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Keywords:

  • D2R agonist;
  • dystonia;
  • G gamma 7 KO mice;
  • pramipexole;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Gγ7 is enriched in striatum and forms a heterotrimeric complex with Gαolf/Gβ, which is coupled to D1 receptor (D1R). Here, we attempted to characterize the pathophysiological, neurochemical, and pharmacological features of mice deficient of Gγ7 gene. Gγ7 knockout mice exhibited age-dependent deficiency in rotarod behavior and increased dystonia-like clasping reflex without loss of striatal neurons. The neurochemical basis for the motor manifestations using immunoblot analysis revealed increased levels of D1R, ChAT and NMDA receptor subunits (NR1 and NR2B) concurrent with decreased levels of D2R and Gαolf, possibly because of the secondary changes of decreased Gαolf/Gγ7-mediated D1R transmission. These behavioral and neurochemical changes are closely related to those observed in Huntington's disease (HD) human subjects and HD model mice. Taking advantage of the finding of D2R down-regulation in Gγ7 knockout mice and the dopamine-mediated synergistic relationship in the control of locomotion between D2R-striatopallidal and D1R-stritonigral neurons, we hypothesized that D2-agonist pramipexole would reverse behavioral dyskinesia caused by defective D1R/Gαolf signaling. Indeed, the rotarod deficiency and clasping reflex were reversed by pramipexole treatment under chronic administration. These findings suggest that Gγ7 knockout mice could be a new type of movement disorders, including HD and useful for the evaluation of therapeutic candidates.

Abbreviations used
Amyg

amygdala

BDNF

brain-derived neurotrophic factor

Cere

cerebellum

ChAT

choline acetyltransferase

Cor

cortex

D1R

dopamine D1 receptor

D2R

dopamine D2 receptor

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

Gγ7

G-protein gamma subunit 7

HD

Huntington's disease

Hip

hippocampus

Hypo

hypothalamus

i.p.

intraperitoneal

Mid

midbrain

MSNs

medium spiny neurons

NR1

NMDA receptor NR1 subunit

NR2A

NMDA receptor NR2A subunit

NR2B

NMDA receptor NR2B subunit

Olf

olfactory bulb

PD

Parkinson's disease

RLS

restless legs syndrome

SNpr

subthalamic nucleus

Str

striatum

Thal

thalamus

Vth

ventrolateral thalamic

WT

wild-type

The dorsal striatum is the major neural network involved in the regulation of motor functions (Yin and Knowlton 2006; Nicola 2007). The striatal projection neurons are central to motor function and loss of its circuitry is associated with neurological diseases such as Huntington's diseases (HD) (Deng et al. 2004), attention-deficit hyperactivity disorder (Comings 2001), Tourette's syndrome (Saka and Graybiel 2003), and schizophrenia (Keshavan et al. 2008). Motor information received from cerebral cortex via glutaminergic input (Calabresi et al. 1992; Yin et al. 2005) is processed by medium-sized spiny GABAergic neurons (Graybiel 2000; Durieux et al. 2009). Dopamine D1 and M4 muscarinic acetylcholine receptor-expressing medium-sized spiny neurons (MSNs) (Bernard et al. 1992; Ince et al. 1997; Jeon et al. 2010) have axonal projections into substantia nigra pars reticulata and globus pallidus interneurons and glutaminergic excitation of these MSNs population transmit wave of inhibition to ventrolateral thalamic (Vth) and substantia nigra pars reticulata (SNpr) and in turn disinhibit motor neurons; ultimately movement is initiated (Jeon et al. 2010). Movement is inhibited through neural network involving dopamine D2 and Adenosine A2 receptors-expressing MSNs (striatopallidal neurons) (Gerfen et al. 1990; Fink et al. 1992). Here, cortical excitation of these MSNs results in inhibition of external globus pallidus neurons and subsequent decrease in their inhibitory influence on the internal globus pallidus and the subthalamic nuclei. Because the internal globus pallidus neurons tonically inhibit Vth and SNpr neurons, stronger inhibition on motor neuron and movement initiation is generated.

