The regulation of neuronal excitability by epilepsy-associated gene Nedd4-2

Epilepsy is the fourth most common neurological disease in the United States, which is characterized by the recurrent seizures in unpredictable frequency with a range of severities and a variety of causes. About 3.4 million people in the United States have epilepsy. Every year, there is an increase of 150,000 of new cases and about 50,000 people die from epilepsy-related causes. Moreover, one in twenty-six of the people in the United States will develop epilepsy during their lifetime. Last but not least, one-third of the patients are drug-resistant. Even for those patients whose seizures could be controlled with medications or other treatments, living with epilepsy is still a big challenge, as they need to face questions about independent capabilities, limitation on driving and uncertainties in employment situations. The overall quality of life and productivity of people with epilepsy and their families need to be improved. Therefore, studying the mechanisms of epileptogenesis is significant to develop better treatments or cure for epilepsy patients. Many patients with neurological disorders suffer from an imbalance in neuronal and circuit excitability and present with seizure or epilepsy as the common comorbidity. The onset of epilepsy can be a result of an acquired brain injury (such as a trauma) or a genetic mutation in some genes. It is widely accepted that the alterations in activity, composition or distribution of ion channels caused by genetic mutations contribute to the onset of epilepsy. DNA sequencing and analysis performed as part of a worldwide research study called Epilepsy 4000 (Epi4k) has revealed over 300 de novo mutations in patients with epilepsy. Among those genes identified, the neural precursor cell expressed developmentally down-regulated gene 4-2, Nedd4-2, was specifically noted. Furthermore, three missense changes in Nedd4-2 have also been identified in families with epilepsy. Nedd4-2 gene encodes a ubiquitin E3 ligase that has high affinity toward binding and ubiquitinating membrane proteins, including ion channels. However, it is currently unknown how Nedd4-2 mediates neuronal circuit activity and how its dysfunction leads to seizures or epilepsies. During my Ph.D. thesis studies, I aimed to characterize the function of Nedd4-2 in regulating neuronal excitability and how epilepsy-associated missense mutations impair such regulation, in order to elucidate the molecular mechanisms underlying Nedd4-2-mediated epileptogenesis. Several neuronal ion channels have been reported as the novel substrates of Nedd4-2, including the major subunit (GluA1) of an ionotropic glutamate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which our lab has identified. AMPA receptors (AMPARs) are the most commonly found receptors in the mammalian nervous system, mediating fast excitatory synaptic transmission. Their distribution and activity are regulated by post-translational modifications, such as ubiquitination and phosphorylation. Studies have shown that GluA1 ubiquitination contributes to its internalization, which, as part of AMPAR trafficking mechanisms, is critical for synaptic depression as well as homeostatic regulation of synaptic strength. Because GluA1 levels affect neuronal activity, and dysregulation of AMPARs has been shown to be linked to epilepsy, we hypothesize that Nedd4-2 plays a role in down-regulating neuronal excitability through fine-tuning of AMPARs, specifically ubiquitinating and degrading GluA1. In Chapter Two, we studied the role of Nedd4-2 in regulation of neuronal activity. Using a genetic mouse model, termed Nedd4-2andi, in which one of the major forms of Nedd4-2 in the brain is selectively deficient, we first demonstrated that Nedd4-2 is involved in the regulation of neuronal network excitability. Measured by a multi-electrode array (MEA) system, the spontaneous neuronal activity in Nedd4-2andi cortical neuron cultures was basally elevated compared to wild-type (WT) cultures. Subsequently, our data indicated that when pharmacologically modulating AMPAR activities, Nedd4-2andi cultures showed less responsive to AMPAR activation, and much more sensitive to AMPAR blockade. Moreover, when performing kainic acid-induced seizures in vivo, we found that elevated seizure susceptibility in Nedd4-2andi mice could be normalized when GluA1 is genetically reduced. In Chapter Three, we focused on characterizing the epilepsy-associated mutations of Nedd4-2. Three Nedd4-2 missense changes in highly conserved residues (S233L, E271A and H515P) were identified in families with epilepsy. Based on our previous findings, we aimed to test the hypothesis that one or more of these mutations could disrupt GluA1 ubiquitination and degradation, resulting in dysregulation of neuronal excitability. To begin with, I generated corresponding Nedd4-2 variants in Nedd4-2 cDNA construct by Site-Directed Mutagenesis. My evidence from HEK cells showed that GluA1 was less ubiquitinated when co-expressed with any of the Nedd4-2 mutants in comparison to WT Nedd4-2. Similar result was observed by in vitro ubiquitination assay as well. Apart from GluA1 ubiquitination, its degradation was also affected by the mutant Nedd4-2s, as GluA1 showed enhanced half-life when co-expressed with any of the Nedd4-2 variants compared to WT Nedd4-2. Furthermore, three epilepsy-associated missense mutations of Nedd4-2 also failed to reduce GluA1 surface expression or spontaneous neuronal activity when compared to WT Nedd4-2. Collectively, our data suggest that impaired GluA1 ubiquitination contributes to Nedd4-2-dependent neuronal hyperactivity and seizures. These findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of Nedd4-2. In Chapter Four, in collaboration with Dr. Kwan Young Lee, a research scientist in Tsai lab, I extended my studies on the function of two major isoforms of Nedd4-2, C2-containing and C2-lacking isoforms, in regulating excitatory synaptic strength. Because single-nucleotide polymorphisms (SNPs) in human Nedd4-2 lead to differential expression of these two isoforms, examining their functional differences may aid our understanding of neuronal excitability regulation and seizure susceptibility in different populations. As the C2 domain is responsible for binding to membranes, we first found that the C2-lacking Nedd4-2 showed a reduced distribution at cell membranes and extremely low affinity in ubiquitinating GluA1. However, meanwhile, 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 PPP3CA (a subunit of calcineurin A), that could mediate excitatory synaptic strength. We then confirmed that PPP3CA is a substrate of C2-lacking Nedd4-2 in HEK cells, as well as by in vitro ubiquitination assay. In addition, the epilepsy-associated mutations in C2-lacking Nedd4-2 showed impaired PPP3CA ubiquitnation. Further studies will be definitely needed to fully characterize the properties and regulation of this Nedd4-2 isoform. In summary, in this dissertation, I studied the molecular mechanisms by which Nedd4-2 gene is associated with epileptogenesis, and provided evidence showing 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. These findings in my dissertation may facilitate the future development of new therapeutic strategies for epilepsy patients who carry mutations of Nedd4-2

