Abnormal Ca2+-calmodulin-dependent protein kinase II function mediates synaptic and motor deficits in experimental parkinsonism

Abstract

The NMDA receptor complex represents a key molecular element in the pathogenesis of long-term synaptic changes and motor abnormalities in Parkinson's disease (PD). Here we show that NMDA receptor 1 (NR1) subunit and postsynaptic density (PSD)-95 protein levels are selectively reduced in the PSD of dopamine (DA)-denervated striata. These effects are accompanied by an increase in striatal levels of alphaCa2+-calmodulin-dependent protein kinase II (alphaCaMKII) autophosphorylation, along with a higher recruitment of activated alphaCaMKII to the regulatory NMDA receptor NR2A-NR2B subunits. Acute treatment of striatal slices with R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride, but not with l-sulpiride, mimicked the effect of DA denervation on both alphaCaMKII autophosphorylation and corticostriatal synaptic plasticity. In addition to normalizing alphaCaMKII autophosphorylation levels as well as assembly and anchoring of the kinase to the NMDA receptor complex, intrastriatal administration of the CaMKII inhibitors KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide) and antennapedia autocamtide-related inhibitory peptide II is able to reverse both the alterations in corticostriatal synaptic plasticity and the deficits in spontaneous motor behavior that are found in an animal model of PD. The same beneficial effects are produced by a regimen of l-3,4-dihydroxyphenylalanine (L-DOPA) treatment, which is able to normalize alphaCaMKII autophosphorylation. These data indicate that abnormal alphaCaMKII autophosphorylation plays a causal role in the alterations of striatal plasticity and motor behavior that follow DA denervation. Normalization of CaMKII activity may be an important underlying mechanism of the therapeutic action of L-DOPA in PD.

Figure 2.

Distinct effects of DA receptor antagonists on αCaMKII autophosphorylation and corticostriatal LTP formation. A, Western blot analysis of total αCaMKII (bottom) and p286-αCaMKII (top) in TIFs obtained from control, 3 μm l-sulpiride-treated, or 10 μm SCH 23390-treated corticostriatal slices; the same amount of protein was loaded per lane (p < 0.01, l-sulpiride vs control; p < 0.05, SCH 23390 vs control). ^*p < 0.01, l-sulpiride versus control; ^**p < 0.05, SCH 23390 versus control. B, TIF proteins from treated corticostriatal slices were immunoprecipitated with an NR2A-NR2B polyclonal antibody. Western blot analysis was performed in the immunoprecipitated (i.p.) material with αCaMKII and NR2A-NR2B antibodies (+73.4 ± 17.5%, p < 0.05, SCH 23390 vs control; -79.5 ± 10.2%, p < 0.01, l-sulpiride vs control). C, Changes in corticostriatal EPSP amplitude after tetanic stimulation under control conditions, in the presence of 3 μm l-sulpiride-treated slices (p < 0.05; control vs l-sulpiride measured 30 min after the induction; n = 23), in 10 μm SCH 23390-treated slices (p < 0.001; control vs SCH 23390 measured 30 min after the induction; n = 20), and after in vivo intrastriatal administration of 3 mm SCH 23390 (p < 0.001; control vs SCH 23390 intrastriatally at 30 min after induction; n = 13). D, Limb-use asymmetry induced in control rats 24 hr after the injection of 3 mm SCH 23390 (^**p < 0.001; SCH 23390 intrastriatally vs sham; n = 8) ipsi, Ipsilateral; contra, contralateral. E, Western blot analysis of active p286-αCaMKII and total αCaMKII in TIFs obtained from treated corticostriatal slices. F, TIF proteins from treated corticostriatal slices were immunoprecipitated with an NR2A-NR2B polyclonal antibody. Western blot analysis was performed in the immunoprecipitated material with αCaMKII and NR2A-NR2B antibodies. WB, Western blot. L-sul, l-sulpiride; SCH, SCH 23390; i.s., intrastriatal; Forsk, forskolin.

Figure 1.

