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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: J Neurochem. 2019 Aug 25;151(3):289–300. doi: 10.1111/jnc.14840

C2-lacking isoform of Nedd4-2 regulates excitatory synaptic strength through GluA1 ubiquitination-independent mechanisms

Jiuhe Zhu 1, Kwan Young Lee 1, Tiffany T Jong 1, Nien-Pei Tsai 1,2,*
PMCID: PMC6819230  NIHMSID: NIHMS1043875  PMID: 31357244

Abstract

Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4–2) is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. Nedd4–2’s substrates include many ion channels and receptors because its N-terminal C2 domain guides Nedd4–2 to the cell membrane. We previously found that Nedd4–2 ubiquitinates the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which leads to reduction of neuronal excitability and seizures in mice. However, despite awareness of a Nedd4–2 isoform with no C2 domain, the functions of this isoform remain elusive. In this study, we showed that the C2-lacking Nedd4–2 has reduced membrane distribution and exhibits reduced affinity toward ubiquitinating GluA1. However, when expressed in primary cortical neurons, we found that the C2-lacking Nedd4–2 exhibits a similar activity toward reducing excitatory synaptic strength as does the C2-containing Nedd4–2. In an attempt to identify novel Nedd4–2 substrates that could mediate excitatory synaptic strength, we used unbiased proteomic screening and found multiple synaptic regulators that were up-regulated in the brain of conditional Nedd4–2 knockout mice, including protein phosphatase 3 catalytic subunit-α (PPP3CA; alternately called calcineurin A-α). We confirmed PPP3CA as a substrate of the C2-lacking Nedd4–2 and showed that all three epilepsy-associated missense mutations of Nedd4–2 disrupted PPP3CA ubiquitination. Altogether, our results revealed novel potential Nedd4–2 substrates and suggest that the C2-lacking Nedd4–2 represses excitatory synaptic strength most likely through GluA1 ubiquitination-independent mechanisms. These findings provide novel information to further our knowledge about Nedd4–2-dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4–2 is compromised.

Keywords: Nedd4-2, Ubiquitination, GluA1, AMPAR, Calcineurin, Epilepsy

Graphical Abstract

Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4–2) is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. This current study revealed that two major isoforms of Nedd4–2 function to reduce excitatory synaptic strength through different mechanisms. The findings provide novel information to further our knowledge about Nedd4–2-dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4–2 is compromised.

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INTRODUCTION

Nedd4–2 is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase (Allen et al. 2013). The mechanism by which Nedd4–2 disruption or mutations lead to seizures and/or epilepsies was unclear until our recent discovery of the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor; AMPAR) as a new substrate of Nedd4–2 (Zhu et al. 2017). Using a mouse model, in which the predominant form of Nedd4–2 (the full-length Nedd4–2) in the brain is deficient, it was shown that both GluA1 and seizure susceptibility were elevated. Most importantly, genetic reduction of GluA1 normalizes seizure susceptibility in Nedd4–2-deficient mice (Zhu et al. 2017). These findings suggest AMPAR is a key substrate of full-length Nedd4–2 in the regulation of brain circuit excitability and introduce a critical role of Nedd4–2 at post-synapses. However, knowledge of other Nedd4–2 isoforms in the brain remains limited.

Another major isoform of Nedd4–2, in which a N-terminal C2 domain is missing, has also been identified in both human and rodent models (Garrone et al. 2009, Dahlberg et al. 2007). This C2-lacking isoform is believed to be expressed through a different mRNA transcript with the amino acids encoded from exons 1 to 6 of the full-length Nedd4–2 transcript were omitted due to the use of a different translation initiation codon (Fu et al. 2013). The C2 domain is known to interact with negatively charged phospholipids and therefore commonly guides the protein to the plasma membrane (Corbalan-Garcia & Gomez-Fernandez 2014). Due to this interaction, C2-containing proteins often interact with membrane-associated proteins, such as ion channels and membrane receptors, and participate in cell–cell communication and intracellular signaling pathways. This is particularly important for the nervous system in which large amount of ion channels are expressed and constantly require dynamic regulation. As our previous studies reported, the C2-containing, full-length, Nedd4–2 interacts with and ubiquitinates GluA1, and negatively regulates excitatory synaptic strength in the hippocampus and cortex (Lee et al. 2018). Nevertheless, it is unclear whether Nedd4–2’s C2-lacking isoform could play a role in excitatory synapses and/or neurons. Because single-nucleotide polymorphisms (SNPs) in human Nedd4–2 lead to differential expression of the C2-containing and C2-lacking isoforms (Garrone et al. 2009, Dahlberg et al. 2007), examining their functional differences may aid our understanding of neuronal excitability regulation and seizure susceptibility in different populations.

In the current study, we showed that the C2-lacking Nedd4–2 showed a reduced distribution at cell membranes and extremely low affinity in ubiquitinating GluA1. Surprisingly, we found that the C2-lacking Nedd4–2 exhibits similar activity toward reducing excitatory synaptic strength when compared to the C2-containing Nedd4–2. Using proteomic screening, we identified multiple potential substrates of the C2-lacking Nedd4–2 in the cytoplasm, including calcineurin A that could mediate excitatory synaptic strength (Woolfrey & Dell’Acqua 2015). Our findings therefore indicate that both C2-containing and C2-lacking Nedd4–2s function to reduce excitatory synaptic strength although most likely via distinct mechanisms. These results further support our conclusion in which we speculated that Nedd4–2-associated neuronal hyperexcitability and seizure susceptibility are critically influenced by elevated excitatory synaptic transmission when the functions of Nedd4–2, both C2-containing and C2-lacking isoforms, are compromised.