The regulation of neuronal excitability by epilepsy-associated gene Nedd4-2

Epilepsy is the fourth most common neurological disease in the United States, which is characterized by the recurrent seizures in unpredictable frequency with a range of severities and a variety of causes. About 3.4 million people in the United States have epilepsy. Every year, there is an increase of 150,000 of new cases and about 50,000 people die from epilepsy-related causes. Moreover, one in twenty-six of the people in the United States will develop epilepsy during their lifetime. Last but not least, one-third of the patients are drug-resistant. Even for those patients whose seizures could be controlled with medications or other treatments, living with epilepsy is still a big challenge, as they need to face questions about independent capabilities, limitation on driving and uncertainties in employment situations. The overall quality of life and productivity of people with epilepsy and their families need to be improved. Therefore, studying the mechanisms of epileptogenesis is significant to develop better treatments or cure for epilepsy patients. Many patients with neurological disorders suffer from an imbalance in neuronal and circuit excitability and present with seizure or epilepsy as the common comorbidity. The onset of epilepsy can be a result of an acquired brain injury (such as a trauma) or a genetic mutation in some genes. It is widely accepted that the alterations in activity, composition or distribution of ion channels caused by genetic mutations contribute to the onset of epilepsy. DNA sequencing and analysis performed as part of a worldwide research study called Epilepsy 4000 (Epi4k) has revealed over 300 de novo mutations in patients with epilepsy. Among those genes identified, the neural precursor cell expressed developmentally down-regulated gene 4-2, Nedd4-2, was specifically noted. Furthermore, three missense changes in Nedd4-2 have also been identified in families with epilepsy. Nedd4-2 gene encodes a ubiquitin E3 ligase that has high affinity toward binding and ubiquitinating membrane proteins, including ion channels. However, it is currently unknown how Nedd4-2 mediates neuronal circuit activity and how its dysfunction leads to seizures or epilepsies. During my Ph.D. thesis studies, I aimed to characterize the function of Nedd4-2 in regulating neuronal excitability and how epilepsy-associated missense mutations impair such regulation, in order to elucidate the molecular mechanisms underlying Nedd4-2-mediated epileptogenesis. Several neuronal ion channels have been reported as the novel substrates of Nedd4-2, including the major subunit (GluA1) of an ionotropic glutamate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which our lab has identified. AMPA receptors (AMPARs) are the most commonly found receptors in the mammalian nervous system, mediating fast excitatory synaptic transmission. Their distribution and activity are regulated by post-translational modifications, such as ubiquitination and phosphorylation. Studies have shown that GluA1 ubiquitination contributes to its internalization, which, as part of AMPAR trafficking mechanisms, is critical for synaptic depression as well as homeostatic regulation of synaptic strength. Because GluA1 levels affect neuronal activity, and dysregulation of AMPARs has been shown to be linked to epilepsy, we hypothesize that Nedd4-2 plays a role in down-regulating neuronal excitability through fine-tuning of AMPARs, specifically ubiquitinating and degrading GluA1. In Chapter Two, we studied the role of Nedd4-2 in regulation of neuronal activity. Using a genetic mouse model, termed Nedd4-2andi, in