Motor performances, synaptic plasticity, and alteration of αCaMKII-PSD-95 binding to the NMDA receptor complex in sham- and 6-OHDA-lesioned striatum. A, A limb-use asymmetry test was performed in sham-operated and 6-OHDA denervated rats (n = 8 in each group). 6-OHDA-lesioned rats preferentially use the limb ipsilateral to the lesion (^***p < 0.0001; 6-OHDA vs sham). ipsi, Ipsilateral; contra, contralateral. B, Coordinated locomotor activity on a rotarod is significantly impaired after dopamine denervation (^**p < 0.001; 6-OHDA vs sham). C, HFS of corticostriatal fibers induced LTP in sham-operated rats (filled circles; p < 0.01; EPSP amplitude after vs before HFS; n = 25) but not in 6-OHDA-lesioned animals (open circles; p > 0.05; EPSP amplitude after vs before HFS; n = 20). D, Effects of 6-OHDA lesioning on the NMDA receptor complex in the rat striatum. Striatal homogenates (left) and TIFs (right) from sham- and 6-OHDA-lesioned animals were analyzed by Western blot analysis with PSD-95, GluR1, NR2A, NR1,αCaMKII, and active p286-αCaMKII antibodies. The same amount of protein was loaded per lane (^*p < 0.05; ^#p < 0.01; 6-OHDA I vs 6-OHDA C; n = 8 for each group). E, TIF proteins from sham- and 6-OHDA-lesioned animals were immunoprecipitated with an NR2A-NR2B polyclonal antibody. Western blot analysis was performed in the immunoprecipitated (i.p.) material with αCaMKII and PSD-95 antibodies. F, Quantitative analysis of Western blot performed on coimmunoprecipitated material. ^*p < 0.01 (6-OHDA I vs sham). G, TIF proteins were phosphorylated under conditions known to maximally activate CaMKII and then immunoprecipitated with anti-NR2A-NR2B (left) or anti-CaMKIIα (right). Autoradiography obtained after immunoprecipitation shows two major phosphorylated protein bands: a 50 kDa band (bottom arrow) pointing to autophosphorylated αCaMKII and a 170 kDa band (top arrow) indicating phosphorylated NR2A-NR2B. Left lanes, Identification by Western blot of NR2A-NR2B and αCaMKII in the immunocomplex. WB, Western blot; I, striatum ipsilateral to the lesion; C, striatum contralateral to the lesion.

Figure 3.

Motor performances, synaptic plasticity, and NMDA receptor complex in sham-operated rats, 6-OHDA-lesioned rats, and lesioned rats after l-DOPA treatment. A, A limb-use asymmetry test was performed on sham- and 6-OHDA-lesioned rats after 1 or 4 d of l-DOPA treatment (n = 8 in each group). Only 4 d of l-DOPA treatment were able to restore the normal limb-use symmetry in 6-OHDA-lesioned animals (^**p < 0.001, 6-OHDA vs sham; ^*p < 0.01, 6-OHDA plus 4 d l-DOPA vs 6-OHDA; not significant, p > 0.05, 6-OHDA plus 4 d l-DOPA vs sham). ipsi, Ipsilateral; contra, contralateral. B, Coordinated locomotor activity on a rotarod is significantly recovered after 4 d of l-DOPA administration (^***p < 0.0001, 6-OHDA vs sham; ^**p < 0.01, 6-OHDA plus 4 d l-DOPA vs 6-OHDA; not significant, p > 0.05, 6-OHDA plus 1 d l-DOPA vs 6-OHDA). C, HFS of corticostriatal fibers induced LTP in chronically l-DOPA-treated rats (filled circles; p < 0.01; EPSP amplitude after vs before HFS; n = 20) but not in 6-OHDA-lesioned animals or in acute l-DOPA-treated rats (open circles and filled triangles, respectively; p > 0.05; EPSP amplitude after vs before HFS; n = 23). D, E, Western (WB) blot analysis of total and p286-αCaMKII in striatal TIFs obtained from sham- and 6-OHDA-lesioned animals in the absence or presence of 1 and 4 d l-DOPA treatment (^*p < 0.01, 6-OHDA vs sham; ^§p < 0.05, 6-OHDA plus 4 d l-DOPA vs 6-OHDA). F, CaMKII activity in striatal TIFs obtained from sham- and 6-OHDA-lesioned animals in the absence or presence of 1 and 4 d l-DOPA treatment (^*p < 0.01, 6-OHDA vs sham; ^§p < 0.01, 6-OHDA plus 4 d l-DOPA vs 6-OHDA).

Figure 4.