MATERIALS AND METHODS

All experiments using animal data followed the guidelines of Animal Care and Use provided by the Illinois Institutional Animal Care and Use Committee (IACUC) and the guidelines of the Euthanasia of Animals provided by the American Veterinary Medical Association (AVMA) to minimize animal suffering and the number of animals used. This study was performed under an approved IACUC animal protocol of University of Illinois at Urbana-Champaign (#14139 and #17075 to N.-P. Tsai.).

Reagents and cell culture

Dimethyl sulfoxide (DMSO) was from Thermo Fisher Scientific (catalog: BP231–100). Tetrodotoxin citrate (TTX) was from Cayman Chemicals (catalog: 14964). Ionomycin was from AdipoGen (catalog: AG-CN2–0416-M001). The antibodies used in this study were purchased from Cell Signaling (anti-Nedd4–2, RRID: AB_1904063; anti-pan-14-3-3, RRID: AB_10860606; anti-Myc, RRID: AB_490778; anti-N-Cadherin, RRID: AB_2798427; anti-HA, RRID: AB_10691311, and anti-Ub, RRID: AB_331292), Millipore (anti-GluA1-N-terminus, RRID: AB_1977459), Santa Cruz Biotechnology (anti-α-tubulin, RRID: AB_628411), and Proteintech (anti-Gapdh, RRID: AB_2107436), and ABclonal (anti-PPP3CA, RRID: AB_2758155). Human embryonic kidney (HEK 293) cells were from ATCC (ATCC #CRL-1573, RRID: CVCL_0045). HEK 293 cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC) and was not authenticated in this study. Cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma, catalog: 10017CV) with 10% Fetal Bovine Serum (JM Bioscience, catalog: 100–500). Cells were used between passages 4 to 25 and kept at 37°C in a humidified incubator containing 5% CO2.

Constructs

The C2-containing, full-length, Nedd4–2 (pCI-HA-Nedd4–2) was obtained from Addgene (RRID: Addgene_27000). The C2-lacking Nedd4–2 was PCR-amplified using pCI-HA-Nedd4–2 as the template and the primer pair (5’-CCGGTACCGAGCGACCCTATACATTTAAG-3’ and 5’-CGAAGCGGCCGCTTAATC-3’) into KpnI/NotI sites of pCI vector. The C2-lacking Nedd4–2 carrying epilepsy-associated mutations were generated with same method above but using pCI-HA-Nedd4–2s that carry those mutations, constructed previously (Zhu et al. 2017), as templates. Amplification was performed in a thermal cycler using the following program: 1 cycle of 3 min at 95°C; 35 cycles of 30 sec at 95°C, 30 sec at 55°C, 2 min 30 sec at 72°C, and 1 cycle of 10 min at 72°C.

In vitro ubiquitination

HA-Ub (Boston Biochem, catalog: U-110), Ubiquitin Activating Enzyme (UBE1) (Boston Biochem, catalog: E-305) and UbcH5b/UBE2D2 (Boston Biochem, catalog: E2–622) were obtained. When HA-tagged C2-lacking Nedd4–2 was produced in HEK cells, 250 μg of total protein lysates were subjected to immunoprecipitation with anti-HA antibody to partially purify HA-tagged C2-lacking Nedd4–2. Recombinant PPP3CA (Sino Biological, catalog: 13670-H07B) was used as substrate for in vitro ubiquitination with C2-lacking Nedd4–2 obtained from transfected HEK cells following a protocol previously described (Woelk et al. 2006).

Mice and genotyping

WT mice in C57BL/6J (RRID: IMSR_JAX:000664), BALB/c (RRID: IMSR_JAX:001026), 129S1 (RRID: IMSR_JAX:002448) background, and Emx1-Cre mice (RRID: IMSR_JAX:022762) in C57BL/6J background were obtained from The Jackson Laboratory. Nedd4–2 floxed mice were generated and obtained from Dr. Hiroshi Kawabe (Max Planck Institute). For genotyping, the primers used to detect Nedd4–2 loxP allele are: 5’-TCCCCACTGCAGTTCCTACC-3’, and 5’-AGCTGCTCAGGCTGAATCACC-3’. PCR was performed using the program as below: 3 min at 94°C; 5 cycles of 20 sec at 94°C, 20 sec at 65°C, 2 min at 72°C; 5 cycles of 20 sec at 94°C, 20 sec at 60°C, 2 min at 72°C; 25 cycles of 20 sec at 94°C, 20 sec at 55°C, 2 min at 72°C; and 10 min at 72°C. The primers used to detect Emx1 allele are 5’-CGGTCTGGCAGTAAAAACTATC-3’ (Emx1-Cre), 5’-GTGAAACAGCATTGCTGTCACTT-3’ (Emx1-Cre); 5’-AAGGTGTGGTTCCAGAATCG-3’ (Wild type), and 5’-CTCTCCACCAGAAGGCTGAG-3’ (Wild type). The PCR program is as below: 2 min at 94°C; 10 cycles of 20 sec at 94°C, 15 sec at 65°C (with 0.5°C per cycle decrease), 10 sec at 68°C; 28 cycles of 15 sec at 94°C 15 sec at 60°C, 10 sec at 72°C; and 2 min at 72°C.