which one of the major forms of Nedd4-2 in the brain is selectively deficient, we first demonstrated that Nedd4-2 is involved in the regulation of neuronal network excitability. Measured by a multi-electrode array (MEA) system, the spontaneous neuronal activity in Nedd4-2andi cortical neuron cultures was basally elevated compared to wild-type (WT) cultures. Subsequently, our data indicated that when pharmacologically modulating AMPAR activities, Nedd4-2andi cultures showed less responsive to AMPAR activation, and much more sensitive to AMPAR blockade. Moreover, when performing kainic acid-induced seizures in vivo, we found that elevated seizure susceptibility in Nedd4-2andi mice could be normalized when GluA1 is genetically reduced. In Chapter Three, we focused on characterizing the epilepsy-associated mutations of Nedd4-2. Three Nedd4-2 missense changes in highly conserved residues (S233L, E271A and H515P) were identified in families with epilepsy. Based on our previous findings, we aimed to test the hypothesis that one or more of these mutations could disrupt GluA1 ubiquitination and degradation, resulting in dysregulation of neuronal excitability. To begin with, I generated corresponding Nedd4-2 variants in Nedd4-2 cDNA construct by Site-Directed Mutagenesis. My evidence from HEK cells showed that GluA1 was less ubiquitinated when co-expressed with any of the Nedd4-2 mutants in comparison to WT Nedd4-2. Similar result was observed by in vitro ubiquitination assay as well. Apart from GluA1 ubiquitination, its degradation was also affected by the mutant Nedd4-2s, as GluA1 showed enhanced half-life when co-expressed with any of the Nedd4-2 variants compared to WT Nedd4-2. Furthermore, three epilepsy-associated missense mutations of Nedd4-2 also failed to reduce GluA1 surface expression or spontaneous neuronal activity when compared to WT Nedd4-2. Collectively, our data suggest that impaired GluA1 ubiquitination contributes to Nedd4-2-dependent neuronal hyperactivity and seizures. These findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of Nedd4-2. In Chapter Four, in collaboration with Dr. Kwan Young Lee, a research scientist in Tsai lab, I extended my studies on the function of two major isoforms of Nedd4-2, C2-containing and C2-lacking isoforms, in regulating excitatory synaptic strength. Because single-nucleotide polymorphisms (SNPs) in human Nedd4-2 lead to differential expression of these two isoforms, examining their functional differences may aid our understanding of neuronal excitability regulation and seizure susceptibility in different populations. As the C2 domain is responsible for binding to membranes, we first found that the C2-lacking Nedd4-2 showed a reduced distribution at cell membranes and extremely low affinity in ubiquitinating GluA1. However, meanwhile, 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 PPP3CA (a subunit of calcineurin A), that could mediate excitatory synaptic strength. We then confirmed that PPP3CA is a substrate of C2-lacking Nedd4-2 in HEK cells, as well as by in vitro ubiquitination assay. In addition, the epilepsy-associated mutations in C2-lacking Nedd4-2 showed impaired PPP3CA ubiquitnation. Further studies will be definitely needed to fully characterize the properties and regulation of this Nedd4-2 isoform. In summary, in this dissertation, I studied the molecular mechanisms by which Nedd4-2 gene is associated with epileptogenesis, and provided evidence showing 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. These findings in my dissertation may facilitate the future development of new therapeutic strategies for epilepsy patients who carry mutations of Nedd4-2

