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The Journal of Neuroscience logoLink to The Journal of Neuroscience
. 2010 Jan 6;30(1):316–324. doi: 10.1523/JNEUROSCI.4974-09.2010

Metabotropic Glutamate Receptor-Mediated Cell Signaling Pathways Are Altered in a Mouse Model of Huntington's Disease

Fabiola M Ribeiro 1,*, Maryse Paquet 1,*, Lucimar T Ferreira 1, Tamara Cregan 1, Patrick Swan 1, Sean P Cregan 1,2, Stephen S G Ferguson 1,2,✉
PMCID: PMC6632544  PMID: 20053912

Abstract

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (Htt). Group I metabotropic glutamate receptors (mGluRs) are coupled to Gαq and play an important role in neuronal survival. We have previously demonstrated that mGluRs interact with Htt. Here we used striatal neuronal primary cultures and acute striatal slices to demonstrate that mGluR-mediated signaling pathways are altered in a presymptomatic mouse model of HD (HdhQ111/Q111), as compared to those of control mice (HdhQ20/Q20). mGluR1/5-mediated inositol phosphate (InsP) formation is desensitized in striatal slices from HdhQ111/Q111 mice and this desensitization is PKC-mediated. Despite of decreased InsP formation, (S)-3,5-dihydroxylphenylglycine (DHPG)-mediated Ca2+ release is higher in HdhQ111/Q111 than in HdhQ20/Q20 neurons. Furthermore, mGluR1/5-stimulated AKT and extracellular signal-regulated kinase (ERK) activation is altered in HdhQ111/Q111 mice. Basal AKT activation is higher in HdhQ111/Q111 neurons and this increase is mGluR5 dependent. Moreover, mGluR5 activation leads to higher levels of ERK activation in HdhQ111/Q111 than in HdhQ20/Q20 striatum. PKC inhibition not only brings HdhQ111/Q111 DHPG-stimulated InsP formation to HdhQ20/Q20 levels, but also causes an increase in neuronal cell death in HdhQ111/Q111 neurons. However, PKC inhibition does not modify neuronal cell death in HdhQ20/Q20 neurons, suggesting that PKC-mediated desensitization of mGluR1/5 in HdhQ111/Q111 mice might be protective in HD. Together, these data indicate that group I mGluR-mediated signaling pathways are altered in HD and that these cell signaling adaptations could be important for striatal neurons survival.

Introduction

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a progressive neuronal cell loss in the caudate–putamen and neocortical regions of the brain, which leads to involuntary body movement, loss of cognitive function, psychiatric disturbance, and death (Young, 2003; Li and Li, 2004). HD patients exhibit a polyglutamine expansion in the N-terminal region of the huntingtin (Htt) protein, which is proposed to cause the neuronal cell loss observed in HD patients (The Huntington's Disease Collaborative Research Group, 1993).

Glutamate is postulated to play an important role in excitotoxic neuronal cell loss (DiFiglia, 1990; Nicoletti et al., 1996; Calabresi et al., 1999). There are two types of glutamate receptors: ionotropic and metabotropic (Conn and Pin, 1997). Ionotropic glutamate receptors are ligand-gated ion channels, whereas metabotropic glutamate receptors (mGluRs) are members of the G-protein-coupled receptor (GPCR) superfamily (Nakanishi, 1994; Pin and Duvoisin, 1995; Conn and Pin, 1997; Dhami and Ferguson, 2006). Group I mGluRs (mGluR1 and mGluR5) are coupled to the activation of Gαq/11 proteins, which stimulate the activation of phospholipase Cβ1 (PLCβ1) resulting in diacylglycerol and inositol-1,4,5-triphosphate formation, release of Ca2+ from intracellular stores and activation of protein kinase C (PKC). mGluR1/5 stimulation also leads to activation of other signaling pathways important for cell survival/proliferation, such as extracellular signal-regulated kinase (ERK) and AKT (Rong et al., 2003; Hou and Klann, 2004; Mao et al., 2005).

Although glutamate receptors are thought to contribute to excitotoxic neuronal loss in HD, it is still unclear whether group I mGluRs activation could delay or accelerate HD, as different reports have published contradictory data (DiFiglia, 1990; Beal et al., 1991; Nicoletti et al., 1996; Bruno et al., 2001; Zeron et al., 2002; Tang et al., 2003; Schiefer et al., 2004). Recently, we determined that group I mGluRs interact with mutant Htt and that mGluR5 signaling was selectively uncoupled as a consequence of this interaction (Anborgh et al., 2005). Thus, it is possible that alterations of receptor-mediated signaling pathways could contribute to protection or exacerbation of cell death cascades in the symptomatic and/or presymptomatic phases of HD. To investigate group I mGluRs activity in HD, we studied different signaling pathways that can be activated by mGluR1/5 in both neuronal primary cultures and acute slices from the striatal brain region of a knock-in (HdhQ111/Q111) mouse model of HD. We find that similar to what was previously reported for striatal neurons derived from YAC128 mice (Tang et al., 2005), Ca2+ signaling is generally disturbed in HdhQ111/Q111 mice. However, we find that mGluR1/5 inositol phosphate (InsP) formation is selectively uncoupled in neuronal cultures and slices derived from young HdhQ111/Q111 mice, as compared to control HdhQ20/Q20 mice, in a PKC-dependent manner. Moreover, basal AKT and mGluR-mediated ERK1/2 phosphorylation is selectively increased in HdhQ111/Q111 mice. Our results indicate that dysregulation of Ca2+ signaling is a common feature of HD mouse models and that mGluR5 signaling pathways are altered in HD and the adaptation in cell signaling favors the activation of pathways that promote striatal neuronal survival.

