14-3-3 Proteins in Glutamatergic Synapses.

The 14-3-3 proteins are a family of proteins that are highly expressed in the brain and particularly enriched at synapses. Evidence accumulated in the last two decades has implicated 14-3-3 proteins as an important regulator of synaptic transmission and plasticity. Here, we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins at glutamatergic synapses. A key challenge for the future is to delineate the 14-3-3-dependent molecular pathways involved in regulating synaptic functions.

1. Introduction

14-3-3 refers to a family of homologous proteins that consist of seven genetic loci or isoforms (β, γ, ε, η, σ, ζ, and τ) in vertebrates. The name 14-3-3 was given based on the fraction number and migration position on DEAE-cellulose chromatography and subsequent starch-gel electrophoresis during its initial biochemical purification process [1]. 14-3-3 proteins exist as homo- or heterodimers, in which each 14-3-3 monomer shares a similar helical structure and forms a conserved concave amphipathic groove that binds to target proteins via specific phosphoserine/phosphothreonine-containing motifs [2-7]. Through protein-protein interactions, 14-3-3 functions by altering the conformation, stability, subcellular localization, or activity of its binding partners. To date, 14-3-3 proteins have been shown to interact with hundreds of proteins and are implicated in the regulation of a multitude of cellular processes [8, 9].

14-3-3 proteins are highly expressed in the brain, comprising ~1% of its total soluble proteins. Thus, it comes to no surprise that 14-3-3 proteins are involved in a variety of neuronal processes, such as neurite outgrowth, neural differentiation, migration and survival, ion channel regulation, receptor trafficking, and neurotransmitter release [10-12]. In addition, 14-3-3 proteins are genetically linked to several neurological disorders, including neurodegenerative diseases (e.g., Parkinson's, Alzheimer's, and Creutzfeldt-Jakob diseases), neurodevelopmental diseases (e.g., Lissencephaly), and neuropsychiatric disorders (e.g., schizophrenia and bipolar disorder) [13-15], thus making them a potential therapeutic target [16, 17]. In recent years, a number of small molecule 14-3-3 modulators have been discovered that could be used to either stabilize or inhibit 14-3-3 protein-protein interactions [18, 19]. However, as 14-3-3 proteins are involved in diverse cellular processes, it is highly desirable to further characterize and develop compounds that have enhanced isoform specificity as well as can selectively modulate the 14-3-3 interaction with a critical target in a particular pathway.

14-3-3 proteins are generally found in the cytoplasmic compartment of eukaryotic cells. In mature neurons, however, certain 14-3-3 isoforms are particularly enriched at synapses, suggesting their potential involvement in synaptic transmissions [20, 21]. Indeed, evidence accumulated in the last two decades reveals that 14-3-3 is an important modulator of synaptic neurotransmissions and plasticity. In this review, we will discuss the functional role of 14-3-3 proteins in the regulation of glutamatergic synapses.

2. Functions of 14-3-3 at the Presynaptic Site

Early evidence that 14-3-3 might regulate synaptic transmission and plasticity came from genetic and functional studies of the fruit fly Drosophila. The gene leonardo encodes 14-3-3ζ, one of the two Drosophila 14-3-3 homologs that is abundantly and preferentially expressed in mushroom body neurons. Mutant leo alleles with reduced 14-3-3ζ proteins exhibit significant deficits in olfactory learning and memory, suggesting a functional role of 14-3-3 in these processes [22]. A subsequent study further determined that the 14-3-3ζ protein progressively accumulates to the synaptic boutons during maturation of the neuromuscular junction (NMJ), where it colocalizes with the synaptic vesicles containing the neurotransmitter glutamate [23]. Based on electrophysiological analyses, Leonardo mutants show impaired presynaptic functions at NMJ, including reduced endogenous excitatory junctional currents (EJCs), impaired transmission fidelity, and loss of long-term augmentation and posttetanic potentiation (PTP). The evoked transmission deficit in leo is more severe under lower external Ca2+ concentration, suggesting a possible defect in Ca2+-dependent presynaptic transmission in the absence of 14-3-3ζ proteins.