MSNs in the striatum receive cortical glutaminergic inputs as well as dopaminergic input (Shen et al. 2008). In terms of locomotion, synergy exists between dopaminergic activities on dopamine D1 receptor (D1R)- and dopamine D2 receptor (D2R)-expressing striatal GABA neurons (Aoyama et al. 2000; Pavese et al. 2003). It is reported that D1R and D2R, which couples to stimulatory Gαolf and inhibitory Gi/o, respectively, are down-regulated in HD human subjects and experimental R6/1 HD mice model (Desplats et al. 2006; Hodges et al. 2006). Gγ7 is enriched in striatum and forms a heterotrimeric complex with Gαolf/Gβ, which is coupled to D1 receptor (Betty et al. 1998; Wang et al. 2001; Schwindinger et al. 2003) and its genetic deletion causes a marked down-regulation of Gαolf (Schwindinger et al. 2003). As HD human subjects and R6/1 HD mice also show a down-regulation of Gγ7 and Gαolf (Desplats et al. 2006; Hodges et al. 2006), we speculated a possibility that Gγ7 KO could be a mouse model for the motor dysfunction that occurs with several types of movement disorders, including HD.

In this study, we first determined whether loss of Gγ7 in the striatum (Danielson et al. 1994; Schwindinger et al. 2003) has noticeable effects on motor function. Here, we showed that Gγ7 KO mice exhibited age-dependent motor dysfunction without a significant loss of striatal projection neurons. Then we studied the possible underlying neurochemical mechanisms. Finally, we provided evidence that the behavioral dyskinesia in Gγ7 KO mice is reversed during chronic administration of D2R agonist pramipexole.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Animals

All male mice (6–30-week-old) were kept in a room with a temperature of 21 ± 2°C with ad libitum access to a standard laboratory diet and tap water in standard animal cages in 12-h light/dark cycle (lights on at 8:00 a.m.). All procedures used in this study were approved by the Institutional Animal Care Committee (Nagasaki University, Nagasaki, Japan).

Genotyping for Gng7-Cre knock-in mice

The mice genotype was confirmed by PCR using primers 5′-GGCGACGTTGTTAGTACCTGAC-3′, 5′-ATCCCTGAACATGTCCATCAGGTTC-3′, and 5′-TATAGGTACCCAGAAGTGAATTCGGTTCGC-3′. Extracted DNA samples were amplified for 35 cycles consisting of 95°C (30 s), 60°C (20 s), 72°C (30 s) and PCR amplifications were repeated in duplicate. PCR samples were separated by electrophoresis (2% of agarose gel, 170 V, 30 min).

Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from each brain regions; striatum, olfactory bulb, cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, amygdala, pons, and cerebellum using TRIzol (Invitrogen, Carlsbad, CA, USA), and 500 ng of RNA was used for cDNA synthesis. Quantitative real-time PCR was performed with qPCR MasterMix Plus for SYBR Green I (Eurogentec, Seraing, Belgium) using the ABI Prism 7000 sequence detection system (Applied Biosystem, Carlsbad, CA, USA). The following primers were used: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TATGACTCCACTCACGGCAAAT-3′ (forward) and 5′-GGGTCTCGCTCCTGGAAGAT-3′ (reverse) Gng7, 5′-GGCCGATGATGTCAGGTACT-3′ (forward) and 5′-TGCTCACAGTAGCCCATCAG-3′ (reverse). Samples were amplified for 45 cycles consisting of 95°C (15 s), 60°C (1 min), and PCR amplifications were repeated in duplicate. GAPDH was used as an internal control for normalization. In all cases, the validity of amplification was confirmed by the presence of a single peak in the melting temperature analysis and by linear amplification with increasing number of PCR cycles.

