Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice

Authors


Address correspondence and reprint requests to Dr Shigeki Moriguchi or Prof. Kohji Fukunaga, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi 980-8578, Japan. E-mails: shigeki@m.tohoku.ac.jp; kfukunaga@m.tohoku.ac.jp

Abstract

J. Neurochem. (2012) 120, 541–551.

Abstract

Parkinson’s disease (PD) patients frequently reveal deficit in cognitive functions during the early stage in PD. The dopaminergic neurotoxin, MPTP-induced neurodegeneration causes an injury of the basal ganglia and is associated with PD-like behaviors. In this study, we demonstrated that deficits in cognitive functions in MPTP-treated mice were associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and impaired long-term potentiation (LTP) induction in the hippocampal CA1 region. Mice were injected once a day for 5 days with MPTP (25 mg/kg i.p.). The impaired motor coordination was observed 1 or 2 week after MPTP treatment as assessed by rota-rod and beam-walking tasks. In immunoblotting analyses, the levels of tyrosine hydroxylase protein and CaMKII autophosphorylation in the striatum were significantly decreased 1 week after MPTP treatment. By contrast, deficits of cognitive functions were observed 3–4 weeks after MPTP treatment as assessed by novel object recognition and passive avoidance tasks but not Y-maze task. Impaired LTP in the hippocampal CA1 region was also observed in MPTP-treated mice. Concomitant with impaired LTP induction, CaMKII autophosphorylation was significantly decreased 3 weeks after MPTP treatment in the hippocampal CA1 region. Finally, the reduced CaMKII autophosphorylation was closely associated with reduced AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) phosphorylation in the hippocampal CA1 region of MPTP-treated mice. Taken together, decreased CaMKII activity with concomitant impaired LTP induction in the hippocampus likely account for the learning disability observed in MPTP-treated mice.

Abbreviations used:
ACSF

artificial CSF

AMPAR

α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid receptor

CaMKII

calcium/calmodulin-dependent protein kinase II

DA

dopamine

ERK

extracellular signal-regulated kinase

fEPSPs

field excitatory post-synaptic potentials

GluR1

AMPA-type glutamate receptor subunit 1

LTP

long-term potentiation

MPP+

1-methyl-4-phenylpyridinium

NMDAR

NMDA receptor

PD

Parkinson’s disease

PKC

protein kinase C

SNc

substantia nigra pars compacta

TH

tyrosine hydroxylase

Parkinson’s disease (PD) is progressive neurodegenerative disorder and causes motor dysfunctions such as slowness of movement, rigidity, balance dysfunction, and resting tremor. In addition to the motor dysfunctions, psychological dysfunctions including cognitive deficits, depression and anxiety are also seen in PD patients (Hornykiewicz 1963; Pillon et al. 1989; Cummings 1992; Walsh and Bennett 2001). The dysregulation of motor coordination in PD patients is partly caused by degeneration of dopaminergic nigrostriatal pathway in the striatum (Hornykiewicz and Kish 1987). However, mechanisms underlying the psychological dysfunction are largely unknown.

MPTP is a potent dopaminergic neurotoxin. It causes markedly depletion of striatal dopamine (DA) through degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) (Heikkila et al. 1984a,b; Sonsalla and Heikkila 1986; Ricaurte et al. 1987; Jackson-Lewis et al. 1995). Monoamine oxidase-B enzymes convert MPTP to 1-methyl-4-phenylpyridinium (MPP+) in dopaminergic neurons uptaken through dopamine transporter. MPP+ inhibits the complex-1 in mitochondria and results in decreased ATP levels and elevation of oxidative stress and in turn causes dopaminergic neuronal cell death (Smeyne and Jackson-Lewis 2005). Systemic administration of MPTP in mice is useful experimental model for early phase of PD which is mainly characterized by cognitive dysfunction and motor impairment (Tanila et al. 1998; Perry et al. 2004; Ferro et al. 2007; Reksidler et al. 2007; Zhu et al. 2011).

