| Annotated protein: | E3 ubiquitin-protein ligase NEDD4 (EC 2.3.2.26) (HECT-type E3 ubiquitin transferase NEDD4). Gene symbol: NEDD4. Taxonomy: Rattus norvegicus (Rat). Uniprot ID: Q62940 |
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| SynGO gene info: | SynGO data @ NEDD4 |
| Ontology domain: | Biological Process |
| SynGO term: | regulation protein catabolic process at postsynapse (GO:0140252) |
| Synapse type(s): | hippocampus, glutamatergic |
| Annotated paper: | Schwarz LA, et al. "Activity-dependent ubiquitination of GluA1 mediates a distinct AMPA receptor endocytosis and sorting pathway" J Neurosci. 2010 Dec 8;30(49):16718-29 PMID:21148011 |
| Figure(s): | Figure 4, Figure 5, Figure 6B-C, Figure 7 |
| Annotation description: | Figure 4. Nedd4-1 interacts with and regulates surface expression of GluA1. Literal: "When we resolved these IPs on Western blots and probed them with anti-GluA1 antibodies, we found GluA1 to be present in the Nedd4-1 precipitates. Similarly, when we immunoprecipitated GluA1 from mature hippocampal lysates, we found Nedd4-1 to be present in the GluA1 precipitates (Fig. 4A). Together, these results indicate that Nedd4-1 associates with GluA1, providing an opportunity for it to ubiquitinate and regulate the trafficking of GluA1-containing AMPARs in hippocampal neurons. "Neurons, at DIV15-18, were infected with Sindbis virus expressing either GFP alone (control) or coexpressing GFP with HA-tagged Nedd4-1. Infection time was limited to 18-22 h to limit cell toxicity. Cells were then labeled with antibodies directed against surface GluA1, permeabilized, and labeled with anti-HA antibodies to detect Nedd4-1-positive cells. Compared with uninfected neurons or GFP-control cells, neurons expressing HA-Nedd4-1 showed a significant loss in surface GluA1 immunofluorescence from the dendritic plasma membrane (1 ± 0.03 for GFP, 0.7 ± 0.03 for Nedd4-1, *p <0.001, unpaired Student's t test), indicating that increased expression of Nedd4-1 decreased surface populations of GluA1-containing AMPARs (Fig. 4B)" "To determine whether the Nedd4-1-induced decrease in surface AMPARs affected synaptic AMPARs, we recorded spontaneous mEPSCs from GFP (control) or Nedd4-1-infected neurons. We observed a significant decrease in mEPSC amplitude in neurons expressing Nedd4-1 compared with control neurons (23.4±1.4 pA for GFP, 17.6±1.3 pA for Nedd4-1) (Fig. 4C--F), whereas the mEPSC frequency was not significantly different between conditions (0.69 ± 0.24 s interevent interval for GFP, 0.59 ± 0.15 s for Nedd4-1) (Fig. 4G)." Figure 5. Overexpression of Nedd4-1 causes increased trafficking of surface GluA1-containing AMPARs to the lysosome. Literal: "we briefly surface labeled hippocampal neurons with anti-GluA1 antibodies before the addition of GFP- or Nedd4-1-expressing Sindbis virus. To one set of infected neurons, we also applied leupeptin (200μg/ml) to block lysosomal degradation. After 18-22 h, we fixed the neurons, blocked any remaining surface GluA1 antibody with unconjugated secondary antibodies, permeabilized the cells, and labeled the population of internalized GluA1. GFP-expressing neurons, with or without leupeptin, showed minimal levels of internalized GluA1 immunofluorescence (Fig. 5A,B). However, in neurons expressing Nedd4-1 in which lysosomal degradation was inhibited by leupeptin, there was a dramatic accumulation of internalized GluA1 in both the soma and dendrites. This accumulation did not occur in Nedd4-1-infected neurons if leupeptin was not added (1.8 ± 0.18 for Nedd4-1 ± leupeptin to 1 ± 0.13 for control, 1 ± 0.07 for control ± leupeptin, and 0.9 ± 0.10 for Nedd4-1). A significant portion of internalized GluA1 puncta colocalized with late endosomal/lysosomal compartments, visualized with anti-Lamp1 