Article Figures & Data
Figures
- Figure 1.
Proteasome protein degradation and ATP hydrolysis kinetics are significantly altered in Rpt6 S120A and S120D KI mice. A, Representative Western blot analysis of proteasomes purified using Ubl/UIM method, and verification of phospho-Rpt6 antibody specificity to WT Rpt6 with expected cross reaction with S120D Rpt6 (performed for each purification). B, Native gel fluorescent activity assay and tandem Western blotting with two bands representing singly and doubly capped proteasomes (performed for each purification). C, Fluorescent peptidase activity assay; S120A has significantly decreased activity (p = 0.04, post hoc Tukey, n = 12), S120D shows increased activity (p < 0.001, post hoc Tukey, n = 12), and ANOVA indicates significant difference within the group (F = 25.26, p < 0.001). D, Malachite green ATPase assay; S120A displays significantly lower activity (p = 0.007, post hoc Tukey, n = 12), while S120D shows increased activity (p = 0.37, post hoc Tukey, n = 12), and the one-way ANOVA indicates significant difference within the group (F = 11.21, p < 0.001). *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 2.
Basal synaptic transmission and LTP are unaltered in Rpt6 S120A and S120D KI mice. A, Input/output curve in slices from WT (n = 12 slices from 7 mice) and S120A mice (n = 17 slices from 7 mice), depicting increasing fEPSP responses to increased amplitude of Schaffer collateral stimulation with no difference between groups (two-way ANOVA, interaction: F(5,147) = 0.21; p = 0.96). B, Input/output curve in acute hippocampal slices from WT (n = 11 slices from 7 mice) and S120D mice (n = 11 slices from 7 mice), with no differences between groups (two-way ANOVA, interaction: F(5,115) = 0.02; p = 0.99). C, Two successive stimuli with short separation delivered to the Schaffer collateral of acute hippocampal slices leads to enhancement of fEPSP amplitude (paired pulse facilitation) in WT (n = 15 slices from 7 mice) and S120A mice (n = 16 slices from 7 mice), with similar facilitation in both groups (two-way ANOVA, interaction: F(4,145) = 0.22; p = 0.93). D, Paired pulse facilitation in WT (n = 10 slices from 7 mice) and S120D mice (n = 9 slices from 7 mice), showing no differences between groups (two-way ANOVA, interaction: F(4,85) = 0.15; p = 0.96). E, Delivery of four 1-s trains (20 s apart) of 100-Hz stimulation causes potentiation of fEPSP amplitude in acute hippocampal slices from WT (n = 9 slices from 7 mice) and S120A mice (n = 6 slices from 6 mice). F, LTP induction in response to four 1-s 100-Hz trains in WT (n = 9 slices from 9 mice) and S120D mice (n = 10 slices from 10 mice). G, LTP induction in response to a single 1-s 100-Hz train in slices from WT (n = 6 slices from 4 mice) and S120A animals (n = 7 slices from 4 mice). H, LTP induction in response to a single 1-s 100-Hz train in WT (n = 4 slices from 4 mice) and S120D animals (n = 6 slices from 4 mice). For E–H, filled squares and triangles indicate stimulated (LTP) pathway while open symbols denote control pathway. Graphs depict mean ± SEM for each current intensity, pulse separation, or time point.
- Figure 3.