It is widely recognized that glutamate acting via the NMDA receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid receptor (AMPAR) accounts for excitatory synaptic transmission in the mammalian brain. NMDAR is involved in various physiological processes including synaptic plasticity, learning, and memory (Bliss and Collingridge 1993; Danysz et al. 1995). Long-term potentiation (LTP) induction is a state of increasing synaptic transmission following high frequency stimulation and is known to be as a model for neuronal events underlying learning and memory (Bliss and Collingridge 1993; Malenka 1994). Long-term synaptic plasticity is regulated by NMDAR function in the CA1 region of the hippocampus (Harris et al. 1984; Mulkey and Malenka 1992; Bliss and Collingridge 1993).

Calcium/calmodulin-dependent protein kinase II (CaMKII) has a critical role in LTP induction (Fukunaga et al. 1993; Hudman and Schulman 2002; Lisman et al. 2002). CaMKII is highly enriched in post-synaptic densities of excitatory synapses and becomes constitutively active through autophosphorylation, thereby increasing synaptic efficacy (Fukunaga et al. 1993; Lledo et al. 1995; Wang and Kelly 1995; Barria et al. 1997; Giese et al. 1998; Lisman et al. 2002). Facilitating synaptic efficacy by CaMKII requires up-regulation of post-synaptic AMPAR function by direct phosphorylation (McGlade-McCulloh et al. 1993; Barria et al. 1997) and Trafficking of AMPAR into post-synaptic membranes (Shi et al. 1999; Poncer et al. 2002; Song and Huganir 2002).

Like CaMKII, protein kinase C (PKC) is also essential for hippocampal LTP induction (Collingridge et al. 2004). Phosphorylation of NMDAR subunit 1 mediated by PKC accounts for up-regulation of NMDAR function and is likely required for CaMKII-dependent LTP enhancement (Tingley et al. 1993). Similarly, extracellular signal-regulated kinase (ERK) is thought to play crucial roles in hippocampal LTP maintenance through phosphorylation of the cAMP-responsible element-binding protein (Xia and Storm 2005).

In this present study, we addressed crucial relevance of reduced CaMKII autophosphorylation and impaired hippocampal LTP induction in MPTP-treated mice as PD disease model.

Materials and methods

Animals

Male mice (C57BL/6N) of 8–9 weeks (Nippon SLC, Hamamatsu, Japan) were housed in cages with free access to food and water at a constant temperature (23 ± 1°C) and humidity (55 ± 5%) with a 12-h light/dark cycle (09:00–21:00 hours). All experimental procedures using animals were approved by the Committee on Animal Experiments at both Tohoku University.

Drug treatment

Mice were treated once a day for 5 days with MPTP (25 mg/kg, i.p.) and subjected to behavioral and neurochemical analyses 1–4 weeks after MPTP treatment.

Measurement of locomotor activity

To measure locomotor activity during 1 h period, mice housed individually in standard plastic cages were positioned in an automated open-field activity monitor using digital counters with an infrared sensor (DAS system, Neuroscience).

Rota-rod task

Rota rod task consisted of a base platform and an iron rod of 3 cm diameter and 30 cm length, with a non-slippery surface. This rod was divided into three equal sections by two disks, thus enabling three mice to walk on the rod at the same time at the speed of 4/min. Animals were placed on the rotating drum up to 5 min. Intervals between the mounting of the mice on the rod and falling off of it were recorded as the performance time.

Beam-walking task

The beam-walking task consisted of a rectangular shaped base (870 mm × 200 mm × 17 mm, medium density fibreboard), with Formica cover. A Vertical stand (310 mm × 160 mm × 5 mm, Perspex), was fixed to the left side of the base. The stand supported a black ‘goal box’ (155 mm × 160 mm × 5 mm, Perspex), with a matt surface on all the inside faces. A horizontal rod (500 mm × 5 mm diameter, dowelled) was fixed between the base of the ‘goal box’ and a vertical stainless steel pole (315 mm ×5 mm), inverted by 90°. Further support was provided to the steel pole by a Perspex upright which prevented sideways movement of the beam. The dowelled rod was graduated/cm from 0 to 50 cm.

Y-maze task

Spontaneous alteration behavior in a Y-maze was assessed as spatial reference memory task. The apparatus consisted of three identical arms (50 × 16 × 32 cm) made by black plexiglas. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded manually. An alternation was defined as entries into all three arms on consecutive choices. The number of maximum alternations was then the total number of arms entered minus two, and the percentage of alternation was calculated as actual alternations/maximum alternations × 100. In addition, the total number of arms entered during the session was also determined.