Materials and Methods

Materials

(S)-3,5-dihydroxylphenylglycine (DHPG) was purchased from Tocris Cookson. Carbachol (carbamylcholine chloride) and dopamine were from Sigma-Aldrich. Bisindolylmaleimide I (Bis I) was purchased from Calbiochem. myo-[3H]Inositol was acquired from PerkinElmer Life Sciences. The Dowex 1-X8 (formate form) resin with 200–400 mesh was purchased from Bio-Rad. Bovine serum albumin (BSA) was obtained from BioShop Canada. Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody was from Bio-Rad. ECL Western blotting detection reagents were from GE Healthcare. EZ-Link Sulfo-NHS-SS-Biotin and immobilized NeutrAvidin beads were from Pierce Biotechnology. Fura-2 and Live/Dead viability assays were from Invitrogen. Rabbit anti-mGluR1 and anti-mGluR5 antibodies were from Millipore. Rabbit anti-phospho ERK, anti-phospho AKT, anti-ERK, and anti-AKT antibodies were from Cell Signaling Technology. Rabbit anti-actin antibody was from Santa Cruz Biotechnology. All other biochemical reagents were purchased from Sigma-Aldrich.

Mouse model

STOCK-Htttm2Mem/J (HdhQ20/Q20) and STOCK-Htttm5Mem/J (HdhQ111/Q111) knock-in mice (Wheeler et al., 1999) and mGluR5 knock-out mice B6;129-Grm5tm1Rod/J (mGluR5−/−) (Lu et al., 1997) were purchased from Jackson Laboratory. HdhQ111/Q111/mGluR5−/− mice were obtained by crossing HdhQ111/Q111 and mGluR5−/− mice. Mice were housed in an animal care facility at 23°C on a 12 h light/12 h dark cycle with food and water provided ad libitum. Animal care was in accordance with the University of Western Ontario Animal Care Committee.

Neuronal primary culture preparation

Neuronal cultures were prepared from the striatal region of E15 mouse embryo brains. Animal procedures were approved by The University of Western Ontario Animal Care Committee. After dissection, striatal tissue was submitted to trypsin digestion followed by cell dissociation using a fire-polished Pasteur pipette. Cells were plated on poly-l-ornithine coated dishes in neurobasal media supplemented with N2 and B27 supplements, 2 mm glutamax, 50 μg/ml penicillin, and 50 μg/ml streptomycin. Cells were incubated at 37°C and 5% CO2 in a humidified incubator and cultured for 10 to 12 d in vitro (DIV) with media replenishment every 4 d.

Inositol phosphate formation assay

Neuronal primary culture.

Inositol lipids were radiolabeled by incubating the striatal neuronal cultures from either HdhQ20/Q20 or HdhQ111/Q111 mice overnight with 1 μCi/ml myo-[3H]inositol in Neurobasal medium. Unincorporated myo-[3H]inositol was removed by washing cells with Hank's balanced salt solution (HBSS). Cells were preincubated for 1 h in HBSS at 37°C and then preincubated in 500 μl of the same buffer containing 10 mm LiCl for an additional 2 min at 37°C. Cells were then incubated in the presence of increasing concentrations of DHPG for 5 min at 37°C, as indicated in the figures. The reaction was stopped on ice by the addition of 500 μl of perchloric acid and then neutralized with 400 μl of 0.72 m KOH, 0.6 m KHCO3. Total [3H]inositol incorporated into cells was determined by counting the radioactivity present in 50 μl of cell lysate. Total inositol phosphate was purified from cell extracts by anion exchange chromatography using Dowex 1-X8 (formate form) 200–400 mesh anion exchange resin. [3H]Inositol phosphate formation was determined by liquid scintillation.

Acute brain slices.

InsP formation in striatal slices was measured as previously described (Alagarsamy et al., 2005). Dissected striata from age-matched HdhQ20/Q20, HdhQ111/Q111, or mGluR5−/− mice were cross-chopped with a McIlwain tissue chopper with a slice thickness of 350 μm. Resulting striatal prisms were recovered in ACSF (127 mm NaCl, 2 mm KCl, 10 mm glucose, 1.2 mm KH2PO4, 26 mm NaH2CO3, 1 mm MgSO4, 1 mm CaCl2, pH 7.4) gassed with 95% O2/5% CO2 and incubated in a shaking bath at 37°C for 30 min. 20 μl of gravity-packed prisms were aliquoted in duplicate samples, gassed, and incubated with 3 μCi/ml myo-[3H]inositol in a shaking bath at 37°C for 90 min. The prisms were gassed again and incubated in a shaking bath at 37°C for an additional 15 min in presence of 10 mm LiCl before they were stimulated with DHPG, dopamine or carbachol for 20 min in the same conditions. The reaction was stopped by adding 900 μl of a 2:1 mix of chloroform:methanol and incubating for 15 min at room temperature. 300 μl of chloroform was then added and samples were mixed vigorously. Total [3H]inositol incorporated into slices was determined by counting the radioactivity present in 200 μl of the hydrophobic layer. 700 μl of top aqueous layer was added to Dowex 1-X8 (formate form) 200–400 mesh anion exchange resin. Columns were washed three times with water and twice with 60 mm ammonium formate. Samples were eluted with 200 mm ammonium formate 0.1 m formic acid in scintillation vials containing scintillation fluid. [3H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system (Dhami et al., 2002, 2005).

