Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus
Understanding how local synaptic events are translated into changes in gene expression is a crucial question in neuroscience . Synapses and nuclei are efficiently connected by bidirectional communication routes that enable transfer of information ( Fainzilber et al . , 2011; Panayotis et al . , 2015 ) and regulate the transcription of genes associated with long-term structural changes of neuronal excitability ( Karpova et al . , 2012 ) . NMDAR activation plays a key role in this regard ( Hardingham and Bading , 2010 ) . NMDARs are heteromeric ionotropic channels that are essential for excitatory neurotransmission ( Paoletti et al . , 2013; Sanz-Clemente et al . , 2013 ) . NMDARs differ in their subunit composition and , in the forebrain , they can be either di- or tri-heteromeric tetramers consisting of two GluN1 and either one or two GluN2A or GluN2B subunits ( Paoletti et al . , 2013 ) . GluN2A-containing NMDARs are mainly localized at the postsynaptic membrane , at the core of the postsynaptic density ( PSD ) , while the GluN2B-containing NMDARs are also prominently present at extrasynaptic sites ( Hardingham and Bading , 2010 ) . In addition , numerous reports have indicated that GluN2A- and GluN2B-containing NMDARs may play different roles in the modulation of synaptic plasticity and in central nervous system ( CNS ) disorders ( Paoletti et al . , 2013 ) . These differences in receptor function are probably linked to the cytoplasmic C-tail of the two subunits , which are fairly different and contain specific motifs that bind to PSD-associated scaffolding proteins and to proteins involved in the downstream signal transduction of receptor activation ( Sanz-Clemente et al . , 2013; Sun et al . , 2016 ) . It is generally accepted that rises in synaptic , somatic and nuclear Ca2+ rapidly regulate gene expression by Ca2+-sensing mechanisms ( Bading , 2013 ) . Recent work showed that following a fast genomic response to sustained rises in Ca2+ , a considerably slower process that depends on the nuclear import of proteins released from synapses couples local synaptic events to more specific gene expression programs ( Jordan and Kreutz , 2009; Ch'ng and Martin , 2011; Karpova et al . , 2012 ) . The classical active nuclear import pathway involves the binding of importin α isoforms to nuclear localization signal ( NLS ) bearing cargo proteins . Interestingly , neuronal importins are present at synapses in association with NMDARs and the PSD ( Dieterich et al . , 2008; Jeffrey et al . , 2009 ) and they are able to translocate to the nucleus in response to NMDAR activation ( Thompson et al . , 2004; Dieterich et al . , 2008 ) . Proteomic studies have identified many proteins in purified synaptosomes that contain a NLS domain , and in the past 10 years , an impressive number of potential synaptonuclear messenger proteins have been characterized ( Ch'ng et al . , 2012; Karpova et al . , 2013; Kaushik et al . , 2014 ) . Many of these proteins contain a bona fide NLS and , in some cases , their binding with importin α was shown to be essential for long-distance transport as importin α can serve as an adaptor for nuclear trafficking of cargo in association with a dynein motor ( Karpova et al . , 2012; Ch'ng et al . , 2012 ) . In recent studies , it was shown that Jacob , a Caldendrin-binding protein abundantly expressed in limbic brain and cortex ( Dieterich et al . , 2008 ) , encodes and transduces the synaptic and extrasynaptic origin of GluN2B-containing NMDAR signals to the nucleus and elicits the divergent transcriptional responses after activation of these receptors ( Dieterich et al . , 2008; Karpova et al . , 2013 ) . These findings point to the fascinating possibility that specific NMDAR signals are encoded at synaptic sites and decoded in the nucleus by long-distance trafficking of protein messengers . Here , we identified Ring Finger protein 10 ( RNF10; Seki et al . , 2000 ) as a novel synaptonuclear protein messenger , localized at the PSD and specifically associated to the cytoplasmic tail of GluN2A but not GluN2B subunit of NMDARs . After stimulation of synaptic GluN2A-containing NMDARs , RNF10 binds to importin α1 for nuclear long-distance transport . Notably , we show that long-term potentiation ( LTP ) induces nuclear translocation of RNF10 , its interaction with the transcription factor Meox2 and the modulation of gene expression . Most interestingly , RNF10-regulated gene expression appears to feed back to synaptic function .
In order to identify new binding partners for GluN2A , we performed a yeast two-hybrid screening using the C-terminal domain ( aa 839–1461 , without the aa 1462–1464 PDZ-binding sequence ) as bait . We obtained a number of positive clones including RNF10 ( Seki et al . , 2000; Hoshikawa et al . , 2008 ) . In Schwann cells , RNF10 has a function in the transcriptional regulation of myelin formation ( Hoshikawa et al . , 2008 ) . However , very little is known about the neuronal function of this protein ( Seki et al . , 2000; Lin et al . , 2005; Hoshikawa et al . , 2008; Malik et al . , 2013 ) . Using specific glial ( GFAP ) and neuronal ( MAP2 ) markers , we confirmed that RNF10 is expressed in both glia ( Figure 1A ) and neurons ( Figure 1B ) . Interestingly , in neurons RNF10 displayed a nuclear and somatodendritic distribution ( Figure 1B ) . Moreover , transfected GFP-RNF10 displayed a prominent co-localization with PSD-95 and GluN2A at the dendritic spines of hippocampal neurons ( Figure 1C ) . Similarly , analysis of endogenous RNF10 in DIV14 primary hippocampal neurons showed clustered RNF10 immunolabeling along dendrites and most puncta co-localized with GluN2A ( Figure 1D , left panels ) and PSD-95 ( Figure 1D , right panels ) . PSDs from the rat hippocampus were purified to confirm the subcellular distribution of RNF10 by a biochemical approach . Subcellular fractionation demonstrated that RNF10 is associated with synaptic fractions and that it is prominently present in PSD fractions ( Figure 1E ) . Finally , immunofluorescence analysis of the CA1 region of the adult rat hippocampus revealed the presence of an intense signal for RNF10 in the soma and nuclei together with a punctate staining along MAP2-positive dendrites ( Figure 1F ) . 10 . 7554/eLife . 12430 . 003Figure 1 . RNF10 subcellular distribution in neurons . ( A , B ) Mixed primary hippocampal cultures ( DIV14 ) immunolabeled with antibodies for RNF10 ( red ) , glial marker GFAP ( A; green ) or the neuronal marker MAP2 ( B; green ) , and Dapi ( blue ) to stain the nucleus; scale bar: 20 μm . ( C ) Dendrite of hippocampal neuron transfected with GFP-RNF10 ( DIV7 ) and immunolabeled at DIV14 for GFP ( green ) , GluN2A ( red ) and PSD-95 ( blue ) ; scale bar: 3 μm . ( D ) High-magnification confocal images of neuronal dendrites ( DIV14 ) immunolabeled for endogenous RNF10 ( green ) and GluN2A ( red; left panels ) or PSD-95 ( red; right panels ) ; scale bar: 4 μm . ( E ) RNF10 and markers of the presynaptic ( synaptophysin ) and postsynaptic compartment ( PSD-95 , GluN2A ) were analyzed by WB in various subcellular compartments ( H: Homogenate fraction; S1: supernatant 1; P1: nuclear fraction; S2: cytosolic fraction 2; P2: crude membrane fraction 2; Syn: synaptosomal fraction; PSD1: Triton Insoluble postsynaptic fraction; PSD2: postsynaptic density fraction ) . ( F ) Representative confocal images of adult rat hippocampal CA1 pyramidal layer sections showing immunohistochemical labeling for RNF10 ( red ) , MAP2 ( green ) , and Dapi ( blue ) ; scale bar: 40 μm . ( G ) Confocal images of dendrites from hippocampal neurons ( DIV14 ) transfected at DIV7 with shGluN2A or scramble vector and immunolabeled for GluN2A; scale bar: 4 μm . The histogram shows the quantification of GluN2A integrated density in dendrites ( n=7 , **p=0 . 0069 scramble vs shGluN2A; unpaired Student’s t-test ) . ( H ) GluN2A silencing induces a reduction of RNF10 enrichment at the glutamatergic synapse . Confocal images of primary hippocampal neurons transfected with pGFP-V-RS-scramble ( left panels ) or with pGFP-V-RS-shGluN2A ( right panels ) plasmids and immunolabeled ( DIV14 ) for RNF10 ( green ) and PSD-95 ( red ) ; scale bar: 4 μm . The histogram shows the quantification of RNF10 co-localization with PSD-95-positive puncta ( n=30 , ***p<0 . 001; unpaired Student’s t-test ) . ( I ) Confocal images of dendrites from hippocampal neurons ( DIV14 ) transfected at DIV7 with shGluN2A or scramble vector and immunolabeled for surface GluN2A ( blue ) and RNF10 ( red ) ; scale bar: 4 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 003 Interestingly , GluN2A silencing in primary hippocampal neurons ( Figure 1G ) induced a significant decrease in RNF10 synaptic levels , as indicated by the reduction of RNF10 co-localization with PSD-95 ( Figure 1H ) . Notably , the remaining dendritic RNF10 in shGluN2A neurons co-localized with the surface GluN2A pool unaffected by the knock down ( Figure 1I ) . However , no modification of RNF10 nuclear level was observed following GluN2A silencing ( data not shown; n=30; p=0 . 5491; shGluN2A vs scramble; unpaired Student’s t-test ) . Different experimental approaches were used to substantiate the yeast two-hybrid data and to confirm the interaction between RNF10 and GluN2A . Co-immunoprecipitation ( co-i . p . ) studies performed from hippocampal P2 crude membrane fractions indicated a specific interaction of RNF10 with GluN2A but not with GluN2B subunit of the NMDARs ( Figure 2A ) . No signal for GluN2A or GluN2B was obtained by using anti-synaptophysin as an irrelevant antibody or in the absence of antibody in the co-i . p . assay ( Figure 2A ) . To further validate these findings we performed similar experiments with the GluN2B-associated synapse-to-nucleus messenger Jacob ( Dieterich et al . , 2008; Karpova et al . , 2013 ) . Indeed , the affinity-purified pan-Jacob antibody preferentially co-i . p . GluN2B from rat brain homogenate . Only a very faint band of GluN2A was detected in the complex with Jacob ( Figure 2B ) , which might potentially represent synaptic tri-heteromeric GluN1/GluN2A/GluN2B NMDARs . 10 . 7554/eLife . 12430 . 