Regulation of Ube3A by neuronal activity. (A) qRT-PCR analysis of Ube3A mRNA extracted from hippocampal neurons at E18 + 10 days in vitro (DIV) stimulated for five hours with the indicated agent (Glut. = glutamate; Bic. = bicuculline). Data are means +/- SEM from three independent experiments. * indicates statistical significance in pairwise comparison to control: P < 0.01 T-test. (B) Western blot analyses of Ube3A and beta-tubulin. Protein lysates were collected from E18 + 10 DIV hippocampal neurons following stimulation with 55 mM KCl for seven hours. Three independent experiments were performed and a representative Western blot is shown. (C) qRT-PCR examining Ube3A and GAPDH mRNA levels in hippocampi of mice placed in standard laboratory cages (control) or in cages with novel objects (novel environment). The expression of Ube3A and GAPDH is normalized to the expression of beta-tubulin which serves as an internal standard. Data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance in pairwise comparison: P < 0.05 T-test. (D) Chromatin immunoprecipitation with control or anti-MEF2 antibodies. PCR amplification is performed on genomic regions corresponding to the promoter regions of the three Ube3A transcripts. (E) qRT-PCR analysis of the three Ube3A transcripts in hippocampal neurons transduced with lentivirus expressing either control shRNA or shRNAs targeting MEF2A and MEF2D. Neurons were stimulated with 55 mM KCl for six hours before mRNA was harvested. Data are plotted as fold induction of stimulated cells over unstimulated cells. Data are presented as mean +/-SEM from three independent experiments. * indicates statistical significance in pairwise comparison: P < 0.01 T-test. (F) Western blot analyses of MEF2D, MEF2A, Ube3A, and the loading control Vav2. Protein lysates were collected from hippocampal neurons at E18 + 10 DIV. Neurons were uninfected or transduced with lentivirus encoding a control shRNA or shRNA targeting MEF2A and MEF2D at E18 + 3 DIV. This experiment was performed three times independently and a representative Western blot is shown here. See also Figure S1.

Arc is a Ube3A substrate. (A) Sequence alignment of Arc (amino acids 255-318) and HHR23A (amino acids 233-290). Identical residues are in red and similar residues are in blue. We would like to note that as the UBD may represent a sequence that encodes a particular protein folding structure, a strict one-to-one map of specific residues is not observed. (B) In vitro binding experiments using recombinant Arc, ArcΔUBD, and GST-tagged Ube3A. (C) Quantitative analysis of in vitro binding experiments using recombinant Arc, or ArcΔUBD, and Ube3A. Western blotting was performed using an anti-Arc antibody. Percentage binding refers to the percent of Arc bound to Ube3A relative to the input. Data are presented as mean +/-SEM from three independent experiments. (D) In vitro ubiquitination assay of Arc in the presence of Ubiquitin (Ub), and/or Ube3A. (E) Western blot analysis using anti-Arc, anti-Ube3A, or anti-actin antibodies on lysates from HEK293T cells transfected with the indicated constructs. (F) Western blot analysis of protein lysates prepared from the hippocampi of wild type and Ube3A knockout mice which had been injected with kainic acid. Western blots performed with anti-MeCP2, anti-phospho-MeCP2, and anti-Arc antibodies as indicated. Three individual experiments representing at least five animals per genotype were performed and a representative example is shown. (G) Quantification of Arc protein by Western blot analysis of protein lysates prepared from hippocampi of wild type and Ube3A knockout mice which had been exposed to an enriched environment. Data represent mean +/- SEM from four animals of each genotype. * denotes significance in pairwise comparison to control: P < 0.01 T-test. (H) Quantification of Arc protein by Western blot analysis of protein lysates prepared from synaptosomes isolated from hippocampi of wild type and Ube3A knockout mice which had been injected with kainic acid. Data represent mean +/- SEM from three animals of each genotype. * denotes significance in pairwise comparison to control: P < 0.05 T-test. (I) Real-time quantitative PCR analysis of Arc mRNA extracted from wild type and Ube3A knockout mice seized with kainic acid used in part (F). Data are presented as mean +/- SEM from three independent experiments. See also Figure S2.

