PKC epsilon Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95

Protein kinase C epsilon (PKCε) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCε induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCε and PSD-95.We show that the PKCε activators DCPLA-ME and bryostatin 1 induce phosphorylation of PSD-95 at the serine295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine-295 residue in PSD-95 abolished PKCεinduced membrane accumulation. Knockdown of either PKCε or JNK1 prevented PKCε activatormediated membrane accumulation of PSD-95. PKCε directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCε, JNK, or CaMKII activity prevented the effects of PKCε activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCε and phosphorylated PSD-95 (p-PSD-95) coincided with increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCε also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30% and 44% respectively. Prolonged activation of PKCε increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCε promotes synaptogenesis by activating PSD-95 phosphorylation directly, through JNK1 and CaMKII, and also by inducing expression of PSD-95 and synaptophysin. Protein kinase C epsilon (PKCε) is one of the novel PKC isotypes and is characterized as a calcium independent and phorbol ester/diacylglycerol–sensitive serine/threonine kinase. Among the novel PKCs, PKCε is the most abundant species in the central nervous system, mediating various neuronal functions (1-2). In neuroblastoma cells overexpression of PKCε, but not PKCα, βII or δ leads to neurite outgrowth through interaction of actin filaments and the C1 domain of PKCε (3-5). The actin binding site of PKCε is also implicated in exocytosis of neurotransmitters (6). PKCε is essential for many types of learning and memory (7-8) and neuroprotection (9-13). Neuronal contact with astrocytes also promotes global synaptogenesis through PKCε signaling (14). PKCε activation has been shown to promote the maturation of dendritic synapses during associative learning (9). PKCε activation also protects against neurodegeneration (10,15). Phosphorylation of long-tailed AMPARs GluA4 and GluA1 by PKC promotes their surface expression (16-17). PKC activation induces protein synthesis required for long-term memory (12,18). PKCε activation is also required for HuDmediated mRNA stabilization of neurotrophic factors (19) and ApoE mediated epigenetic http://www.jbc.org/cgi/doi/10.1074/jbc.M116.730440 The latest version is at JBC Papers in Press. Published on June 21, 2016 as Manuscript M116.730440 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A ril 7, 2018 hp://w w w .jb.org/ D ow nladed from PKCε induces synaptogenesis via PSD-95 2 regulation of BDNF (20). PKC activation induces translocation of calcium/calmodulin-dependent kinase II (CaMKII) to synapses (21) where it participates in PSD-95-induced synaptic strengthening (22). PKC also promotes NMDA receptor trafficking by indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs (23). Thus, a number of studies have suggested that PKC activators such as bryostatin and dicyclopropanated linoleic acid methyl ester (DCPLA-ME) may be useful therapeutic candidates for the treatment of Alzheimer's disease (AD) and other causes of synaptic loss such as ischemia, stroke, and Fragile X syndrome (56,14,24). Some of these benefits have been attributed to induction of neurotrophic factors such as BDNF or the activation of anti-Aβ repair pathways and anti-apoptotic activity (10,13,20,25). However, the biochemical mechanisms by which PKCε induces synaptogenesis and mediates neuroprotection are still not fully understood. At excitatory synapses, the postsynaptic density is characterized by an electron-dense thick matrix that contains key molecules involved in the regulation of glutamate receptor targeting and trafficking (26). PSD-95 is an abundant scaffold protein in excitatory synapses, where it functions to cluster proteins such as glutamate receptors on the postsynaptic membrane and couples them to downstream signaling molecules, thereby inducing the surface expression and synaptic insertion of glutamate receptors (27-29). In addition to its role in synaptic function, PSD-95 has also been proposed to affect synapse maturation and stabilization (30-32) and thus, synapse number. Phosphorylation of the serine-295 residue of PSD95 enhances the synaptic accumulation of PSD-95 and its ability to recruit surface α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and potentiate excitatory postsynaptic currents (33). In the present study, we examined the role of PKCε signaling and PKCε activators in PSD-95 regulation and induction of