Western blot

The brain samples were sonicated in ice-cold sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris-HCl, pH6.8, 2% sodium dodecyl sulfate, 10% glycerol, 1 μM p-amidinophenyl methanesulfonyl fluoride hydrochloride. Total protein (20 μg) extracted from each brain region was separated on SDS-polyacrylamide gel (10%). Primary antibodies were used at the following dilutions: goat anti-Gαolf polyclonal antibody (1 : 200; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Gγ7 polyclonal antibody (1 : 200; Santa Cruz Biotechnology), rabbit anti-Gi1/2 polyclonal antibody (1 : 400; Abnova, Taipei, Taiwan), rabbit anti-Gαs polyclonal antibody (1 : 200; Santa Cruz Biotechnology), rabbit anti-Gαo polyclonal antibody (1 : 200; Santa Cruz Biotechnology), rabbit anti-Gα11 polyclonal antibody (1 : 200; Santa Cruz Biotechnology), rabbit anti-Gα13 polyclonal antibody (1 : 200; Santa Cruz Biotechnology), mouse anti-NR1 antibody (1 : 500; Upstate, Lake Placid, NY, USA), rabbit anti-NR2A antibody (1 : 500; Millipore, Billerica, MA, USA), rabbit anti-NR2B antibody (1 : 500; Chemicon, Temecula, CA, USA), goat anti-BDNF polyclonal antibody (1 : 200; Santa Cruz Biotechnology), goat anti-ChAT polyclonal antibody (1 : 400; Chemicon), and rabbit anti-β-tubulin polyclonal antibody (1 : 1000; Santa Cruz Biotechnology). Horseradish peroxidase-labeled anti-mouse IgG and horseradish peroxidase-labeled anti-rabbit IgG were used as secondary antibodies at dilutions of 1 : 2000. Immunoreactive bands were detected using an enhanced chemiluminescent substrate (SuperSignal West Pico or Dura Chemiluminescent Substrate; Pierce Chemical, Dallas, TX, USA).

Histological assessment

Under deep pentobarbital anesthesia (50 mg/kg, i.p.), mice were perfused transcardially with 20 mL of potassium-free phosphate-buffered saline (K+-free Phosphate Buffered Saline, pH 7.4), followed by 50 mL of a 4% paraformaldehyde solution in potassium-free PBS. Brains were isolated, post-fixed for 3 h, and cryoprotected overnight in a 25% sucrose solution. Tissues were fast frozen in cryo-embedding compound in a mixture of ethanol and dry ice and stored at −80°C until used. Brain was cut on a cryostat at a thickness of 10 μm, and the sections were thawed in the 0.1% sodium azide stock solution at 4°C until used. To confirm the total cell numbers, brain sections were stained with Hoechst 33342 to identify nuclei. The total cell numbers per 880 μm × 660 μm fields of the striatum regions were counted in two sections per mouse under a fluorescence microscope at 20× magnification. Neuronal cell loss was assessed histologically by Nissl staining (0.1% cresyl violet) of brain sections at the level of the striatum. Nissl-positive striatal neurons per 880 μm × 660 μm fields of the striatum regions were counted in two sections per mouse under a light microscope at 20× magnification.

Behavioral analyses

Measurement of clasping reflex

To analyze limbs dystonia, the clasping behavior was assessed as previously reported (Wacker et al. 2009). The degree of clasping behavior induced by tail-suspension was evaluated as follows; 0 (no clasp), 1 represents mild clasp (fore or hind-limbs press on the stomach) 2 represents severe clasp (both fore- and hind-limbs touch and press on the stomach).

Rotarod test

The rotarod apparatus (MK0610A, Muromachi KIKAI, Tokyo, Japan) was used to measure fore- and hind-limbs motor coordination. During the training period, each mouse was placed on the rotarod at a constant speed (20 rpm) for maximum of 60 s, and the latency to fall from the rotarod within this time period was recorded. Mice were trained for three consecutive days, receiving four trials per day with a 1 h intertrial interval. Testing was performed every 2 weeks, starting at 6-weeks old. Each mouse had 60 s trial at constant speeds of 10, 20, 30, and 40 rpm. The mean latency to fall from the rotarod (for the four trials at each speed level) was recorded as previously described (Carter et al. 1999).

Drug treatments

Pramipexole hydrochloride was obtained as Bi-Sifrol® Tablets from Boehringer Ingelheim, Ingelheim, Germany. The tablets were suspended in physiological saline and intraperitoneally administered at 0.01 mg/kg. To evaluate the acute effect, single dose of pramipexole was administrated after behavioral study of pre-treatment. Chronic effect was observed following pramipexole administration once a day for five consecutive days. Behavioral tests were performed 24 h after the last administration.

