Figure 8.
Densin is not required for localization of CaMKII in spines, but loss of densin alters activity-dependent autophosphorylation of CaMKII. A, Representative images of hippocampal neurons cultured from wild-type, densin knock-out, GluN1 knock-out, and densin/GluN1 double knock-out mice, immunostained for αCaMKII (green) and PSD-95 (red). B, Loss of either densin or NMDARs (two postulated high-affinity CaMKII binding sites in the PSD) resulted in only small reductions in the intensity of CaMKII immunostaining that colocalized with PSD-95, whereas loss of both proteins resulted in a ∼50% reduction in the intensity of CaMKII immunostaining colocalizing with PSD-95. The graph shows the intensity of staining for CaMKII colocalized with PSD-95 puncta in densin knock-out, GluN1 knock-out, and densin/GluN1 double knock-out cultures normalized to wild-type levels (densin knock-out, 90.4 ± 9.1% of wild type, n = 4 densin knock-out embryos; GluN1 knock-out, 86.3 ± 1.6% of wild type, n = 4 GluN1 knock-out embryos, **p < 0.01; densin/GluN1 knock-out, 49.2 ± 7.2% of wild type, n = 3 densin/GluN1 double knock-out embryos, *p < 0.05, one-sample t test). C, Autophosphorylation of CaMKII (Thr286) in response to synaptic activity is increased in densin knock-out (KO) compared with wild-type (Wt) cultures. Top shows representative immunoblots of homogenates of hippocampal cultures that were treated with bicuculline (10 μm)/glycine (10 μm) for 0.25, 1, 3, or 5 min. Homogenates were fractionated by SDS-PAGE and stained with anti-phospho-CaMKII antibody (C, untreated control) as described under Materials and Methods. The graph shows the average intensity of phospho-CaMKII immunostaining (normalized to total CaMKII) after treatment with bicuculline/glycine, expressed as a percentage of the untreated control. The increase in phospho-CaMKII over untreated control levels is significantly greater in densin knock-out cultures compared with wild-type after treatment with bicuculline/glycine for 3 min (wild type, 147 ± 12%; densin knock-out, 200 ± 23% of control; *p < 0.05, ANOVA) and 5 min (wild type, 150 ± 23%; densin knock-out, 202 ± 23% of control; *p < 0.05, ANOVA; n = 9 wild-type and 9 densin knock-out embryos). Error bars represent SEM. D, Steady-state autophosphorylation of CaMKII is reduced in densin knock-out compared with wild-type hippocampal neurons. There was no difference in the amount of total CaMKII in wild-type and knock-out neurons (knock-out, 96 ± 7.67% of wild type, n = 9 littermate pairs). However, a significantly smaller fraction of CaMKII was phosphorylated on the threonine-286 residue of CaMKII in the knock-out compared with wild-type neurons (knock-out, 74 ± 6.3% of wild type, n = 9 littermate pairs, **p < 0.01, one-sample t test; hypothetical mean = 100).