomembanes. Based on these results, we propose that the interplay between gephyrin- and Cb-interacting proteins and phosphatases is crucial for interconversion of PI3P and thereby the trafficking of Cb-gephyrin complexes toward the plasma membrane, where the latter might be stabilized through interactions with additional proteins, such as NL2 and NL4 (7, 9). In line with this idea, a recent report identified a mechanism for phosphoinositide interconversion from PI3P to PI4P at endosomes en route to the plasma membrane and showed that this interconversion enables the subsequent recruitment of the exocyst tethering complex (23). Interestingly, the small GTPase TC10, which interacts in its active GTP-bound state with Cb (8), also binds to one of the components of the exocyst complex, Exo70, and promotes translocation of Exo70 to the plasma membrane (48).

FIGURE 13.
Tentative model for the role of PI3P-containing early/sorting endosomes in the assembly of the gephyrin scaffold at postsynaptic membranes. A, intracellularly, gephyrin trimers bind to Cb in its closed conformation. At postsynaptic sites, the open conformation ...

In line with our finding that PI3K-C3 is important for gephyrin clustering at inhibitory postsynapses, overexpression of hVps34, the human homologue of PI3K-C3, in hippocampal neurons led to significant increases in the density and size of perisomatic gephyrin clusters. Similarly, overexpression of the SH3 domain-deficient Cb isoform, which strongly binds PI3P (9,11), causes increases in the density and size of perisomatic gephyrin clusters (49, 50). This indicates that the interaction of Cb and gephyrin with PI3P-containing endosomes is of particular importance for the regulation of the size of perisomatic synapses, which typically produce a stronger inhibitory drive than the smaller synapses localized on dendrites (51). Furthermore, mutations in both PI3K-C3 and gephyrin have been implicated in bipolar disorder and schizophrenia (52, 53), and both phosphoinositides and gephyrin are potential targets for the therapeutic effects of lithium in bipolar disorder (52, 54). Thus, our findings also provide a new view on signaling pathways related to neuropsychiatric disorders, linking them to PI3K-C3 function, PI3P generation, and postsynaptic gephyrin clustering.

In summary, this study identifies PI3P as an efficient regulator of Cb-dependent gephyrin and GABAAR clustering and indicates the involvement of early/sorting endosomes during the membrane activation process (55) that is required for gephyrin nucleation and scaffold formation at the postsynaptic plasma membrane of developing inhibitory synapses.

Experimental Procedures

Plasmids and Oligonucleotide Sequences

The GFP-gephyrin and mRFP-gephyrin constructs used have been described previously (56, 57). The pEGFP-N1 vector was purchased from Clontech. The GFP-2×FYVE construct was provided by Dr. Marino Zerial (Dresden, Germany), and vectors encoding mCherry-MTM1-CAAX and mCherry alone (38) were provided by Dr. Volker Haucke (Berlin, Germany). A plasmid encoding the Myc-tagged 72-5ptase (41) was provided by Dr. Christina A. Mitchell (Victoria, Australia), and the plasmid encoding GFP-hVps34 was provided by Dr. Matthias Wymann (Basel, Switzerland). The pcDNA 6.2-GW/EmGFP and pcDNA 6.2-GW/EmGFP-miR-neg.Cntrl plasmid were included in the BLOCK-iT polymerase II miR RNAi expression vector kit (Invitrogen). The negative control sequence without 5′-overhangs was 5′-GAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT-3′. The following single-stranded DNA oligonucleotides encoding the pre-miRNAs of PI3K-C3, PI3K-C2α, and PI3K-C2β used in this study were purchased from Invitrogen, Germany: PI3K-C3: forward, 5′-TGCTGTCAAGAAGGTACAAAGATCCTGTTTTGGCCACTGACTGACAGGATCTTTACCTTCTTGA-3′, and reverse, 5-CCTGTCAAGAAGGTAAAGATCCTGTCAGTCAGTGGCCAAAACAGGATCTTTGTACCTTCTTGAC-3′; PI3K-C2α, forward, 5′-TGCTGTACAAATGATGGTTCAAGGTGGTTTTGGCCACTGACTGACCACCTTGACATCATTTGTA-3′, and reverse, 5′-CCTGTACAAATGATGTCAAGGTGGTCAGTCAGTGGCCAAAACCACCTTGAACCATCATTTGTAC-3′; PI3K-C2β: forward, 5′-TGCTGATCACAAACCGACTAGGGAAGGTTTTGGCCACTGACTGACCTTCCCTACGGTTTGTGAT-3′, and reverse, 5′-CCTGATCACAAACCGTAGGGAAGGTCAGTCAGTGGCCAAAACCTTCCCTAGTCGGTTTGTGATC-3′.