Primary neuron cultures

Primary neuron cultures were made from mixed-sex mice aged at p0–p1 as described previously (Liu et al. 2017) and maintained in NeuralA basal medium (Thermo Fisher, catalog: 10888022) supplemented with B27 supplement (Invitrogen, catalog: 17504001), GlutaMax (final concentration at 2 mM; Invitrogen, catalog: 35050061), and Cytosine β-D-arabinofuranoside (AraC, final concentration at 2 μM; Sigma, catalog: C1768–100MG). Each set of cultures was made by pooling 4–6 cortices before plating into multiple 35-mm petri dishes depending on the need of each experimental. Each experiment was performed using sister cultures made from the same litter. The culture medium was changed 50% on days-in-vitro (DIV) 2 and every 3–4 days thereafter until the experiments on DIV 14–16.

Proteomics

The proteomic screening was conducted using cytoplasmic fractions of WT and Nedd4–2 conditional knockout mouse brains with label-free analyses provided by Bioproximity. In brief, after adding trypsin at a ratio of 1:50 to the samples, and the solution was incubated at 37°C overnight. The peptides were then extracted, lyophilized and resuspended in 2–20 μL of 0.1% formic acid. Ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) was then carried out by Easy-nLC1200 and HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer. The raw data files were analyzed and searched against uniprot-Mus musculus protein databases.

Membrane protein extraction

Membrane protein extraction assay was conducted by the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, catalog: 89842) and according to manufacturer’s protocol with minor modification. Cortical neuron cultures at DIV 14 or whole brains were harvested in 1 mL of Cell Wash Solution. After washing and a centrifuge at 300×g for 5 min, cell or tissue pellet was resuspended in 500 μL of Permeabilization Buffer. The cell or tissue suspension was then incubated at 4 °C for 10 min with constant rotation. Permeabilized cells were subjected to a centrifuge for 15 min at 16,000×g at 4°C. The supernatant containing cytoplasmic proteins was transferred to a clean tube and saved as cytoplasmic fractions. The pellet was resuspended in 300 μL of Solubilization Buffer by pipetting up and down, followed by an incubation of 10 min at 4°C with constant rotation. After another centrifuge at 16,000×g for 15 min at 4℃, the supernatant containing solubilized membrane and membrane-associated proteins was collected.

Whole-cell patch-clamp recording

Whole-cell patch-clamp recordings were performed, as we previous described (Jewett et al. 2018, Tsai et al. 2017), at room temperature (23–25°C) in a submersion chamber continuously perfused with artificial cerebrospinal fluid (aCSF) containing (as in mM): 119 NaCl, 2.5 KCl, 4 CaCl2, 4 MgCl2, 1 NaH2PO4, 26 NaHCO3 and 11 D-Glucose, saturated with 95% O2/5% CO2 (pH 7.4, 310 mOsm). aCSF was supplemented with 1 μM TTX and 100 μM picrotoxin for mEPSC measurements to block action potentials and GABAA receptors, respectively. Whole cell recording pipettes (~4–6 MΩ) were filled with intracellular solution containing (as in mM): 130 K-gluconate, 6 KCl, 3 NaCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.4 Na-GTP, 14 Tris-phosphocreatine, 2 QX-314 (pH 7.25, 285 mOsm). Neurons at DIV 14–16 were used for electrophysiological analyses. Membrane potential was clamped at –60 mV. Neurons were excluded in analyses when the resting membrane potential was > –45 mV, access resistance was > 30 MΩ, or if access resistance changed by > 20%. All recordings were performed with Clampex 10.6 and Multiclamp 700B amplifier interfaced with Digidata 1440A data acquisition system (Molecular Devices). Recordings were filtered at 1 kHz and digitized at 10 kHz. Miniature excitatory postsynaptic currents (mEPSCs) were analyzed with Mini Analysis Program (Synaptosoft) with a 5-pA threshold level.

Transfection, immunoprecipitation and western blotting

For transient transfection, HEK 293 and primary cortical neurons were transfected using Lipofectamine 3000 (Invitrogen, catalog: L3000015). Specifically for neurons, cells were transfected at DIV 11 (for electrophysiology experiments) for 60 minutes followed by replacing the culture medium with conditioned medium to minimize toxicity produced by Lipofectamine. For immunoprecipitation (IP), HEK cell lysates were obtained by sonicating pelleted cells in an IP buffer (50 mM Tris, pH 7.4, 120 mM NaCl, 0.5% Nonidet P-40). One hundred μg of total protein was incubated 2 hours at 4 °C with 0.5 μg antibodies. Protein A/G agarose beads (from Santa Cruz Biotechnology, RRID: AB_10201400) were added for another hour followed by washing with IP buffer three times. Samples of immunoprecipitation or cell lysates were then mixed in a buffer (40% Glycerol, 240 mM Tris/HCl pH 6.8, 8% sodium dodecyl sulfate, 0.04% bromophenol blue and 5% β-mercaptoethanol) and boiled for 5 min. After cooling down on ice for another 5 min, the samples were loaded onto 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. For Western blotting, after SDS-PAGE, the gel was transferred onto a polyvinylidene fluoride membrane. After blocking with a 1% Bovine Serum Albumins (from Thermo Fisher Scientific, catalog: BP9706100) solution in TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20), the membrane was incubated with primary antibody overnight at 4 °C, followed by a 30-min washing with PBS on the next day. The membrane was then incubated with an HRP-conjugated secondary antibody (from Cell Signaling, RRID: AB_330924) in 5% non-fat milk in TBST for 1 hour at room temperature, followed by another 30-min washing. Finally, the membrane was developed with an ECL Chemiluminescent Reagent.