Statistical analysis

The differences between multiple groups were analyzed using a one-way anova with the Tukey-Kramer multiple comparison post hoc analysis (Figs 2-5, 7, 8 and Figure S1). For comparisons of changes in clasping score with age, the data were analyzed using two-way anova with genotype and age as main factors, and significant interactions were then assessed with the post hoc Tukey-Kramer test (Fig. 2d). Significance was set at < 0.05, < 0.01, and ##< 0.01. All results are expressed as means ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Gγ7 expression profile in selected brain regions of Gng7-Cre knock-in mice

Gng7-Cre knock-in mice, which had been developed by inserting Cre gene with stop codon within the exon 4 containing the translational initiation site of Gng7 gene (Kishioka et al. 2009) were genotyped by PCR using the primer sequence given in the materials and methods section (Fig. 1a). The PCR-genotyping data showed 495 bp (WT-Gγ7), successful Cre knock-in mice showed 570 bp (homozygous Gγ7 knockout; KO) band while 495 bp/570 bp (heterozygous Gγ7 KO) bands. The loss of Gγ7 in Gng7-Cre knock-in (Gng7-Cre (Cre/Cre)) mice was confirmed by western blot (Fig. 1b). Striatum-specific expression of Gng7 mRNA expression was shown in wild type (100%), modest (below 20% of the striatal value) level of expression was observed in hippocampus, amygdala, thalamus, and hypothalamus, and lowest in olfactory bulb, cerebral cortex, midbrain, pons, and cerebellum. No transcription of Gng7 gene was observed throughout brain regions of Gng7-Cre (Cre/Cre) (homozygous Gγ7 KO) mice (Fig. 1c). In this manuscript, Gng7-Cre (Cre/Cre) mice were designated as Gγ7 KO mice.

image

Figure 1. Eliminate the gene and protein expression of Gγ7 in Gng7-Cre knock-in mouse. (a) The genotype was identified by PCR using a DNA extracted from mouse ear. PCR products obtained with primers described above of ear biopsy DNA from pups of Gng7-Cre (+/Cre) × Gng7-Cre (+/Cre) cross. Lane 1, wild type (Gng7-Cre (+/+)) mouse with a single 495 bp band. Lane 2, heterozygous (Gng7-Cre (cre/+)) mouse with 495 bp and 570 bp bands. Lane 3, Gng7 homologous knock out (Gng7-Cre (Cre/Cre)) mouse with a single 570 bp band. (b) Western blot analysis of proteins prepared from striatum of wild type and Gng7 homozygous knockout (KO) mouse. The upper panel shows Gng7 homozygous KO mouse is complete deficiency of Gγ7 level. The lower panel shows β-tubulin as an internal control. (c) Gng7 mRNA expressions levels in the striatum, olfactory bulb, cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, amygdala, pons, and cerebellum are measured. The mRNA expression levels were assessed using quantitative real-time PCR and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data are calculated as percentages of striatum expression level and expressed as the means ± SEM from at least three mice.

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Age-dependent clasping behavior in Gγ7 KO mice

Body weight can be a clinical sign of biomechanical functions and motor development. Because the regulator of G-protein signaling protein 9-2 (RGS 9-2) regulates feeding through striatal D2R (Johnson and Kenny 2010; Waugh et al. 2011) and mu-opioid receptor (Le Merrer et al. 2009) signaling, we measured body weights of wild-type (WT), Gγ7 heterozygous, and homozygous KO mice. From the results of no significant differences in body weight among these mice during 30 weeks (Fig. 2a), we excluded the contribution of biomechanical difference to pathognomonic symptom in Gγ7 deficient mice. As shown in Fig. 2b, Gγ7 KO mice at 11–30-week-old showed abnormal involuntary movements like dystonia as determined by limbs clasping (Fig. 2c). The clasping score progressed with age from 11-week-old of Gγ7 homozygous KO mice, while the score in Gγ7 heterozygous KO mice substantially progressed from 20-week-old. However, WT mice showed no clasping behaviors during 30 weeks. The clasping score in Gγ7 homozygous and heterozygous KO mice were significantly increased (vs. WT mice of the same age) with increasing age (Fig. 2d). In mice of 6–10-week-old, there was no significant difference in the clasping scores of WT, Gγ7 homozygous and heterozygous KO mice. The variation in the clasping score was present in mice of 11–20-week-old, with Gγ7 homozygous KO mice group showing significantly increased clasping score compared with WT control. The highest clasping score values were observed between 21 and 30-week-old, with both homozygous and Gγ7 heterozygous KO mice group exhibiting significantly increased clasping reflex compared with WT control. In addition, clasping reflex of Gγ7 homozygous and heterozygous KO mice progressed in an age-dependent manner.