Additional file 2: Table S1: of Feedback modulation of neural network synchrony and seizure susceptibility by Mdm2-p53-Nedd4-2 signaling

Summary of normalized MEA measurements of spike rate and synchrony index over 48 h of recording after elevation of neuronal activity. (DOCX 15 kb

Additional file 1: Figure S1: of Feedback modulation of neural network synchrony and seizure susceptibility by Mdm2-p53-Nedd4-2 signaling

Primary cortical neuron cultures treated with PTX along with or without Nutlin-3 or Pifithrin-ι do not exhibit altered cell viability or apoptosis. (DOCX 78 kb

Epilepsy-associated gene <i>Nedd4-2</i> mediates neuronal activity and seizure susceptibility through AMPA receptors

<div><p>The <u>n</u>eural precursor cell <u>e</u>xpressed <u>d</u>evelopmentally <u>d</u>own-regulated gene <u>4</u>–2, <i>Nedd4-2</i>, is an epilepsy-associated gene with at least three missense mutations identified in epileptic patients. <i>Nedd4-2</i> encodes a ubiquitin E3 ligase that has high affinity toward binding and ubiquitinating membrane proteins. It is currently unknown how <i>Nedd4-2</i> mediates neuronal circuit activity and how its dysfunction leads to seizures or epilepsies. In this study, we provide evidence to show that <i>Nedd4-2</i> mediates neuronal activity and seizure susceptibility through ubiquitination of GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, (AMPAR). Using a mouse model, termed <i>Nedd4-2</i>^<i>andi</i>, in which one of the major forms of <i>Nedd4-2</i> in the brain is selectively deficient, we found that the spontaneous neuronal activity in <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures, measured by a multiunit extracellular electrophysiology system, was basally elevated, less responsive to AMPAR activation, and much more sensitive to AMPAR blockade when compared with wild-type cultures. When performing kainic acid-induced seizures <i>in vivo</i>, we showed that elevated seizure susceptibility in <i>Nedd4-2</i>^<i>andi</i> mice was normalized when GluA1 is genetically reduced. Furthermore, when studying epilepsy-associated missense mutations of <i>Nedd4-2</i>, we found that all three mutations disrupt the ubiquitination of GluA1 and fail to reduce surface GluA1 and spontaneous neuronal activity when compared with wild-type Nedd4-2. Collectively, our data suggest that impaired GluA1 ubiquitination contributes to Nedd4-2-dependent neuronal hyperactivity and seizures. Our findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of <i>Nedd4-2</i>.</p></div

Three epilepsy-associated missense mutations of Nedd4-2 reduce GluA1 ubiquitination.