Novel object recognition task

This task is based on the tendency of rodents to discriminate a familiar from new object. Mice were individually habituated to an open-field box (35 × 25 × 35 cm) for two consecutive days. The experimenter scoring behavior was blinded to the treatment. During the acquisition phases, two objects of the same material were placed in a symmetric position in the center of the chamber for 5 min. One hour after the acquisition phase training, one of the objects was replaced by a novel object, and exploratory behavior was again analyzed for 5 min. After each session, objects were thoroughly cleaned with 75% ethanol to prevent odor recognition. Exploration of an object was defined as rearing on the object or sniffing it at a distance of less than 1 cm, touching it with the nose, or both. Successful recognition of a previously explored object was reflected by preferential exploration of the novel object. Discrimination of spatial novelty was assessed by comparing the difference between time of exploration of the novel and familiar object and the total time spent exploring both objects, which made it possible to adjust for differences in total exploration time.

Step-through passive avoidance task

Training and retention trials of passive avoidance task were conducted in box which consists of dark (25 × 25 × 25 cm) and light (14 × 10 × 25 cm) compartments. The floor was constructed of stainless steel rods. The floor rods in the dark compartment were connected to an electronic stimulator (Nihon Kohden, Tokyo, Japan). Mice were habituated to the apparatus the day before passive avoidance acquisition. On the training trials, a mouse was placed in the lighted compartment of box. When the mouse entered the dark compartment, the door was closed and an inescapable electric shock (1 mA for 500 ms) was delivered from the grid floor. The mouse was then removed from the apparatus 30 s after receiving the shock. The same procedure without the footshock was repeated after an interval of 24 h to assess the level of retention.

Electrophysiology

Preparation of hippocampal slices was performed as described (Moriguchi et al. 2008). Briefly, brains were rapidly removed from ether-anesthetized male mice (C57BL/6N) and hippocampi dissected out. Transverse hippocampal slices (400 μm thick) prepared using a vibratome (microslicer DTK-1000) were incubated for 2 h in continuously oxygenated (95% O2, 5% CO2) artificial CSF (ACSF) at room temperature (28°C). After a 2-h recovery period, a slice was transferred to an interface recording chamber and perfused at a flow rate of 2 mL/min with ACSF warmed to 34°C. Field excitatory post-synaptic potentials (fEPSPs) were evoked by a 0.05 Hz test stimulus through a bipolar stimulating electrode placed on the Schaffer collateral/commissural pathway and recorded from the stratum radiatum of CA1 using a glass electrode filled with 3 M NaCl. Recording was performed using a single-electrode amplifier (CEZ-3100; Nihon Kohden, Tokyo, Japan), and the maximal value of the initial fEPSP slope was collected and averaged every 1 min (three traces) using an A/D converter (PowerLab 200; AD Instruments, Castle Hill, Australia) and a personal computer. After a stable base-line was obtained, high frequency stimulation of 100 Hz with a 1-s duration was applied twice with a 20-s interval and test stimuli were continued for the indicated periods.