Antibodies

The following primary antibodies were used for immunocytochemistry: monoclonal mouse anti-gephyrin (mAb7a, Connex, 1:3000); polyclonal guinea pig anti-VIAAT (Synaptic Systems, 1:2000); polyclonal rabbit anti-VIAAT, affinity-purified (131003, Synaptic Systems, 1:500); monoclonal mouse anti-EEA1 (BD Transduction Laboratories, 1:1000); and polyclonal rabbit anti-hemagglutinin (HA) (Zymed Laboratories Inc., Invitrogen 1:2000). The polyclonal guinea pig anti-GABAAR-α2 subunit antibody (1:1000) was kindly provided by Dr. Jean-Marc Fritschy (Zurich, Switzerland). The following secondary antibodies were used for immunocytochemistry: Alexa Fluor 488, 555, or 633 goat anti-mouse or goat ant- rabbit IgG (Invitrogen, 1:2,000). The following primary antibodies were used for Western blotting: polyclonal rabbit anti-PIK3C3 (PAB12073, Abnova, 1:500); monoclonal mouse anti-PIK3C2A (anti-PI3-kinase p170, BD Transduction Laboratories, 1:1000); monoclonal mouse anti-PIK3C2B (Sigma, 1:1000); monoclonal mouse anti-actin (clone AC-40, Sigma, 1:2000); and monoclonal mouse anti-β-tubulin (clone TUB 2.1, Sigma, 1:2500). Bound primary antibodies were visualized on Western blots using horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-mouse or goat anti-rabbit IgGs (Jackson ImmunoResearch, 1:10,000).

Generation and Expression of Pre-microRNA (Pre-miRNA) Constructs

Annealing of single-stranded oligonucleotides (see under “Plasmids and Oligonucleotide Sequences”), cloning of the double-stranded oligonucleotides into pcDNATM6.2-GW/EmGFP-miR, and transformation into One Shot TOP10 chemically competent Escherichia coli bacteria was performed according to the manufacturer's instructions (BLOCK-iT polymerase II miR RNAi Expression Vector Kits, Invitrogen). All plasmids were confirmed by automated DNA sequencing using the EmGFP forward (5′-GGCATGGACGAGCTGTACAA-3′) and the miRNA reverse (5′-CTCTAGATCAACCACT-TTGT-3′) sequencing primers. Knockdown efficiencies were determined by Western blotting analysis, as described previously (8), and densitometric scanning of the relative band intensities of the proteins of interest in lysates from stable transfectants of Rat2 cells (ATCC CRL-1764; Rattus norvegicus fibroblasts) expressing the corresponding miRNAs for PI3K-C3, PI3K-C2α, or PI3K-C2β, as compared with the lysates of cells stably expressing the control miRNA. RNAi knockdowns in hippocampal neurons were performed by transfecting the cultures at DIV 4 with the corresponding miRNA plasmids using the CalPhos mammalian transfection kit (Clontech, Takara Bio). After fixation at DIV 14, the cultures were immunostained as described below.