Statistical Analysis

At least 3 litters, 4–6 mice each, were used in each experiment. Due to the design of our research, no blinding was performed. The data presented in this study have been tested for normality using Kolmogorov-Smirnov Test. Statistical methods to determine significance along with sample numbers were indicated in each figure legend. In brief, Student’s t-test was used for paired samples and one sample t-test was used for conditions where experimental groups are normalized to control groups. One-way ANOVA with post-hoc Tukey HSD (Honest Significant Differences) test was used for multiple comparisons. A Grubbs’ outlier test was conducted on the data and, as a result, one data point in Fig. 5C was removed. Prism software (RRID: SCR_002798) was used for analysis. Differences are considered significant at the level of P < 0.05.

Figure 5. Calcium influx does not promote membrane association of, or GluA1 ubiquitination by, C2-lacking Nedd4–2.

Figure 5.

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(A) Western blots of Nedd4–2, N-Cadherin and Gapdh from membrane and cytoplasm fractions of WT cortical neuron cultures treated with DMSO or ionomycin (1 μM) for 1 hour. (n = 3 sets of cell culture dishes from 3 independent preparations). (B) Western blots of Nedd4–2, N-Cadherin and Gapdh from membrane and cytoplasm fractions of HEK cells transfected with C2-containing or C2-lacking Nedd4–2 for 48 hours followed by DMSO or ionomycin (1 μM) treatment for 1 hour (n = 3 sets of cell culture dishes from 3 independent preparations). (C) Western blots of ubiquitin (Ub) after immunoprecipitation using anti-GluA1 antibody from HEK cells transfected with GluA1+C2-containing Nedd4–2 or GluA1+C2-lacking Nedd4–2 for 48 hours followed by DMSO or ionomycin (1 μM) treatment for 1 hour (n = 9 sets of cell culture dishes from 5 independent preparations). Data were analyzed using Student’s t-test and represented as mean ± SEM with *p < 0.05 and ns: non-significant.

RESULTS

C2-lacking Nedd4–2 has reduced association with membranes.

Studies have reported the observation of two major Nedd4–2 splice variants (isoforms 1 and 2) in human and rodent models (Garrone et al. 2009, Dahlberg et al. 2007). The proteins encoded by these two transcripts are predicted to be roughly 20 kDa different in molecular weight. The long-form, encoded by isoform 1, contains a N-terminal C2 domain while the short-form, encoded by isoform 2, does not (Fig. 1A). We confirmed the co-existence of these two protein isoforms in multiple commonly used mouse models, including C57BL/6J, BALB/c, and 129S1 mice (black, white, and brown mice, respectively) (Fig. 1B). We chose C57BL/6J for the rest of our experiments due to the same genetic background as other transgenic mouse models that will be used later in this study.

Figure 1. C2-lacking Nedd4–2 has reduced association with membranes in comparison to C2-containing Nedd4–2.

Figure 1.

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(A) A diagram showing two alternative protein products from the Nedd4–2 gene and their domains. (B) Western blots of Nedd4–2 and Gapdh from total brain lysates of C57BL/6J, BALB/c and 129S1 mice. (C) Western blots of Nedd4–2, N-Cadherin and Gapdh from membrane fractions of C57BL/6J mouse brains. For the analysis, a one-sample t-test was used (n = 4 mouse brains). Data are represented as mean ± SEM with *p<0.05, ns: non-significant.

The C2 domain is involved in targeting proteins to cell membranes (Corbalan-Garcia & Gomez-Fernandez 2014), and Nedd4–2 has many membrane-associated substrates reported previously (Manning & Kumar 2018). In order to determine whether the C2-lacking isoform of Nedd4–2 indeed showed less association with membranes, we isolated total membrane fractions from wildtype (WT) C57BL/6J mouse brains. As shown, in comparison to C2-lacking Nedd4–2, the C2-containing Nedd4–2 showed much higher distribution in membrane-enriched fractions, supporting the possibility that the C2-containing and C2-lacking Nedd4–2s function differently in the brain.

C2-lacking Nedd4–2 represses excitatory synaptic strength.

We previously showed that the C2-containing Nedd4–2 is capable of ubiquitinating the GluA1 subunit of AMPA receptors (Zhu et al. 2017) and reducing excitatory synaptic strength (Lee et al. 2018). In order to determine whether and the way in which the C2-lacking Nedd4–2 functions to affect excitatory synaptic transmission, we transiently expressed C2-containing or C2-lacking Nedd4–2 into primary cortical neuron cultures prepared from WT C57BL/6J mice. Forty-eight hours after transfection, we used whole-cell patch-clamp recording in order to obtain miniature excitatory post-synaptic currents (mEPSC). As shown in Fig. 2, neurons receiving either C2-containing or C2-lacking Nedd4–2 showed significant reductions in mEPSC amplitude without changes in mEPSC frequency (Supplemental Figure S1) when compared to control neurons receiving only green fluorescence protein (GFP). These results indicate that C2-lacking Nedd4–2 can function to limit excitatory synaptic strength in a manner similar to C2-containing Nedd4–2.