Ube3A-mediated degradation of Arc affects AMPAR cell surface expression. (A) In vitro ubiquitination assay of Arc or a version of Arc in which all lysine residues are mutated to arginine (ArcΔK) in the presence of Ubiquitin (Ub), Ube3A or Ube3A C833A (C833A). Western blotting analysis was performed with an anti-Arc antibody. (B) Quantitative Western blot analysis of protein lysates from HEK293T cells transfected with the indicated constructs. Western blots were performed using an anti-Arc antibody, and the signals were normalized to an actin loading control. (C) Quantitative Western blot analysis of protein lysates from HEK293T cells transfected with the indicated constructs. Western blots were performed using an anti-Flag antibody to detect EphA4, and the resultant values were normalized to an actin loading control. As previously reported Cbl-B promotes the degradation of EphA4 (Sharfe et al., 2003). Cbl-B-mediated degradation of EphA4 is not inhibited by Ube3A C833A, even though Ube3A and Cbl-B can employ the same E2 conjugating enzyme when ubiquitinating substrates. (D) Quantification of surface AMPAR expression for E18 + 17 DIV hippocampal neurons transfected with GFP and vector control, Ube3A, or Ube3A C833A plasmids. At least 30 neurons were imaged for each condition and data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.05, ANOVA, with Bonferroni correction for multiple comparison. (E) Quantification of surface AMPA receptor expression on E18 + 14 DIV hippocampal neurons transfected at 10 DIV with vector control, Arc, Ube3A + Arc, ArcΔUBD, or ArcΔUBD + Ube3A. Data are presented as mean +/- SEM from three independent experiments. * denotes statistical significance P < 0.05, ANOVA, with Bonferroni correction for multiple comparison. (F) Quantification of surface AMPAR expression on hippocampal neurons transfected with vector control, Ube3A RNAi, Arc RNAi, Ube3A RNAi and scrambled control Arc RNAi, or Ube3A RNAi and Arc RNAi. Data are presented as mean +/- SEM from three independent experiments. * denotes statistical significance P < 0.05, ANOVA, with Bonferroni correction for multiple comparison. See also Figures S4 and S5.

Analysis of synaptic function in the hippocampi of Ube3A knockout mice. (A) Representative traces of currents evoked while holding the neuron at -70 or +40 mV to measure AMPAR or NMDAR-mediated currents, respectively. Examples are shown from a control (left) and Ube3A knockout (right) neuron. Currents are scaled by the current amplitude measured between 50 and 70 ms after the peak of the evoked current at +40 mV to highlight the relative changes in AMPAR-mediated current. (B) A summary histogram of AMPA/NMDA receptor-mediated current ratios presented as the geometric mean +/- SEM. At least 15 cells were analyzed per condition. * p < 0.05 by students t-test of the geometric means for each neuron. (C) Representative mEPSC traces of hippocampal neurons from wild type (top) and Ube3A knockout neurons (bottom). (D) Quantification of mEPSC frequency from wild type (black line) and Ube3A knockout (red line) mice. Data are presented as cumulative probability plots of interevent intervals and represent recordings from at least 14 neurons from at least three independent animals of each genotype. A significant difference was observed between wild type and Ube3A knockout mice, P < 0.01 by KS test. (E) Quantification of mEPSC amplitude from wild type (black line) and Ube3A knockout (red line) mice. Data are presented as cumulative probability plots and represent recordings from at least 14 neurons from at least three independent animals of each genotype. No statistically significant difference was observed between wildtype and Ube3A knockout mice by KS test. See also Figure S7.

Identification of a Ube3A binding domain. (A) Analysis of ubiquitinated proteins in wild type and HA-ubiquitin mice. Western blots using an anti-Ubiquitin antibody were performed on cell lysates (WCE) or anti-HA immunoprecipitates from hippocampal mouse brain lysates prepared from wild type (WT) or HA-ubiquitin transgenice (HA) mice. * indicates the presence of free ubiquitin. (B) Analysis of ubiquitinated proteins in wild type and HA-ubiquitin mice. Western blots using an anti-HA antibody were performed on cell lysates (WCE) or anti-HA immunoprecipitations from hippocampal mouse brain lysates from wild type (WT) or HA-ubiquitin transgenic (HA) mice. * indicates the presence of free ubiquitin. (C) Quantification of the relative abundance of ubiquitinated Sacsin in the brain of wild type and Ube3A knockout mice. No peptides were detected corresponding to ubiquitinated Sacsin in Ube3A knockout mice. (D) Sequence alignment of human Sacsin and human HHR23A. Identical residues are in red and similar residues are in blue. (E) Quantitative analysis of in vitro binding experiments using recombinant HHR23A, a version of HHR23A lacking the Ube3A binding domain (ΔHHR23A), and Ube3A. Western blotting was performed using an anti-HHR23A antibody. Data are presented as mean +/- SEM from three independent experiments.