004Figure 2 . RNF10 interaction with GluN2A-containing NMDARs . ( A ) Co-immunoprecipitation ( co-i . p . ) assay performed in P2 crude membrane fractions by using antibodies against PSD-95 , RNF10 , synaptophysin ( Syn ) and GluN2A . WB analysis shows the levels of GluN2A ( left panel ) and GluN2B ( right panel ) in the co-immunoprecipitated material . No ab lane: control lane in absence of antibodies during the co-i . p . assay . ( B ) Jacob is a part of the GluN2B receptor complex . Affinity purified pan-Jacob antibodies co-immunoprecipitate GluN2B . ( C ) Co-i . p . assay performed by using an anti-RNF10 antibody from COS-7 cell extracts transfected with HA-GluN1 and GFP-GluN2A or GFP-GluN2B . WB analysis was performed by using anti-GFP and anti-RNF10 antibodies . No ab lane: control lane in absence of antibodies during the co-i . p . assay . ( D ) COS-7 cells expressing RNF10 were transfected with HA-GluN1 and GFP-GluN2A or GFP-GluN2B constructs and immunolabeled for GFP ( green ) , GluN1 ( blue ) , Dapi ( cyan ) and endogenous RNF10 ( red ) ; scale bar: 10 μm . ( E ) In situ detection of proximity between RNF10 and GluN2A ( red ) along MAP2 ( green; left panels ) or GFP-positive ( green; right panels ) dendrites . In control experiments ( - ) , primary hippocampal neurons were labeled with only RNF10 primary antibody and thus only unspecific PLA signals are generated; scale bars: 5 μm ( MAP2 ) and 3 μm ( GFP ) . ( F , G ) COS-7 cells expressing RNF10 were transfected with GFP-GluN2A ( right panels ) or GFP-GluN2B ( left panels ) constructs and immunolabeled for GFP ( green ) , Dapi ( cyan ) and endogenous RNF10 ( red ) ; scale bar: 10 μm . The histogram shows the quantification of RNF10 integrated density ( i . d . ) expressed as cytoplasm/nucleus ratio ( n=10; ***p<0 . 001; one-way ANOVA , followed by Bonferroni post-hoc test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 004 In addition , cell lysates from COS-7 cells transfected with HA-GluN1 and GFP-GluN2A or GFP-GluN2B constructs were immunoprecipitated with anti-RNF10 and immunoblotted for GFP . The results confirmed that GluN2A interacts with RNF10 , whereas GluN2B failed to associate with RNF10 ( Figure 2C ) . To corroborate these results , we transfected HA-GluN1 and either GFP-GluN2A or GFP-GluN2B constructs in heterologous cells ( COS-7 ) endogenously expressing RNF10 . Immunofluorescence studies revealed the presence of RNF10 aggregates with a high co-localization degree with GluN2A but not with GluN2B ( Figure 2D; 73 . 8% ± 2 . 9% GluN2A/RNF10 vs 31 . 3 . % ± 1 . 7% GluN2B/RNF10; p<0 . 001 , n=15; unpaired Student’s t-test ) . Finally , RNF10 clustering with GluN2A at synapses was confirmed by proximity ligation assay ( PLA ) . As shown in Figure 2E , a large number of PLA signals were detected when the two antibodies RNF10 and GluN2A were used indicating these two proteins are in close proximity ( <40 nm ) to each other . As expected , PLA signals were distributed along MAP2-positive dendrites ( Figure 2E , left panels ) and at the top of GFP-positive dendritic spines ( Figure 2E , right panels ) . For control experiments , only RNF10 primary antibody was used and no PLA signal was generated ( Figure 2E ) . Notably , transfection of COS-7 cells with GluN2A but not GluN2B induces a highly significant redistribution of endogenous RNF10 from the nucleus to the cytoplasm , thus suggesting that GluN2A traps RNF10 outside the nucleus ( Figure 2F–G ) . Overall , these results demonstrate that RNF10 is a component of the excitatory PSD and that it is specifically associated with GluN2A-containing NMDARs . We next mapped the binding interface of the interaction between RNF10 and GluN2A using truncation constructs in pull-down and co-i . p . experiments . The pull-down assay performed from adult rat brain tissue using glutathione S-transferase ( GST ) -GluN2A C-terminal domain fusion proteins ( Figure 3A ) revealed that RNF10 interacts with the GluN2A cytoplasmic tail ( CT; aa 839–1464 ) but failed to bind the distal part of the GluN2A C-terminus ( aa 1049–1464 ) ( Figure 3B , upper panel ) . In addition , GluN2A ( 839–991 ) failed to interact with RNF10 ( Figure 3B , lower panel ) , thus indicating GluN2A ( 991–1049 ) is needed for the formation of the GluN2A/RNF10 complex . 10 . 7554/eLife . 12430 . 005Figure 3 . RNF10 N-terminal domain interacts with the juxtamembrane region of GluN2A C-tail . ( A ) Scheme showing GST-GluN2A fusion proteins used in the pull-down assay . ( B ) GST and GST-GluN2A fusion proteins were incubated in a pull-down assay with rat hippocampal extracts . WB analysis was performed with RNF10 antibody . ( C ) Confocal images of COS-7 cells transfected with GFP-GluN2A ( 1–1049 ) and immunolabeled for GFP ( green ) , Dapi ( cyan ) and RNF10 ( red ) ; scale bar: 10 μm . ( D ) Co-i . p . assay performed from lysates of COS-7 cells transfected with GFP-GluN2A or GFP-GluN2A ( 1–1049 ) . WB analysis was performed by using a GFP antibody ( JL-8 ) . ( E ) GST and GST-RNF10 full-length ( FL ) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts in presence or absence of calcium ( 2 mM ) . WB analysis was performed with GluN2A antibody . ( F ) GST and GST-GluN2A ( 839–1464 ) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts in presence or absence of calcium ( 2 mM ) . WB analysis was performed with RNF10 and CaM antibodies . ( G ) GST and GST-GluN2A ( 839–1049 ) fusion proteins were incubated in a pull-down assay with rat hippocampal extracts with CaM ( 0 . 1 μM ) in the presence or absence of calcium ( 2 mM ) . WB analysis was performed with RNF10 and CaM antibodies . ( H ) Co-i . p . assay performed from lysates of COS-7 cells transfected with GFP , GFP-GluN2A or GFP-GluN2B in the presence or absence of calcium ( 2 mM ) /CaM ( 0 . 1 μM ) . WB analysis was performed by using RNF10 and CaM antibodies . ( I ) Co-i . p assay performed by using a GFP antibody from lysates of COS-7 cells transfected with RNF10 FL , RNF10 ( 221–802 ) and GFP-GluN2A . WB analysis was performed by using a Myc antibody . No ab lanes: control lanes in absence of antibodies during the co-i . p . assay . ( J ) Co-i . p assay performed by using a Myc antibody from lysates of COS-7 cells transfected with RNF10 truncation mutants and GFP-GluN2A . WB analysis was performed by using a GFP antibody ( JL-8 ) . ( K , L ) COS-7 cells expressing RNF10 were transfected with GFP-GluN2A and Myc-RNF10 FL or Myc-RNF10 ( 221–802 ) constructs and immunolabeled for GFP ( green ) , Myc ( red ) and Dapi ( blue ) ( G ) ; scale bar: 10 μm . The histogram ( H ) shows the quantification of Myc/EGFP co-localization index [n=10; ***p<0 . 001 RNF10 FL vs RNF10 ( 221–802 ) ; unpaired Student’s t-test] . ( M ) GST and GST-RNF10 fusion proteins were incubated in a pull-down assay with rat hippocampal extracts . WB analysis was performed with GluN2A antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 005 To strengthen these results , GFP-GluN2A ( 1–1049 ) truncation mutant ( bearing a stop codon at aa 1049 ) was transfected in COS-7 cells to evaluate its capability to interact with endogenous RNF10 ( Figure 3C , D ) . Immunofluorescence assay revealed a high GFP-GluN2A ( 1–1049 ) /RNF10 clustering leading also to RNF10 redistribution from the nucleus to the cytoplasm ( Figure 3C; cytoplasm/nucleus ratio 0 . 98 ± 0 . 04 vs 0 . 26 ± 0 . 02 GFP-GluN2A ( 1–1049 ) vs untransfected , p<0 . 001 , n=10; unpaired Student’s t-test ) , similarly to what has been described above for GFP-GluN2A construct ( see Figure 2 ) . Accordingly , RNF10 co-immunoprecipitated with both GFP-GluN2A and GFP-GluN2A ( 1–1049 ) from COS-7 lysates ( Figure 3D ) , confirming that RNF10 interacts with the GluN2A juxta-membrane aa 839–1049 region . Interestingly , GluN2A ( 991–1029 ) domain previously shown to be responsible for a calcium-dependent binding of the NMDAR subunit with Calmodulin ( CaM; Bajaj et al . , 2014 ) . Based on this consideration , we first evaluated whether calcium ( Ca2+ ) could modulate GluN2A/RNF10 interaction . Using a pull-down assay we found that the presence of free Ca2+ ( 2 mM ) in the buffer significantly reduced GluN2A binding to GST-RNF10 FL ( Figure 3E; -37 . 8% ± 6 . 2%; p<0 . 01 ) . Similarly presence of free Ca2+ ( 2 mM ) in the buffer significantly reduced RNF10 binding to GST-GluN2A ( 839–1464 ) ( Figure 3F; -65 . 4 ± 8 . 8%; p<0 . 05 ) . As previously reported ( Bajaj et al . , 2014 ) , Ca2+ induced also the binding of CaM to the GluN2A fusion protein ( Figure 3F ) . In addition , co-incubation with exogenous CaM completely prevented the interaction between RNF10 and GluN2A in the pull-down assay ( Figure 3G ) only in the presence of Ca2+ in the buffer ( Bajaj et al . , 2014 ) . Similarly , a co-i . p . assay from COS-7 cells transfected with GFP , GFP-GluN2A or GFP-GluN2B confirmed the capability of Ca2+/CaM to disrupt RNF10/GluN2A complex ( Figure 3H ) . The RNF10 protein contains a binding sequence for the transcription factor Mesenchyme Homeobox 2 ( Meox2; Meox2 Binding Domain , MBD , aa 101–185; Lin et al . , 2005 ) , a Ringer Finger Domain ( RFD , aa 225–270 ) and two putative nuclear localization sequences ( NLS1 , aa 591–599 and NLS2 , aa 784–791 ) . Several Myc-RNF10 mutants were prepared and co-transfected with GluN2A in COS-7 cells in order to identify the RNF10 domain responsible for the interaction with the NMDAR subunit . The RNF10 ( 221–802 ) construct failed to interact with GluN2A as demonstrated by both co-i . p . ( Figure 3I ) and co-localization ( Figure 3K , L ) assays when compared to RNF10 full length ( FL ) . Conversely , all RNF10 truncation mutants bearing the RNF10 ( 1–221 ) domain co-immunoprecipitated with GFP-GluN2A ( Figure 3J ) . Finally , pull-down assay performed from adult rat brain tissue using GST-RNF10 fusion proteins confirmed that the RNF10 N-terminal region 1–221 is crucial for the binding to GluN2A ( Figure 3M ) . RNF10 is a member of the Ring Finger Protein family , which has been generally implicated in development , transcriptional regulation , signal transduction , DNA repair and oncogenesis ( Saurin et al . , 1996 ) . However , the neuronal function of RNF10 is still unknown ( Lin et al . , 2005; Malik et al . , 2013 ) . To understand the role of RNF10 in neurons , we silenced RNF10 expression by using a short hairpin ( sh ) RNF10 knock-down or a scramble sequence ( as control ) in primary hippocampal cultures . We tested three different sequences of RNF10 shRNA ( shRNF10; see Materials and methods ) ; shRNF10 that led to the highest level of RNF10 downregulation ( >90%; TRCN0000041128 ) was selected and used in all experiments . Confocal imaging of hippocampal neurons transfected with shRNF10 or scramble plasmids demonstrated a significant effect of RNF10 knock-down on dendritic spine morphology ( Figure 4A–E ) . In particular , RNF10 silencing produced a significant reduction in dendritic spine density ( Figure 4B ) without any effect on dendritic spine length ( Figure 4C ) or dendritic spine head width ( Figure 4D ) . For a more detailed morphological analysis , dendritic spines were categorized according to their shape ( mushroom , thin and stubby ) using a validated classification method ( Bourne and Harris , 2008 ) . However , no effect of shRNF10 in the proportion of dendritic spine subtypes was observed ( Figure 4E ) . Notably , the effect of RNF10 silencing on dendritic spine density was fully rescued by co-expressing a wild-type human variant of RNF10 resistant to shRNA ( see Materials and methods; flag-RNF10; Figure 4A–E ) . Interestingly , overexpression of RNF10 per se had no effect on dendritic spine morphology ( data not shown ) . We then verified whether the loss of spines following RNF10 protein knock-down was correlated with an altered expression of the main components of the excitatory synapse . To this end , we infected primary hippocampal neurons ( Figure 4F , left panels ) and organotypic hippocampal slices ( Figure 4F , right panels ) with pLKO-shRNF10 lentivirus or scramble sequence as a control . As expected , RNF10 silencing produced a significant reduction of RNF10 protein level compared to dissociated neurons and slices treated with scramble construct ( Figure 4F ) . Most importantly , RNF10 silencing resulted in a significant decrease of GluN2A , PSD-95 and the GluA1 subunit of the AMPA receptor ( AMPAR ) protein levels in the total cell homogenate of pLKO-shRNF10-infected dissociated neurons and organotypic slices ( Figure 4F ) . 