synaptogenesis in cultured neurons and CA1 hippocampal slices. We report that PKCε activation induces membrane translocation and phosphorylation of PSD-95 at the serine-295 residue, coinciding with an increased number of synapses. Our data suggest that an important mechanism by which PKCε induces synaptogenesis is by increasing the phosphorylation of PSD-95 at the postsynaptic site and by regulating the expression of synaptophysin at the presynaptic site. RESULTS PKC activation prevents degradation of primary human neuronsPKCε is present in high concentration in central neuronal tissues and has been implicated in broad spectrum neuronal functions. To determine the effect of PKCε activation on survival and maintenance, primary human neurons were treated for 40 days with two different PKC activators (bryostatin 1 and DCPLA-ME, which are relatively specific for PKCε) (13,34-36). Culture media and activators were changed every three days. Cells were imaged from three independent wells every five days and neurite positive cells were counted from 508μm field images. Cells treated with either DCPLA-ME (100nM) or bryostatin 1 (0.27nM) showed an improved survival with increased neuritic branching (Fig. 1A). Untreated cells showed degeneration and 50% cell loss by 36 days, while the treated cells remained healthy for at least 40 days (Fig. 1B). The number of viable neuritepositive cells was also significantly higher at 40 days (F (2, 6)=705.4; ANOVA, P<0.0001) in the activator treated cells than untreated cells (bryostatin 1: 369.7 ± 12.2; DCPLA-ME: 334.7 ± 1.8; untreated 109.7 ± 6.4). Prolonged PKCε activation prevents loss of synaptic proteinsWe quantified the mRNA expression of PKCε, PSD-95 and synaptophysin at 40 days in untreated and PKCε activator-treated neurons. At 40 days the mRNA levels of PKCε (F(3,8) = 18.3; P=0.0006) and PSD-95 (F(3,8) = 44.6; P<0.0001) were significantly higher in the PKCε activator-treated cells compared to 40 day control cells (Fig. 1C, D). Synaptophysin mRNA showed no significant change in between treated and untreated groups (Fig. 1E). We also quantified the protein expression of phosphorylated PSD-95 (pPSD-95), PSD-95 and synaptophysin at 40 days in untreated and PKCε activator-treated neurons by immunoblot (Fig. 1F). Expression levels of PKCε (F(3,8) = 16.60; P<0.001), p-PSD95 (F(3,8) = 66.83; P<0.0001), PSD-95 (F(3,8) = 21.22; P<0.001) and synaptophysin were significantly higher in the 40 day PKCε activatortreated cells compared to 40 day control cells (Fig. by gest on A ril 7, 2018 hp://w w w .jb.org/ D ow nladed from PKCε induces synaptogenesis via PSD-95 3 1G-J). Moreover, protein expression levels of PKCε, PSD-95 and synaptophysin showed a marked decrease in 40-day untreated cells compared to 1-day cells, even after correction for total protein, while PKCε activation prevented the time-dependent loss. This indicates an essential role of PKCε in maintenance of synapses and preserving normal levels of both PSD-95 and synaptophysin. Bryostatin 1 and DCPLA-ME specifically activate PKCεWe then investigated whether this phenomenon is specific to PKCε or whether other PKC isozymes are involved. PKC translocation to the plasma membrane generally has been considered the hallmark of activation and frequently has been used as a surrogate measure of PKC isoform activation in cells (37). Expression levels of PKCα, PKCε and PKCδ in the soluble (cytosol) and particulate (membrane) were measured by immunoblot at 1hr, 4hr and 24hr after either bryostatin 1 (0.27 nM) or DCPLA-ME (100 nM) treatment (Fig. 2A, C). Both DCPLAME and bryostatin 1 increased membrane translocation of PKCε but not PKCα or PKCδ (Fig. 2B,D), confirming that both the compounds activate PKCε but not PKCα or PKCδ. PKCε activation induces membrane translocation of phosphorylated PSD-95 (serine 295)Phosphorylation of PSD-95 on Serine-295 is known to promote localization of PSD-95 in the postsynaptic density (PSD), strengthening the excitatory synapse (33). To determine whether time-dependent PKCε activation has an effect on localization and expression of p-PSD-95, we measured the expression of p-PSD-95 in the soluble and particulate fractions of the primary human neurons at 1hr, 4hr and 24hr post PKC activator treatment (Fig. 2E, F). PKCε activation increased the level of p-PSD-95 in the particulate fraction of both bryostatin 1 (F (3,8)=4.9; ANOVA, P=0.03) and DCPLA-ME treated cells (F (3,8)=11.7; ANOVA, P=0.003) (Fig. 2F). The total PSD-95 expression in whole cell lysate from primary human neurons was unchanged among different groups (Fig. 2E). At 4hr p-PSD-95 levels were significantly higher in bryostatin 1 (156.4 ± 14.9 %; P=0.01