Figure 2. C2-lacking Nedd4–2 suppresses excitatory synaptic transmission.

Figure 2.

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(A-C) Patch-clamp recording from WT mouse cortical neurons transfected with GFP, GFP+C2-containing Nedd4–2 and GFP+C2-lacking Nedd4–2 for 48 hours. Representative miniature excitatory postsynaptic current (mEPSC) traces (A), cumulative probability of mEPSC amplitude (B) and quantification of mEPSC amplitude (C) are shown (n = 14–16 cells from 3 independent cultures). Data were analyzed using a one-way ANOVA with Tukey test and represented as mean ± SEM with *p < 0.05, **p < 0.01, and ns: non-significant.

To further determine whether the effect of C2-lacking Nedd4–2 on mEPSC amplitude is dependent on C2-containing Nedd4–2, we expressed C2-lacking Nedd4–2 in the absence of C2-containing Nedd4–2. To this end, we first obtained Nedd4–2 conditional knockout (cKO) mice by crossing Nedd4–2 floxed mice (Nedd4–2f/f) with Emx1-Cre mice. Emx1-Cre can confer Nedd4–2 reduction in the cortex and hippocampus beginning as early as embryonic 10.5 (E10.5) (Gorski et al. 2002, Young et al. 2007). The reduction of Nedd4–2 in Nedd4–2 cKO (Nedd4–2f/f Emx1+) cultures was confirmed in Figure 3A. We subsequently expressed C2-containing or C2-lacking Nedd4–2 into Nedd4–2 cKO cultures, followed by mEPSC recordings. As shown (Fig. 3B–D and Supplemental Figure S2), both C2-containing and C2-lacking Nedd4–2 are capable of reducing mEPSC amplitude without changes in mEPSC frequency. These data conclude that the effect of C2-lacking Nedd4–2 isoform on reducing mEPSC amplitude is independent of C2-containing Nedd4–2.

Figure 3. C2-lacking Nedd4–2 suppresses excitatory synaptic transmission independent of C2-containing Nedd4–2.

Figure 3.

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(A) Representative western blots of Nedd4–2 from Nedd4–2 WT (Nedd4–2f/f Emx1-) and Nedd4–2 cKO (Nedd4–2f/f Emx1+) cortical neuron cultures (DIV 14). (B-D) Patch-clamp recording from Nedd4–2 cKO mouse cortical neurons transfected with GFP, GFP+C2-containing Nedd4–2 and GFP+C2-lacking Nedd4–2 for 48 hours. Representative miniature excitatory postsynaptic current (mEPSC) traces (B), cumulative probability of mEPSC amplitude (C) and quantification of mEPSC amplitude (D) are shown (n = 22–23 cells from 4 independent cultures). Data were analyzed using a one-way ANOVA with Tukey test and represented as mean ± SEM with *p < 0.05, and ns: non-significant.

C2-lacking Nedd4–2 has low activity toward ubiquitinating GluA1

Because C2-containing Nedd4–2 ubiquitinates GluA1, it is logical to speculate that C2-lacking Nedd4–2 may also ubiquitinate GluA1 to repress excitatory synaptic strength as we showed in Fig. 2. To this end, we employed human embryonic kidney (HEK 293) cells, which do not express Nedd4–2 or GluA1 endogenously, to determine whether C2-lacking Nedd4–2 can ubiquitinate GluA1. As shown in Fig 4A, in comparison to being co-expressed with C2-containing Nedd4–2, the GluA1 co-expressed with C2-lacking Nedd4–2 exhibited significantly lower ubiquitination. Because GluA1 ubiquitination negatively affects the amount of GluA1 on the cell membrane (Lin et al. 2011, Schwarz et al. 2010), we also determined the amount of GluA1 associated with membrane when co-expressed with either isoform of Nedd4–2s. As shown in Figure 4B, in comparison to being co-expressed with C2-containing Nedd4–2, the GluA1 co-expressed with C2-lacking Nedd4–2 exhibited a significantly higher level in the membrane fractions. These results conclude that C2-lacking Nedd4–2 has a relatively low affinity for ubiquitinating GluA1.

Figure 4. C2-lacking Nedd4–2 exhibits significantly lower activity toward ubiquitinating GluA1.

Figure 4.

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(A) Western blots of ubiquitin (Ub) after immunoprecipitation using anti-GluA1 antibody from HEK cells transfected with GluA1, GluA1+C2-containing Nedd4–2 or GluA1+C2-lacking Nedd4–2 for 48 hours (n = 5 sets of cell culture dishes from 4 independent preparations). (B) Western blots of GluA1, Nedd4–2, N-Cadherin and Gapdh from total cell lysates and membrane fractions of HEK cells transfected with GluA1 along with C2-containing or C2-lacking Nedd4–2 for 48 hours (n = 4 sets of cell culture dishes from 2 independent preparations). (C) Western blots of Nedd4–2 after co-immunoprecipitation of 14-3-3 using lysates from HEK cells transfected with C2-containing or C2-lacking Nedd4–2 for 48 hours (n = 4 sets of cell culture dishes from 4 independent preparations). Data were analyzed using Student’s t-test and represented as mean ± SEM with *p < 0.05.