Immunoblotting analysis

Preparation of striatum or hippocampal slices was performed as described (Moriguchi et al. 2008). Briefly, brains were rapidly removed from ether-anesthetized male mice (C57BL/6N) and striatum or hippocampi were dissected out. Transverse striatum or hippocampal slices (400 μm thick) prepared using a vibratome (microslicer DTK-1000) were incubated for 2 h in continuously oxygenated (95% O2, 5% CO2) ACSF at room temperature (28°C). Slices were transferred to a plastic plate cooled on ice to dissect out the CA1 region, CA3 region and dentate gyrus (DG) from hippocampus. Tissues were frozen in liquid nitrogen and stored at −80˚C for immunoblotting analysis. Striatum or Hippocampal CA1, CA3 and DG samples were homogenized in 70 μL of homogenizing buffer containing 50 mM Tris–HCl (pH 7.4), 0.5 % Triton X-100, 4 mM EGTA, 10 mM EDTA 1 mM Na3VO4, 40 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 50 μg/mL leupeptin, 25 μg/mL pepstatin A, 50 μg/mL trypsin inhibitor and 1 mM dithiothreitol. Insoluble material was removed by a 10-min centrifugation at 17 400 g. After determining protein concentration in supernatants using Bradford’s solution, samples were boiled 3 min in Laemmli’s (1970) sample buffer. Samples containing equivalent amounts of protein were subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. Proteins were transferred to an Immobilon PVDF membrane for 2 h at 70 V. After blocking with TTBS solution (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 2.5% bovine serum albumin for 1 h at 28°C, membranes were incubated overnight at 4°C with anti-phospho CaMKII, (1 : 5000; Fukunaga et al. 1988), anti-CaMKII (1 : 5000; Fukunaga et al. 1988), anti-phospho-synapsin I (Ser-603) (1 : 2000; Chemicon, Temecula, CA, USA), anti-synapsin 1 (1 : 2000; Fukunaga et al. 1992), anti-phospho-AMPA-type glutamate receptor subunit 1 (GluR1; Ser-831) (1 : 1000; Upstate, Lake Placid, NY, USA), anti-GluR1 (1 : 1000; Chemicon), anti-phospho-PKCα (Ser-657) (1 : 2000; Upstate), anti-phospho-mitogen-activated protein (MAP) kinase (diphosphorylated ERK 1/2) (1 : 2000; Sigma, St Louis, MO, USA), anti-tyrosine hydroxylase (1 : 2000; Chemicon), and anti-β-tubulin (1 : 5000; Sigma). Bound antibodies were visualized using the enhanced chemiluminescence detection system (Amersham Life Science, Buckinghamshire, UK) and analyzed semiquantitatively using the NIH Image program.

Other chemicals

MPTP was purchased from Sigma-Aldrich (St Louis, MO, USA).

Statistical analysis

Data were presented as means ± SEM. Multiple comparisons were performed by one-way analysis of variance (anova) with Scheffe’s test. A value of < 0.05 was considered statistically significant.

Results

Impairment of motor coordination and decreased striatal tyrosine hydroxylase protein levels in MPTP-treated mice

We first tested effect of MPTP treatment on motor coordination assessed by rota-rod and beam-walking tasks in mice. Mice were treated once a day for 5 days with MPTP (25 mg/kg, i.p.) and measured 1–4 weeks after MPTP treatment. In rota-rod task, rota-rod latency significantly decreased 2 weeks after MPTP treatment [saline-treated mice (4 weeks): 280.8 ± 16.7% of control, = 7: MPTP-treated mice (4 weeks): 184.1 ± 44.6% of control, = 7] (Fig. 1a). Similarly, impaired step also significantly increased 1 week after MPTP treatment by beam walking task [saline-treated mice (4 weeks): 2.3 ± 0.4% of control, = 7: MPTP-treated mice (4 weeks): 4.6 ± 0.6% of control, = 7] (Fig. 1b). Thus, impairments of motor coordination were observed in MPTP-treated mice. In addition, we measured the basal locomotor activity during 1 hr both MPTP-treated mice and saline-treated mice. There are not difference the basal locomotor activity between MPTP-treated and saline-treated mice (Fig. 1c). As a reduced tyrosine hydroxylase (TH) level is reported in MPTP-treated mice (Heikkila and Sonsalla 1987), we measured protein levels of TH in the striatum from MPTP-treated mice. In immunoblot analyses, the reduced TH protein was detected 1 week and continuously until 4 weeks after MPTP treatment in the striatum compared with control mice (4 weeks: 56.7 ± 7.4% of control, = 4) (Fig. 1d and e).

Figure 1.

 Abnormal motor coordination and decreased protein level of striatal tyrosine hydroxylase (TH) in MPTP-treated mice. (a) Analyses of motor coordination with rota-rod task in MPTP-treated mice compared with saline-treated mice. (b) Analyses of motor coordination with beam walking task in MPTP-treated mice compared with saline-treated mice. (c) Measurement of basal locomotor activity in MPTP-treated mice and saline-treated mice. (d) Representative images of immunoblots using antibody against TH. (e) Quantitative analyses of protein level of striatal TH as analyzed by densitometry. Data are expressed as the seconds (a), number (b) and percentage (e) of value of saline-treated mice. *< 0.05; **< 0.01 versus the control of saline-treated mice.