10 . 7554/eLife . 12430 . 006Figure 4 . RNF10 silencing induces molecular and morphological modifications of the glutamatergic synapse . ( A ) Confocal images of primary hippocampal neurons ( DIV14 ) transfected at DIV7 with pGIPZ-scramble , shRNF10 and shRNF10 plus flagRNF10 and immunolabeled for GFP ( green ) ; scale bar: 5 μm . ( B-E ) Histograms showing the quantification of dendritic spine density ( B ) ( n=6–10; *p<0 . 05 , scramble vs shRNF10; ***p<0 . 001 , shRNF10 vs shRNF10 + flagRNF10; one-way ANOVA , followed by Tukey post-hoc test ) , dendritic spine length ( C ) , dendritic spine head width ( D ) and dendritic spine type ( E ) . ( F ) WB analysis from homogenates of primary hippocampal neurons ( DIV14 ) and organotypic hippocampal slices ( DIV14 ) lentivirally infected with pGIPZ-scramble sequence ( scramble ) as control or with pLKO-shRNF10 ( shRNF10 ) . The histogram shows the quantification of the expression levels of GluN2A , GluA1 , PSD-95 and GluN2B in shRNF10-infected neurons and slices , normalized on tubulin and expressed as % of scramble ( n=6; *p<0 . 05; **p<0 . 01; unpaired Student’s t-test ) . ( G ) mRNA expression levels of genes associated with synaptic transmission or dendritic spine morphology by real-time PCR from DIV14 organotypic hippocampal slices lentivirally infected ( DIV4 ) with pGIPZ-scramble sequence ( scramble ) as control or with pLKO-shRNF10 ( shRNF10 ) ( n=4 , ***p<0 . 001; **p<0 . 01; *p<0 . 05; unpaired Student’s t-test ) . ( H ) WB for ArhGef6 , ArhGap4 , Ophn1 and tubulin from cell lysates of organotypic hippocampal slices infected with pGIPZ-scramble or with pLKO-shRNF10 . The histogram shows the quantification of protein levels from shRNF10 samples with respect to pGIPZ-scramble , following normalization on tubulin ( n=3 , *p<0 . 05; **p<0 . 01; unpaired Student’s t-test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 006 To learn more about the effect of RNF10 knock-down on gene expression , we performed a microarray analysis from organotypic hippocampal slices virally infected with pLKO-shRNF10 lentivirus in order to identify RNF10 target genes . Interestingly , heat map of differentially expressed genes ( Figure 5A ) , gene ontology analysis ( Figure 5B ) and real-time PCR validation ( Figure 4G ) showed that RNF10 silencing modulated the expression of several genes involved in excitatory synaptic transmission and dendritic spine morphology ( Vogt et al . , 2007; Michaluk et al . , 2011; Ramakers et al . , 2012 ) . Furthermore , we confirmed by WB analysis the effect of viral infection with pLKO-shRNF10 on protein levels of some of the RNF10 target genes , such as Ophn1 , ArhGap4 and ArhGef6 ( Figure 4H ) . 10 . 7554/eLife . 12430 . 007Figure 5 . Heat map and gene ontology of differentially expressed genes identified in microarray experiments . ( A ) Heat map of differentially expressed genes . Expression data are reported as log2 and blue color indicates high expression values and red color low expression value . Sc represents the different scramble control ( 1–4 ) , while Sh represents the shRNA against RNF10 ( 5–8 ) . ( B ) Gene ontology analysis of biological processes and enriched pathways analysis of differentially regulated genes in absence of RNF10 . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 007 The synaptic and nuclear localization in neurons , the presence of two different NLS motifs and its interaction with GluN2A and Meox2 ( Lin et al . , 2005 ) suggest that RNF10 could be a novel synaptonuclear protein messenger . To validate this hypothesis , we assessed whether the modulation of neuronal activity affects the subcellular localization of RNF10 and its association with the interacting proteins . We first enhanced synaptic excitatory activity with the GABA-A receptor antagonist Bicuculline ( 50 μM ) in the presence of the K+ channel blocker 4-AP ( 2 . 5 mM; Hardingham et al . , 2002 ) ( 'Bic' treatment ) . Enhanced excitatory activity significantly decreased RNF10 immunoreactivity along dendrites as compared to untreated neurons , without affecting PSD-95 ( Figure 6A ) labeling and induced a significant increase in RNF10 nuclear staining ( Figure 6D , left panels ) . This effect was not correlated to an alteration of RNF10 total protein amount as determined by WB analysis performed with neuronal lysates ( data not shown; +7 . 8% ± 8 . 6% , cLTP vs . control; p>0 . 05 , n=6 ) . To further confirm that RNF10 accumulates in the nucleus following long-distance protein transport , we performed time-lapse confocal imaging of RNF10 fused to photoconvertible tdEOS ( Figure 6B , C ) . We photoconverted RNF10-tdEOS in distal dendrites from green-to-red emission using 405 nm irradiation immediately after Bic treatment and tracked the red fluorescence signal over 90 min . Bic treatment induced a significant increase in RNF10-tdEOS photoconverted fluorescence intensity in the nucleus compared to untreated neurons accompanied by a decline in distal dendrites ( Figure 6B–C ) . 10 . 7554/eLife . 12430 . 008Figure 6 . Activation of synaptic NMDARs triggers RNF10 translocation to the nucleus . ( A ) Hippocampal neurons ( DIV14 ) were incubated for 24 hr with 50 μM Bicuculline and 2 . 5 mM 4-AP ( 'Bic' ) and immunolabeled for RNF10 ( green ) and PSD-95 ( red ) . The histogram shows the quantification of RNF10 levels along dendrites 24 hr after treatment ( n=30 , ***p<0 . 001; unpaired Student’s t-test ) ; scale bar: 4 μm . ( B , C ) Bic treatment induces RNF10-tdEOS translocation from distal dendrites to the nucleus in hippocampal neurons . ( B ) The histogram shows a significant increase in RNF10-tdEOS photoconverted fluorescent intensities in the nucleus following Bic treatment ( n=6; p<0 . 05 Bic vs . control , from 40' to 90'; unpaired Student’s t-test ) . ( C ) , left panels: Baseline confocal image of RNF10-tdEOS expressing hippocampal neuron illuminated sequentially with 488 nm and 555 nm laser excitation wavelengths showing no emitted signal in the red spectra ( ex555nm; left panels ) . Distal dendrite ( ROI ) selected for photoconversion was illuminated with UV laser ( 405 nm wavelengths ) repetitively through the image z-stack . ( C ) , right panels: Depicted are confocal max intensity projection images at respective time points in control after Bic treatment or in control ( untreated ) neurons; scale bar: 20 μm . ( D ) Hippocampal neurons ( DIV14 ) were treated with Bic in presence of the GluN2A inhibitor NVP-AAM007 at different concentrations ( 50 and 300 nM ) , immunolabeled for RNF10 ( green ) and stained with Dapi ( blue ) . The histogram shows the quantification of RNF10 integrated density in the nucleus expressed as % of control neurons ( n=10 , ***p<0 . 001 control vs Bic , Bic vs Bic+NVP 50 nM and Bic vs Bic+NVP 300 nM; one-way ANOVA followed by Bonferroni post-hoc test ) ; scale bar: 10 μm . ( E ) Hippocampal neurons ( DIV14 ) treated with 'Syn' ( 50 μM Bicuculline; 2 . 5 mM 4-AP; 5 μM Ifenprodil , 8 hr ) , with 'Extrasyn#1' or 'Extrasyn#2' protocols ( see Materials and methods ) , immunolabeled for RNF10 ( green ) and stained with Dapi ( blue ) . Histogram showing the quantification of RNF10 integrated density in the nucleus expressed as % of control neurons ( n=8 , *p<0 . 05 Extrasyn#2 vs Syn , **p<0 . 01 Syn vs Extrasyn#1 and Syn vs . control; one-way ANOVA followed by Bonferroni post-hoc test ) ; scale bar: 10 μm . ( F ) Depicted are representative laserscans averaged from three confocal sections of the nucleus of DIV16 hippocampal primary neurons immunolabeled with affinity purified antibodies against pan-Jacob ( rabbit ) and co-labeled with anti-MAP2 antibodies as a neuronal specific marker . Neuronal nuclei are outlined with the DNA stain Hoechst 34580 . Scale bar: 10 µm . Relative fluorescence intensities of Jacob 30 min of synaptic stimulation with and without selective inhibitors were normalized to untreated non-stimulated control ( n=31–70 , ***p<0 . 001; one-way ANOVA followed by Bonferroni post-hoc test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 008 Interestingly , the co-incubation of Bic with the GluN2A-specific inhibitor NVP-AAM007 ( NVP , 50 nM or 300 nM ) led to a significant reduction of RNF10 nuclear accumulation compared to Bic treatment alone also at a concentration of 50 nM , which is known to preferentially block di-heteromeric GluN1/GluN2A and not tri-heteromeric GluN1/GluN2A/GluN2B NMDARs ( Figure 6D; Foster et al . , 2010 ) . Similarly , Bic dependent increase in RNF10 nuclear staining was reduced by the co-incubation with the NMDAR blocker MK801 ( data not shown ) . Moreover , hippocampal neurons ( DIV14 ) were treated with Bicuculline ( 50 μM ) and 4-AP ( 2 . 5 mM ) in presence of the GluN2B antagonist ifenprodil ( 5 µM ) , corresponding to a well-validated protocol to obtain a specific activation of synaptic NMDARs ( Syn; Figure 6E , left panel; Hardingham et al . , 2002 ) . Synaptic NMDARs stimulation induced a significant increase in RNF10 nuclear accumulation ( Figure 6E , left panels ) . On the other hand , two different protocols known to activate extrasynaptic NMDARs ( Hardingham et al . , 2002; Karpova et al . , 2013 ) failed to induce the translocation of RNF10 from dendrites to the nucleus ( Figure 6E , right panel ) . In control experiments , we found that enhanced synaptic activity with Bicuculline ( 50 µm ) and 4-AP ( 2 . 