Effect of the CaMKII inhibitor KN-93 on motor performances, synaptic plasticity, and the NMDA receptor complex of DA-denervated animals. A, A limb-use asymmetry test was performed in sham-operated, 6-OHDA denervated, and 6-OHDA plus KN-93 animals (n = 8 in each group). 6-OHDA-lesioned rats preferentially use the limb ipsilateral to the lesion (^***p < 0.0001; 6-OHDA vs sham). CaMKII inhibitor reversed the parkinsonian-like motor features in the lesioned animals 24 hr after the injection, although the effect was lost 72 hr after the injection (not significant, p > 0.05, 6-OHDA plus 24 hr KN-93 vs sham; ^***p < 0.0001, 6-OHDA plus 72 hr KN-93 vs sham). ipsi, Ipsilateral; contra, contralateral. B, Coordinated locomotor activity on a rotarod is significantly impaired after dopamine denervation but recovers to normal levels (i.e., sham group) in KN-93-injected 6-OHDA rats 24 hr but not 72 hr after the injection (^**p < 0.001, 6-OHDA vs sham; not significant, p > 0.05, 6-OHDA plus 24 hr KN-93 vs sham; ^**p < 0.001, 6-OHDA plus 72 hr KN-93 vs sham). C, HFS of corticostriatal fibers induced LTP in sham-operated rats (filled circles; p < 0.01; EPSP amplitude after vs before HFS; n = 25) but not in 6-OHDA-lesioned animals (open circles; not significant, p > 0.05; EPSP amplitude after vs before HFS; n = 20). Intrastriatal injection of CaMKII inhibitor was able to restore normal LTP 24 hr (open diamonds) but not 72 hr (filled diamonds) after the injection (24 hr KN-93, p < 0.01, EPSP amplitude after vs before HFS, n = 20; 72 hr KN-93, not significant, p > 0.05, EPSP amplitude after vs before HFS, n = 15). D, Western blot (WB) analysis of αCaMKII, active p286-αCaMKII, NR1, and PSD-95 in striatal TIFs from sham- and 6-OHDA-lesioned animals in the absence or presence of 24 or 72 hr KN-93 treatments (^*p < 0.001, 6-OHDA vs sham; ^**p < 0.01, 6-OHDA plus 24 hr KN-93 vs 6-OHDA; ^***p < 0.01, 6-OHDA plus 72 hr KN-93 vs sham). E, TIF proteins from sham- and 6-OHDA-lesioned animals in the absence or presence of 24 or 72 hr KN-93 treatments were phosphorylated under conditions known to maximally activate CaMKII and then immunoprecipitated with anti-NR2A-NR2B. Autoradiography obtained after immunoprecipitation shows a phosphorylated 50 kDa protein band pointing to autophosphorylated αCaMKII. Three independent experiments were performed on different TIF preparations and replicated three times in each TIF preparation (p < 0.01, 6-OHDA vs sham; not significant, p > 0.05, 6-OHDA plus 24 hr KN-93 vs sham; p < 0.01, 6-OHDA plus 24 hr KN-93 vs 6-OHDA; p < 0.01, 6-OHDA plus 72 KN-93 vs sham). i.s., Intrastriatal; i.p., immunoprecipitated.

Figure 5.

Effect of Ant-AIP-II on motor performances, synaptic plasticity, and the NMDA receptor complex of DA-denervated animals. A, A limb-use asymmetry test was performed in sham-operated rats, 6-OHDA denervated animals, and 6-OHDA-lesioned rats receiving a striatal unilateral injection of Ant-AIP-II (n = 8 in each group). 6-OHDA-lesioned rats preferentially use the limb ipsilateral to the lesion (^**p < 0.001; 6-OHDA vs sham). Ant-AIP-II peptide reversed the parkinsonian-like motor features in the lesioned animals (not significant, p > 0.05; 6-OHDA plus Ant-AIP-II vs sham). ipsi, Ipsilateral; contra, contralateral. B, Coordinated locomotor activity on a rotarod is significantly impaired after dopamine denervation but recovers to normal levels (i.e., sham group) after unilateral AIP-2 injection (^*p < 0.01, 6-OHDA vs sham; not significant, p > 0.05, 6-OHDA plus Ant-AIP-II vs sham). C, Intrastriatal injection of AIP-2 was able to restore normal LTP in 6-OHDA denervated rats (sham, filled circles, p < 0.01, EPSP amplitude after vs before HFS, n = 25; 6-OHDA, open circles, not significant, p > 0.05, EPSP amplitude after vs before HFS, n = 20; 6-OHDA plus Ant-AIP-II, open diamonds, p < 0.01, EPSP amplitude after vs before HFS, n = 20). D, Western blot analysis of active p286-αCaMKII in striatal TIFs from sham- and 6-OHDA-lesioned animals in the absence or presence of 24 hr of Ant-AIP-II treatment (^*p < 0.01, 6-OHDA vs sham; ^**p < 0.01, 6-OHDA plus Ant-AIP-II 24 hr vs 6-OHDA). E, TIF proteins from sham- and 6-OHDA-lesioned animals in the absence or presence of 24 hr of Ant-AIP-II treatment were phosphorylated under conditions known to maximally activate CaMKII and then immunoprecipitated with anti-NR2A-NR2B. Representative autoradiography obtained after immunoprecipitation shows two major phosphorylated protein bands: a 50 kDa band (bottom arrow) pointing to autophosphorylated αCaMKII and a 170 kDa band (top arrow) indicating phosphorylated NR2A-NR2B (^*p < 0.005, 6-OHDA vs sham; ^**p < 0.005, 6-OHDA plus Ant-AIP-II vs 6-OHDA). i.s., Intrastriatal.