Reduction of CaMKIIα(Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the striatum from MPTP-treated mice

We next examined which of protein kinases are affected in the striatum by MPTP treatment. CaMKIIα (Thr-286) autophosphorylation significantly decreased 1 week after MPTP treatment without changes in CaMKII protein levels (1 week: 26.7 ± 6.1% of control, = 4; 4 weeks: 46.2 ± 11.8% of control, = 4) (Fig. 2a and b). By contrast, PKCα (Ser-657) autophosphorylation and ERK phosphorylation were not affected in the striatum of MPTP-treated mice (Fig. 2a and b). In addition, we analyzed phosphorylation of GluR1 (Ser-831) and synapsin I (Ser-603) as post-synaptic and pre-synaptic substrates, respectively, for CaMKII in the striatum. Like the reduced CaMKII autophosphorylation, GluR1 (Ser-831) phosphorylation significantly decreased 1 week after MPTP treatment without changes in GluR1 protein levels in the stratum (1 week: 58.6 ± 4.6% of control, = 4; 4 weeks: 47.7 ± 4.2% of control, = 4) (Fig. 2c and d), whereas synapsin I (Ser-603) phosphorylation was not changed.

Figure 2.

 Reduction of CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the striatum from MPTP-treated mice. (a) Representative images of immunoblots using antibodies against autophosphorylated CaMKIIα (Thr-286) (50 kDa), CaMKIIα (50 kDa), autophosphorylated PKCα (Ser-657) and phosphorylated ERK. (b) Quantitative analyses of autophosphorylated CaMKIIα (Thr-286) and PKCα (Ser-657) and phosphorylated ERK as analyzed by densitometry. (c) Representative images of immunoblots using antibodies against phosphorylated synapsin I (Ser-603), synapsin I, GluR1 (Ser-831) and GluR1. (d) Quantitative analyses of phosphorylated synapsin I (Ser-603), synapsin I, GluR1 (Ser-831) and GluR1 as analyzed by densitometry. Data are expressed as the percentage of value of saline-treated mice. **< 0.01 versus the control of saline-treated mice.

Deficits in cognitive function assessed by novel object recognition and passive avoidance tasks but normal spatial memory in Y-maze task in MPTP-treated mice

We next tested novel object recognition task to define impairment of cognitive function (Prickaerts et al. 2002). In training session, there is no difference in discrimination index using the same objects 1–4 weeks after MPTP or saline treatment (data not shown). After a 1-h retention time, the saline-treated mice discriminated between the familiar and novel objects in the test session throughout the test periods of 1–4 weeks, whereas MPTP-treated mice failed to discriminate between the familiar and novel objects at 3 and 4 weeks after MPTP treatment (Fig. 3a). In Y-maze task, the spontaneous alteration performance and total number of arms entries were measured 1–4 weeks after MPTP or saline treatment. The percentage of alterations of behavior the number of total arm entries were not changed in MPTP-treated mice compared with those in saline-treated mice (Fig. 3b and c). We also confirmed contextual memory impairment at 4 weeks after MPTP treatment, by fear-conditioned passive avoidance task. There were no significant differences in the latency time until entering dark compartment without electrical foot shock (Fig. 3d). However, the latency until entering the darkened compartment in where electric shock was given were significantly decreased 5 days after electric shock in MPTP-treated mice as compared with that in saline-treated mice, suggesting an impairment of contextual memory (saline-treated mice: 266.4 ± 23.0% of control, = 5; MPTP-treated mice: 169.6 ± 7.6% of control, = 5).

Figure 3.

 Deficits in cognitive function by novel object recognition task and passive avoidance task. (a) The discrimination index of object exploration was shown as times to interest familiar and novel objects in the test session after 1 h of training session at 1–4 weeks after saline and MPTP treatment in mice. (b) Total arm entry in Y-maze task was measured 1–4 weeks after MPTP treatment. (c) Alternation in Y-maze task was measured 1–4 weeks after MPTP treatment. (d) Latency time in passive avoidance task was measured 4 weeks after MPTP treatment. (e) The latency time on retention trials in MPTP-treated mice as compared with that in saline-treated mice. Data are expressed as compared the percentage (a), seconds (e) of value of saline-treated mice. *< 0.05; **< 0.01 versus the control of saline-treated mice.