image

Figure 2. Gγ7 KO mice exhibit progressive self-clasping reflex. (a) Body weight of Gγ7 homo and hetero KO mice almost did not change compared with wild type littermate mice over a period of at least 30 weeks. < 0.05, versus WT littermate mice. Limbs clasping phenotype was scored upon mouse suspension by the tail for 30 s. (b) Representative pattern of limbs clasping reflex. (c) A score of 0 represents no clasping behavior like a wild type mouse, 1 occurs mild claps in which only the fore or hind-limbs press into the stomach, 2 occurs a severe clasp in which both fore and hind-limbs touch and press into the stomach. (d) Age-dependent manifestation of clasping behavior in homo and hetero KO mice. Data are expressed as the means ± SEM from experiments using at least five mice per each genotype groups. < 0.05 and < 0.01, versus WT littermate mice. ##p < 0.01, compared to 6-10-week-old mice with the same genotype.

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Motor incoordination with rotarod test

To analyze motor incoordination of Gγ7 KO mice, we performed the standard rotarod test. This test is useful for monitoring progressive motor dysfunction, such as a R6/2 model of HD (Carter et al. 1999). In the rotarod test, mice were put on the rod, and the latency to fall from the rotating rod at different speeds (10, 20, 30, and 40 rpm) was measured. As shown in Fig. 3a–d, the latency to fall from rotarod in WT mice was constant without variation at speeds of 10 or 20 rpm throughout 30 weeks. Gγ7 KO mice, on the other hand, showed a significant decline in the ability of performance as the rotation rate increased. In addition, this motor incoordination of Gγ7 KO mice progressed in an age-dependent manner.

image

Figure 3. Impaired motor coordination in Gγ7 homo KO mice. Deletion of Gγ7 shows motor incoordination using rotarod test that was tested four consecutive 60 s trial at constant speeds of 10 (a), 20 (b), 30 (c) and 40 rpm (d). The mean latency to fall the rotarod (for the three trials at each speed level) was recorded. Gγ7 KO mice spend significantly less latency to fall on rotarod than their WT littermates at each speed. Data are expressed as the means ± SEM from experiments using at least five mice per each genotype groups. < 0.05 and < 0.01, versus WT littermate mice.

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Gγ7 ablation is not associated with striatal neuron degeneration

It is reported that neurodegeneration is often observed in HD human subjects and R6/2 HD model mice (Vonsattel 2008; Samadi et al. 2013), but it is not always a crucial feature for HD, as there are reports that morphologic abnormalities in N171-82Q mice, a representative HD model mice, are not enough to be considered evidence of cell death in light microscopy, and degenerative changes varied in severity in electron microscopy (Schilling et al. 1999; Yu et al. 2003). As shown in Fig. 4a, indeed, there was no apparent cellular loss in KO mice. In the Nissl staining, the number of neurons in the striatum was approximately 50% of the total cells. The number of Nissl-positive neurons is also comaparable between WT and KO mice (Fig. 4b). On the other hand, there was no significant difference in the expression level of brain-derived neurotrophic factor (BDNF), which is closely related to survival/neurodegeneration status, between WT and Gγ7 homozygous KO mice (Fig. 4c).

image

Figure 4. Deficiency in Gγ7 has an insignificant effect on total cell number and neural cell number using Hoechst-33342 and Nissl staining. (a) Number of total cell count of Gγ7 KO mice and their WT littermates using Hoechst staining. A comparison showed no significant differences of total cell number in the striatum using Hoechst staining. (b) Nissl staining is made the calculation of neural cell number, Nissl-positive neuron shown as bluish-violet. The striatal neural cell numbers are no significantly different between genotypes. (c) Expression of brain-derived neurotrophic factor (BDNF) in the striatum. There is no change for the expression level of striatal BDNF. Data are expressed as the means ± SEM.