Transfection and Immunostaining of Hippocampal Neurons

Cultures of hippocampal neurons were prepared from embryonic day 18 (E18) rats. Hippocampi were treated with trypsin in Hanks' balanced salt solution (Gibco, Life Technologies, Inc.) for 15 min at 37 °C and then dissociated by trituration. Cells were plated on poly-l-lysine-coated glass coverslips at a density of 120,000 cells/ml in Neurobasal medium supplemented with B27 (Gibco), GlutaMax (Gibco), and penicillin/streptomycin (Roche Applied Science). Neurons were transfected at different DIV, as indicated under “Results,” using the CalPhosTM mammalian transfection kit (Clontech). Immunocytochemistry with the antibodies listed above was performed as described previously (11). Images were collected with an inverse Leica DMIRE2 microscope equipped with a ×63 oil-immersion objective and connected to a Leica TCS SP2 AOBS confocal laser scanning setup (Leica Microsystems) or with an AxioImager Z1 equipped with a Zeiss apochromat ×63 objective and an Apotome module (Zeiss). Acquired images were processed identically using the ImageJ software package (rsb.info.nih.gov). Single channels were recorded using the same standardized threshold levels. Subsequently, a binary image was generated, and immunoreactive puncta were counted automatically by the ImageJ software, as described previously (8, 9).

PI3P-AM/PI4P-AM Treatment and Time-lapse Imaging of Transfected Hippocampal Neurons

Neurons were cultured on poly-l-lysine-coated 35-mm glass bottom ibidi plates (ibidi GmbH) and transfected as described above. The synthesis of caged PI3P-AM, PI3P-AM, and PI4P-AM has been described previously (21). PIP-AM derivatives were dissolved in DMSO to make a stock solution of 50 mm. To achieve optimal dispersion of the AM compounds, appropriate amounts of the 50 mm stock solutions or DMSO only were mixed with Pluronic F-127 (Invitrogen) at a 1:0.5 (v/v) ratio and added to the imaging medium (Neurobasal-A, phenol red-free, Invitrogen, Karlsruhe, Germany) to a final concentration of 50 μm of the AM compounds. The final concentration of DMSO was 0.1%. The neuronal cultures were washed once and kept in imaging medium during the subsequent procedures. Image stacks of transfected hippocampal neurons were taken with an inverse Leica DMIRE2 microscope equipped with a ×63 oil-immersion objective and connected to a Leica TCS SP2 AOBS (acousto-optical beam splitter) confocal laser scanning setup (Leica Microsystems) 10 min prior to treatment. Media were replaced by imaging media containing DMSO/pluronic, 50 μm PI3P-AM/pluronic, or 50 μm PI4P-AM/pluronic, and the cultures were incubated at 37 °C in 5% CO2 for another 2 h. Subsequently, new image stacks of the same identified neurons were collected, or the cultures were processed directly for immunocytochemistry, as described previously (8).

Cultures of Hippocampal and Striatal Autaptic Neurons

Micro-island cultures of hippocampal and striatal neurons were prepared and cultured as reported previously (34, 58). Briefly, astrocytes for autaptic cultures were obtained from mouse cortices dissected from P0 wild type animals and enzymatically digested for 15 min at 37 °C with 0.25% (w/v) trypsin/EDTA (Gibco, Life Technologies, Inc.). Astrocytes were plated in T75 culture flasks in DMEM (Gibco, Life Technologies, Inc.) containing 10% fetal bovine serum (Gibco), penicillin (100 units/ml)/streptomycin (100 μg/ml; Roche Applied Science), MITO (BD Biosciences), and grown for 7–10 DIV. Subsequently, astrocytes were trypsinized and plated at a density of ~30,000 cells/well onto 32-mm diameter glass coverslips, which had been coated with agarose (Sigma) and stamped using a custom-made stamp to generate 200 × 200 μm substrate islands with a coating solution containing poly-d-lysine (Sigma), acetic acid, and collagen (BD Biosciences). Hippocampi and striata from P0 mice were isolated and digested for 60 min at 37 °C in DMEM containing 25 units/ml papain (Worthington), 0.2 mg/ml cysteine, 1 mm CaCl2, and 0.5 mm EDTA (Sigma). After washing, the dissociated hippocampal and striatal neurons were seeded onto the micro-island plates in pre-warmed Neurobasal Medium (Gibco, Life Technologies, Inc.) supplemented with B27 (Gibco, Life Technologies, Inc.), GlutaMax (Gibco, Life Technologies, Inc.), and penicillin (100 units/ml)/streptomycin (100 μg/ml; Roche Applied Science) at a density of ~4000 cell/well. The neurons were allowed to differentiate for 9–11 days prior to recording, and only islands containing single neurons were examined.