Impairment of long-term potentiation (LTP) in the hippocampal CA1 region from MPTP-treated mice

As the hippocampus-dependent contextual memory in passive avoidance task was impaired without impairment of spatial memory in Y-maze task, we measured the hippocampal LTP in the CA1 region in MPTP-treated mice 4 weeks after MPTP treatment. In control slices at 4 weeks in saline-treated mice, twice high-frequency stimulation (100 Hz) of Schaffer collateral/commissural pathways induced LTP in the hippocampal CA1 region, which lasted over 60 min (167.6 ± 13.1% of the baseline at 60 min, = 5) (Fig. 4a–c). As expected, a significant reduction of LTP was observed at 4 weeks in MPTP-treated mice (123.1 ± 3.2% of the baseline at 60 min, = 5) (Fig. 4a–c). In addition, input-output relationship was recorded compared with MPTP-treated mice and saline-treated mice. The input-output relationship at 0.1–1.0 mA was not affected by MPTP treatment compared with control mice (Fig. 4d). As shown in Fig. 4e, the ratios of second to the first fEPSPs with various inter-pulse intervals were indistinguishable between MPTP-treated mice and saline-treated mice (Fig. 4e).

Figure 4.

 Impairment of NMDAR-dependent long-term potentiation (LTP) in the hippocampal CA1 region. (a) Representative fEPSPs were recorded from the CA1 region in saline-treated mice and MPTP-treated mice. (b) Changes in slope of fEPSPs following HFS recorded in the CA1 region was attenuated in MPTP-treated mice as compared with that in saline-treated mice. (c) Changes in slope of fEPSPs following HFS were represented in saline-treated mice and MPTP-treated mice. (d) Input–output relationship was recorded in saline-treated mice and MPTP-treated mice. High intensity stimulated responses does not affect between saline-treated mice and MPTP-treated mice. (e) The rations of second to the first fEPSPs with various inter-pulse intervals were indistinguishable among MPTP-treated mice and saline-treated mice. Data are expressed as the percentage of value of saline-treated mice. *< 0.05; **< 0.01 versus the control of saline-treated mice.

Reduction of CaMKIIα(Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA1 region from MPTP-treated mice

Consistent with LTP impairment in MPTP-treated mice, CaMKIIα (Thr-286) autophosphorylation significantly decreased 3 weeks after MPTP treatment without changes in CaMKII protein levels (3 week: 82.4 ± 7.0% of control, = 4; 4 weeks: 73.0 ± 3.4% of control, = 4) (Fig. 5a and b). By contrast, PKCα (Ser-657) autophosphorylation and ERK phosphorylation were not changed in the hippocampal CA1 region from MPTP-treated mice (Fig. 5a and b).

Figure 5.

 Reduction of CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA1 region from MPTP-treated mice. (a) Representative images of immunoblots using antibodies against autophosphorylated CaMKIIα (Thr-286) (50 kDa), CaMKIIα (50 kDa), autophosphorylated PKCα (Ser-657) and phosphorylated ERK. (b) Quantitative analyses of autophosphorylated CaMKIIα (Thr-286) and PKCα (Ser-657) and phosphorylated ERK as analyzed by densitometry. (c) Representative images of immunoblots using antibodies against phosphorylated synapsin I (Ser-603), Synapsin I, phosphorylated GluR1 (Ser-831) and GluR1. (d) Quantitative analyses of phosphorylated synapsin I (Ser-603) and GluR1 (Ser-831) as analyzed by densitometry. Data are expressed as the percentage of value of saline-treated mice. *< 0.05; **< 0.01 versus the control of saline-treated mice.

As increased phosphorylation of GluR1 (Ser-831) by CaMKII results in an increase in single-channel conductance of AMPA receptors (Derkach et al. 1999), we measured GluR1 (Ser-831) phosphorylation. Like reduced CaMKII autophosphorylation, GluR1 (Ser-831) phosphorylation significantly decreased 3 weeks after MPTP treatment in the hippocampal CA1 region, whereas synapsin I (Ser-603) phosphorylation (Yamagata 2003) was not changed 3 weeks after MPTP treatment (Fig. 5c and d).