Cell surface biotinylation assay

Plasma membrane proteins of striatal prisms prepared as described above were biotinylated with sulfo-NHS-SS-biotin for 1 h on ice, as described previously (Ribeiro et al., 2007). To quench the biotinylation reaction, slices were washed and incubated for 30 min with cold 100 mm glycine in HBSS, followed by 3 washes with cold HBSS. Slices were then lysed in RIPA buffer (0.15 m NaCl, 0.05 m tris-HCl, pH 7.2, 0.05 m EDTA, 1% Nonidet P40, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (1 mm AEBSF and 10 μg/ml of both leupeptin and aprotinin). Biotinylated proteins were separated from nonbiotinylated proteins by NeutrAvidin bead pull-down from equivalent amounts of total cellular protein from each sample. Biotinylated proteins were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes.

Immunoblotting

Membranes were blocked with 10% milk in wash buffer (150 mm NaCl, 10 mm Tris-HCl, pH 7.0, and 0.05% Tween 20) for 1 h and then incubated with rabbit anti-mGluR5 (1:4000), rabbit anti-mGluR1 (1:1000), or rabbit anti-actin (1:10,000) antibodies in wash buffer containing 3% milk overnight. Membranes were rinsed three times with wash buffer and then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1:10,000 in wash buffer containing 3% skim milk for 1 h. Membranes were rinsed three times with wash buffer and incubated with ECL Western blotting detection reagents.

ERK and AKT activation experiments

Neuronal primary culture.

Neuronal primary cultures from HdhQ20/Q20 and HdhQ111/Q111 embryos were incubated in HBSS for 30 min at 37°C. Cultures were then stimulated with 10 μm DHPG for 0, 2 or 5 min at 37°C. When 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was used, it was added before and kept during DHPG stimulation. Neurons were lysed in RIPA buffer containing protease inhibitors (1 mm AEBSF and 10 μg/ml of both leupeptin and aprotinin). 50 μg of total cellular protein for each sample were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes. Immunoblot was performed as described above. Membranes were blocked with 10% milk in wash buffer for 1 h and then incubated with either rabbit anti-phospho ERK (1:1000) or rabbit anti-phospho AKT (S473) (1:1000) antibodies in wash buffer containing 3% milk overnight. Membranes were probed with secondary horseradish peroxidase-conjugated goat anti-rabbit and incubated with ECL Western blotting detection reagents as described above. Antibodies were then stripped and membranes were incubated with anti-ERK (1:1000) and anti-AKT (1:1000) overnight and probed with secondary antibody to determine total kinase expression. Immuno-band intensity was quantified and the number of pixels of ERK and AKT phospho-bands was divided by the number of pixels of total ERK and AKT, respectively, to normalize phosphorylation levels of kinases to total kinase expression.

Acute brain slices.

350 μm thick coronal slices from age-matched HdhQ20/Q20 and HdhQ111/Q111 mouse brains were prepared using a Vibratome system (Leica). The striatal region was dissected and slices were recovered in ACSF gassed with 95% O2/5% CO2 and incubated at 30°C for 90 min. Slices were transferred to tubes in duplicate samples, gassed, and kept at 30°C for 30 min. After this incubation, slices were stimulated with DHPG at 30°C for 30 min. Slices were snap-frozen in ethanol/dry ice bath and lysed in RIPA buffer containing protease and phosphatase inhibitors. 100 μg of total cellular protein for each sample were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes. Immunoblot was performed as described above to detect phospho- and total-ERK.

Cell death assay

Neurons were incubated for 24 h with either vehicle or drug (250 μm glutamate, 100 μm DHPG, 1 μm Bis I, or 100 μm DHPG along with 1 μm Bis I), as indicated in Figure 7 legend, and cell death was determined by Live/Dead viability assay, as described previously (Fortin et al., 2001). Briefly, neurons were stained with Calcein-AM (2 μm) and ethidium homodimer (2 μm) for 15 min and the fraction of live (Calcein-AM positive) and dead (ethidium positive) cells were determined. Neurons were visualized by fluorescence microscopy (IX70; Olympus) and images were captured with a CCD camera (Q-Imaging) and Northern Eclipse software (Empix Imaging). Images were captured and scored by a blinded observer and a minimum of 300 cells were analyzed per well in duplicate. Dead cells were expressed as a percentage of the total number of cells.

Figure 7.

Figure 7.

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Inhibition of PKC-mediated mGluR5 desensitization causes an increase in cell death. Graph shows percentage of drug-induced cell death in striatal neurons from HdhQ20/Q20/mGluR5+/+, HdhQ111/Q111/mGluR5+/+ or HdhQ111/Q111/mGluR5−/− mice. Striatal neuronal cultures were incubated with 250 μm glutamate, 100 μm DHPG, 1 μm Bis1 or 100 μm DHPG +1 μm Bis1 for 24 h. Data represent the means ± SEM of six to seven independent experiments. Asterisks indicate significant difference in HdhQ111/Q111/mGluR5+/+ cell death as compared to HdhQ20/Q20/mGluR5+/+ and HdhQ111/Q111/mGluR5−/− neuronal cell death (p < 0.05).