Following those studies in Drosophila NMJs, the involvement of 14-3-3 proteins in the presynaptic site of glutamatergic synapses was further investigated in the vertebrate nervous system. One potential mechanism is thought to be mediated by 14-3-3 binding to RIM1α, an active zone protein that is essential for presynaptic short- and long-term plasticity [24, 25]. Early biochemical studies have provided the first evidence that 14-3-3 binds to RIM1α through its N terminal domain, raising the possibility that 14-3-3 regulates neurotransmitter release and synaptic plasticity through the regulation of RIM1α [26]. A later study further confirmed this protein-protein interaction and identified that PKA phosphorylation of serine-413 at RIM1α (pSer413) is critical for 14-3-3 binding [27]. Moreover, electrophysiological assays in cultured cerebellar neurons suggested that recruitment of 14-3-3 to RIM1α at pSer413 is required for a presynaptic form of long-term potentiation (LTP) at granule cell and Purkinje cell synapses in the mouse cerebellum [27-29]. However, apparently contradictory evidence came from later efforts to examine the involvement of 14-3-3 and RIM1α interaction in presynaptic long-term plasticity using in vivo animal models. In one of these studies, a line of knock-in mice was generated to substitute RIM1α serine-413 with alanine (S413A), thereby abolishing RIM1α phosphorylation at S413 and 14-3-3 binding. Surprisingly, electrophysiological examination of the RIM1α S413A knock-in mice failed to detect a significant defect in presynaptic LTP, either at parallel fiber or mossy fiber synapses [30]. In agreement with this finding, an acute in vivo rescue experiment showed that deficits of mossy fiber LTP in RIM1α−/− mice can be rescued by expression of the phosphorylation site-deficient mutant of RIM1α (S413A) [31]. Thus, it remains unclear whether 14-3-3 binding to S413 phosphorylated RIM1α plays a significant role in the regulation of presynaptic long-term plasticity.

A better-understood action of 14-3-3 at the presynaptic site is its role as the modulator of ion channels [32, 33], which include voltage-gated calcium (Ca2+) channels that play a central role in neurotransmitter release by mediating Ca2+ influx at nerve terminals [34]. In particular, CaV2.2 channels undergo cumulative inactivation after a brief, repetitive depolarization, thus markedly impacting the fidelity of synaptic transmission and short-term synaptic plasticity [35, 36]. 14-3-3 modulates inactivation properties of CaV2.2 channels through its direct binding to the channel pore-forming α1B subunit. In cultured rat hippocampal neurons, inhibition of 14-3-3 proteins in presynaptic neurons augments short-term depression, likely through promoting the closed-state inactivation of CaV2.2 channels (Figure 1) [37]. As 14-3-3 binding can be regulated by specific phosphorylation of the α1B subunit, this regulatory protein complex may provide a potential mechanism for phosphorylation-dependent regulation of short-term synaptic plasticity.

Figure 1

14-3-3 regulates presynaptic short-term plasticity by modulating CaV2.2 channel properties. 14-3-3 binding reduces cumulative inactivation of CaV2.2 channels and sustains Ca2+ influx and neurotransmitter release (a). Inhibition of 14-3-3 accelerates CaV2.2 channel inactivation and enhances short-term synaptic depression (b).

3. Functions of 14-3-3 at the Postsynaptic Site

The role of 14-3-3 at the postsynaptic site emerged more recently from the studies of various 14-3-3 mouse models. One of them, the 14-3-3 functional knockout (FKO) mice, was generated by transgenic expression of difopein (dimeric fourteen-three-three peptide inhibitor) that antagonizes the binding of 14-3-3 proteins to their endogenous partners in an isoform-independent manner, thereby disrupting 14-3-3 functions [38-41]. Transgene expression is driven by the neuronal-specific Thy-1 promoter which produces variable expression patterns in the brains of different founder lines, making it possible to assess the behavioral and synaptic alterations associated with expression of the 14-3-3 inhibitor in certain brain regions [42, 43]. Inhibition of 14-3-3 proteins in the hippocampus impairs associative learning and memory behaviors and suppresses long-term potentiation (LTP) at hippocampal CA3-CA1 synapses of the 14-3-3 FKO mice [41]. Through comparative analyses of two different founder lines with distinct transgene expression patterns in the subregions of the hippocampus, it was further determined that postsynaptic inhibition of 14-3-3 proteins may contribute to the impairments in LTP and cognitive behaviors. These observations thus revealed a postsynaptic function for 14-3-3 proteins in regulating long-term synaptic plasticity in mouse hippocampus.

What might be the molecular targets of 14-3-3 proteins at the postsynaptic site of hippocampal synapses? In the 14-3-3 FKO mice, there is a significant reduction of the NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons which express the 14-3-3 inhibitor. Consistently, the level of NMDA receptors (NMDARs), particularly GluN1 and GluN2A subunits, is selectively reduced in the postsynaptic density (PSD) fraction of 14-3-3 FKO mice that exhibit deficits in cognitive behaviors and hippocampal LTP [41]. Considering the critical role that NMDARs play in mediating LTP at hippocampal CA3-CA1 synapses [44], 14-3-3 proteins likely exert their effects on postsynaptic sites through the regulation of NMDA receptors, either directly or indirectly (Figure 2).

Figure 2

14-3-3 regulates NMDA receptors and actin dynamics at postsynaptic sites. (1) 14-3-3 proteins facilitate targeting of NMDARs to the postsynaptic density, thereby regulating long-term potentiation; (2) 14-3-3 proteins might promote spinogenesis by facilitating F-actin formation.