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NMDA receptor subunits up-regulation in Gγ7 KO mice

As it is reported that there is an increased sensitivity through NR1/NR2B-complexed NMDARs in HD model mice (Zeron et al. 2002; Fan et al. 2012), we attempted to examine the striatal levels of NR1, NR2A, and NR2B in WT, Gγ7 heterozygous and homozygous KO mice. In the western blot, Gγ7 homozygous KO mice showed a significance increase in NR1 and NR2B but not NR2A levels, compared with WT ones, while Gγ7 heterozygous KO mice also showed partial and equivalent increase in NR1 and NR2B but not NR2A levels, as shown in Fig. 5.

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Figure 5. Measurement of NMDA receptor subunits in the striatum. The expression levels of NMDA receptor NR1 subunit (NR1), NR2A subunit (NR2A), and NR2B subunit (NR2B) proteins in the striatum were quantitated using western blot analysis. Data are expressed as the means ± SEM from experiments using at least five mice per each genotype groups.

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Striatal Gγ7 gene ablation causes down-regulation of striatal Gαolf

Dopamine binding elicits different G-protein coupling and transduction mechanisms in D2R-striatopallidal and D1R-striatonigral GABAergic neurons (Rinken et al. 2001; Wang et al. 2001). It is interesting to note that deficiency of Gγ7 is associated with the down-regulation of Gαolf protein (Wang et al. 2001; Schwindinger et al. 2003), possibly through decreased half-life of Gβ (Wang et al. 1999). Our data established that reduced Gαolf protein is striatum-specific when compared with other selected brain regions (striatum, olfactory bulb, cerebral cortex, hippocampus, thalamus, hypothalamus, midbrain, amygdala, pons, and cerebellum) using western blot (Fig. 6). Furthermore, as D1R selectively couples Gαolf, we determined that reduced expression of Gαolf did not interfere with the expression levels of Gα subunits (Gαs, Gαo, Gαq/11, and Gα13) in the striatum and other selected brain regions. Our data showed clearly that only Gαolf expression is reduced in Gγ7 KO mice without noticeable expression changes in other Gα subunits. The motor manifestation in Gγ7 KO mice can be traced to aberrant Gαolf signaling with little or no contribution from other Gα subunits.

image

Figure 6. Expression of the G-protein α subunits, Gαolf, Gαs, Gαo, Gαq/11, and Gα13 in several brain regions including striatum (Str), olfactory bulb (Olf), Cortex (Cor), hippocampus (Hip), thalamus (Thal), hypothalamus (Hypo), midbrain (Mid), amygdala (Amyg), pons, and cerebellum (Cere) of Gγ7 homo KO mice and their WT littermates mice. It should be noted that the expression level of Gαolf is prominently reduced in Gγ7 homo KO mice in the striatum but other brain regions. There is no change for the expression levels of other G-protein α subunits. β-tubulin is used as an internal control.

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Disruption of balance between striatal dopaminergic and cholinergic transmission in Gγ7 KO mice

The protein expression of Gαolf was significantly decreased in the striatum of Gγ7 homozygous KO mice (30-week-old), but no significant change was observed in Gγ7 heterozygous mice (Fig. 7). In contrast, D1R was up-regulated in Gγ7 homozygous KO mice. These results suggest that this alteration is a compensatory mechanism, because Gαolf has an ability to couple with D1R. Furthermore, we examined expression levels of Gαi1/2 and Gαi1/2-coupled D2R. Although there was no apparent change in the level of Gαi1/2, D2R was dramatically decreased in Gγ7 homozygous KO mice. We speculate that down-regulation of D2R might coordinate the balance of signaling between D1R and D2R. In the absence of dopaminergic signaling, M4 receptors are desensitized and recycled through β-arrestin-endosome pathway (Hashimoto et al. 2008). We found that choline acetyltransferase (ChAT) was significantly up-regulated in Gγ7 heterozygous and homozygous KO mice, compared with WT mice (Fig. 7). The increase in ChAT would be a positive indication of compensation in response to desensitized or low levels of surface M4 muscarinic acetylcholine receptors.

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Figure 7. Gγ7 homo KO mice show expression change of motor function-related proteins in the striatum. The expression levels of Gαolf, dopamine D1 receptor (D1R), Gαi1/2, dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) proteins in the striatum were quantitated by western blot. Data are expressed as the means ± SEM from experiments using at least five mice per each genotype groups. < 0.05 and < 0.01, versus WT littermate mice.