Measurement of [Ca2+]i

Neuronal primary cultures from HdhQ20/Q20 and HdhQ111/Q111 embryos seeded on 12 mm coverslips were loaded with 0.2 μm fura-2 acetoxymethyl ester (AM) for 20 min at 37°C. Coverslips were mounted on a Nikon inverted microscope and perfused with HBSS at 1–3 ml/min at room temperature. Neurons were stimulated with either 10 μm DHPG or 100 μm carbachol and illuminated with alternating 345 and 380 nm light using a Deltascan system (Photon Technology International), with the 510 nm emission detected using a photometer. Baseline (3–5 min) measurements were obtained before pulse of agonist. DHPG-induced Ca2+ responses were measured in either normal HBSS or Ca2+-free HBSS (omitted CaCl2 from HBSS and supplemented with 100 μm EGTA). [Ca2+]i was calibrated using the method of Grynkiewicz et al. (1985).

Quantitative RT-PCR

RNA was isolated using Trizol reagent as per manufacture's instructions (Invitrogen) and RNA concentration was measured on a spectrophotometer. Quantitative RT-PCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) and 40 ng of RNA was used. RT-PCR was performed on a Chromo4 detection system (MJ Research/Bio-Rad) and changes in gene expression were determined by the Δ(ΔCt) method using the ribosomal S12 transcript for normalization. Data are reported as fold increase in mRNA levels for each sample relative to that of 4 months old HdhQ20/Q20 mice. All PCRs exhibited high amplification efficiency (>90%) and the specificity of PCR products were confirmed by sequencing. Primer sequences used for gene specific amplification are available on request.

Data analysis

Nonsaturated, immunoreactive mGluR5 bands from cell surface biotinylation assay and ERK and AKT bands were quantified by scanning densitometry using Scion Image software. Means ± SEM are shown for the number of independent experiments indicated in the figure legends. GraphPad Prism software was used to analyze data for statistical significance and for curve fitting. Statistical significance was determined by ANOVA testing followed by post hoc Multiple Comparison testing.

Results

mGluR1/5 mediated InsP formation is attenuated in HdhQ111/Q111 mice

Both mGluR1 and mGluR5 are expressed in medium sized spiny striatal neurons, which are the neurons that die selectively in HD (Vonsattel et al., 1985; Romano et al., 1995). Previously, we demonstrated that mGluR5 was the primary group I mGluR expressed in primary cultured striatal neurons (12–15 DIV) obtained from E15 embryos (Ribeiro et al., 2009). To determine which group I mGluR was the primary receptor responsible for DHPG-stimulated InsP formation in adult mice, we stimulated striatal slices from either wild-type or mGluR5−/− mice with 5 or 50 μm DHPG and determined InsP formation. Striatal slices from mGluR5−/− mice exhibited only ∼20–30% of wild-type DHPG-mediated InsP formation, indicating that mGluR5 was the primary Gq/11-coupled mGluR in the striatum of adult mice (Fig. 1A).

Figure 1.

Figure 1.

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DHPG-stimulated InsP formation is decreased in HdhQ111/Q111 mice. A, Shown is mGluR1/5-stimulated inositol phosphate formation in response to either 5 or 50 μm DHPG for 15 min at 37°C in striatal slices from either mGluR5−/− or wild-type mice. Data represent the means ± SEM of three independent experiments, expressed as percentage of DHPG-stimulated wild-type slice maximum response. Asterisks indicate significant differences as compared to wild-type slices at the corresponding agonist concentration (p < 0.05). B, Shown is DHPG-stimulated inositol phosphate formation in striatal neurons stimulated with increasing concentrations of agonist for 5 min at 37°C. The data points represent the means ± SEM of five independent experiments, expressed as percentage of HdhQ20/Q20 maximum DHPG-stimulated response. C, Shown is mGluR1/5-stimulated inositol phosphate formation in response to either 5 or 50 μm DHPG for 15 min at 37°C in striatal slices from either HdhQ20/Q20 or HdhQ111/Q111 mice. Data represent the means ± SEM of five independent experiments, expressed as percentage of DHPG-stimulated HdhQ20/Q20 slice maximum response. Asterisks indicate significant differences as compared to HdhQ20/Q20 slices at the corresponding agonist concentration (p < 0.05).

To determine whether mGluR1/5 activity was altered in HD, we measured InsP formation in primary cultured striatal neurons that were stimulated with increasing concentrations of DHPG (Fig. 1B). The maximum response for DHPG-stimulated InsP formation in HdhQ111/Q111 neurons was 74.7 ± 9.5% of that of HdhQ20/Q20 neurons, and the half-maximal effective concentration (EC50) for the agonist was the same in neurons from both HdhQ111/Q111 and HdhQ20/Q20 mice (Fig. 1B). In acute striatal slices from 2- to 4-month-old HdhQ20/Q20 or HdhQ111/Q111 mice treated with either 5 or 50 μm DHPG, InsP formation was significantly reduced in HdhQ111/Q111 mice, as compared to that of HdhQ20/Q20 mice (Fig. 1C). There was no difference in basal InsP formation between HdhQ111/Q111 and HdhQ20/Q20 mice (data not shown). The reduction in InsP formation in HdhQ111/Q111 mice was specific to mGluR1/5, as slices stimulated with either 100 μm carbachol, a cholinergic agonist, or 1 mm dopamine exhibited similar levels of InsP formation in both HdhQ20/Q20 and HdhQ111/Q111 striatal slices. Carbachol promoted 120 ± 16% and 124 ± 13% InsP formation over basal in HdhQ20/Q20 and HdhQ111/Q111 striatal slices, respectively; and dopamine stimulated 36 ± 6% and 31 ± 5% InsP formation over basal in HdhQ20/Q20 and HdhQ111/Q111 striatal slices, respectively. Thus, only agonist-stimulated mGluR5 InsP formation was reduced in HdhQ111/Q111 striatal slices.