Nedd4–2 is known to interact with multiple isoforms of the adaptor protein 14-3-3 (Liang et al. 2006, Liang et al. 2008, Ichimura et al. 2005) and we have previously shown that C2-containing Nedd4–2 requires 14-3-3 in order to interact with GluA1 (Zhu et al. 2017, Lee et al. 2018). In order to determine whether C2-lacking Nedd4–2 has a reduced interaction with 14-3-3, we expressed C2-containing or C2-lacking Nedd4–2 into HEK cells followed by co-immunoprecipitation with an anti-pan 14-3-3 antibody. As shown in Fig. 4C, C2-lacking Nedd4–2 exhibited significantly lower association with 14-3-3 in comparison to C2-containing Nedd4–2. Together, our results suggest that C2-lacking Nedd4–2 shows significantly reduced activity toward ubiquitinating GluA1, which could be due to a reduced interaction with one or multiple isoforms of the adaptor protein 14-3-3.

Nedd4–2’s sub-cellular distribution and substrate recognition are known to be mediated by calcium (Ca2+) influx (Boehmer et al. 2003, Wang et al. 2010). This is particularly apparent for C2-containing Nedd4–2 because the C2 domain binds to Ca2+, which subsequently leads to enhanced membrane interaction with C2-containing Nedd4–2 (Garrone et al. 2009). In order to determine whether GluA1 ubiquitination by C2-containing and C2-lacking Nedd4–2s is modulated by Ca2+ influx, we utilized ionomycin, a Ca2+ ionophore, which has been shown to efficiently affect membrane distribution of Nedd4–2 in Xenopus (Garrone et al. 2009). We first aimed to evaluate whether ionomycin treatment facilitated the targeting of Nedd4–2 to membrane compartments in neurons. As shown in Fig. 5A, primary cortical neuron cultures treated with ionomycin (1 μM, 1 hr) exhibited no significant difference in distribution of C2-containing and C2-lacking Nedd4–2 in the membrane fractions. Similar results were obtained when we employed HEK cells transfected with C2-containing or C2-lacking Nedd4–2 followed by ionomycin treatment for 1 hr (Fig. 5B). These results suggest that the membrane distribution of Nedd4–2 might be differentially regulated in mammalian cells, as opposed to the Xenopus cells (Garrone et al. 2009). However, when we studied GluA1 ubiquitination in HEK cells, we found that GluA1 ubiquitination was significantly elevated when GluA1 was co-expressed with C2-containing but not C2-lacking Nedd4–2 (Fig. 5C). The results suggest that despite the absence of significant changes in Nedd4–2’s association with membranes, Ca2+ influx promotes C2-containing Nedd4–2-mediated ubiquitination of GluA1. This could potentially be resulted from the crosstalk between Ca2+ and 14-3-3, as the functions of 14-3-3 are known to be affected by Ca2+ (Zhitomirsky et al. 2018, Li et al. 2019). The results, together with our findings in Fig. 4, led to our hypothesis in which C2-lacking Nedd4–2 represses excitatory synaptic strength most likely through GluA1 ubiquitination-independent mechanisms.

Unbiased proteomic screening identifies potential substrates of Nedd4–2 in regulating synaptic strength

In order to identify potential mechanisms by which C2-lacking Nedd4–2 represses excitatory synaptic strength, we decided to search for novel Nedd4–2 substrates using an unbiased proteomic approach. To this end, we obtained cytoplasmic fractions from whole brains of Nedd4–2 WT (Nedd4–2f/f Emx1-Cre−) and Nedd4–2 cKO (Nedd4–2f/f Emx1+) littermate mice (Fig. 6A) for quantitative proteomic profiling. From two pairs of Nedd4–2 WT and cKO littermate mice, we obtained quantitative measurements for a total of 414 proteins that were detected in all four mice. Among those proteins, 34 proteins exhibited at least a two-fold increase, and 15 proteins exhibited at least a 50% reduction in Nedd4–2 cKO compared to WT (Fig 6B and Supplemental Table S1). Further, among those proteins with obvious changes in expression levels, eight of them have known functions that affect excitatory synaptic transmission through pre-synaptic and/or post-synaptic mechanisms (Fig. 6C). Up-regulation of some or all of these proteins in the Nedd4–2 cKO brain could explain altered excitatory synaptic strength in neurons transfected with Nedd4–2. Further studies would be needed to characterize the relationship between Nedd4–2 and each of its potential substrates.

Figure 6. Proteomic screening to identify potential substrates of Nedd4–2 in the cytoplasm.

Figure 6.

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(A) Western blots of Nedd4–2, N-Cadherin and Gapdh from total lysates and cytoplasmic fractions from Nedd4–2 WT (Nedd4–2f/f Emx1-) and Nedd4–2 cKO (Nedd4–2f/f Emx1+) mouse brains. (B) A pie chart summarizing the proteomic screening results using the cytoplasmic fractions from two pairs of Nedd4–2 WT and Nedd4–2 cKO mouse brains. (C) A heat map showing the levels of 8 proteins potentially involved in regulation of excitatory synaptic transmission and regulated by Nedd4–2.