Ube3A regulates AMPAR function. (A) Quantification of plasma membrane expression of AMPARs on E18 + 14 DIV hippocampal neurons transfected at 10 DIV with GFP and vector control, either of two shRNAs targeting Ube3A (RNAi 1 or 2), scrambled control shRNA (scRNAi 1 or 2), a form of Ube3A that is resistant to Ube3A shRNA (Ube3Ares) or Ube3A shRNA and Ube3A that is RNAi resistant (Ube3Ares + RNAi 2). At least 35 neurons were imaged for each condition. Data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.05, ANOVA using a Bonferroni correction for multiple comparisons. (B) Quantification of plasma membrane expression of NMDA receptors on E18 + 14 DIV hippocampal neurons transfected at 10 DIV with GFP and vector control, either of two shRNAs targeting Ube3A (RNAi 1 or 2), or a scrambled control shRNA (scRNAi 1). At least 20 neurons were imaged for each condition, and data are presented as mean +/- SEM from three independent experiments. (C) Same as in (A) except only GluR1 puncta that co-localize with PSD95 are counted. At least 29 neurons were imaged for each condition, and data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.05, ANOVA using a Bonferroni correction for multiple comparisons. (D) Quantification of internalized GluR1 receptors from E18 + 14 DIV hippocampal neurons transfected at 10 DIV with GFP plus vector, Ube3a shRNA, or control scrambled shRNA. Data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.05, ANOVA using a Bonferroni correction for multiple comparisons. (E) Representative mEPSC traces of control transfected (top) or Ube3A RNAi transfected neurons (bottom) used for analysis in (F) and (G). (F) Quantification of mEPSC interevent interval (the time between mEPSC events and thus inversely proportional to mEPSC frequency) from E18 + 14 DIV hippocampal neurons transfected as in part (A). Data are presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.01, t-test. (G) Quantification of mEPSC amplitude from E18 + 14 DIV hippocampal neurons transfected as in part (A). Data are presented as mean +/- SEM from three independent experiments. See also Figure S3.

Ube3A knockout mice have fewer synaptically expressed AMPARs. (A) Quantification of plasma membrane expression of AMPARs on P2 + 12 DIV hippocampal neurons isolated from wild type (WT) and Ube3A knockout (KO) animals transfected at 8 DIV with GFP. At least 40 neurons were imaged for each condition, and data are normalized to wild type and presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.01, T-test. (B) Quantification of plasma membrane expression of NMDA receptors on P2 + 12 DIV hippocampal neurons isolated from wild type (WT) and Ube3A knockout (KO) animals transfected at 8 DIV with GFP. At least 24 neurons were imaged for each condition, and data are normalized to wild type and presented as mean +/- SEM from three independent experiments. (C) Quantification of plasma membrane expression of AMPA receptors on P2 + 12 DIV hippocampal neurons isolated from wild type (WT) and Ube3A knockout (KO) animals transfected at 8 DIV with GFP and either vector control, scrambled control shRNAs, or shRNAs targeting Arc. At least 28 neurons were imaged for each condition, and data are normalized to wild type transfected with control and presented as mean +/- SEM from three independent experiments. * indicates statistical significance P < 0.01, ANOVA, with Bonferroni correction for multiple comparisons. (D) Quantification of the number of co-localized GluR1 and SV2 puncta in wild type and Ube3A knockout hippocampi. Data are presented as mean +/- SEM from three independent animals for each genotype. * indicates statistical significance P < 0.01 T-test. (E) Quantification of the number of co-localized NR1 and SV2 puncta in wild type and Ube3A knockout hippocampi. Data are presented as mean +/- SEM from three independent animals for each genotype. P > 0.05, T-test. (F) Analysis of the ratio of the density of GluR1 puncta that co-localize with SV2 to the density of NR1 puncta that co-localize with SV2 obtained from (D) and (E). * indicates statistical significance P < 0.01 T-test. (G) Quantitative Western blot analysis of protein lysates prepared from the hippocampi of P21 wild type and Ube3A knockout mice using anti-NR1 (left panel) and anti-GluR1 (right panel) antibodies. Band intensity was normalized to the intensity of actin to control for differences in protein concentration. Data are presented as mean +/- SEM from three independent experiments. See also Figure S6.