HD is a progressive neurodegenerative disorder and its symptoms first appear in midlife, although the mutated Htt protein is present since the beginning of the patient's life (Vonsattel et al., 1985; The Huntington's Disease Collaborative Research Group, 1993; Vonsattel and DiFiglia, 1998). It is possible that either protective pathways are activated or that cell death signals are attenuated in the presymptomatic phase of the disease. To test whether mGluR1/5-mediated InsP formation was altered with age, we determined InsP formation in HdhQ20/Q20 and HdhQ111/Q111 mice of different age groups. mGluR1/5-mediated InsP formation was attenuated in presymptomatic HdhQ111/Q111 mice (up to 9 months old mice), as compared to age matched HdhQ20/Q20 mice, when slices were stimulated with either 5 μm (Fig. 2A) or 50 μm DHPG (Fig. 2B). However, this difference in InsP formation between HdhQ20/Q20 and HdhQ111/Q111 mice was not present in mice older than 11 months (Fig. 2A,B). Interestingly, the extent of DHPG-stimulated InsP formation significantly waned in the HdhQ20/Q20 mice with such that at 11 months of age InsP formation was indistinguishable from the HdhQ111/Q111 mice. These data indicated that Group I mGluR signaling can be differently modulated in HdhQ111/Q111 mice and that its modulation can vary with age.

Figure 2.

Figure 2.

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The decrease in HdhQ111/Q111 InsP formation is only present in presymptomatic huntingtin mice. Shown is mGluR1/5-stimulated inositol phosphate formation in response to either 5 μm (A) or 50 μm (B) DHPG for 15 min at 37°C in striatal slices from either HdhQ20/Q20 or HdhQ111/Q111 mice from different age groups. Asterisks indicate significant differences as compared to HdhQ20/Q20 slices at the corresponding age group (p < 0.05).

mGluR1/5 protein expression is not different between HdhQ20/Q20 and HdhQ111/Q111 mice

The decreased levels of DHPG-stimulated InsP formation in HdhQ111/Q111 mice could be due to diminished mGluR1/5 protein expression levels. To investigate this possibility, we determined mGluR1/5 total cell lysate expression and mGluR5 plasma membrane expression by performing a cell surface biotin labeling assay using striatal slices. mGluR1 overall expression was too low to allow plasma membrane expression measurements. Striatal slices of HdhQ20/Q20 and HdhQ111/Q111 mice from different age groups exhibited the same levels of mGluR1 and mGluR5 protein expression (supplemental Fig. 1A–C, available at www.jneurosci.org as supplemental material). Furthermore, mGluR5 plasma membrane expression was also identical in striatal slices from HdhQ20/Q20 and HdhQ111/Q111 mice (supplemental Fig. 1D, available at www.jneurosci.org as supplemental material). mGluR5 plasma membrane expression was 16 ± 3% and 15 ± 2% of total cell lysate mGluR5 expression in HdhQ20/Q20 and HdhQ111/Q111 striatal slices, respectively. Therefore, the difference in DHPG-stimulated InsP formation between HdhQ20/Q20 and HdhQ111/Q111 mice could not be explained by differences in mGluR1/5 protein expression levels.

Ca2+ release is increased in HdhQ111/Q111 neurons

It was previously demonstrated that Ca2+ release from intracellular stores was dysregulated in medium spiny neurons derived from YAC128 transgenic HD mouse models (Tang et al., 2003, 2005). Thus, we tested whether the dysregulation of Ca2+ signaling observed in the YAC128 HD mouse might be a common feature of HD mouse models and was also observed in HdhQ111/Q111 mice. We used Fura-2 to measure Ca2+ release in response to either 10 μm DHPG or 100 μm carbachol treatments in primary striatal neurons derived from HdhQ20/Q20 and HdhQ111/Q111 mice. Despite the observed decrease in DHPG-mediated InsP formation in HdhQ111/Q111 neurons, DHPG-stimulated Ca2+ release was significantly exaggerated in HdhQ111/Q111 when compared to DHPG-stimulated Ca2+ release in HdhQ20/Q20 neurons (Fig. 3A). Carbachol treatment, which induced similar levels of InsP formation in HdhQ20/Q20 and HdhQ111/Q111 mice, also resulted in increased Ca2+ release in HdhQ111/Q111 neurons when compared to HdhQ20/Q20 neurons (Fig. 3B). Both the area under the curve and the maximum rise in Ca2+ concentration for both DHPG- and carbachol-induced Ca2+ release were significantly elevated in HdhQ111/Q111 neurons when compared to HdhQ20/Q20 neurons (Fig. 3C,D). In the presence of EGTA to chelate extracellular Ca2+, both the area under the curve (268 ± 75 nm.sec and 540 ± 115 nm.sec for HdhQ20/Q20 and HdhQ111/Q111 striatal neurons, respectively) and the maximum rise in Ca2+ concentration for DHPG-stimulated Ca2+ release (6 ± 1 nm and 15 ± 2 nm for HdhQ20/Q20 and HdhQ111/Q111 striatal neurons, respectively) were significantly enhanced in HdhQ111/Q111 neurons when compared to HdhQ20/Q20 neurons. These data suggested that the increased release of Ca2+ was due to the sensitization of Ca2+ release from intracellular stores.

Figure 3.

Figure 3.