C2-lacking Nedd4–2 ubiquitinates PPP3CA

In order to begin testing the possibility that C2-lacking Nedd4–2 ubiquitinates one of the proteins that we identified in Fig. 6C, we chose to study protein phosphatase 3 catalytic subunit-α (PPP3CA) or alternatively called calcineurin A-α. PPP3CA is encoded by an epilepsy-associated gene (Mizuguchi et al. 2018), and has been shown to either positively or negatively affect synaptic strength (Szabo et al. 2010, Kim & Ziff 2014). To first validate that PPP3CA is a potential substrate of Nedd4–2, we examined ubiquitination of endogenous PPP3CA in Nedd4–2 WT and cKO mouse cortices. As shown in Figure 7A, the ubiquitination of PPP3CA is drastically reduced in Nedd4–2 cKO cortex. To further examine the Nedd4–2-mediated ubiquitination of PPP3CA, we co-expressed C2-lacking Nedd4–2 and a Myc-tagged PPP3CA into HEK cells followed by immunoprecipitation with anti-Myc antibody and western blotting with anti-Ub antibody. As shown (Fig 7B), strong PPP3CA ubiquitination was observed when co-expressed with C2-lacking Nedd4–2, and such ubiquitination presumably leads to proteasomal degradation (Supplemental Figure S3). Similar ubiquitination of PPP3CA was also observed when the Myc-tagged PPP3CA was co-expressed with C2-containing Nedd4–2 (Supplemental Figure S4), suggesting PPP3CA is likely a substrate of both C2-containing and C2-lacking Nedd4–2s. To strengthen the conclusion that PPP3CA is a direct substrate of C2-lacking Nedd4–2, we conducted an in vitro ubiquitination assay with full-length, recombinant, His-tagged PPP3CA protein and C2-lacking Nedd4–2 proteins (partially purified from HEK cells). As shown in Figure 7C, the ubiquitination of PPP3CA is elevated in the presence of C2-lacking Nedd4–2. All these results above indicate PPP3CA is a substrate of C2-lacking Nedd4–2.

Figure 7. C2-lacking Nedd4–2 ubiquitinates PPP3CA.

Figure 7.

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(A) Western blots of ubiquitin (Ub) after immunoprecipitation of PPP3CA from Nedd4–2 WT (Nedd4–2f/f Emx1-) and Nedd4–2 cKO (Nedd4–2f/f Emx1+) mouse brain lysates. (B) Western blots of ubiquitin (Ub) after immunoprecipitation using anti-Myc antibody from HEK cells without transfection or transfected with Myc-PPP3CA or Myc-PPP3CA+C2-lacking Nedd4–2 for 48 hours. (C) Western blots of Ub or His after immunoprecipitation with anti-His antibody following in vitro ubiquitination with recombinant PPP3CA. HA-tagged C2-lacking Nedd4–2 used for in vitro ubiquitination were obtained from HEK cells transfected with C2-lacking Nedd4–2 followed by immunoprecipitation with anti-HA antibody. Coomassie blue staining showing the purity of recombinant PPP3CA is shown. (D) Western blots of ubiquitin (Ub) after immunoprecipitation using anti-Myc antibody from HEK cells transfected with Myc-PPP3CA along with WT C2-lacking Nedd4–2 or C2-lacking Nedd4–2 carrying one of the three epilepsy-associated missense mutations for 48 hours (n = 4 sets of cell culture dishes from 4 independent preparations). Data were analyzed using a one-way ANOVA with Tukey test and represented as mean ± SEM with *p < 0.05, ***p < 0.001.

We followed these experiments with additional testing as to whether three reported epilepsy-associated mutations on Nedd4–2 can affect PPP3CA ubiquitination. To this end, we generated expression constructs of C2-lacking Nedd4–2 carrying either of the three missense mutations (S233L, E271A, or H515P). These mutations, located on or near one of the three WW domains (protein-protein interaction domains), have been shown to affect the substrate binding affinity of C2-containing Nedd4–2 (Zhu et al. 2017), and we hypothesize that some or all of these mutations could disrupt PPP3CA ubiquitination. As shown in Figure 7D, in comparison to WT C2-lacking Nedd4–2, PPP3CA exhibited significantly lower ubiquitination when co-expressed with either of the mutant C2-lacking Nedd4–2s. These results indicate that the epilepsy-associated mutations can alter the ubiquitination of substrates of both C2-containing and C2-lacking Nedd4–2s. Because these mutations do not affect the ubiquitination of a voltage-gated sodium channel by Nedd4–2 (Dibbens et al. 2007), it suggests the impairment resulted from these mutations is likely substrate-dependent. Altogether, our findings support the likelihood that C2-lacking Nedd4–2 ubiquitinates one or more synaptic regulators leading to down-regulation of excitatory synaptic strength, and epilepsy-associated mutations of Nedd4–2 disrupt such regulation.