<p>(<b>A</b>_<b>1</b>) Western blots of Ubiquitin (Ub) or GluA1 after immunoprecipitation using anti-GluA1 antibody from HEK cells transfected with GluA1 along with HA-tagged WT or mutant Nedd4-2 for 48 hours. Quantification of ubiquitinated GluA1 by the entire area of smear from 100–250 kDa is shown on the right (<b>A</b>_<b>2</b>) (n = 4, one-way ANOVA with post-hoc Tukey test). (<b>B</b>_<b>1</b>) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following <i>in vitro</i> ubiquitination with recombinant GluA1. HA-tagged WT or mutant Nedd4-2s used for <i>in vitro</i> ubiquitination were obtained from HEK cells transfected with one of the Nedd4-2s followed by immunoprecipitation with anti-Nedd4-2 antibody. Quantification of ubiquitinated GluA1 by the entire area of smear (<b>B</b>_<b>2</b>) and Coomassie blue staining showing the purity of recombinant GluA1 (<b>B</b>_<b>3</b>) are shown (n = 4, one-way ANOVA with post-hoc Tukey test). (<b>C</b>_<b>1</b>) Western blots of GluA1 and Nedd4-2 after cycloheximide treatment over an 8-hr time course. HEK cells were transfected with GluA1 and HA-tagged WT or mutant Nedd4-2s for 48 hours. Cells were then treated with cycloheximide (100 μg/ml) to inhibit protein translation and follow protein degradation. Representative western blots after 0- and 8-hr cycloheximide treatment (<b>C</b>_<b>1</b>) and time courses of GluA1 (<b>C</b>_<b>2</b>) and Nedd4-2 (<b>C</b>_<b>3</b>) levels after cycloheximide treatment are shown. Analyses were performed by comparing GluA1 or Nedd4-2 level at each time point between cultures receiving different Nedd4-2s (n = 4, one-way ANOVA with post-hoc Tukey test). For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.</p

Nedd4-2 mediates spontaneous neuronal and synaptic activity.

<p>(<b>A</b>_<b>1</b>) A diagram showing two alternative protein products (isoforms 1 and 2) from the <i>Nedd4-2</i> gene, and (<b>A</b>_<b>2</b>) western blot results of Nedd4-2 and Gapdh from brain lysates of WT or <i>Nedd4-2</i>^<i>andi</i> mice at age 4-weeks old. (<b>B</b>) Quantification of spontaneous spike rate (left) and representative raster plots of spontaneous spikes from 1-min recording (right) of WT or <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures at DIV 13–14. (<b>C</b>) Quantification of average spontaneous spike amplitude (left) and representative average traces of 1-min recording (right) of WT or <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures at DIV 13–14. The black lines represent the average of all the spikes within representative 1-min recordings. (<b>D</b>) Patch-clamp recording from WT or <i>Nedd4-2</i>^<i>andi</i> cortical neurons at DIV 14. Representative mEPSC traces and quantification of mEPSC amplitude and frequency are shown (n = 15 for both WT or <i>Nedd4-2</i>^<i>andi</i> neurons). Data are analyzed by Student’s <i>t</i>-test and represented as mean ± SEM with *p<0.05, **p<0.01.</p

Acute AMPAR activation triggers stronger elevation of spontaneous neuronal activity in WT than <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures.