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Amelioration of motor dysfunctions by chronic pramipexole treatments

To ameliorate motor dysfunction of Gγ7 KO mice, we chose dopamine D2R agonist pramipexole. Pramipexole is a representative frontline therapeutic in the treatment of motor dysfunction-associated diseases such early PD and restless leg syndrome (RLS) (Antonini et al. 2010; Hogl et al. 2011; Luo et al. 2011). Pramipexole administered acutely to mice (30-week-old), at 0.01 mg/kg had no significant improvement on motor functions, because no significant difference in limbs clasping was observed pre-treatment and post-treatment up to 5 h (Figure S1a). Similarly, latency to fall from rotarod at 20, 30, and 40 rpm in Gγ7 KO mice (Figure S1b) was not significantly altered between pre-treatment and post-treatment conditions. When pramipexole was administered once a day for five consecutive days no apparent behavioral changes including clasping were observed 24 h after the last administration in WT mice (Fig. 8a and b). In contrast, the treatments markedly attenuated the clasping behavior of Gγ7 KO mice. In addition, the attenuation was observed until day 7 after the cessation of pramipexole treatment. In the rotarod test, on the other hand, the shorter latency at 20 and 30 rpm to fall in Gγ7 KO mice was significantly reversed by consecutive pramipexole treatments, but not until 7 day after the cessation of treatments (Fig. 8c).

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Figure 8. Ameliorate motor symptoms in the 30-week-old Gγ7 homo KO mice by repeated intraperitoneally administration of pramipexole. (a) Intraperitoneal injections of pramipexole (0.01 mg/kg) were given once daily from consecutive 5 days. Behavioral analyses were assessed before treatment, after consecutive 5 days and 7 days after drug withdrawal. (b) Ameliorate of clasping reflex by pramipexole treatment. There is no significantly effect for clasping behavior in the WT mice using pramipexole treatment. Clasping score was quantitated and displayed lower bar graph. (c) The rotarod tests, at 10, 20, 30, and 40 rpm, were assessed motor coordination in the same schedule. All data represent the means ± SEM from five to twelve mice. < 0.05 and < 0.01 versus the pre-treated group.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Activities of the GABAergic D2R-striatopallidal and D1R-striatonigral neurons finely regulate thalamo-cortical motor functions (Ena et al. 2011) through reciprocal regulation of basal ganglia output. D1R-striatonigral neurons promote movement as they transmit wave of inhibitory transmission through their monosynaptic projections to the substantia nigra (reticulata) and the medial globus pallidus when activated by glutaminergic input from cerebral cortex. D2R-striatopallidal neurons relay cortical glutaminergic input through an indirect pathway and projections into the lateral globus pallidus. They reach substantia nigra (reticulata) and the internal globus pallidus by synaptic relay through the subthalamic nucleus where they inhibit movement (Hamani et al. 2004). Loss or lesion of any of the composite cells involved in neural network results in motor dysfunction. Loss of synaptic connection between excitatory glutaminergic projections originating from layer 5 of the cortex and the striatal neurons has been reported in HD-associated dyskinesia (Castellanos et al. 1996; Cicchetti et al. 2011). Similarly, dysfunctional striosomal organization (Saka and Graybiel 2003) and dysregulation of striatal dopamine release was identified in Schizophrenia-associated motor dysfunction and Tourette's syndrome, respectively (Abi-Dargham et al. 1998; Keshavan et al. 2008).

In this study, we revealed altered expression of D1R and D2R in striatum of Gγ7 KO mice. In the WT mice, Gγ7 was highly expressed and specifically distributed in the striatum, which are consistent with previous evidence revealed by in situ hybridization (Betty et al. 1998). Previous reports have suggested that D1R-striatonigral neurons are the resident GABAergic neurons that express Gγ7 in the striatum where it regulates Gαolf-mediated stimulation of adenylyl cyclase following dopamine/D1R activation (Wang et al. 1997, 2001; Schwindinger et al. 2003). On the other hand, D2R-Gi/o mediates inhibitory signaling to striatopallidal GABAergic neurons, which is further connected to pallidonigral GABAergic neurons. Therefore, it is considered that substance nigra (reticulata) receives synergistic inhibitory signals both from former direct D1R pathway and latter indirect D2R pathway in terms of dopaminergic locomotive functions. Thus, the decrease of D1R-mediated striatonigral GABAergic transmission following Gγ7 deletion is finally coupled to a decrease in inputs to motor areas of the thalamus and cortex (Nishi et al. 2011). This change decreases the corticostriatal glutamatergic transmission, which may cause down-regulation of downstream D2R. The up-regulation of NMDA receptor may be caused as a compensatory event. However, details of mechanisms underlying these D2R down-regulation and NMDAR up-regulation should be the next subjects to study.