antibodies, in Nedd4-1-expressing neurons when compared with control neurons (2.8 ± 0.45 for Nedd4-1 + leupeptin to 1 ± 0.22 for control + leupeptin and 0.9±0.12 for Nedd4-1 + leupeptin with random Lamp1 signal) (Fig. 5C). These results indicate that expression of Nedd4-1 in hippocampal neurons targets endogenous AMPARs to late endosomal/lysosomal compartments." Figure 6 B, C. Loss of Nedd4-1 inhibits AMPA-mediated but not NMDA-mediated endocytosis of GluA1-containing AMPARs Literal: "To test this, we examined AMPA-induced internalization ofAMPARs in control and Nedd4-1 shRNA-expressing hippocampal neurons. As expected, AMPA produced robust internalization of GluA1 in control neurons (1 ± 0.10 for control to 1.6 ± 0.19 for control ± AMPA) (Fig. 6B,C). In contrast, Nedd4-1 shRNAexpressing neurons showed a significant inhibition of AMPAinduced AMPAR internalization (0.7±0.12 for RNAi and 0.5± 0.07 for RNAi + AMPA). Strikingly, RNAi-expressing neurons were still able to internalize GluA1 in response to NMDA at levels similar to control neurons exposed to NMDA (1±0.10 for RNAi to 1.3 ± 0.10 for RNAi ± NMDA and 1.4 ± 0.10 for control + NMDA) (Fig. 6B,D). Importantly, coexpression of pSuper-RNAi with Nedd4-1 resist rescued the ability of the neurons to endocytose GluA1-containing AMPARs in response to AMPA (1 ± 0.12 for Nedd4-1resist to 1.4 ± 0.16 for Nedd4- 1resist + AMPA,*p<0.05, unpaired Student's t test) (Fig. 6B). This indicates that Nedd4-1 mediates AMPAR internalization after direct AMPAR activation but is not required for NMDA-dependent endocytosis and suggests that Nedd4-1 is crucial for mediating AMPAR internalization and trafficking to the lysosome." Figure 7. Ubiquitination of GluA1-containing AMPARs is upregulated in aged neurons but blocked by loss of Nedd4-1. Literal: "We infected neurons at DIV9 with Nedd4-1 RNAi lentivirus or lentivirus expressing GFP alone as a control. At DIV14, neurons were treated with AMPA (100 μM, 10 min) and lysed, and GluA1 was isolated by immunoprecipitation (Fig. 7A,B). Whereas AMPA induced significant ubiquitination of GluA1 in DIV14 control neurons as observed previously (Fig. 1), ubiquitination of GluA1 was completely abolished in cultures lacking Nedd4-1 (3.0 ± 0.4 for DIV14 AMPA to 1 ± 0.2 for DIV14 control and 1.3 ± 0.4 for DIV14 RNAi ± AMPA). While performing these experiments, we observed that, in older neurons (DIV35), GluA1 was significantly ubiquitinated under control conditions, at levels similar to ubiquitination of GluA1 induced by application of AMPA in younger neurons (3.4 ± 1 for DIV35 control and 2.8 ± 0.7 for DIV35 AMPA to 3.0±0.4 for DIV14 AMPA). Furthermore, this increase in GluA1 ubiquitination in older neurons was still dependent on the presence of Nedd4-1, because loss of Nedd4-1 as a result of expression of Nedd4-1 RNAi lentivirus for 5 d resulted in significantly decreased GluA1 ubiquitination levels similar to that of younger untreated neurons (1.0 ± 0.3 for DIV35 RNAi + AMPA to 1 ± 0.2 for DIV14 control). These results suggest that factors, such as increased AMPAR activation or Nedd4-1 ligase activity, may change in neurons as they age, resulting in increased Nedd4-1- mediated AMPAR ubiquitination." |
| Evidence tracking, Biological System: | Cultured neurons |
| Evidence tracking, Protein Targeting: | RNAi / shRNA Over-expression |
| Evidence tracking, Experiment Assay: | Confocal Electrophysiology (generic) Western blot |
| Annotator(s): | Chiara Verpelli (ORCID:0000-0003-2949-9725) Carlo Sala (ORCID:0000-0003-0662-9523) |
| Lab: | CNR Neuroscience Institute Milan and Dept. of Biotechnology and Translational Medicine, University of Milan, 20129 Milan, Italy |
| SynGO annotation ID: | 3679 |
| Dataset release (version): | 20231201 |
| View annotation as GO-CAM model: |  |