DISCUSSION

Our current study explored the functions of a C2-lacking isoform of Nedd4–2 in neurons. We found that despite a similar function toward reducing excitatory synaptic strength as the C2-containing Nedd4–2, the C2-lacking Nedd4–2 exhibits a much lower activity toward ubiquitinating GluA1. This is consistent with our data showing significantly reduced interaction between C2-lacking Nedd4–2 and 14-3-3, the adaptor protein necessary for Nedd4–2-dependent GluA1 ubiquitination (Zhu et al. 2017). Using an unbiased proteomic approach, we identified several potential new substrates of Nedd4–2 and confirmed one of them, PPP3CA, as a new substrate of the C2-lacking Nedd4–2. When PPP3CA is ubiquitinated, it is presumably degraded in the proteasome. However, to mechanistically understand other downstream effects when PPP3CA is ubiquitinated by Nedd4–2, a future study to generate mutant PPP3CAs that cannot be ubiquitinated by Nedd4–2 after mapping out the lysine residue(s) ubiquitinated by Nedd4–2 would be particularly important. In the current study, we showed that three epilepsy-associated mutations of Nedd4–2 disrupted PPP3CA ubiquitination, suggesting that dysregulation of PPP3CA, which is encoded by an epilepsy-associated gene, may contribute to Nedd4–2-associated epilepsy. Our data also suggest that those epilepsy-associated mutations on Nedd4–2, which are carried in both C2-containing and C2-lacking Nedd4–2 transcripts, may have much stronger effects on dysregulating excitatory synaptic strength than we initially expected as we now know that both isoforms function to repress synaptic strength. Although our data suggest that the C2-lacking Nedd4–2 does not directly ubiquitinate GluA1, many of the potential substrates of Nedd4–2 that we identified could indirectly affect the trafficking or stability of AMPARs, leading to altered excitatory synaptic strength. For example, PPP3CA is involved in GluA1 dephosphorylation (Kam et al. 2010), which is known to affect GluA1/AMPAR function (Goel et al. 2011, Machado et al. 2018). Adducin-1 is another protein shown to regulate AMPAR through modulation of its stability (Vukojevic et al. 2012). As a future direction, it would be of particular importance to further study the role of C2-lacking Nedd4–2 in AMPAR trafficking and stability, so we may develop therapies to properly treat patients who carry mutations in Nedd4–2.

Our data showed that C2-lacking Nedd4–2 primarily resides in non-membrane compartments as we predicted based on the lipid-binding property of C2 domain. Using proteomic screening, we indeed identified multiple potential substrates of Nedd4–2 that are primarily located in the cytoplasm (Fig. 6C and Supplemental Table S1). Among those, 14-3-3 epsilon is particularly interesting. 14-3-3 is known to be crucial to Nedd4–2-dependent ubiquitination (Liang et al. 2006, Liang et al. 2008, Ichimura et al. 2005, Zhu et al. 2017). 14-3-3 epsilon being a potential substrate of Nedd4–2 indicates a possibility that the C2-lacking Nedd4–2 may regulate the substrate recognition of both C2-lacking and C2-containing Nedd4–2s through ubiquitination of its own adaptor protein. To determine whether this possibility adds another layer of regulation toward GluA1 ubiquitination and synaptic hyperexcitability, we would need future work to first determine which isoform of 14-3-3 is more critical to Nedd4–2-dependent ubiquitination of GluA1. Although 14-3-3 epsilon and many other proteins identified in our proteomic screening may contribute to Nedd4–2-associated hyperexcitability when Nedd4–2 is compromised in epilepsy patients, we may still miss many other yet-to-be identified substrates of Nedd4–2 due to the limitation of proteomic approaches along with the low expression levels that are involved in regulation of synaptic strength. In addition, many studies have confirmed that protein ubiquitination can affect protein functions without causing protein degradation (Miranda & Sorkin 2007). With this notion, we may be missing substrates of Nedd4–2 that are functionally altered without being up-regulated in Nedd4–2 cKO mice. Other types of approaches, such as use of co-immunoprecipitation followed by mass spectrometry analyses, may be useful for identifying binding partners that could be substrates of Nedd4–2.

Because of membrane-binding capacity of Nedd4–2, many membrane proteins have been identified as the direct substrates of Nedd4–2 in various organs, including brain, lung, and kidney (Manning & Kumar 2018). Specifically in the brain, several ion channels, such as voltage-gated sodium channels Nav1.6 and voltage-gated potassium channels Kv7.2/Kv7.3 (KCNQ2/3), have been shown to be ubiquitinated by Nedd4–2 (Ekberg et al. 2007, Goel et al. 2015, Schuetz et al. 2008) (Ekberg et al. 2014) and could be involved in Nedd4–2-dependent excitability regulation. Although the C2-lacking Nedd4–2 may not be in a close proximity with those ion channels on cell membranes, it remains to be determined whether C2-lacking Nedd4–2 can interact with endocytosed ion channels and subsequently regulates their ubiquitination and overall neuronal excitability. Although our current study provides the first evidence to demonstrate the function of C2-lacking Nedd4–2 in neurons, future studies are clearly needed in order to further characterize the properties and regulation of this Nedd4–2 isoform.

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ACKNOWLEDGEMENTS

We sincerely thank Dr. Hiroshi Kawabe for providing us the Nedd4–2 floxed mice. This work is supported by the startup fund provided by the School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, by Brain and Behavior Research Foundation NARSAD Young Investigator Grant (27018 to N-P.T.) and by National Institute of Health (R01NS105615 to N-P.T.).

Abbreviations used:

AMPA

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

DMSO

dimethyl sulfoxide

GluA1

glutamate receptor subunit 1

HA

hemagglutinin

HEK

human embryonic kidney

IP

immunoprecipitation

mEPSC

miniature excitatory post-synaptic current

Nedd4–2

neural precursor cell expressed developmentally down-regulated gene 4-like

PPP3CA

Protein Phosphatase 3 Catalytic Subunit-α

RRID

Research Resource Identifier (see scicrunch.org)

Ub

ubiquitin

Footnotes

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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