Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome

Abstract

Angelman syndrome (AS) is a disorder of human cognition characterized by severe mental retardation and epilepsy. Recently, a mouse model for AS (Ube3a maternal null mutation) was developed that displays deficits in both context-dependent learning and hippocampal long-term potentiation (LTP). In the present studies, we examined the molecular basis for these LTP and learning deficits. Mutant animals exhibited a significant increase in hippocampal phospho-calcium/calmodulin-dependent protein kinase II (CaMKII), specifically at sites Thr(286) and Thr(305), with no corresponding change in the levels of total CaMKII. In addition, mutants show a reduction in CaMKII activity, autophosphorylation capability, and total CaMKII associated with postsynaptic density. These findings are the first to implicate misregulation of CaMKII as a molecular cause for the neurobehavioral deficits in a human learning disorder.

Fig. 1.

The LTP deficit in m⁻/p⁺ mice is NMDA receptor dependent. Baseline synaptic responses of wild-type (m⁺/p⁺) (○) and maternal-deficient mutant (m⁻/p⁺) (●) animals were measured before application of LTP-inducing HFS (indicated byarrows) to hippocampal area CA1. A, Slices were maintained at 32°C, and LTP was induced with a single set of HFS consisting of two trains of 100 Hz stimulation for 1 sec separated by 20 sec. B, Slices were maintained at 32°C, and NMDA receptor-dependent LTP was induced with three sets of HFS, with each set separated by 10 min. C, NMDA receptor-independent LTP was induced with three sets of HFS consisting of 200 Hz stimulation for 1 sec separated by 4 min. The black bar represents the application of the NMDA receptor antagonist AP-5. All results are graphed as the percentage of potentiation standardized to the baseline recording. Dashed linesrepresent the 100% mark of baseline synaptic responses. Data represent mean ± SEM.

Fig. 2.

Kinase concentrations in m⁻/p⁺ mutant hippocampus.A, A schematic diagram of a postsynaptic neuron showing potential candidates (shaded hexagons) for LTP and learning disruption observed in E6-AP-deficient mice. B, Quantitative Western blot analysis on hippocampal homogenates from wild-type and m⁻/p⁺ mutants probed for total PKC, PKA [α catalytic (α Cat.), αII regulatory (αII Reg.), and βI regulatory (βI Reg.) subunits], ERK (p42 and p44 MAP kinase), and CaMKII. C, Top, Representative Western blot showing no change in total CaMKII but an increase in P-Thr²⁸⁶ αCaMKII in m⁻/p⁺ mouse hippocampal homogenates. Bottom, Quantitative Western blot analysis of hippocampal homogenates for protein levels of phospho-PKC, phospho-PKA, phospho-p42 ERK, and phospho-αCaMKII. A significant increase was detected in m⁻/p⁺mice only for total P-Thr²⁸⁶ αCaMKII (WT, 100 ± 7.1%, n = 10; m⁻/p⁺, 162 ± 16.2%,n = 10; p = 0.0024). Results shown are the percentage difference of m⁻/p⁺ protein levels compared with that of wild type. Dashed line represents control level of 100%. *p < 0.05. Data represent mean ± SEM. PLC, Phospholipase C; DAG, diacylglycerol; MEK, mitogen and extracellular signal regulated kinase.

Fig. 3.

Increased phosphorylated Thr²⁸⁶αCaMKII corresponds to reduced post hocautophosphorylation and kinase activity. A,Top, Representative autoradiograph from hippocampal homogenates corresponding to α and β catalytic subunits of CaMKII in the presence of [γ-³²P]ATP before and after Ca²⁺/calmodulin stimulation. Bottom, Quantitative analysis of the total Ca²⁺/CaM-dependent CaMKII autophosphorylation in hippocampal homogenates of wild-type animals (■) or m⁻/p⁺ mutants (▪) shows a significant reduction in total CaMKII autophosphorylation after activation with calcium and calmodulin (wild type, 100 ± 5.5%,n = 6; m⁻/p⁺, 71 ± 8.9,n = 7; p = 0.002).Bars represent the percentage of quantified signal compared with that of wild types. B, Hippocampal homogenates from wild-type animals (■) or m⁻/p⁺ mutants (▪) were assayed for basal CaMKII activity and CaMKII activity after activation with Ca²⁺ and CaM. The m⁻/p⁺ mutants (n = 6) show no significant difference in basal phosphotransferase activity compared with wild-type controls (n = 7). Lower total activity after CaMKII activation in the presence of Ca²⁺ and CaM was seen in m⁻/p⁺ mutants (wild type, 0.33 ± 0.05, n = 6; m⁻/p⁺, 0.487 ± 0.04,n = 7; p = 0.048). Data represent mean ± SEM. N/D, Not detected;Wt, wild type. *p < 0.05.