5 mM ) resulted in an increase in nuclear Jacob as reported previously ( Figure 6F; Behnisch et al . , 2011; Karpova et al . , 2013 ) . This increase was abolished in the presence of the GluN2B antagonist ifenprodil ( 5 µM ) , but not with GluN2A antagonist NVP-AAM077 ( 50 nM ) indicating that Jacob dissociates from the synapses following activation of GluN2B but not di-heteromeric GluN2A containing NMDAR ( Figure 6F ) . The import of proteins from the cytosol into the nucleus through the nuclear pore complex depends on the binding of importins to a specific nuclear localization sequence ( NLS; Thompson et al . , 2004; Jordan and Kreutz , 2009; Karpova et al . , 2012 ) . According to this scheme , importins function as adapter molecules by binding NLS-bearing proteins . Components of the classical nuclear import machinery are present at synapses in association with NMDARs and importins translocate to the nucleus in response to NMDAR stimulation ( Thompson et al . , 2004; Jeffrey et al . , 2009; Marfori et al . , 2011; Ch'ng et al . , 2012 ) . Analysis of the RNF10 sequence showed the presence of two putative NLS motifs ( NLS1: aa 591–599; NLS2: aa 784–791; Seki et al . , 2000; Marfori et al . , 2011 ) . We therefore investigated the neuronal localization of RNF10 by transfection of RNF10 FL and RNF10 truncated mutants lacking one [Myc-RNF10 ( 1–611 ) ] or both NLS motifs [Myc-RNF10 ( 1–590 ) ] in primary hippocampal neurons . Myc-RNF10 FL showed an intense nuclear accumulation ( as the endogenous protein; Figure 7A ) . However , both truncated mutants failed to localize to the nucleus as indicated by a very low co-localization with the nuclear marker Dapi ( Figure 7A ) . In particular , also the longest Myc-RNF10 ( 1–611 ) construct lacking only the C-terminal NLS2 domain accumulated in the cell soma but did not show a marked nuclear localization ( Figure 7A ) , thus suggesting that the second NLS2 motif could be responsible for RNF10 nuclear translocation in neurons . Notably , the overexpression of Myc-RNF10 ( 1–611 ) construct produced a significant reduction in the density of PSD-95-positive puncta along dendrites ( Figure 7B , upper panels ) similar to that observed following RNF10 silencing ( Figure 7B , lower panels ) , indicating that this protein fragment can act in a dominant negative manner . 10 . 7554/eLife . 12430 . 009Figure 7 . RNF10 translocates into the nucleus through NLS2-dependent interaction with importin α1 . ( A ) Confocal images of primary hippocampal neurons transfected with Myc-RNF10 full-length ( FL ) or Myc-RNF10 truncated mutants and immunolabeled for Myc ( green ) and Dapi ( blue ) ; scale bar: 20 μm . ( B ) Primary hippocampal neurons transfected with Myc , Myc-RNF10 ( 1–611 ) , pGIPZ-scramble or with shRNF10 were immunolabeled ( DIV14 ) for PSD-95 . The histograms show the quantification of PSD-95-positive puncta expressed as % of Myc ( upper histogram ) or scramble ( lower histogram ) transfected neurons ( n=50–88 , ***p<0 . 001 , Myc-RNF10 ( 1–611 ) vs Myc and shRNF10 vs scramble; unpaired Student’s t-test ) ; scale bar: 4 μm . ( C ) Confocal images of dendrites of hippocampal neurons ( DIV14 ) immunolabeled for RNF10 ( red ) , surface GluN2A ( green ) and importin α1 ( blue ) ; scale bar: 4 μm . ( D ) Importin α1 and PSD-95 protein levels were analyzed by means of WB analysis in various subcellular compartments purified from rat hippocampal tissue ( H: Homogenate fraction , S1: supernatant 1 , P1: nuclear fraction , S2: cytosolic fraction 2 , P2: crude membrane fraction 2 , Syn: synaptosomal fraction , PSD1: Triton insoluble postsynaptic fraction , PSD2: postsynaptic density fraction ) . ( E ) Representative co-i . p . assay showing the interaction between RNF10 and importin α1 in hippocampal tissue homogenate ( upper and lower panel ) and PSD fraction ( lower panel ) . No Ab lane: control lane in absence of the antibody . WB analysis was performed with RNF10 and importin α1 antibodies . ( F ) RNF10-tagged with GFP was co-expressed with multiple importin α isoforms ( KPNA1-KPNA6 ) tagged with tagRFP in HEK293T cells . RNF10-GFP was immunoprecipitated from cell extract using anti-GFP MicroBeads . Co-immunoprecipitated importinerase - α isoforms were detected in complex with RNF10 using anti-tagRFP antibodies . ( G ) Co-i . p . assay performed by using an importin α1 antibody from cell homogenates of control ( C ) neurons or treated with Bic . WB analysis was performed with RNF10 antibody . The histogram shows the quantification of RNF10/importin α1 interaction expressed as % of control ( n=3 , *p<0 . 05; unpaired Student’s t-test ) . ( H ) Representative co-i . p . assay from cell homogenates of control ( C ) neurons or treated with Bic . WB analysis was performed with RNF10 antibody . The histogram shows the quantification of RNF10 interaction with GluN2A and Meox2 expressed as % of control ( n=3 , ***p<0 . 001 , Meox2 , Bic vs control; **p<0 . 01 , GluN2A , Bic vs control; unpaired Student’s t-test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 009 Based on these results , we next evaluated a possible RNF10 interaction with importins in hippocampal neurons . RNF10 displayed a prominent co-localization with importin α1 ( KPNA2 gene product; see Zienkiewicz et al . , 2013 ) and surface GluN2A along dendrites of DIV14 primary hippocampal neurons ( Figure 7C ) . In agreement with previous data about other importins ( Thompson et al . , 2004; Jeffrey et al . , 2009; Marfori et al . , 2011; Ch'ng et al . , 2012 ) , analysis of importin α1 subcellular distribution showed its presence in purified PSDs from rat hippocampus ( Figure 7D ) . Immunoprecipitation experiments performed revealed that RNF10 interacts with importin α1 in neuronal cells ( Figure 7E , upper panel ) . Interestingly , RNF10 co-precipitation with importin α1 was detected both in PSD and homogenate fraction but it was much more prominent in PSDs ( Figure 7E , lower panel ) . In order to verify the capability of RNF10 to interact also with other importins , we performed heterologous co-immunoprecipitation experiments of RNF10 fused to GFP with six importin α isoforms ( KPNA1-KPNA6 gene products ) fused to tag-RFP . As shown in Figure 7F , RNF10 can interact with majority of importin α isoforms except importin α3 ( KPNA4 gene product ) . Bic treatment significantly increased RNF10/importin α1 association ( Figure 7G ) . Interestingly , also the RNF10 interaction with the transcription factor Meox2 was significantly increased by Bic treatment ( Figure 7H ) . On the contrary , Bic treatment significantly decreased RNF10/GluN2A interaction compared to control neurons ( Figure 7H ) , which is in agreement with the reduction of RNF10 labeling along dendrites ( see Figure 6A ) . Taken together these findings indicate that enhancing synaptic activity of di-heteromeric GluN2A-containing NMDARs results in the dissociation of RNF10 from the cytoplasmic tail of GluN2A at postsynaptic sites and a tighter association with neuronal importin and importin-dependent nuclear translocation , followed by an association of RNF10 with the transcription factor Meox2 . LTP is widely considered as one of the major cellular mechanisms that underlie learning and memory ( Bliss and Collingridge , 1993 ) and requires de novo protein synthesis and gene transcription for its maintenance ( Smolen et al . , 2012 ) . We next asked whether RNF10 translocates to the nucleus from distal dendrites following induction of LTP in mature hippocampal neuronal cultures . For induction of LTP we used two established protocols ( Deisseroth et al . , 1996; Otmakhov et al . , 2004; Oh et al . , 2006 ) . First , we employed 18s@50Hz field stimulation , which is known to produce a stable synaptic potentiation in cultured hippocampal neurons ( Deisseroth et al . , 1996 ) and in parallel we performed time-lapse confocal imaging of RNF10 fused to photoconvertible tdEOS ( Figure 8A–C ) . The rise in intracellular Ca2+ concentration upon LTP-inducing stimuli was verified by Fluo-4-AM fluorescence ( data not shown ) . We photoconverted RNF10-tdEOS in distal dendrites ( Figure 8A ) and tracked the red fluorescence signal over time . We observed a significant increase in RNF10-tdEOS photoconverted green-to-red fluorescence intensity in the nucleus when compared to unstimulated neurons ( Figure 8B , C ) accompanied by a concomitant decline in distal dendrites upon stimulation ( Figure 8C , lower panels ) . This emphasizes that the synaptodendritic but not the somatic pool of RNF10 largely contributes to its nuclear transport upon LTP-inducing stimuli . Thus , RNF10 accumulates in the nucleus following long-distance protein transport . 10 . 7554/eLife . 12430 . 010Figure 8 . LTP induction triggers RNF10 translocation to the nucleus . ( A–C ) RNF10-tdEOS translocates from distal dendrites to the nucleus in mature hippocampal neurons upon high-frequency 18s@50Hz field synaptic stimulation . Distal dendrites ( ROIs ) selected for photoconversion were illuminated with UV laser ( 405 nm wavelengths ) repetitively through the image z-stack ( image t0` ) . ( A ) Representative images of control and stimulated RNF10-tdEOS expressing hippocampal neurons illuminated with 568 nm laser excitation wavelength 1 min following photoconversion . ( B ) The histogram shows a significant increase in RNF10-tdEOS photoconverted fluorescent intensities in the nucleus following high frequency 18s@50Hz field synaptic stimulation ( n=9 , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001; unpaired Student’s t-test ) . ( C ) Depicted are confocal max intensity projection images of control and stimulated RNF10-tdEOS expressing hippocampal neurons at respective time points after stimulation . Lower panels: magnified image sequence of UV illuminated dendritic segment with multiple spines is represented showing the decrease in distal dendrites upon field stimulation . All experiments were performed in a presence of anisomycin ( 7 . 5 µM ) . ( D ) Confocal images of dendrites of hippocampal neurons ( DIV14 ) transfected with GFP ( green ) to visualize neurites and immunolabeled for RNF10 ( red ) and PSD-95 ( blue ) . The histogram shows the quantification of RNF10 signal in dendrites 2 hr after induction of cLTP ( see Materials and methods ) expressed as % of control ( n=5 , **p<0 . 01; unpaired Student’s t-test ) ; scale bar: 4 μm . ( E ) Co-i . p . assay from hippocampal extracts performed with an antibody against GluN2A . WB analysis was performed using antibody for RNF10 and GluN2A . The graph shows the effect of cLTP induction on RNF10 interaction with GluN2A expressed as % of control ( n=4 , ***p<0 . 001; unpaired Student’s t-test ) . ( F ) Representative confocal images of hippocampal neurons ( DIV14 ) after the induction of cLTP in the presence or absence of 7 . 5 μM anisomycin and immunolabeled for RNF10 ( green ) and Dapi ( blue ) ; scale bar: 10 μm . ( G ) WB analysis for RNF10 from P1 nuclear fraction purified from hippocampal neurons after the induction of cLTP in the presence or absence of 7 . 5 μM anisomycin . The histogram shows the quantification of RNF10 integrated density normalized on Histone-H3 ( n=4 , *p<0 . 05 , control vs cLTP and control vs cLTP+Anisomycin; one-way ANOVA , followed by Tukey post-hoc test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 010 We next used a validated chemical LTP ( cLTP ) protocol ( Otmakhov et al . , 2004; Oh et al . , 2006 ) , which is known to activate synaptic NMDARs in primary hippocampal neurons . As expected , induction of cLTP leads to an increase of CREB-Ser133 ( P-CREB; Bito et al . , 1996 ) and GluA1-Ser845 ( P845-GluA1; Marcello et al . , 2013 ) phosphorylation ( data not shown ) . Under these experimental conditions , cLTP induction produced a significant decrease in endogenous RNF10 immunostaining along dendrites ( Figure 8D ) and a concomitant significant increase of RNF10 localization in the nucleus as revealed by western blotting in a crude nuclear fraction ( Figure 8G ) and co-localization assays with the nuclear marker Dapi ( Figure 8F and Figure 9B ) . Consistent with the above-described results ( see Figure 7H ) , cLTP detached RNF10 from GluN2A ( Figure 8E ) . These effects were not correlated to an alteration of RNF10 total protein abundance as determined by WB analysis performed with neuronal lysates ( data not shown; +9 . 