Electrophysiological Recordings on Cultured Hippocampal and Striatal Autaptic Neurons

Cells were whole-cell voltage-clamped at −70 mV using a MultiClamp 700B amplifier (Axon Instruments, Molecular Devices) under the control of the Clampex program 10.1 (Molecular Devices). All analyses were performed using AxoGraph X version 1.3.1 (AxoGraph Scientific). The cells were recorded between DIV 9 and 11. Postsynaptic currents were evoked by depolarizing stimuli from −70 to 0 mV for 2 ms. The response to triggered release of the RRP was measured after application of 0.5 m hypertonic sucrose solution. The Pvr was calculated by dividing the charge transfer during an AP-evoked response through the charge transfer measured during a response to hypertonic sucrose solution (reflecting the apparent RRP). Miniature spontaneous postsynaptic currents were recorded in the presence of 300 nm tetrodotoxin. The number of synaptic vesicles of eIPSC and the total number of primed SVs were calculated by dividing the charge transfer of the evoked release and the response to 0.5 m hypertonic sucrose solution, respectively, through the charge transfer of mIPSCs (33, 34). Glutamate- and GABA-induced whole-cell currents were recorded after exogenous application of 100 μm glutamate or 3 μm GABA for 1 or 3 s, at a holding voltage of −70 mV. Mean amplitudes of responses to exogenously applied glutamate and GABA in PI3P-AM-treated neurons were normalized to those of DMSO-treated control cells in the same set of experiments. The extracellular solution contained 140 mm NaCl, 2.4 mm KCl, 10 mm Hepes, 10 mm glucose, 4 mm CaCl2, and 4 mm MgCl2 (320 mosmol/liter), pH 7.3. The patch-pipette solution for recordings on autaptic neurons contained 136 mm KCl, 17.8 mm Hepes, 1 mm EGTA, 4.6 mm MgCl2, 4 mm NaATP, 0.3 mm Na2GTP, 15 mm creatine phosphate, and 5 units/ml phosphocreatine kinase (315–320 mosmol/liter), pH 7.4. The series resistance was compensated by ~35–60%. All extracellular solutions were applied with a custom-built fast flow system consisting of an array of flow pipes controlled by a stepper motor that allows complete and rapid solution exchange with time constants of ~30 ms. Pressure was on for 20 ms. All chemicals, except for TTX (Tocris Bioscience), were purchased from Sigma.

Generation, Transfection, and Treatment of the Flp-In T-Rex-GFP-Gephyrin HEK293 Cell Line

The Flp-In T-Rex system (Invitrogen) was used to generate a HEK 293 cell line exhibiting TET-inducible expression of GFP-gephyrin. The following components of the Flp-In T-Rex system were purchased from Invitrogen: (i) Flp-In T-Rex HEK 293 cell line (catalogue no. R780-07), containing two independently integrated plasmids, the pFRT/lacZeo plasmid, which introduces a single FRT site into the genome and stably expresses the lacZ-Zeocin fusion gene under the control of the SV40 early promoter, and the pcDNA6/TR plasmid, stably expressing the Tet repressor gene under the control of the constitutive human cytomegalovirus (CMV) immediate-early enhancer/promoter; (ii) the pcDNA5/FRT/TO expression vector for cloning the GFP-gephyrin cDNA (56) into its KpnI/NotI sites. This expression vector expresses the gene of interest under the control of TET-regulated hybrid human CMV/TetO2 promoter and contains the hygromycin resistance gene with a FRT site embedded in the 5′-coding region and (iii) the pOG44 plasmid, which constitutively expresses the Flp recombinase under the control of the human CMV promoter. Generation of the Flp-In T-Rex-GFP-gephyrin HEK 293 cell line was performed according to the manufacturer's instructions (Flp-In System, catalogue no. K6010-01, Invitrogen). The cells were cultured and maintained in DMEM (Gibco, Life Technologies, Inc.), 10% (v/v) fetal calf serum (Gibco), 50 units/ml penicillin and 50 units/ml streptomycin (Roche Applied Science), 15 μg/ml blasticidin (Invitrogen), and 200 μg/ml hygromycin B (Invitrogen) at 37 °C and 5% CO2. GFP-gephyrin expression was induced by the addition of 1 μg/ml tetracycline (Invitrogen) to the medium. Under these conditions, further incubation of the cells for 3–4 h was sufficient to induce the expression of GFP-gephyrin. For transfection, cells were plated in 24-well plates on 12-mm coverslips. Sterile coverslips were coated with poly-l-lysine (Sigma, 0.001% (w/v), in Dulbecco's PBS, PAA Laboratories) for at least 2 h and washed three times with Dulbecco's PBS before plating. Medium was exchanged to DMEM devoid of supplements prior to transfection, at ~80% confluency. For transfections, 100 ng of mCherry-empty vector and 100 ng of Myc-ΔSH3CbII or MycSH3(+)CbII cDNA were used per well. Cells were transfected using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) following the manufacturer's protocol. DMEM containing 10% (v/v) fetal calf serum and antibiotics were added 2 h after transfection. GFP-gephyrin expression was induced 10 h after transfection by adding 1 μg/ml tetracycline to the media. After 4 h, the cells were washed once and kept for an additional 2 h in imaging medium (Neurobasal-A, phenol red-free, Invitrogen) containing 50 μm PI3P-AM or DMSO only, as described above (see under “PI3P-AM/PI4P-AM Treatment and Time-lapse Imaging of Transfected Hippocampal Neurons”). Subsequently, the cells were fixed and stained as described previously (15). Stainings were inspected under an AxioImager Z1 equipped with a Zeiss apochromat ×63 objective (Zeiss, Göttingen, Germany). For GFP-gephyrin, exposure times were kept constant at 170 ms. Quantifications were performed using the ImageJ software package (rsb.info.nih.gov).