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Agonist-stimulated Ca2+ release is higher in HdhQ111/Q111 than in HdhQ20/Q20 neurons. A, B, Graphs show intracellular concentration of Ca2+ in HdhQ20/Q20 and HdhQ111/Q111 striatal neurons stimulated with either 10 μm DHPG (A) or 100 μm carbachol (B) for 20 s (indicated by the bar). C, Graph shows the area under the curve (AUC) of the integrated Ca2+ responses to agonist stimulation in HdhQ20/Q20 and HdhQ111/Q111 neurons. D, Graph shows the amplitude of agonist-stimulated Ca2+ transients quantified as the maximal rise of [Ca2+]i above basal levels in HdhQ20/Q20 and HdhQ111/Q111 neurons. Data represent the means ± SEM of five independent experiments and a total of 80 cells analyzed. Asterisks indicate significant differences as compared to HdhQ20/Q20 response (p < 0.05).

Decreased InsP formation in HdhQ111/Q111 mice is PKC dependent

HdhQ20/Q20 and HdhQ111/Q111 exhibited the same levels of mGluR1/5 protein expression at the cell surface and Ca2+ release was dysregulated in HdhQ111/Q111 neurons. Therefore, we hypothesized that mGluR1/5 InsP formation in HdhQ111/Q111 striatal neurons may be the consequence of increased PKC-mediated receptor desensitization. Group I mGluRs was previously shown to be desensitized by PKC (Catania et al., 1991). Therefore, we examined whether mGluR1/5-stimulated InsP formation in HdhQ111/Q111 striatal slices might be altered by the treatment of striatal slices with drugs that either activate or inactivate PKC. Similar to what we demonstrated in the earlier experiments, DHPG-stimulated InsP formation in striatal slices prepared from 2- to 4-month-old HdhQ111/Q111 mice was significantly attenuated when compared to slices prepared from 2- to 4-month-old HdhQ20/Q20 mice (Fig. 4A). The observed differences in InsP formation in striatal slices derived from HdhQ111/Q111 versus HdhQ20/Q20 mice was not affected by the activation of PKC with the phorbol ester PMA (Fig. 4A). However, the treatment of slices prepared from 2- to 4-month-old mice with bisindolylmaleimide I (Bis I) to inhibit PKC abolished the difference in InsP formation observed between HdhQ20/Q20 and HdhQ111/Q111 slices (Fig. 4A). It was previously shown that NMDA receptor activation could lead to resensitization of mGluR5 signaling (Alagarsamy et al., 1999). Therefore, we tested whether the treatment of slices derived from 2- to 4-month-old mice with 10 μm NMDA would reverse the PKC-mediated desensitization observed in HdhQ111/Q111 mice. NMDA treatment increased DHPG-mediated InsP formation in both HdhQ20/Q20 and HdhQ111/Q111 mice, but InsP formation remained reduced in HdhQ111/Q111 slices, as compared to that of HdhQ20/Q20 slices (Fig. 4A). The difference in DHPG-stimulated InsP formation between HdhQ20/Q20 and HdhQ111/Q111 was only eliminated when slices were incubated with both 10 μm NMDA and 1 μm Bis I (Fig. 4A). No difference in DHPG-stimulated InsP formation was observed in slices derived from 11- to 14-month-old HdhQ20/Q20 and HdhQ111/Q111 mice following any drug treatments, except that NMDA treatment increased DHPG-stimulated InsP formation in slices from either mouse line (Fig. 4B). The increased PKC-mediated mGluR1/5 desensitization observed in HdhQ111/Q111 mice could be the consequence of both Ca2+ dysregulation and alterations in PKC expression in HdhQ111/Q111 mice. To test whether PKC expression was altered in HdhQ111/Q111 mice, we performed real time RT-PCR to determine expression of different Ca2+-regulated PKC isoforms (PKC α, β, and γ) in striatal tissue obtained from HdhQ20/Q20 and HdhQ111/Q111 mice from different age groups (4-, 9-, and 13-month-old mice). No difference in PKC mRNA expression was observed between HdhQ20/Q20 and HdhQ111/Q111 mice from any of the age groups tested (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Together, these data indicated that the difference in mGluR1/5-dependent InsP formation between HdhQ20/Q20 and HdhQ111/Q111 mice was due to increased PKC-mediated desensitization in HdhQ111/Q111 mice that may occur as the consequence of dysregulated Ca2+ release from intracellular stores.

Figure 4.

Figure 4.

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The decrease in InsP formation in HdhQ111/Q111 mice is PKC dependent. A, B, Shown is mGluR1/5-stimulated inositol phosphate formation in response to 50 μm DHPG for 15 min at 37°C in striatal slices from either HdhQ20/Q20 or HdhQ111/Q111 mice that were 2–4 (A) or 11–14 months old (B). Vehicle alone or vehicle plus indicated drug [PMA, NMDA, bisindolylmaleimide I (Bis1), or NMDA+Bis1] were added to slices 15 min before DHPG stimulation. Data represent the means ± SEM of six independent experiments, expressed as percentage of DHPG-stimulated HdhQ20/Q20 slices (control). Asterisks indicate significant differences as compared to matched treated HdhQ20/Q20 slices (p < 0.05).