5% ± 8 . 7% , cLTP vs . control; p>0 . 05 , n=8 ) . Finally , we used anisomycin to exclude the possibility that de novo RNF10 protein synthesis could account for the nuclear accumulation of RNF10 following cLTP ( Figure 8F , G ) . Anisomycin treatment did not prevent nuclear accumulation of RNF10 in this assay ( Figure 8F , G ) . 10 . 7554/eLife . 12430 . 011Figure 9 . NLS2 peptide but not anysomicin blocks RNF10 accumulation in the nucleus after induction of cLTP . ( A ) Confocal images from living hippocampal neurons transfected with dTomato ( DIV10 ) and treated with TAT-TF2 or NLS2-TF2 coniugated peptide ( DIV14 ) . Samples were illuminated with 543 nm and 488 nm to visualize respectively neurons and peptide . The representative image shows the presence of peptide ( green ) within the neuron ( red , DTOMATO ) , demonstrating the capability of crossing the plasmatic membrane; scale bar: 10 μm . ( B ) Representative co-i . p . assay performed by using anti-RNF10 antibody and showing the interaction between RNF10 and importin α1 in primary hippocampal neurons ( DIV14 ) treated with NLS2 peptide ( active ) or TAT ( control ) peptide . No IgG lane: control lane in absence of the antibody . WB analysis was performed with importin α1 and RNF10 antibodies . The histogram shows the quantification of importin α1 interaction with RNF10 expressed as % of control ( TAT; n=3; *p<0 . 05; unpaired Student’s t-test ) . ( C , E ) Hippocampal neurons ( DIV14 ) were treated with NLS2 peptide ( active ) or TAT ( inactive ) peptide for 24 hr and then cLTP was induced in the presence of the same peptides . Confocal images show the immunolabeling for RNF10 ( green ) and the staining for Dapi ( blue ) in the nucleus ( C ) or PSD-95 ( red ) along dendrites ( E ) ; scale bars: 10 μm ( C ) and 4 μm ( E ) . ( D , F ) The histograms show the quantification of RNF10 signal in the nucleus ( D ) and along dendrites ( F ) after the induction of cLTP in the presence of TAT or NLS2 peptides expressed as % of control [n=10 , 11 ( D ) , n=17–19 ( F ) ]; *p<0 . 05; **p<0 . 01; *p<0 . 001; one-way ANOVA , followed by Tukey post-hoc test ) . ( G , H ) WB analysis for RNF10 from P1 crude nuclear fraction purified from hippocampal neurons after the induction of cLTP in the presence of NLS2 or TAT peptide ( G ) . The histogram ( H ) shows the quantification of RNF10 integrated density normalized on Histone-H3 and expressed as % of control ( n=4 , *p<0 . 05; one-way ANOVA , followed by Tukey post-hoc test ) . ( I ) Representative co-i . p . assay performed with an antibody against RNF10 from cell lysates of hippocampal neurons following induction of cLTD . WB analysis was performed using antibody for RNF10 and GluN2A . ( J ) Confocal images of the soma of hippocampal neurons ( DIV14 ) treated with a protocol to induce cLTD and then immunolabeled for RNF10 ( green ) and stained with Dapi ( blue ) . Scale bar: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 011 The RNF10 ( 1–611 ) protein fragment , lacking the NLS2 motif , fails to localize to the nucleus ( Figure 7A ) . Taking this into account , we induced cLTP in the presence of an exogenously added RNF10 NLS2 peptide , thus competing with endogenous RNF10/importin binding . RNF10 NLS2 peptide is mostly composed of basic residues having a very high similarity with the most common cell-permeable peptides ( CPPs ) such as the TAT peptide ( Farkhani et al . , 2014 ) . However , before the evaluation of the effect of NL2 peptide on RNF10 subcellular distribution , we first verified the ability of this peptide to act as a CPP , that is , that it can cross the plasma membrane and enter neurons . As shown in Figure 9A , we detected TideFluorTM-tagged TAT and NLS2 peptides into neurons to a similar extent , thus indicating the capability of the NLS2 peptide to work as a CPP . Notably , NLS2 peptide induced a significant reduction of RNF10/importin α1 interaction when compared to treatment with TAT ( used as control peptide; Figure 9B ) . The NLS2 peptide completely prevented the LTP-dependent increase in nuclear RNF10 , as indicated by immunofluorescence ( Figure 9C , D ) and by immunoblot analyses of RNF10 protein levels in a P1 crude nuclear fraction ( Figure 9G , H ) . Importantly , treatment with NLS2 peptide also prevented the decrease in dendritic RNF10 labeling observed after induction of cLTP ( Figure 9E , F ) . Overall , these results further confirm that NLS2-dependent binding to importin represents a key step for RNF10 trafficking to the nucleus . Conversely , induction of chemical long-term depression ( cLTD ) did not modify RNF10 binding to GluN2A ( Figure 9I; +0 . 9% ± 4 . 8% , cLTD vs . control; p>0 . 05 ) and the nuclear distribution of RNF10 in hippocampal neurons ( Figure 9J ) . We next asked whether RNF10 translocation from dendritic spines to the nucleus produced a modulation of the protein level of Meox2/RNF10 target genes . First , we analyzed the expression of p21WAF1/cip1 , a known Meox2 target gene ( Lin et al . , 2005; Malik et al . , 2013 ) . Bic treatment increased p21WAF1/cip1 levels as evaluated by immunofluorescence ( Figure 10A , B ) and Western blot analysis ( Figure 10C ) . Up-regulation of p21WAF1/cip1 was significantly attenuated by co-treatment with the NMDAR blocker MK801 ( Figure 10A–C ) . Similarly , stimulation of synaptic NMDARs induced an increase in p21WAF1/cip1 that was blocked by bath application of MK801 ( Figure 10D ) . Notably , the increase in p21WAF1/cip1 protein levels following stimulation of synaptic NMDARs was completely prevented by RNF10 silencing ( Figure 10E ) , indicating that modulation of p21WAF1/cip1 expression is RNF10-dependent . In addition , the same effect on p21WAF1/cip1 levels was observed following induction of cLTP . Again , the increased expression of p21WAF1/cip1 was prevented by RNF10 silencing ( Figure 10F ) . Finally , RNF10 silencing decreased the expression of the newly identified RNF10 target gene Ophn1 ( see Figure 4G , H ) also after synaptic stimulation ( Figure 10G ) . 10 . 7554/eLife . 12430 . 012Figure 10 . RNF10 nuclear trafficking regulates the expression level of specific target genes . ( A ) Hippocampal neurons ( DIV14 ) were treated with Bic in the presence or absence of MK801 , immunolabeled for MAP2 ( red ) , p21WAF1/cip1 ( green ) and stained with Dapi ( blue ) ; scale bar: 10 μm . ( B ) Histogram showing the quantification of p21WAF1/cip1 signal in the nucleus expressed as % of control ( n=15 , ***p<0 . 001 Bic vs control and Bic+MK801 vs Bic; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( C ) WB analysis from cell lysates of hippocampal neurons treated with Bic in presence or absence of MK801 . The histogram shows the quantification of p21WAF1/cip1 integrated density normalized on tubulin ( n=5 , **p<0 . 01 Bic vs control and Bic+MK801 vs Bic; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( D ) WB analysis for p21WAF1/cip1 and tubulin performed on cell lysates from DIV14 hippocampal neurons treated for synaptic stimulation ( Syn; see Materials and methods ) in the presence or absence of MK801 . The histogram shows the quantification of p21WAF1/cip1 levels normalized on tubulin ( expressed as integrated density ) ( n=5 , **p<0 . 01 , Syn vs control; *p<0 . 05 Syn+MK801 vs Syn; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( E ) WB analysis for RNF10 , p21WAF1/cip1 and tubulin performed on cell lysates from hippocampal neurons virally infected with pLKO-shRNF10 or control vector and treated at DIV14 for Syn . The histogram shows the effect of RNF10 silencing on p21WAF1/cip1 expression levels normalized on tubulin ( n=4 , *p<0 . 05 , Syn vs control and Syn+shRNF10 vs Syn; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( F ) WB analysis for p21WAF1/cip1 and tubulin from hippocampal extracts following induction of cLTP in the presence or absence of viral infection with pLKO-shRNF10 . The graph shows the modulation of p21WAF1/cip1 levels ( following normalization on tubulin ) ( n=5 , *p<0 . 05 , cLTP vs control , **p<0 . 01 , cLTP + pLKO-shRNF10 vs cLTP; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( G ) WB analysis for Ophn1 and tubulin performed on cell lysates from hippocampal neurons virally infected with pLKO-shRNF10 or control vector and treated at DIV14 for Syn . The histogram shows the effect of RNF10 silencing on Ophn1 expression levels normalized on tubulin ( n=3 , *p<0 . 05 , Syn+shRNF10 vs Syn; one-way ANOVA , followed by Bonferroni post-hoc test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 012 Altogether these results indicate that stimulation of synaptic NMDARs or induction of LTP leads to RNF10 translocation to the nucleus , where it binds to Meox2 and stimulates the expression of RNF10/Meox2 target genes ( Lin et al . , 2005; Malik et al . , 2013 ) , such as p21WAF1/cip1 and Ophn1 . We finally analyzed whether RNF10 silencing leads not only to a decrease of dendritic spine density ( Figure 4A–E ) but also to an alteration of the plastic properties of synapses . To this end , we recorded miniature EPSCs ( mEPSCs ) in hippocampal neurons before and for 2 hr after cLTP induction . In basal conditions , down-regulation of RNF10 led to a significant decrease of the frequency ( Figure 11A , C ) but not the amplitude ( Figure 11A , B ) of mEPSCs . Notably , no alteration of mEPSCs frequency was observed by co-expressing shRNF10 with the wild-type human variant of RNF10 resistant to shRNA ( FlagRNF10; Figure 11A , C ) . Furthermore , while in control neurons cLTP induction resulted in a long-lasting increase of the amplitude and frequency of mEPSCs , this effect was totally prevented in neurons transfected with shRNF10 ( Figure 11D , E ) . Again , co-transfection of FlagRNF10 fully rescued a physiological long-lasting increase of the amplitude ( Figure 11D ) and frequency ( Figure 11E ) of mEPSCs . 10 . 7554/eLife . 12430 . 013Figure 11 . Down-regulation of RNF10 expression prevents cLTP expression . ( A ) mEPSCs recorded at -60 mV before ( basal ) , and after cLTP induction in hippocampal neurons transfected or not ( CT ) with shRNF10 or with shRNF10 + FlagRNF10 . ( B , C ) Basal amplitude ( B ) and frequency ( C ) of mEPSCs , prior cLTP induction ( **p<0 . 01 , shRNF10 vs CT ) . ( D , E ) Time-course of amplitude ( D ) and frequency ( E ) of mEPSC following cLTP induction ( *p<0 . 05 , shRNF10 vs CT ) . ( F , H ) Effects of RNF10 overexpression on cLTP-induced modifications of dendritic spine morphology . Confocal images of hippocampal neuron dendrites ( DIV14 ) transfected at DIV7 with GFP or GFP-RNF10 , treated for cLTP induction and immunolabeled for PSD-95 ( F ) or GluA1 ( H ) ; scale bar: 4 μm . The histograms show the quantification of PSD-95 ( F ) and GluA1 ( H ) clusters width ( PSD-95: n=6 , ***p<0 . 001; GluA1: n=8–10 , *p<0 . 05; one-way ANOVA , followed by Bonferroni post-hoc test ) . ( G , I ) Effects of RNF10 silencing on cLTP-induced modifications of dendritic spine morphology . Confocal images of hippocampal neuron dendrites ( DIV14 ) transfected at DIV7 with scramble sh or shRNF10 , treated for cLTP induction and immunolabeled for PSD-95 ( G ) or GluA1 ( I ) ; scale bar: 4 μm . The histograms show the quantification of PSD-95 ( G ) and GluA1 ( I ) clusters width ( PSD-95: n=6 , ***p<0 . 001; GluA1: n=8–10 , ***p<0 . 001; one-way ANOVA , followed by Bonferroni post-hoc test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12430 . 013 We then tested whether different levels of RNF10 expression could affect not only spine density ( see Figure 4A–E ) but also interfere LTP-dependent modulation of dendritic spine size ( Bosch and Hayashi , 2012 ) . To this end , we overexpressed or silenced RNF10 in hippocampal neurons ( DIV7 ) followed by induction of cLTP ( DIV14 ) . As expected , cLTP increased significantly PSD-95 cluster width in control neurons ( Figure 11F , G ) . RNF10 overexpression produced an increase in PSD-95 clusters width also in absence of stimulation and occluding any further increase of PSD-95 clusters width following induction of cLTP ( Figure 11F ) . On the other hand , RNF10 silencing blocked any cLTP-dependent modification of PSD-95 clusters ( Figure 11G ) . Evaluation of GluA1 synaptic cluster size confirmed that RNF10 silencing precludes any increase in GluA1 clusters width induced by cLTP ( Figure 11I ) .
