Figure 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).