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

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

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

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

<i>Nedd4-2</i>^<i>andi</i> cortical neuron cultures are more sensitive to acute AMPAR blockade than WT cultures in reduction of spontaneous neuronal activity.

<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 (DMSO) or NBQX (2 μM) treatment for 15 min. (Vehicle: n = 9 and 5 for WT and <i>Nedd4-2</i>^<i>andi</i>; NBQX: n = 8 and 5 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 (DMSO) or NBQX (2 μM) treatment for 15 min (Vehicle: n = 9 and 5 for WT and <i>Nedd4-2</i>^<i>andi</i>; NBQX: n = 8 and 5 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<0.01, 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