Reduction of CaMKIIα(Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA3 and DG region from MPTP-treated mice

As the hippocampal CA3 and DG excitatory glutamatergic inputs are required for memory consolidation and maintenance (O’Reilly and McClelland 1994), we also examined CaMKII and GluR1 phosphorylation in the CA3 and DG regions of MPTP-treated mice. In the CA3 region, both CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation were significantly decreased 3–4 weeks after MPTP treatment (CaMKIIα (Thr-286), 3 week: 49.8 ± 2.9% of control, = 4; 4 weeks: 57.9 ± 4.2% of control, = 4; GluR1(Ser-831), 3 week: 56.3 ± 2.5% of control, = 4; 4 weeks: 55.8 ± 3.4% of control, = 4) (Fig. 6a and b). Interestingly, in the DG region, both CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation were significantly decreased 1 week after MPTP treatment [CaMKIIα (Thr-286), 1 week: 71.7 ± 10.8% of control, = 4; 4 weeks: 51.5 ± 5.3% of control, = 4; GluR1(Ser-831), 1 week: 78.0 ± 0.6% of control, = 4; 4 weeks: 63.9 ± 5.5% of control, = 4] (Fig. 6c and d), suggesting that the DG neurons are more susceptible for MPTP treatment.

Figure 6.

 Reduction of CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation in the hippocampal CA3 and DG region from MPTP-treated mice. (a) Representative images of immunoblots using antibodies against autophosphorylated CaMKIIα (Thr-286) (50 kDa), CaMKIIα (50 kDa), phosphorylated GluR1 (Ser-831) and GluR1 in the CA3 region as analyzed by densitometry. (b) Quantitative analyses of autophosphorylated CaMKIIα (Thr-286) and GluR1 (Ser-831) in the CA3 region as analyzed by densitometry. (c) Representative images of immunoblots using antibodies against autophosphorylated CaMKIIα (Thr-286) (50 kDa), CaMKIIα (50 kDa), phosphorylated GluR1 (Ser-831) and GluR1 in the DG region as analyzed by densitometry. (d) Quantitative analyses of autophosphorylated CaMKIIα (Thr-286) and GluR1 (Ser-831) in the DG region as analyzed by densitometry. Data are expressed as the percentage of value of saline-treated mice. *< 0.05; **< 0.01 versus the control of saline-treated mice.

Discussion

PD is characterized by a severe DA deficiency in the striatum and is caused by a selective and progressive degeneration of dopaminergic neurons in the SNc (Adolfsson et al. 1979; Hornykiewicz and Kish 1987; Jellinger 1987; Kish et al. 1988). Like PD, MPTP is a potent dopaminergic neurotoxin and produces markedly depletion of striatal DA and degeneration of dopaminergic neurons in SNc (Heikkila et al. 1984a,b; Sonsalla and Heikkila 1986; Ricaurte et al. 1987; Jackson-Lewis et al. 1995). MPTP is a lipophilic compound that crosses the blood-brain barrier and is metabolized to MPP+ by the enzyme monoamine oxidase-B. MPP+ selectively causes death of dopaminergic neurons in SNc (Nicotra and Parvez 2000). MPTP treatment in animal cause abnormal motor coordination and decreased rearing frequencies (Reksidler et al. 2007, 2008). In the present study, we confirmed abnormal motor coordination as assessed by rota-rod task and beam walking task in MPTP treated mice (Fig. 1). Deficit in motor performance was also observed by beam walking task (Quinn et al. 2007), overall rod performance (Rozas et al. 1998), stride length (Fernagut et al. 2002) and pole test (Ogawa et al. 1985). Consistent with reduced TH protein levels in the present study, Hong et al. (2007) reported that TH-immunoreactive neurons in the SNc and TH-immunoreactive fibers in the striatum were reduced in MPTP-treated mice. Furthermore, MPTP injection reduces the number of TH-positive neurons in the SNc within 7 days (Zhu et al. 2011). In addition to abnormal motor coordination, hippocampal synaptic transmission and synaptic plasticity are disturbed in PD (Liu et al. 2007; Wang et al. 2008; Kitada et al. 2009; Hanson et al. 2010). Our data show that MPTP causes cognitive deficit as assessed by novel object recognition task and passive avoidance task, but not Y-maze task (Fig. 3). Similar with our experiments, MPTP treatment causes cognitive impairment in animals as tested by active avoidance task (Da Cunha et al. 2001; Gevaerd et al. 2001), Morris water maze task (Miyoshi et al. 2002; Perry et al. 2004; Reksidler et al. 2007) and novel recognition task in monkey (Schneider et al. 2000). However, there are no extensive studies regarding mechanisms underlying impairment of motor coordination and cognitive function in MPTP-treated animals.

