Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus

Essential revisions:

1) The authors show in Figure 4 that RNF10 knockdown decreases spine density and levels of certain synaptic proteins. Measuring how RNF10 knockdown affects mEPSCs and mIPSCs would strengthen the conclusions. A rescue condition should be included. Ideally, this experiment should be done using cultured slices or virus-infected brain slices rather than dissociated neurons to increase the relevance of the results to more intact circuits.

We agree with the reviewers that an evaluation of RNF10 function in more intact circuits is of great relevance. Accordingly, we have received very recently from KOMP Repository the RNF10 ko mice (RNF10^{tm1a(KOMP)Wtsi}) for in vivo/ex vivo studies that will represent the ideal follow up of the present manuscript.

In order to increase the relevance of results obtained in dissociated neurons we replicated the western blotting analysis shown in Figure 4F also from organotypic hippocampal slices infected with pLKO-shRNF10 lentivirus or scramble sequence as a control. As shown in Figure 4F of the revised version of the manuscript, RNF10 silencing resulted in a significant decrease of GluN2A, PSD-95 and GluA1 protein levels in the total cell homogenate of both pLKO-shRNF10-infected dissociated neurons and organotypic slices. Similarly, we also show that RNF10 silencing lead to a significant reduction of the expression of OphN1 expression in experiments performed both in organotypic slices (Figure 4G, H) and dissociate neurons (Figure 10H).

Following the reviewers’ indications, electrophysiological experiments have been repeated with the addition of a rescue condition, namely the co-transfection of a sh-resistant form of RNF10 (Flag-RNF10). As shown in Figure 11 of the revised version of the manuscript, the effect of RNF10 silencing on mEPSCs frequency was fully rescued by co-expressing Flag-RNF10 resistant to shRNA (Figure 11A, C). Furthermore, co-transfection of FlagRNF10 fully rescued a physiological long-lasting increase of the amplitude (Figure 11D) and frequency (Figure 11E) of mEPSCs as observed following induction of LTP.

2) It is unclear how RNF10 dissociates from GluN2A by NMDAR activation. If any mechanism were involved, would it be also involved in the enhanced interaction between RNF10 and importins/Meox2? These mechanisms should be explored to some extent or discussed.

As shown in Figure 3B and Figure 3E-H of the revised version of the manuscript we have performed additional experiments to address this important issue. Firstly, we have prepared novel GST-GluN2A fusion proteins to identify more precisely the GluN2A domain involved in the interaction with RNF10. As shown in Figure 3B we found that GluN2A(991-1049) is needed for the binding to RNF10. This GluN2A intracellular domain contains the GluN2A(991-1029) domain that has been recently identified as an atypical high affinity calcium/calmodulin binding site (Bajaj G, et al., 2014, Biochem Biophys Res Commun 444: 588-94). Notably, the formation of GluN2A/calmodulin complex was strictly dependent to the presence of calcium. In agreement with these results, here we show by in vitro pull-down assay (Figure 3E-G) and co-IP from transfected COS-7 cells (Figure 3H) that incubation with calcium/calmodulin leads to a complete disruption of the GluN2A/RNF10 complex. Overall, these new experiments indicate the existence of a competition between RNF10 and calmodulin for the binding to GluN2A that can take place following NMDA receptor activation and consequent calcium influx.