NMDARs contribute to excitatory neurotransmission, but NMDARs-mediated hyperactivation causes excitotoxic neuronal death. Recently, it is reported that mthtt-induced neurodegeneration of striatal MSNs in HD is mediated by the selective enhancement of NR1/NR2B-complexed NMDAR activity (Zeron et al. 2002; Fan et al. 2012). Our results indicated a similar pattern in 30-week-old Gγ7 KO mice exhibiting significantly increased NR1 and NR2B subunits expression in the striatum compared with WT littermates. In contrast, Gγ7 KO mice did not show any neuronal degeneration and/or death in the striatum. These results suggested that Gγ7 KO mice had a risk factor, which are identified as leading cause of neural death through NMDARs expression. Thus, there is a possibility that the neural damages occur after 30-week-old or over in Gγ7 KO mice.

Regarding the involvement of Gγ7 and Gαolf in the pathology of HD, there are reports that decreased expression of Gγ7 and Gαolf was observed in the striatum of R6/1 HD mice model and in HD human subjects (Hodges et al. 2006), while increased Gαolf and Gγ7 expression levels were documented in the putamen in PD model mice (Corvol et al. 2004). Thus, the pathophysiology of Gγ7 KO mice seems to be closely related to HD models rather than PD models.

Besides cortical glutaminergic and dopaminergic modulation, MSNs have intrinsic cholinergic innervations (Bonsi et al. 2011). Aberrant cholinergic transmission is a known contributor to the pathophysiology of HD (Lange et al. 1992; Smith et al. 2006) and movement disorders associated with PD (Bonsi et al. 2011). At cellular level, D1R-striatonigral neurons express M4 muscarinic acetylcholine receptors (Jeon et al. 2010). Acetylcholine antagonizes dopaminergic transmission through D1R-mediated direct pathway and limits the motor response to dopamine agonists (Guo et al. 2010). Increased ChAT level detected in Gγ7 KO mice compared with WT mice possibly indicates increased acetylcholinergic input through D1R-striatonigral neurons, resulting in further debilitation of motor functions.

As synergistic relationship exists between D2R-striatopallidal neurons of the indirect pathway and D1R-striatonigral neurons of direct pathway in terms of dopamine-induced locomotion (Kelly et al. 1998), we tested whether pramipexole a D2R agonist would reverse the motor dysfunction initiated by defective D1R signaling. Our result demonstrated that chronically administered pramipexole improved motor incoordination. Although, pramipexole has been used as a frontline therapeutic to treat early PD and RLS with significant improvement of motor symptoms for many years (Takahashi et al. 2008; Hogl et al. 2011; Luo et al. 2011), this result opens further possibility for HD treatment.

In conclusion, loss of striatal network has been reported in several cases of motor disorder, we demonstrated that blunted D1R response through decreased Gαolf expression is sufficient to initiate progressive motor dysfunction without striatal neuron loss as shown in Gγ7 KO mice. We also demonstrated that D2R agonist pramipexole reversed the deficiency in both rotarod behavior and clasping phenotype in Gγ7 KO mice. Pramipexole may be potentially evaluated for trials in treatment of patients with of motor dysfunctions especially HD dyskinesia.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank H. Kurosu, H. Matsunaga, and T. Eihara for technical advice and assistance. Parts of this study were supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (MEXT) (to H.U.: 08062874). We have no conflict of interest to declare.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12149-sup-0001-FigS1.docxWord document20KFigure S1. Acute effect of pramipexole for motor symptoms by single intraperitoneally administration.
jnc12149-sup-0001-FigS1.tifimage/tif3322K 

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