This report describes a novel synapse-to-nucleus signaling pathway that specifically links activation of synaptic GluN2A-containing NMDARs to nuclear gene expression . We demonstrate that RNF10 is highly expressed in the nucleus as well as at synapses , where it is part of the NMDAR complex and directly interacts with the cytoplasmic tail of the GluN2A subunit of NMDARs . RNF10 dissociates from the NMDAR complex in an activity-dependent manner and we provide compelling evidence for the importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus . The intracellular C-terminal domain of GluN2A contains several target sequences for downstream signaling and scaffolding molecules ( Sanz-Clemente et al . , 2013; Sun et al . , 2016 ) . Among others , RNF10 and CaM share the same GluN2A binding region ( aa 991–1029; Bajaj et al . , 2014 ) . Our results indicate a preferential formation of RNF10/GluN2A complex in resting conditions that is disrupted in favor of CaM/GluN2A complex following synaptic activity-dependent calcium influx . Overall , our data show that RNF10 binding to GluN2A plays a key role for RNF10 anchoring at the excitatory synapse but also interfere with the formation of CaM/NMDAR complex . Moreover , the presence of RNF10 in close proximity to the receptor channel allows for a rapid calcium-dependent dissociation of RNF10/GluN2A complex following NMDAR activation and for the subsequent formation of RNF10/importin complex . Here , we show that interaction of RNF10 with importin α1 represents a key step for its nuclear translocation . However , we also describe that RNF10 can interact with the majority of importin α isoforms including the importin α5 previously described to be associated with the NMDAR complex ( Jeffrey et al . , 2009 ) . Taken together , these findings suggest that synaptonuclear trafficking of RNF10 is involved in the control of gene expression , which is necessary for LTP-type synaptic plasticity in hippocampal neurons . Very few studies are available addressing the role of RNF10 in the CNS , and they mainly address its association with the transcription factor Meox2 , which regulates cell proliferation and differentiation ( Seki et al . , 2000; Lin et al . , 2005; Hoshikawa et al . , 2008; Malik et al . , 2013 ) . A very recent study performed in P19 carcinoma cell line and in mouse cerebellar granule cells suggests that RNF10 acts as a positive regulator of neuronal differentiation , as its knockdown reduced the number of cells expressing early and late neuronal markers ( Malik et al . , 2013 ) . Notably , microarray , subsequent real-time PCR and western blotting analysis allowed us to find , among others , novel RNF10 target genes known to be involved in the regulation of the excitatory synapse function and morphology ( Vogt et al . , 2007; Michaluk et al . , 2011; Bagni et al . , 2012; Ramakers et al . , 2012; Pavlowsky et al . , 2012 ) . RNF10 silencing induced a dramatic up-regulation of MMP9 expression and a concomitant reduction of Arhgef6 , ArhGap4 and Ophn1 levels . Intriguingly , all these genes are mutated or dysregulated in intellectual disability syndromes in humans and/or in the corresponding mouse models ( Vogt et al . , 2007; Michaluk et al . , 2011; Ramakers et al . , 2012 ) , which are characterized by various alterations in dendritic spines , thus suggesting a key role in synaptic effects observed following RNF10 silencing . In this view , modulation of RNF10 expression in hippocampal neurons demonstrated its relevant role in regulating dendritic spine morphology under resting conditions as well as following activity-dependent plasticity . In particular , RNF10 silencing leads to a significant decrease in spine density that is fully rescued by co-transfection of shRNA-resistant RNF10 construct . As a direct consequence of this dramatic morphological event , we also observe a concomitant reduction in the expression of proteins almost exclusively localized at the excitatory PSD , such as PSD-95 , AMPARs GluA1 subunit and NMDARs GluN2A subunit . Notably , overexpression of the RNF10 ( 1–611 ) construct unable to translocate to nucleus leads to a significant reduction in PSD-95 positive puncta along dendrites . This result strongly indicates that an impaired RNF10-mediated synaptonuclear signaling and not a reduction of RNF10 synaptic levels is responsible for the reduction of dendritic spine density observed following RNF10 silencing . The present and other studies indicate that NMDAR complex is likely a very rich source of protein messengers that are capable of trafficking to the nucleus . Most important , the present study provides convincing evidence that different NMDAR signals will indeed induce the nuclear import of different messengers . Several reports have documented that NMDARs activation leads to a differential modulation of nuclear gene expression , depending upon their localization ( Hardingham and Bading , 2010 ) . In general , activation of synaptic NMDARs promotes the expression of synaptic plasticity-related genes , whereas activation of extrasynaptic NMDARs has been correlated to CNS pathological conditions ( Hardingham and Bading , 2010 ) . The protein messenger Jacob , following long-distance transport and nuclear import , can encode and transduce the synaptic and extrasynaptic origin of GluN2B NMDAR signals to the nucleus and might dock a NMDA-receptor-derived signalosome to nuclear target sites in a stimulus-dependent manner ( Karpova et al . , 2013 ) . Notably , here we show that activation of synaptic but not extrasynaptic NMDARs leads to an increase in RNF10 nuclear localization . Similarly to what was previously described for Jacob ( Behnisch et al . , 2011 ) , we observe that RNF10 moves to the nucleus after induction of LTP but not LTD , modulating the expression of Meox2 target genes . However , in contrast to Jacob , inhibition of GluN2A- but not GluN2B-containing NMDARs is sufficient to block RNF10 trafficking to the nucleus , its interaction with the transcription factor Meox2 and the expression of the Meox2 target gene p21WAF1/cip1 . Another intriguing finding includes the observation that the transcription factor CREB2 also known as ATF4 transits to the nucleus only after induction of NMDAR-dependent cLTD but not cLTP ( Lai et al . , 2008 ) . Transcriptional regulators at synaptic sites like CRTC1 can also be phosphorylated by synaptic signals in a complex manner and translocation of CRTC1 to the nucleus might link specific types of stimuli to specific changes in gene expression . Overall our data together with the findings from other recent reports ( Kaushik et al . , 2014; Rishal and Fainzilber , 2014; Panayotis et al . , 2015 ) , suggest that synapse-to-nucleus protein transport provide specific ways to inform the nucleus about very different types of synaptic activity . In this context , RNF10 represents a novel synaptonuclear protein messenger responsible for long-lasting re-shaping of dendritic spines as observed after specific synaptic stimuli and required for postsynaptic modifications needed to convey LTP induction .
Post-synaptic densities ( PSDs ) from rat hippocampus were isolated as previously reported ( Gardoni et al . , 1998 ) . To purify the postsynaptic Triton-insoluble fraction ( TIF ) , cell lysates were centrifuged at 13 , 000 g for 15 min at 4°C . The resulting pellet was resuspended in 150 mM KCl , 0 . 5% Triton and spun at 100000 g for 1 hr at 4°C . The final pellet ( TIF ) was homogenized with a glass-glass potter in 20 mM Hepes buffer containing Complete . All purifications were performed in the presence of complete sets of protease and phosphatase inhibitors ( Roche Diagnostics , Monza , Italy ) Protein content of the samples has been quantified by using Bio-Rad ( Hercules , CA , USA ) protein assay . After measuring protein concentration , all samples have been standardized at 1 μg/μl concentration and the same protein amount loaded in each lane for western blot analysis . Organotypic hippocampal slice cultures were prepared as previously described ( Pellegrini Giampietro et al . , 1999 ) . Hippocampal neuronal primary cultures were prepared from embryonic day 18–19 ( E18-E19 ) rat hippocampi as previously described ( Piccoli et al . , 2007 ) . Neurons were transfected at DIV7 using calcium-phosphate method . For induction of 'Bic' treatment: hippocampal neurons ( DIV14 ) or organotypic hippocampal slices ( DIV14 ) were incubated with Bicuculline ( 50 μM; Tocris ) and 2 . 5 mM 4-Aminopyridine ( 4-AP;Tocris ) in Neurobasal medium supplemented with B27; 'Syn' treatment: stimulation of synaptic NMDA receptors was obtained by treating hippocampal neurons at DIV14 with 50 μM Bicuculline ( Tocris ) , 2 . 5 mM 4-AP and 5 μM ifenprodil in Neurobasal medium supplemented with B27; 'Extrasyn#1' treatment: stimulation of extrasynaptic NMDA receptors was obtained by pretreating hippocampal neurons at DIV14 with 50 μM Bicuculline , 2 . 5 mM 4-AP and 10 μM MK801 for 30 min , washing them with Neurobasal medium supplemented with B27 , incubating with 50mM KCl ( Carlo Erba ) for 10 min and then with 40 μM Glutamate ( Sigma ) ; 'ExtraSyn#2' treatment was performed as previously described by Karpova and co-workers ( 2013 ) . cLTP ( Otmakhov et al . , 2004; Oh et al . , 2006 ) : Primary neuronal cultures at DIV14 were first incubated in ACSF ( 125 mM NaCl , 2 . 5 mM KCl , 1 mM MgCl2 , 2 mM CaCl2 , 33 mM D-glucose and 25 mM HEPES , pH 7 . 3 ) for 30 min , followed by 16 min of stimulation with 0 . 05 mM Forskolin ( Sigma Aldrich ) , 0 . 