NMDARs are heterotetramers composed of two obligatory GluN1 subunits and two regulatory subunits derived from GluN (GluN2A-2D) and GluN3 subunits [45, 46]. 14-3-3 is known to promote surface expression of NMDA receptors in cerebellar neurons through its interaction with PKB-phosphorylated GluN2C subunits [47]. A more recent study also showed that inhibiting endogenous 14-3-3 proteins using difopein greatly attenuate GluN2C surface expression in cultured hippocampal neurons [48]. However, it remains to be determined whether 14-3-3 proteins directly interact with other subunits of NMDAR and have similar effects on their surface expression. Alternatively, 14-3-3 might indirectly regulate the PSD level of NMDARs by modulating other critical steps of NMDAR synaptic trafficking, such as dendritic transport and synaptic localization [45, 49]. Therefore, further studies are needed to better understand the exact mechanism underlying 14-3-3 proteins' regulation of NMDA receptors. Interestingly, the synaptic level of certain 14-3-3 isoforms is reduced in GluN1 knockdown mice, but not by subchronic administration of an NMDAR antagonist in wild-type mice [50]. It raises a possibility that a reciprocal regulation between 14-3-3 and NMDARs may take place at the postsynaptic site.

14-3-3 proteins also modulate other glutamate receptors at the postsynaptic membrane. For example, 14-3-3 interacts with GluK2a, a subunit of the kainate receptor (KAR) that mediates postsynaptic transmission, synaptic plasticity, and neuronal excitability [51]. 14-3-3 binding slows desensitization kinetics of GluK2a-containing KARs. In 14-3-3 FKO mice, expression of the 14-3-3 inhibitor in CA3 neurons leads to a faster decay of KAR-EPSCs at hippocampal mossy fiber-CA3 synapses [52]. This study provides another potential mechanism by which 14-3-3 proteins regulate synaptic functions at the postsynaptic site.

In addition to modulating the level and biophysical properties of postsynaptic glutamate receptors, 14-3-3 functions by regulating synaptogenesis. In the 14-3-3 FKO mice, there is a reduction of both dendritic complexity and spine density in the cortical and hippocampal neurons where the 14-3-3 inhibitor is extensively expressed [53]. A similar reduction in dendritic spine density was observed in 14-3-3ζ-deficient mice in BALB/c background [54, 55]. On the contrary, overexpressing 14-3-3ζ in rat hippocampal neurons significantly increases spine density [56]. Collectively, studies on these animal models provide in vivo evidence for a significant role of 14-3-3 proteins in promoting the formation and maturation of dendritic spines.

While the molecular mechanism for 14-3-3 dependent regulation of synaptogenesis remains elusive, several in vitro studies have proposed 14-3-3 proteins as important regulators of cytoskeleton and actin dynamics, which are critical for controlling the shape, organization, and maintenance of dendritic spines in postsynaptic neurons [57, 58]. Earlier studies showed that 14-3-3ζ regulates actin dynamics through its direct interaction with phosphorylated cofilin (p-cofilin) [57]. Cofilin is a major actin depolymerizing factor. Reduction of p-cofilin enhances the activity of cofilin, promotes the turnover of actin filaments, and consequently destabilizes dendritic spines [55, 56]. Moreover, a different group identified cofilin and its regulatory kinase LIM-kinase 1 (LIMK1) as binding partners of 14-3-3ζ and suggested that interactions with the C-terminal region of 14-3-3ζ inhibit the binding of cofilin to F-actin [59]. However, a direct interaction between 14-3-3 and cofilin/p-cofilin was challenged by a later study, in which Sudnitsyna et al. demonstrated that 14-3-3 only weakly interacts with cofilin, and they suggested that 14-3-3 proteins most likely regulate actin dynamics through other regulatory kinases such as LIMK1 or slingshot 1 L phosphatase (SSH) [60]. In fact, 14-3-3ζ has been shown to directly bind with phosphorylated SSH and lower its ability to bind F-actin [58].

More recently, Toyo-oka et al. showed that 14-3-3ε and 14-3-3ζ bind to δ-catenin and potentially regulate actin dynamics through δ-catenin [11, 61]. Catenin activates the Rho family of GTPase that results in the phosphorylation and activation of LIMK1. Loss of 14-3-3 proteins results in stabilization of δ-catenin through the ubiquitin-proteasome system, thereby decreasing LIMK1 activity and reducing p-cofilin level. Therefore, it is possible that 14-3-3 proteins may promote F-actin formation and spinogenesis by interacting with multiple elements in the regulatory pathways of the actin polymerization/depolymerization cycles (Figure 2).

4. Conclusion

The glutamatergic synapses mediate the majority of excitatory neurotransmission in the mammalian brain. Regulation of the property and connectivity of glutamatergic synapses represents a major mechanism for activity-dependent modification of synaptic strength and is critical for higher brain functions. 14-3-3 proteins have emerged as one of the important modulators at these synapses. It is particularly interesting that 14-3-3 binding and function are generally regulated by phosphorylation, which is a well-established molecular process underlying synaptic plasticity. Thus, 14-3-3 can potentially integrate multiple signaling pathways and plays a significant role in dynamic modification of glutamatergic synapses. As demonstrated by recent animal models, 14-3-3 deficiencies in rodent brain often result in the onset of abnormal behaviors, which might correspond to symptoms of neurological disorders.