We first found that CaMKII autophosphorylation and GluR1 (Ser-831) phosphorylation significantly decreased in the striatum from MPTP-treated mice (Fig. 2). Ba et al. (2006) reported that GluR1 (Ser-831) phosphorylation significantly reduce in the plasma membrane of lesioned striatal neurons from PD model of 6-Hydroxydopamine-treated mice. Our results confirmed that the reduced GluR1 (Ser-831) phosphorylation is due to reduced post-synaptic CaMKII activity in the striatum. Like impaired motor coordination observed 1 week after MPTP treatment, the marked reduction of CaMKII autophosphorylation and GluR1 phosphorylation was observed 1 week after MPTP treatment without changes in ERK and PKC activities. Therefore, reduced CaMKII activity suggests impairment of glutamatergic inputs in cortico-striatal pathways and partly accounts for the motor coordination impairment observed in MPTP-treated mice.

Concomitant with cognitive deficits reported in MPTP-treated mice, NMDAR-dependent LTP in the hippocampal CA1 region was impaired in MPTP-treated mice (Fig. 4). Dopaminergic neurons from ventral tegmental area and/or substantia nigra directly project to the hippocampus (Gasbarri et al. 1994). MPTP-mediated DA depletion is known to impair the induction of hippocampal LTP (Zhu et al. 2011). Likewise, dopaminergic D1 receptor agonist treatment potentiates the hippocampal LTP through the pre-synaptic growth-associated protein, GAP-43 protein (Williams et al. 2006). Thus, MPTP-mediated DA neuron degeneration likely causes impairment of hippocampal LTP.

We for the first time defined that impairment of dopaminergic innervation to the hippocampus causes marked reduction CaMKII in the hippocampal CA1, CA3 and DG. Interestingly, reduction of CaMKII autophosphorylation and GluR1 (Ser-831) phosphorylation was observed 1 week after MPTP treatment in DG and 3–4 weeks after MPTP treatment in CA1 and CA3 (Figs 5 and 6). Thus, DA depletion induced by MPTP causes impairment of hippocampal DG region in early stage similar to the striatum. By contrast, the impairment of hippocampal CA1 or CA3 region was observed in the late stage, when the cognitive impairment is observed in novel object recognition task. As the impairment of CaMKII activity and the CA1 LTP were partial and moderate compared with the hippocampal slices from control mice, the spatial memory impairment in Y-maze task is not observed 1 to 4 week after MPTP treatment (Fig. 3c). However, the cognition in novel object recognition task was largely impaired in MPTP-treated mice and closely associated with reduced CaMKII activities in the hippocampal CA1 and CA3 regions. Interestingly, in the passive avoidance task, MPTP-treated mice are indistinguishable in the entrainment process during first 3 days tested but failed to maintain the memory in the later phase when compared with control mice. The storage or retrieval processes may be impaired in MPTP-treated mice and the other brain regions may also be affected by dopaminergic neurodegeneration. Indeed, impairments of memory storage and retrieval in forebrain CaMKII knockout mice were not associated with the hippocampal LTP but associated with impaired cortical LTP (Frankland et al. 2001). In this context, the observation of reduced CaMKII and GluR1 phosphorylation in the cortex is also important (data not shown).

In addition to CaMKII, PKC and ERK are also essential for hippocampal LTP induction (Collingridge et al. 2004; Xia and Storm 2005). In this present study, our data shows that phosphorylation of PKCα and ERK were unchanged in the CA1 from MPTP-treated mice. Thus, PKC and ERK activities are not required for cognitive deficits observed in MPTP-treated mice. Taken together, we conclude that reduction of post-synaptic CaMKII activity and impaired LTP induction are critical for cognitive deficits observed in MPTP-treated mice.

Acknowledgements

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health and Welfare of Japan (22390109 to KF; 20790398 to SM) and the Pharmacological Research Foundation, Tokyo (to SM), the Research Foundation for Pharmaceutical Sciences (to SM), the Smoking Research Foundation (to KF), Takeda Science Foundation (to SM), NISHINOMIYA Basic Research Fund (Japan) (to SM) and HIROMI Medical Research Foundation (to SM).

Disclosure/conflict of interest

The authors have no conflict of interest.

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