1 mM Picrotoxin ( Tocris ) and 100 nM Rolipram ( Calbiochem ) in ACSF ( no MgCl2 ) to induce NMDAR-dependent cLTP . Control was incubated in ACSF . After stimulation , neurons were replaced in regular ACSF for 2 hr . cLTD ( Marcello et al . , 2013 ) : primary neuronal cultures at DIV14 were first incubated in ACSF , followed by incubation in ACSF ( in presence of MgCl2 ) and 50 μM NMDA ( Sigma Aldrich ) for 10 min . Neurons were then replaced in regular ACSF for 1 hr to induce cLTD . For treatment with 'NLS2 peptide' hippocampal neurons ( DIV14 ) were treated with active ( NLS2: RKRKRQKQK ) or control ( TAT: GRKKRRQRRRPQ ) peptide for 24 hr and then treated to induce cLTP the presence of the same peptides . COS-7 cells were maintained in DMEM + Glutamax medium ( GIBCO-BRL ) supplemented with 10% Fetal Bovine Serum ( FBS ) ( GIBCO-BRL ) and Pen/Strep ( GIBCO-BRL ) . The day before transfection , COS-7 cells were placed in a 6 wells multiwell ( for cells lysis ) or 12 wells multiwell ( for cells immunostaining ) , then cells were transfected using lipofectamine method ( Invitrogen ) . After 36 hr , COS-7 cells were lysed for co-immunoprecipitation and western blotting or fixed for immunostaining . RNF10-tagged with GFP was co-expressed with importin α isoforms ( KPNA1-KPNA6 ) tagged with tagRFP in HEK293T cells . Thirty-six hours post-transfection proteins were extracted with RIPA buffer containing phosStop ( 50 mM Tris pH=8 , 1% NP-40 , 0 . 5% NO deoxycholate , 0 . 1% SDS , 150 mM NaCl , 1% Triton X-100 , protease inhibitor ) . RNF10-GFP was immunoprecipitated from cell extract using anti-GFP MicroBeads ( MACSMolecular ) . Co-immunoprecipitated importinerase-α isoforms were detected in complex with RNF10 using anti-tagRFP antibodies ( Evrogen ) . Real-time quantitative PCR ( qPCR ) was performed as previously described ( De Fabiani et al . , 2003 ) . The following gene expression assay kits from Life Technologies have been used: Rn01409258_g1 for S100a11; Rn01504461_g1 for Rac2; Rn01526492_m1 for Apbb1ip; Rn01422083_m1 for Il18; Rn01430875_g1 for Timp1; Mm00623991_m1 for Itgb8; Rn01513693_m1 for Thbs1; Rn00580728_m1 for Cd36; Rn00579162_m1 for Mmp9 . For microarray experiments , RNA was analyzed by the Genopolis Consortium using an Affimetrix platform ( GEO accession number GSE69267 ) . Data was primarily analyzed using the TAC and Partek Genomic suite softwares . The Robust Multichip Average ( RMA ) method was employed to calculate probe set intensity ( Irizarry et al . , 2003 ) . Differentially-expressed genes whose fold change was higher or equal than 1 . 75 ( up-regulated genes ) and lower or equal to 0 . 65 ( down-regulated genes ) with a p value lower than 0 . 05 were selected as significantly modulated . The expression value of each probe is divided by the mean value of the scramble controls and the log2 of this ratio has been reported . Gene Ontology biological process and enriched pathways analyses were performed using Panther software ( http://www . pantherdb . org/ ) . Hippocampi from adult rats were homogenized at 4°C in an ice-cold buffer containing 0 . 32 M Sucrose , 1 mM Hepes , 1 mM NaF , 0 . 1 mM PMSF , 1 mM MgCl in presence of protease inhibitors ( Complete , GE Healthcare , Mannheim , Germany ) and phosphatase inhibitors ( PhosSTOP , Roche Diagnostics GmbH , Mannheim , Germany ) , using a glass-Teflon homogenizer . Homogenates were then centrifuged at 1000 g for 5 min at 4°C , to remove nuclear contamination and white matter . The supernatant was collected and centrifuged at 13 , 000 g for 15 min at 4°C . The resulting pellet was resuspended in hypotonic buffer ( 1 mM Hepes with Complete ) and referred as P2 crude membrane fraction . Aliquots of proteins from homogenate , P2 crude membrane fractions or PSDs were incubated overnight at 4°C in RIPA buffer containing 50mM Tris HCl ( pH 7 . 2 ) , 150 mM NaCl , 1% NP-40 , 0 . 5% deoxycholic acid , 0 . 1% sodium dodecyl sulphate ( SDS ) in a final volume of 150 µl with the antibody . Pre-incubation with 1% SDS was used for co-i . p . experiments performed in PSDs ( Gardoni et al . , 2001 ) . As a control , one sample was incubated in the absence of the antibody ( no IgG lane ) and another sample in presence of an irrelevant antibody . Protein A-sepharose beads ( Sigma-Aldrich ) were added and incubation was continued for 2 hr , at room temperature , with shaking . Beads were collected by centrifugation and washed three times with RIPA buffer before adding sample buffer for SDS-PAGE and boiling for 5 min . Beads were collected by centrifugation , all supernatants were applied onto 7%–12% SDS-PAGE and revealed by western blotting . PLA was performed as previously described ( Söderberg et al . , 2006; Augusto et al . , 2013 ) in primary hippocampal neurons . Cells were fixed with 4% PFA - 4% Sucrose for 5 min at 4ºC . Cells were then rinsed in PBS , permeabilized with 0 . 1% Triton X-100 in PBS for 15 min at room temperature and blocked with 5% BSA in PBS for 30 min at room temperature . Subsequently , cells were incubated with goat polyclonal anti-RNF10 antibody ( 1:200; Santa-Cruz ) and rabbit polyclonal anti-GluN2A antibody ( 1:200; Lifetechnologies ) overnight at 4ºC . After washing in PBS , the cells were incubated for 1 hr at 37ºC with the PLA secondary probes anti-goat Plus and anti-rabbit Minus ( Olink Bioscience ) . Afterward , the cells were washed twice with Duolink II Wash Buffer A ( Olink Bioscience ) and incubated with the ligation-ligase solution ( Olink Bioscience ) for 30 min at 37ºC . After a new rinse , the cells were incubated with DNA polymerase ( 1:80; Olink Bioscience ) in the amplification solution ( Olink Bioscience ) for 100 min at 37°C . After washes in consecutive decreasing concentrations with Duolink II Wash Buffer B ( Olink Bioscience ) , the cells were then incubated with chicken polyclonal anti-GFP/rabbit polyclonal anti-MAP2 ( 1:300/1:400; Milipore/Milipore ) overnight at 4ºC . After washing in PBS , the cells were incubated for 1 hr at room temperature with secondary goat anti-chicken-AlexaFluor 488/goat anti-rabbit-AlexaFluor 647 ( Lifetechnologies ) . The cells were washed with PBS and mounted on slides with Fluoroshield mounting medium ( Sigma ) . The C-terminal tail of the GluN2A subunit was subcloned downstream of glutathione S-transferase ( GST ) in the BaMHI and HindIII sites of the expression plasmid pGEX-KG by PCR using the Pfu polymerase ( Stratagene ) on a GluN2A cDNA template ( kind gift from S . Nakanishi ) . GST-GluN2A fusion proteins containing the cytoplasmic domain of GluN2A ( 839–1464 or 1049–1464 or 1244–1461 or 1244–1464 ) were expressed in Escherichia coli and purified on glutathione agarose beads ( Sigma Aldrich , St . Louis , MO ) as previously described ( Gardoni et al . , 2001 ) . Three hundred micrograms of proteins were incubated with GST to a final volume of 1 ml with Tris Buffered Saline solution for 45 min with shaking . Samples were centrifuged at 1000 rpm for 5 min and the pellets were discarded . Supernatants were incubated for 2 hr with GST fusion proteins or GST alone . Beads were washed four times with TBS and 0 . 1% Triton X-100 . Bound proteins were resolved by SDS-PAGE and subjected to immunoblot analysis . For co-localization and morphological studies , treated hippocampal neurons were fixed 7 min in 4% paraformaldehyde plus 4% sucrose in PBS at room temperature; then extensively washed with PBS supplemented with CaCl2 and MgCl2 ( PBS-C . M ) , permeabilized with 0 . 2% Triton X100 in PSB-C . M and blocked for 2 hr at room temperature with 5% BSA in PBS-C . M . Primary and secondary antibodies were applied in 5% BSA in PBS-C . M . Cells were chosen randomly for quantification from different coverslips . Fluorescence images were acquired by using Zeiss Confocal LSM510 Meta system with a sequential acquisition setting at 1024x1024 pixels resolution; for each image two up to four 0 . 5μm sections were acquired and a z projection was obtained ( Malinverno et al . , 2010 ) . All images were acquired standing the signals within the linear range in order to perform a reliable quantification ( avoiding the presence of any saturated pixel ) and to compare appropriately all experimental conditions . For analysis of dendritic spine morphology , primary hippocampal neurons were transfected with GFP-containing constructs at DIV7 . Cells were fixed and immunolabeled for GFP at DIV14 . The SP5 CLSM system ( Leica-Microsystems , Mannheim , Germany ) equipped with Diode ( 405 nm ) , Argon ( 488 nm ) and Diode Pumped Solid State ( 561 nm ) lasers was used for time-lapse imaging of RNF10 fused to tdEOS . Images were taken with HCX APO L20x/1 . 00W objective ( Leica , Germany ) . Along the z-axis at list 10 optical sections with focus depth of 300–400 nm were taken in order to cover the complete volume of imaged neurons . Quick change 18 mm Chamber with Field Stimulation ( RC-49MFS , Warner Instruments ) was used for mounting coverslips with living overexpressing RFN10-tdEOS neurons in Neurobasal medium ( Gibco ) on the microscope stage . Master-8/U 8-channel programmable pulse generator ( Science Products ) was used to generate timing and two channels were synchronized to produce complex patterns of pulses ( 1 ms pulse duration ) with inter pulse interval of 20 ms . To eliminate the distortion and thereby to achieve a rectangular pulse permitting accurate measurement , the constant-current stimulus isolation unit ( SIU-102 , Warner Instruments ) was used in the output of a stimulus generator . The LTP was induced by applying current pulses 18s@50 Hz via field stimulation electrode . Response of cultured hippocampal neurons on field synaptic stimulation was verified by Ca2+-imaging with Fluo-4-AM ( 1 µM ) ( data not shown ) . Miniature EPSCs were recorded on DIV14 hippocampal neurons at room temperature , at a holding potential of -60 mV . The recording pipettes had resistance of around 5 MΩ when filled with the following medium ( in mM ) : 140 CsCl , 0 . 5 CaCl2 , 20 EGTA , 10 HEPES , 10 D-glucose , pH 7 . 2 and osmolarity of 300 mOsm . The high concentration of EGTA avoided slow Ca2+-dependent desensitization of NMDARs . Neurons were perfused continuously with the following external medium ( in mM ) : 140 NaCl , 2 CaCl2 , 3 KCl , 10 HEPES , 10 D-glucose , 0 . 01 glycine , 0 . 01 bicucullin , 0 . 0003 tetrodotoxin , pH 7 . 4 and osmolarity of 330 mOsm . Currents were recorded through an Axopatch 200B amplifier , filtered at 1 kHz , digitized at 3 kHz and analyzed using the pClamp 10 . 0 software of Axon Instrument ( Molecular Devices , Sunnyvale , CA ) . Acquisition and quantification of western blotting was performed by means of computer-assisted imaging ( ChemiDoc system and Image lab 4 . 0 software; Bio-Rad ) . Western blotting experiments have been normalized on tubulin or H3 levels , depending if they have been performed from total cell lysate ( homogenate ) or P1 nuclear fraction , respectively . Data obtained by pull-down assays were normalized on the amount of GST fusion protein . The data from all quantifications were analyzed using Graphpad Prism software and levels and values were expressed as mean ± standard error of the mean ( SEM ) . Co-localization analysis was performed using Zeiss AIM4 . 