Spine outgrowth dynamics and excitatory synaptic structure are not altered in KI mice. A, Images of dendrites from EGFP-expressing CA1 pyramidal neurons from WT and S120A animals at 7–9 DIV before and after the addition of vehicle (veh) or bicuculline (bic) (30 μm) at t = 0 (black arrow). Yellow arrowheads indicate new spines. B, Quantification of number of spines gained per 10 μm with addition of vehicle or bicuculline, in WT (n = 6 and 6 neurons, respectively, with one neuron per slice) and S120A (n = 7 and 6 neurons, respectively, with one neuron per slice). Bicuculline induced significantly more spine outgrowth than vehicle in both WT and S120A neurons (p = 0.02 and p = 0.04, respectively), but no significant difference was observed in bic-induced outgrowth between WT and S120A (p = 0.79 with t test; ANOVA of interaction, F(1,21) = 0.197, p = 0.66). C, Quantification of normalized spine gain (% of vehicle) in neurons from WT and S120A slices, illustrating similar bic-induced spine outgrowth in both genotypes (p = 0.89 with t test). D, Representative proximal dendritic segments from hippocampal pyramidal neurons in fixed slices obtained from WT and homozygous S120A mutant littermates, after filling by targeted microinjection of Alexa Fluor 594 Hydrazide. E, Basal spine density quantified by counting the individual number of spine heads on 20-μm segments of secondary dendritic branches in 63× confocal stacks (WT: n = 56 segments from 12 cells; S120A: n = 51 segments from 10 cells), showing no significant difference in density (p = 0.83). F, Quantification of mean Rpt6 fluorescence within dendritic spines of cultured WT and S120D neurons, identified using the GFP channel (p = 0.90; n = 19 and 20 dendrites from 10 neurons each). G, Quantification of mean PSD-95 fluorescence within spines (p = 0.34; n = 19 and 20 dendrites from 10 neurons each). H, Example z-stacked immunofluorescent images of GFP-filled (S. virus) cultured hippocampal neurons (DIV21–DIV24) from WT and S120D mice stained with Rpt6 (red) and PSD-95 (blue) antibodies. Scale bar: 5 μm. *p < 0.05; n.s. = not significant (p > 0.05).
- Figure 4.
Contextual and cued fear conditioning are unaltered in Rpt6 S120A and S120D KI mice. A, Activity during a 2-min baseline and activity during a 2-s shock in WT (n = 8) and S120A (n = 16) mice, demonstrating normal baseline activity (p = 0.62) but slightly attenuated shock reactivity (ANOVA, F(1,22) = 4.33, p < 0.05) in S120A mice. B, Same as A, but for WT (n = 13) and S120D (n = 21), depicting similar baseline activity (p = 0.27) but reduced shock reactivity (ANOVA, F(1,32) = 10.48, p < 0.005). C, Percent time spent freezing to three sequential tone presentations in a novel context, during post-training (24 h) test; S120A and S120D KI mice show similar amounts of freezing compared with respective WT mice (p = 0.21, p = 0.28, respectively). D, Percent time spent freezing to initial training context during 5 min test, 24 h after training; S120A and S120D mice show normal amounts of freezing (p = 0.73, p = 0.77, respectively). *p < 0.05; ***p < 0.001; n.s. = not significant (p > 0.05).
Extended Data
Extended Data Figure 1-1
Generation of Rpt6 S120A and S120D KI mice. A, The original targeting strategy for Rpt6 S120D mutant mice was previously described in Gonzales et al. (2018). Rpt6 S120A mutant mice were made at the same time using the same targeting strategy to create the phospho-ablated mutant. Codon 120 in exon 5 of the PSMC5 (Rpt6) gene was mutated from AGC to GCC (S120 to ala; Gonzales et al., 2018; see also Materials and Methods). A, Tail genomic DNA was analyzed by PCR screening for genotyping and to verify deletion of the Neo cassette. B, Representative electropherograms confirming the presence of the mutation in homozygous Rpt6 S120A and S120D mutant male mice. C, Representative images of Nissl-stained fixed whole-brain coronal sections (with higher magnification of the hippocampus) of 60-d-old mice. Download Figure 1-1, PDF file.
Extended Data Figure 4-1
Performance on the elevated plus maze was not impaired in S120 KI mice. A, Total distance travelled during elevated plus maze assay, demonstrating no differences between WT (n = 9) and S120A (n = 8) mice (p = 0.16, t test). B, Same as A, for WT (n = 13) and S120D (n = 14; p = 0.80, t test). C, Percent time spent in each arm of the elevated plus maze (open vs closed) did not differ between S120A (n = 8) and WT (n = 9) mice (p = 0.82 and p = 0.08, open and closed, respectively, post hoc Bonferroni). D, Same as C, for S120D (n = 14) and WT (n = 13; p = 0.17 and p = 0.30, open and closed, respectively, post hoc Bonferroni). Download Figure 4-1, PDF file.
In this issue
