| Annotated protein: | SH3 and multiple ankyrin repeat domains protein 3 (Shank3) (Proline-rich synapse-associated protein 2) (ProSAP2) (SPANK-2). Gene symbol: SHANK3. Taxonomy: Mus musculus (Mouse). Uniprot ID: Q4ACU6 |
| antibody wiki: | |
| SynGO gene info: | SynGO data @ SHANK3 |
| Ontology domain: | Biological Process |
| SynGO term: | modulation of chemical synaptic transmission (GO:0050804) |
| Synapse type(s): | striatum, glutamatergic |
| Annotated paper: | Wang X, et al. "Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism" Nat Commun. 2016 May 10;7:11459 PMID:27161151 |
| Figure(s): | Figure 5, Figure 6, Figure 7, Supplementary Figure 6b-g and Supplementary Fig. 7a-f |
| Annotation description: | Figure 5, Excitatory synapses in striatal neurons are impaired. Literal: "we characterized the intrinsic excitability of MSNs by measuring spike frequency in response to depolarizing steps of current injection. Delta4-22-/- MSNs showed enhanced excitability, compared with Delta4-22+/- and Delta4-22+/+ neurons (Fig. 5a), while no genotype difference was observed for resting membrane potential (Fig. 5b). Delta4-22-/- mice displayed a marked reduction in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) (Fig. 5c,d), consistent with decreased spine density and number of glutamatergic synapses on MSNs in these mice. In contrast, the mean sEPSC amplitude was not affected by Shank3 deficiency (Fig. 5e,f). We used high-frequency stimulation (HFS)-induced long-term depression (LTD) to probe synaptic plasticity. LTD was significantly reduced in Delta4-22-/- mice, compared with the Delta4-22+/+ and Delta4-22+/- mice (Fig. 5g). These results indicate that multiple aspects of synaptic function are impaired in Delta4-22-/- striatal neurons." "Golgi staining revealed that spine density in the striatum of Delta4-22-/+ mice was reduced, but not in the hippocampus (Fig. 5h,i). Electron microscopic examination showed that the postsynaptic density (PSD) was significantly shorter and thinner in striatal synapses of Delta4-22-/- compared with Delta4-22+/+ mice (Fig. 5j-l). There was a similar trend for smaller PSD structures in hippocampal and cortical synapses (data not shown for cortical synapses)." Figure 6 and Supplementary Figure 6b-g, the mGluR5-Homer scaffolds complex is altered in striatal medium spiny neurons (MSNs). Literal: "we examined synaptic proteins in PSD fractions from Delta4-22 striata and hippocampi (Supplementary Fig. 6b). Pan-SAPAP and SAPAP3 levels were decreased only in striatum, while GluN2A was reduced only in hippocampus (Supplementary Fig. 6b). The most prominent changes in Delta4-22-/- mice were for the Homer family proteins (Fig. 6a-c). Homer1b/c was markedly decreased (18% of +/+) in striatum and mildly reduced in hippocampus (77% of +/+) relative to Delta4-22+/+ mice. There was no significant change for Homer1a in either brain region. In the Delta4-22-/- mice, Homer2 was significantly decreased in both brain areas (48% of +/+ in striatum; and 78% of +/+ in hippocampus). Homer3 (the least abundant isoform) was undetectable in striatum, but was reduced in the Delta4-22-/- hippocampus (20% of +/+). Unexpectedly, we found that mGluR5 was substantially increased in striatum (165% of +/+), but not in Delta4-22-/- hippocampus. These results indicate region-specific alterations in PSD proteins in Delta4-22-/- brains, especially in the striatum." "The mRNA levels for Homer1a and Homer1b/c in Delta4-22-/- striatum and hippocampus were similar to +/+ controls (Fig. 6d). Although total Homer1b/c protein was slightly decreased in Delta4-22-/- striatum (Fig. 6e,f), this is unlikely to explain the drastic reduction of Homer1b/c in the PSD. Sub-fractionation studies revealed a marked increase of Homer1b/c in the cytosolic fraction from Delta4-22-/- striatum (379% of +/+), while that