Fig. 4.

Increased phosphorylated Thr³⁰⁵CaMKII in m⁻/p⁺ mutants.A, Left blot, Western blot analysis showing that P-Thr^305/306 CaMKII serum recognizes purified CaMKII activated with Ca²⁺/CaM, but not nonactivated CaMKII. Right blot, Specificity for the Thr³⁰⁵ autophosphorylation site was tested with autophosphorylation reactions with hippocampal homogenates from wild-type mice in the absence [control (Ctrl)] or presence (+Ca²⁺/CaM) of calcium and calmodulin. Control Western blots show an increase in immunoreactivity for activated CaMKII corresponding to the α and β subunits of CaMKII. Addition of antigen completely blocked recognition of phospho-Thr^305/306 CaMKII in control and activated homogenates (peptide block). B,Top, Representative Western blot analysis of hippocampal homogenates probed for P-Thr^305/306 CaMKII (right blot). The same blot was then stripped and reprobed for total αCaMKII (left blot).Bottom, Quantitative Western blot analysis shows no change in total αCaMKII but an increased Thr³⁰⁵αCaMKII phosphorylation in m⁻/p⁺ mutants in whole hippocampal homogenates [wild type (Wt), 100 ± 12, n = 7; m⁻/p⁺, 133.2 ± 3.3,n = 7; p = 0.02]. Data represent mean ± SEM. *p < 0.05.

Fig. 5.

PSD-associated CaMKII and autophosphorylated CaMKII. A, B, Top, Representative Western blot for PSD-associated total CaMKII (A) and P-Thr²⁸⁶ and P-Thr³⁰⁵ CaMKII (B).A, Bottom, PSD fractions isolated from the hippocampus of WT (■) or m⁻/p⁺ mutants (▪) show a reduction of ∼50% in total PSD-associated CaMKII (WT, 100.0 ± 4.2, n = 3; m⁻/p⁺, 54.6 ± 6.7,n = 4; p = 0.045).B, Bottom, P-Thr²⁸⁶CaMKII shows an ∼2.5-fold increase in the PSD fraction (WT, 100.0 ± 1.9, n = 3; m⁻/p⁺, 264.3 ± 36.4,n = 4; p = 0.012). P-Thr³⁰⁵ CaMKII levels in the PSD were detectable but showed no significant differences between wild-type and m⁻/p⁺ mice. Data represent mean ± SEM. *p < 0.05.

Fig. 6.

Increased immunoreactivity to phosphorylated CaMKII at Thr²⁸⁶ in Angelman mouse hippocampus. Phosphorylated CaMKII at Thr²⁸⁶ was detected immunohistochemically in the hippocampi of wild-type (A,C, E) and Angelman (B,D, F) mice. Increases in immunoreactivity are seen in the stratum pyramidale, stratum oriens, and stratum radiatum of CA1 and CA3 as well as the molecular layer and granule cell layer of the dentate gyrus. A,B, 100× magnification of the hippocampus.C, D, 200× magnification of area CA1.E, F, 200× magnification of area CA3.

Fig. 7.

Decreased PP1/PP2A activity in m⁻/p⁺ mutants. A, Quantitative Western blot analysis showing no change in total protein levels of PP1 and PP2A from hippocampal homogenates of m⁻/p⁺ mutant animals compared with WT controls. The dashed line indicates wild-type protein levels. B, Phosphatase activity was measured for PP1/PP2A using a PP1/PP2A-specific phosphopeptide (K-R-pT-I-R-R). Phosphatase activity was significantly reduced in the m⁻/p⁺ mutants (▪) (0.659 ± 0.08 pmol · min⁻¹ · μg⁻¹protein; n = 5) compared with wild-type controls (■) (1.758 ± 0.27 pmol · min⁻¹ · μg⁻¹protein; n = 8; p = 0.009). Data represent mean ± SEM. *p < 0.05.