mGluR5 can activate protective signaling pathways in HdhQ111/Q111 neurons

Given that both InsP formation and Ca2+ release in response to mGluR1/5 activation is altered in HdhQ111/Q111 mice, we tested whether other signaling pathways activated by mGluRs might be altered in the HdhQ111/Q111 mice. For example, mGluR1/5 activation were previously shown to promote the phosphorylation and activation of both ERK1/2 and AKT (Datta et al., 1999; Grewal et al., 1999; Kandel and Hay, 1999). Primary cultured striatal neurons from HdhQ20/Q20 mouse embryos exhibited increased ERK1/2 phosphorylation following 5 min, but not 2 min, DHPG stimulation (Fig. 5A,B). However, an increase in ERK1/2 phosphorylation in HdhQ111/Q111 neurons was observed as early as within 2 min DHPG stimulation, indicating that mGluR-mediated ERK1/2 activation occurs more rapidly in HdhQ111/Q111 neurons, as compared to that of HdhQ20/Q20 neurons (Fig. 5A,B). Moreover, ERK1/2 activation in response to 10 μm DHPG treatment for 2 and 5 min was significantly greater in HdhQ111/Q111 neurons when compared with HdhQ20/Q20 neurons (Fig. 5A,B). The enhanced activation of ERK1/2 in HdhQ111/Q111 neurons by DHPG was mGluR5-dependent, as the treatment of neurons with the mGluR5 selective antagonist MPEP (10 μm) mitigated the observed difference in ERK1/2 phosphorylation between HdhQ20/Q20 and HdhQ111/Q111 neurons (Fig. 5A,B). Similar results were obtained in striatal slices, as an increase in ERK1/2 phosphorylation was observed following DHPG stimulation of HdhQ111/Q111 striatal slices, as compared to that of HdhQ20/Q20 striatal slices (Fig. 5C,D).

Figure 5.

Figure 5.

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mGluR5-stimulated ERK1/2 phosphorylation is more elevated in HdhQ111/Q111 than in HdhQ20/Q20 mice. A, Shown is a representative immunoblot for phospho- (upper panel) and total-ERK expression (lower panel) in primary cultured striatal neurons from either HdhQ20/Q20 or HdhQ111/Q111 mice stimulated with 10 μm DHPG for 0, 2, or 5 min in presence or absence of 10 μm MPEP. 50 μg of cell lysate is used for each sample. B, Graph shows the densitometric analysis of phospho-ERK normalized to total-ERK expression in primary cultured striatal neurons. Data represent the mean ± SEM of six independent experiments, expressed as percentage of basal ERK phosphorylation in HdhQ20/Q20 neurons. Asterisks indicate significant differences as compared to matched treated HdhQ20/Q20 neurons (p < 0.05). # indicates significant difference as compared to basal ERK1/2 phosphorylation levels (p < 0.05). C, Shown is a representative immunoblot for phospho- (upper panel) and total-ERK expression (lower panel) in striatal slices from either HdhQ20/Q20 or HdhQ111/Q111 mice stimulated with 100 μm DHPG for 30 min. 100 μg of cell lysate is used for each sample. D, Graph shows the densitometric analysis of phospho-ERK normalized to total-ERK expression in striatal slices. Data represent the mean ± SEM of five independent experiments, expressed as percentage of basal ERK phosphorylation in HdhQ20/Q20 slices. Asterisk indicates significant difference as compared to matched treated HdhQ20/Q20 slices (p < 0.05).

To determine whether mGluR1/5 stimulation resulted in AKT activation in HdhQ111/Q111 striatal neurons, we stimulated neuronal primary cultures with 10 μm DHPG and determined the levels of AKT phosphorylation. 5 min DHPG-stimulation led to an increase in AKT phosphorylation in both HdhQ20/Q20 and HdhQ111/Q111 neurons (Fig. 6A,B). However, basal AKT phosphorylation was significantly higher in HdhQ111/Q111 than in HdhQ20/Q20 neurons, suggesting that this signal transduction pathway was constitutively engaged in the neurons derived from HdhQ111/Q111 mice (Fig. 6A,B). Interestingly, this increase in basal AKT phosphorylation in HdhQ111/Q111 neurons was mGluR5-dependent, as treatment of neurons with 10 μm MPEP reduced basal AKT phosphorylation to HdhQ20/Q20 levels (Fig. 6A,B). Together, these data indicated that mGluR5-dependent activation of cell survival signaling pathways was increased in neurons derived from HdhQ111/Q111 mice, despite the fact that InsP formation via the receptor was uncoupled as a consequence of PKC-mediated desensitization.

Figure 6.

Figure 6.

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mGluR5-dependent basal AKT phosphorylation is higher in HdhQ111/Q111 than in HdhQ20/Q20 neurons. A, Shown is a representative immunoblot for phospho- (upper panel) and total-AKT expression (lower panel) in primary cultured striatal neurons from either HdhQ20/Q20 or HdhQ111/Q111 mice stimulated with 10 μm DHPG for 0, 2, or 5 min in presence or absence of 10 μm MPEP. 50 μg of cell lysate is used for each sample. B, Graph shows the densitometric analysis of phospho-AKT normalized to total-AKT expression in primary cultured striatal neurons. Data represent the mean ± SEM of six independent experiments, expressed as percentage of basal AKT phosphorylation in HdhQ20/Q20 neurons. Asterisks indicate significant differences as compared to matched treated HdhQ20/Q20 neurons (p < 0.05).