in the hippocampus was similar to controls (Fig. 6g,h). In contrast, striatal cytosolic mGluR5 was decreased (70% of +/+), with no genotype differences in the hippocampus (Fig. 6i). Homer1b/c was also examined in Delta4-9 mice. The decreased expression of Homer1b/c in the PSD and the cytosolic accumulation of this protein in Delta4-9-/- striatum were less prominent than in Delta4-22-/- mice (Supplementary Fig. 6c,d,f,h). The increase in cytosolic Homer1b/c in Delta4-22-/- striatum was confirmed by Homer1b/c immunostaining of striatal neurons (Fig. 6j). Similar changes were observed in the NAC but not in the hippocampus or neocortex (Supplementary Fig. 6e,g). Conversely, the co-localization of Homer1b/c with the presynaptic marker Bassoon was significantly decreased in Delta4-22-/- striatum, consistent with decreased Homer1b/c in the PSD (Fig. 6k,l). Homer 1b/c immunostaining completely overlapped with the neuronal marker NeuN (Supplementary Fig. 7g), but only partially overlapped in dopamine D1 receptor (D1R) labeled neurons (defined by D1-td-Tomato) (Supplementary Fig. 7h). Since the striatum is composed primarily of MSNs containing either D1Rs or D2Rs, our findings suggest that mGluR5-Homer scaffolds are likely altered in both D1R- and D2R-positive MSNs." Figure 7 Supplementary Fig. 7a-f, mGlu5 signaling is altered in striatal medium spiny neurons (MSNs) Literal: "To corroborate the increased mGluR5 in striatal PSDs, we immune-stained for mGluR5 and PSD-95 in striatal slices. Co-localization studies revealed their increased association in Delta4-22-/- mice (Fig. 7a,b), supporting the biochemical evidence for elevated mGluR5 in synapses. Using an antibody recognizing the extracellular domain of mGluR5, we found that that surface mGluR5s were unchanged in cortical-striatal neuronal co-cultures (Supplementary Fig. 7a-d). However, the ratio of surface to intracellular mGluR5 was reduced in Delta4-22-/- MSN dendrites but not in soma (Fig. 7c-e), suggesting a selectively increased intracellular pool of mGluR5. The aforementioned mislocalization of Homer1b/c and mGluR5 likely causes disruption of the mGluR5-Homer scaffold, and this is supported by the reduced interaction of Homer1b/c and mGluR5 in Delta4-22-/- striatum (Fig. 7f). To further test if mGluR5-mediated signalling was altered, we examined the phosphorylation state of several kinases in the mGluR5-mediated pathway from Delta4-22-/- striatal and hippocampal slices. (Fig. 7g). Under the baseline condition, levels of p-ERK1/2 and p-S6K were significantly elevated in Delta4-22-/- striatum, but not in hippocampus (Fig. 7h,i). Activation of mGluR5 with the selective group I metabotropic agonist (S)-3,5-dihydroxyphenylglycine (DHPG) enhanced levels of p-ERK1/2 and p-S6K in the striatum in both genotypes. However, the DHPG-induced increase (fold change) of ERK1/2 and S6K phosphorylation was less apparent in Delta4-22-/- striatum (Fig. 7k,l), suggesting a weaker ligand-dependent response for mGluR5. By contrast, there was no genotype difference in p-mTOR level in Delta4-22 striatum and hippocampus before or after DHPG treatment (Fig. 7j,m). Since mGluR5 signalling regulates protein synthesis in hippocampus, we also evaluated protein synthesis in Delta4-22-/- striatum. No genotype difference was observed (Supplementary Fig. 7e,f), though cell type-specific changes cannot be excluded. These results suggest that mGluR5 function may be enhanced at baseline, but the ligand-dependent response of mGluR5 is attenuated in the De4-22-/- striatum." |
| Evidence tracking, Biological System: | Intact tissue |
| Evidence tracking, Protein Targeting: | Genetic transformation (eg; knockout, knockin, mutations) |
| 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: | 3686 |
| Dataset release (version): | 20231201 |
| View annotation as GO-CAM model: |  |