2 software . Statistical evaluations were performed by Student t test or Pearson correlations or , as appropriate , by one-way ANOVA followed by Bonferroni’s or Tukey's as a post-hoc test . When appropriate , experiments have been performed in blind conditions . The following antibodies were used: monoclonal antibody ( mAb ) anti-α-calcium/calmodulin-dependent kinase II ( αCaMKII ) , polyclonal antibody ( pAb ) anti-GluN2A , pAb anti-CREB , mAb anti-Myc , pAb anti-GluA1 and pAb anti-p-CREB ( Ser-133 ) were purchased from Millipore ( Billenca , MA , USA ) ; mAb anti-Meox2 and pAb anti-Synaptotagmin were purchased from Abcam ( Cambridge , MA , USA ) ; mAb anti-GluR2 , mAb anti-GFP , mAb anti-GST and anti-PSD-95 were purchased from NeuroMab ( Davis , CA ) ; mAb anti-α-Tubulin and mAb anti-Flag were purchased from Sigma-Aldrich ( St . Louis , MO ) ; mAb anti-p21 and mAb anti-Rch1 ( importin α1 , KPNA2 gene product ) were purchased from BD Biosciences ( Franklin Lakes , NJ ) ; pAb anti-histone H3 was purchased from Proteintech ( Chicago , IL ) ; pAb anti-RNF10 were purchased from ProteinTech and Santacruz; mAb anti-GluN2B and p-GluA1 ( 845 ) were purchased from Invitrogen ( Carlsbad , CA ) ; pAb anti-BDNF was purchased from Genetex ( Irvine , CA ) ; mAb anti-Map2 was purchased from Immunological Sciences ( Roma , Italy ) ; mAb anti-JL8 was purchased from Clontech ( Mountain View , CA ) ; pAb anti-ArhGap4 was purchased from Novus Biologicals ( Cambridge , UK ) ; mAb anti-GFAP , pAb anti-Ophn1 and pAb anti-ArhGef6 were purchased from Cell Signaling ( Danvers , MA ) . Peroxidase-conjugated secondary anti-mouse Ab was purchased from Pierce ( Rockford , IL ) while peroxidase-conjugated secondary anti-rabbit Ab was purchased from Bio-Rad ( Hercules , CA ) . AlexaFluor secondary Abs were purchased from Invitrogen ( Carlsbad , CA ) . The specificity of the RNF10 antibody ( ProteinTech ) in recognizing RNF10 in western blotting and immunocytochemistry was carefully verified by RNF10 overexpression and silencing in primary hippocampal neurons ( data not shown ) . The specificity of the importin α1 antibody ( BD Biosciences ) in recognizing KPNA2 gene product in western blotting was carefully verified by KPNA1-6 overexpression in HEK293T cells ( data not shown ) . The RNAi Consortium ( TRC ) shRNA target gene set in pLKO . 1 lentiviral vector for rat RNF10 ( accession number NM_001011904 . 1 ) along with scrambled vector were purchased from Open Biosystems ( Thermo Scientific , Milano , Italy ) . The set contained three vectors ( the TRC IDs: TRCN0000041128 , mature antisense TTCAGGTTGATCTTCTTAGGG; TRCN0000041131 , mature antisense TTTCTGTGAATTGGAGCGACG; and TRCN0000041132 , mature antisense ATACCCAGCAATCTCTACCAC ) , all of them were validated by real-time PCR on rat hippocampal primary cultures . Vector TRCN0000041128 showed the highest level of RNF10 downregulation ( >90% , data not shown ) and was used in all further experiments ( designated as shRNF10 ) . To knock down rat GluN2A gene expression , a set of 4 short hairpin RNA ( shRNA ) expression vectors in pGFP-V-RS was purchased from Origene ( Rockville , MD ) . The sequences were as follows: A-gacagcactcctatgataacattctggac; B-tctaccagcaggactggtcacagaacaac; C-tgcgagccagatgacaaccacctcagcat; D-agtagaggtggctgtcagcactgaatcca . A scrambled control from Origene ( Rockville , MD ) was also used as a negative control . Sequence called B showed the best knockdown and was therefore used for further experiments . For the testing and validation of sh-resistant form of RNF10 , rat primary hippocampal cultures were transfected with GFP ( Ctrl ) , rat-shRNF10 , RFP-humRNF10 , and rat-shRNF10 + TRP-humRNF10 . Total RNA was extracted with Tryzol 48 hr after transfection , retrotranscribed ( Improm II , Promega ) and analyzed by quantitative PCR using primers that specifically amplify rat RNF10 or human RNF10 . Transfection with rat-shRNF10 resulted in three fold decrease of endogenous RNF10 mRNA , whereas transfection with human RFP tagged RNF10 resulted in 45-fold increase in mRNA for human RNF10 , the level which was not affected by rat-shRNF10 co-transfection . Interestingly , the overexpression of human RNF10 in rat hippocampal cells abolished effect of shRNF10 on levels of endogenous RNF10 mRNA . The specificity of rat-shRNF10-mediated knock down was further tested using human neuroblastoma line SY-S5Y5 . No effect on both endogenous and everexpressed RNF10 was observed after transfection with rat-shRNF10 . Cloning of pLV-RNF10-EGFP . The cloning of this construct was accomplished in two steps . First , EGFP was amplified from the pRRLSIN . cPPT . hCMV-EGFP . WPRE ( pLV-EGFP ) plasmid to create in frame sequence with XbaI-SalI sites at 5’ of EGFP and XhoI sites at 3’ of EGFP using primers: forward 5’-aagcttTCTAGAgaattcGTCGACATGGTGAGCAAGGGCGAGGA; reverse 5’- aagcttCTCGAGTTACTTGTACAGCTCGTCCAT . Amplificate was digested with XbaI and XhoI enzymes and ligated in XbaI-SalI digested pLV vector creating the intermediate pLV- ( XS ) EGFP bearing XbaI-SalI cassette at 5’ of EGFP . After the sequence was confirmed by sequencing , human RNF10 was excised from pCMV-Tag4-RNF10 plasmid by XbaI and SalI and ligated in XbaI/SalI digested pLV- ( XS ) EGFP yielding pLV-RNF10-EGFP plasmid . For production of lentiviral particles 2 mln of HEK293T producing cells were plated on 100 mm plates ( Costar , Milan , Italy ) in complete medium ( DMEM , Cat . D5671; 2 mM Glutamine; 100 units/ml penicillin and 100 μg/ml streptomycin ( Sigma ) ; 10% Foetal Bovine Serum ( FBS , Immunological Sciences , Milano , Italy ) . For overexpression of RNF10 , the day after plating the cells were transfected with 3μg pL-shRNF10 or pGIPZ-scr and with 2 . 5 μg psPAX2 and 0 . 75 μg pMD2 . G retroviral packaging constructs using Lipofectamine 2000 ( Life Technologies , Milan , Italy ) in 7 ml of complete medium . For RNF10 silencing , the cells were transfected with 3 μg pLV-RNF10-EGFP , and with 2 μg pMDLg/pRRE , 0 . 75 μg pRSV-REV , 11 μg pMD2 . G lentiviral packaging constructs . 48 hr after transfection culture medium was collected , filtered through 0 . 45 μm PVDF filter and precipitated with polyethylene glycol ( PEG; 5x PEG solution contained: 200 g PEG , Sigma Cat . 89510 , 12 g NaCl in 2 mM Tris pH 7 . 5 ) overnight . Precipitates were then collected by centrifugation at 1500 xg for 30 min at 4°C , resuspended in cold HEPES balanced salt solution ( HBSS , Sigma ) , aliquoted and snap frozen . Aliquots were kept at -80°C . Each viral batch was tittered by serial dilution infections of rat hippocampal primary cells by means of real-time PCR ( for RNF10 silencing ) using primers for rat RNF10 ( to evaluate efficacy of downregulation ) : forward 5’-GAACAAGAGACAGGGCAGGT , reverse 5’-GACAGAGGGGTCAGCAGAAA . For experiments , a viral titer was used yielding > 80% of RNF10 downregulation . Virus for overexpression was tittered by FACS , and a titer yielding > 80% EGFP positive cells was used . Y2H was conducted according to the manufacture procedure guidelines using the Mate & Plate™ Library - Mouse Brain ( Normalized ) ( cat # 630488 , TakaraBio/Clontech Europe , France ) . Briefly , the GluN2A ( 839–1461 ) C-tail ( bait ) was cloned in the pGBKT7 plasmid and transformed in the AH109 haploid yeast strain ( MATa ) . This was mated overnight with the Mouse Brain normalized library ( prays ) cloned in pGADT7 transformed in Y187 haploid yeast strain ( MATα ) . Yeast were plated after 24 on selective plate , allowing only the growth of diploid where a protein interaction between the bait and pray protein occurred ( absence of Leucine , Adenine , Tryptophan and Histidine ) . Then the diploid where tested by a colorimetric assay ( α-gal ) to avoid the presence of false positive . Plasmids ( 21 positive clones ) were extracted from the yeast and sequenced . Five genes were identified following sequencing , one of those being RNF10 .
Synapses and nuclei are connected by bidirectional communication mechanisms that enable information transfer encoded by macromolecules . Here , we identified RNF10 as a novel synaptonuclear protein messenger . RNF10 is activated by calcium signals at the postsynaptic compartment and elicits discrete changes at the transcriptional level . RNF10 is enriched at the excitatory synapse where it associates with the GluN2A subunit of NMDA receptors ( NMDARs ) . Activation of synaptic GluN2A-containing NMDARs and induction of long term potentiation ( LTP ) lead to the translocation of RNF10 from dendritic segments and dendritic spines to the nucleus . In particular , we provide evidence for importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus . Notably , RNF10 silencing prevents the maintenance of LTP as well as LTP-dependent structural modifications of dendritic spines .
Brain activity depends on the communication between neurons . This process takes place at the junctions between neurons , which are known as synapses , and typically involves one of the cells releasing a chemical messenger that binds to receptors on the other cell . The binding triggers a cascade of events inside the recipient cell , including the production of new receptors and their insertion into the cell membrane . These changes strengthen the synapse and are thought to be one of the ways in which the brain establishes and maintains memories . However , in order to induce these changes at the synapse , neurons must be able to activate the genes that encode their component parts . These genes are present inside the cell nucleus , which is located some distance away from the synapse . Studies have shown that signals can be sent from the nucleus to the synapse and vice versa , enabling the two parts of the cell to exchange information . Synapses that communicate using a chemical called glutamate have been particularly well studied; but it still remains unclear how the activation of receptors at these “glutamatergic synapses” is linked to activation of genes inside the nucleus at the molecular level . Dinamarca , Guzzetti et al . have now discovered that this process at glutamatergic synapses involves the movement of a protein messenger to the nucleus . Specifically , activation at synapses of a particularly common subtype of receptor , called NMDA , causes a protein called Ring Finger protein 10 ( or RNF10 for short ) to move from the synapse to the nucleus . To leave the synapse , RNF10 first has to bind to proteins called importins , which transport RNF10 into the nucleus . Once inside the nucleus , RNF10 binds to another protein that interacts with the DNA to start the production of new synaptic proteins . Further work is required to identify the molecular mechanisms that trigger RNF10 to leave the synapse . In addition , future studies should evaluate the levels and activity of RNF10 in brain disorders in which synapses are known to function abnormally .