Statistics

Experimental data were evaluated by investigators blind to experimental conditions. Statistical significance was tested using the unpaired two-tailed Student's t test. Values represent means ± S.E. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001); n.s. indicates no significant difference.

Author Contributions

T. P. and H. B. designed the experiments. T. P. performed all experiments with the exception of the electrophysiological recordings, which were designed and performed by H. J .R. and J. S. R. F. P. helped with overexpression of phosphatases, and D. S., R. M., and C. S. synthesized the membrane-permeant phosphoinositides PI3P-AM, caged-PI3P-AM, and PI4P-AM and provided advice on their use. T. P, N. B., and H. B. wrote the manuscript. All authors critically reviewed the results and approved the final version of the manuscript.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Drs. Volker Haucke (Berlin, Germany), Christina A. Mitchell (Victoria, Australia), Matthias Wymann (Basel, Switzerland), and Marino Zerial (Dresden, Germany) for providing expression vectors; Drs. Ioannis Alexopoulos and Miso Mitkovski for advice regarding time-lapse confocal imaging; and Felicitas Vauk for help with some of the experiments.

*This work was supported by German Research Foundation Grants PA2087/1-3 (to T. P.), SFB1190/P10 (to N. B.), and TRR83 (to C. S.). The authors declare that they have no conflicts of interest with the contents of this article.

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Object name is sbox.jpgThis article contains supplemental Movie 1.

3The abbreviations used are:

SV
synaptic vesicle
72-5ptase
72-kDa 5-phosphatase
AM
acetoxymethyl
AP
action potential
Cb
collybistin
DIV
days in vitro
EEA1
early endosome antigen 1
eEPSC
evoked excitatory postsynaptic current
eIPSC
evoked inhibitory postsynaptic currents
GABAAs
GABAA receptor
mEPSC
miniature excitatory postsynaptic current
mIPSC
miniature inhibitory postsynaptic currents
MTM1
myotubularin 1
NL2
neuroligin 2
NL4
neuroligin 4
PH
pleckstrin homology
PI3K-C3
class III phosphatidylinositol 3-kinase
PI3P
phosphatidylinositol 3-phosphate
PSV
primed synaptic vesicle
Pvr
vesicle release probability
RRP
readily releasable pool
TET
tetracycline
TTX
tetrodotoxin
VIAAT
vesicular inhibitory amino acid transporter
SH
Src homology
C
coulomb
PI(3,4)P2
phosphatidylinositol 3,4-bisphosphate
CAAX
aliphatic amino acid
PIP
phosphatidylinositol
mRFP
monomeric red fluorescent protein.

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