<p>(<b>A</b>) Representative raster plots and quantification of spontaneous spikes from 1-min recording (right) of (<b>A</b>_<b>1</b>) WT or (<b>A</b>_<b>2</b>) <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures before and after vehicle (ddH₂O) or AMPA (1 μM) treatment for 15 min. (Vehicle: n = 6 and 9 for WT and <i>Nedd4-2</i>^<i>andi</i>; AMPA: n = 7 and 11 for WT and <i>Nedd4-2</i>^<i>andi</i>) (<b>B</b>) Representative average traces of 1-min recording and quantification of average spontaneous spike amplitude of (<b>B</b>_<b>1</b>) WT or (<b>B</b>_<b>2</b>) <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures before and after vehicle (ddH₂O) or AMPA (1 μM) treatment for 15 min (Vehicle: n = 6 and 9 for WT and <i>Nedd4-2</i>^<i>andi</i>; AMPA: n = 7 and 11 for WT and <i>Nedd4-2</i>^<i>andi</i>). The black lines represent the average of all the spikes within representative 1-min recordings. Data are analyzed by a 2-way ANOVA with post-hoc Tukey test and represented as mean ± SEM. The comparison between treatments or genotypes is described with *p<0.05, ns: non-significant. Significant interaction between treatment and genotype was detected in spontaneous spike rate (A₃; p<0.05) but not amplitude (B₃; p>0.05).</p

Nedd4-2 mediates surface expression of GluA1.

<p>(<b>A</b>) Illustration of GluA1 on the cell membrane and the 4 lysine residues potentially ubiquitinated by Nedd4-2 at the C-terminus of GluA1. (<b>B</b>_<b>1</b>) Western blots of Ubiquitin (Ub) or GluA1 after immunoprecipitation using anti-GluA1 antibody from HEK cells transfected with WT HA-Nedd4-2 along with WT GluA1 or mutant GluA1s (K813R, K819R, K822R, K868R and 4KR) for 48 hours. Quantification of ubiquitinated GluA1 by the area of smear from 100–250 kDa is shown on the right (<b>B</b>_<b>2</b>) (n = 4, one-way ANOVA with post-hoc Tukey test). (<b>C</b>) Western blots of GluA1, N-cadherin, and Actin from WT or <i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures. Proteins from total lysate or after surface biotinylation were as indicated (n = 3, one-sample <i>t</i>-test was performed after normalization to WT groups). For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.</p

Three epilepsy-associated missense mutations of Nedd4-2 disrupt 14-3-3-facilitated GluA1 ubiquitination.

<p>(<b>A</b>_<b>1</b>) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following <i>in vitro</i> ubiquitination with recombinant GluA1 and Nedd4-2, and in the presence or absence of recombinant 14-3-3ε, 14-3-3 inhibitor R18, or Ubiquitin as labeled. Quantification of lanes 1–4 by the entire area of smear from 100–250 kDa (<b>A</b>_<b>2</b>) and Coomassie blue staining showing the purity of recombinant Nedd4-2 and 14-3-3ε (<b>A</b>_<b>3</b>) (n = 4, 2-way ANOVA with post-hoc Tukey test). (<b>B</b>_<b>1</b>) Western blots of Nedd4-2 or 14-3-3 after a co-immunoprecipitation using anti-14-3-3 antibody with the lysate of HEK cells transfected with HA-tagged WT or mutant Nedd4-2s for 48 hours. Input of transfected Nedd4-2s, endogenous 14-3-3, and Tubulin are shown on the bottom. Quantification of immunoprecipitated Nedd4-2 (<b>B</b>_<b>2</b>) is shown on the right (n = 4, one-way ANOVA with post-hoc Tukey test). (<b>C</b>_<b>1</b>) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following <i>in vitro</i> ubiquitination with recombinant GluA1 in the presence or absence of recombinant 14-3-3ε. HA-tagged WT or mutant Nedd4-2s used for <i>in vitro</i> ubiquitination were obtained from HEK cells transfected with one of the Nedd4-2s followed by immunoprecipitation with anti-Nedd4-2 antibody. Right before the washing, 1/10 of reaction mixture was obtained and used as input control shown on the bottom. (<b>C</b>_<b>2</b>) The intensity of ubiquitinated GluA1 by the entire area of smear from 100–250 kDa is normalized to the WT group in the absence of 14-3-3ε (lane 3 on the representative blot C₁). The difference in each group with or without the addition of 14-3-3ε was analyzed by Student’s <i>t</i>-test. For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.</p