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. 2014 Jul 2;34(27):9088–9095. doi: 10.1523/JNEUROSCI.0303-14.2014

Figure 1.

Figure 1.

hnRNP K knockdown impairs dendritic spine density and synaptic function. A, Labeling of endogenous hnRNP K and PSD-95 or VGLUT1 in neurons. Scale bar, 5 μm. The histogram represents the percentage of hnRNP K-positive puncta that colocalized with VGLUT1 (47.41 ± 3.92%) and PSD-95 (61.38 ± 5.64%). B, Analysis of dendrites in neurons transfected with scrambled or siRNA or rescue. Scale bar, 20 μm. Silenced neurons exhibited reduced dendritic length and branching compared with scrambled and rescue neurons (length μm/neuron: scrambled 787.89 ± 44, siRNA 638.63 ± 46.25, rescue 783.08 ± 61.81; number of branching points per neuron: scrambled 26.55 ± 2.71, siRNA 18 ± 1.87, rescue 25.81 ± 1.9; ANOVA and Bonferroni post hoc test, *p < 0.05). C, Analysis of dendritic spine density in neurons transfected with scrambled or siRNA or rescue constructs. Scale bar, 10 μm. In silenced neurons, the dendritic spine density was reduced compared with controls (spines/10 μm: scrambled 4.59 ± 0.26, siRNA 3.01 ± 0.32; ANOVA and Bonferroni post hoc test, **p < 0.01). Rescued neurons exhibited normal spine density. D, Analysis of synaptic markers evaluated by IF (transfected neurons) and WB (infected neurons). Scale bar, 10 μm. The staining intensity was measured along the dendrites of the transfected neurons. Compared with scrambled neurons, the staining intensity for GluA2 and PSD-95 was significantly lower in silenced neurons (GluA2: 0.42 ± 0.08 vs 1 ± 0.14, **p < 0.01; PSD-95: 0.37 ± 0.07 vs 1 ± 0.13 **p < 0.01; ANOVA and Bonferroni post hoc test, values normalized to scrambled), while the staining intensity of VGLUT1 and VGAT was unchanged. Rescued neurons exhibited normal levels of GluA2 and PSD-95. The decrease in GluA2 and PSD-95 staining was reconfirmed by WB analysis of infected neurons (***p< 0.001; *p < 0.05). E, Representative mEPSC recordings (left) and average traces of 50 mEPSCs (right) from neurons transfected with scrambled, siRNA, or rescue. siRNA neurons exhibited a significant decrease in the frequency (scrambled: 2.4 ± 0.6 Hz, siRNA: 0.7 ± 0.2 Hz, **p < 0.01) and slower decay kinetics compared with the scrambled and rescued neurons (tau fast of 4.32 ± 0.79 ms vs 1.92 ± 0.09 and 1.42 ± 0.18 ms, respectively; **p < 0.01 siRNA vs both scrambled and rescue). The curve in red represents the fitting of the decay phase.