PKC-mediated mGluR5 desensitization protects against neuronal cell death

We observed that mGluR5-dependent activation of AKT in the absence of an agonist challenge was increased and agonist-stimulated InsP formation was uncoupled in neurons derived from HdhQ111/Q111 mice. Therefore, we examined whether the alterations in mGluR5 signaling observed in presymptomatic HdhQ111/Q111 mice may contribute to enhanced neuroprotection of HdhQ111/Q111 neurons. We found that cell death in response to the treatment of striatal neurons with 250 μm glutamate for 24 h was not different for cultures derived from HdhQ20/Q20, HdhQ111/Q111, or HdhQ111/Q111/mGluR5−/− mice (Fig. 7). Treatment of HdhQ20/Q20 neurons with 100 μm DHPG or PKC inhibitor (1 μm Bis I), either alone or in the presence of 100 μm DHPG, resulted in no increase in neuronal cell death, as compared to untreated HdhQ20/Q20 neurons (Fig. 7). Furthermore, treatment of striatal neurons derived from HdhQ111/Q111 mice with either 100 μm DHPG or 1 μm Bis I alone failed to induce neuronal cell death (Fig. 7). However, when HdhQ111/Q111 neurons were treated with 100 μm DHPG in presence of a PKC inhibitor, a significant increase in neuronal cell death was observed, as compared to that of matched treated HdhQ20/Q20 neurons (Fig. 7). No increase in neuronal cell death was observed in striatal neuronal cultures derived from HdhQ111/Q111 lacking mGluR5 (−/−) in response to DHPG in the presence of Bis I (Fig. 7). Thus the increased level in neuronal cell death observed in HdhQ111/Q111 mice treated with DHPG and Bis I was mGluR5-dependent, demonstrating that altered mGluR5 signal transduction may be neuroprotective in the presymptomatic phase of the disease.

Discussion

The neuronal cell loss that takes place in the striatum and cortex of HD patients is considered to be the primary cause of HD symptoms and eventual death of HD patients (Vonsattel et al., 1985; Vonsattel and DiFiglia, 1998). It is still not clear why mutant Htt protein leads to selective neuronal cell death and why there is a delayed loss of neurons late in life. mGluR1/5 can signal to activate different pathways that can be either protective or exacerbate neuronal cell death (Nicoletti et al., 1996; Bruno et al., 2001; Tang et al., 2003; Baskys et al., 2005). In the present study, we show that mGluR1/5 signaling pathways are altered in the presymptomatic phase of a HdhQ111/Q111 knock-in mouse model of HD (Fig. 8). DHPG-mediated InsP formation is significantly reduced in HdhQ111/Q111 mice, when compared to HdhQ20/Q20 mice, and this attenuation of InsP signaling is PKC-dependent. Furthermore, activation of protective pathways involving ERK and AKT is augmented in HdhQ111/Q111 neurons, as compared to HdhQ20/Q20 neurons, which indicates an early integral adaptive response to protect against cell death. PKC inhibition leads to increased DHPG-stimulated neuronal cell death in HdhQ111/Q111 neurons, suggesting that PKC-mediated mGluR1/5 desensitization in HdhQ111/Q111 neurons might be protective.

Figure 8.

Figure 8.

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Schematic representation of the proposed model for mGluR1/5 signaling alterations in HD. Shown is a schematic for mGluR1/5 signaling in HdhQ20/Q20 and HdhQ111/Q111 mice. mGluR1/5 are coupled to Gαq/11 proteins, and its activation results in diacylglycerol (DAG) and IP3 formation, release of Ca2+ from intracellular stores and activation of PKC, as well as activation of ERK and AKT. In HdhQ111/Q111 mice, mGluR1/5-mediated IP3 formation is decreased due to increased PKC-mediated mGluR1/5 desensitization. Despite of decreased InsP formation, mGluR1/5-mediated calcium release, as well as ERK and AKT activation are increased in HdhQ111/Q111 mice, as compared to HdhQ20/Q20 mice. This is likely the consequence of an adaptive response in an attempt to keep HdhQ111/Q111 neurons alive in the presymptomatic phase of the disease. Inhibition of PKC leads to increased DHPG-stimulated cell death in neurons derived from HdhQ111/Q111 mice, as compared to HdhQ20/Q20 mice.

DHPG-mediated Ca2+ release is sensitized in HdhQ111/Q111 neurons when compared to HdhQ20/Q20 neuronal cultures, despite the fact that DHPG-mediated InsP formation is reduced in HdhQ111/Q111 neurons. These results are in agreement with published results showing increased Ca2+ release in a YAC128 HD transgenic mouse model (Tang et al., 2005). At least three different mechanisms account for the increase in intracellular Ca2+ release in transgenic HD mouse models: (1) mutated Htt protein may cause the sensitization of the NDMA receptor permeability, resulting in an increase in extracellular Ca2+ entrance into neurons (Chen et al., 1999; Sun et al., 2001); (2) mutated Htt protein may destabilize mitochondrial Ca2+ regulation (Panov et al., 2002; Choo et al., 2004); or (3) mutated Htt protein sensitizes the inositol-1,4,5-triphosphate (IP3) receptor-mediated release of Ca2+ from intracellular stores (Tang et al., 2005). In the present study, HdhQ111/Q111 neurons exhibit enhanced sensitivity for intracellular Ca2+ release in response to DHPG treatment, which may be the underlying mechanism responsible for enhanced ERK phosphorylation in DHPG-treated HdhQ111/Q111 neurons.

mGluR5-mediated ERK activation in striatal neurons occurs as the consequence of either InsP-stimulated Ca2+ release, the activation of Pyk2 or Homer proteins (Mao et al., 2005) (A. A. Nicodemo and S. S. G. Ferguson, unpublished observations). Interestingly, Homer is important for mGluR5-mediated ERK activation in the striatum and spinal cord, but Homer does not appear to contribute to mGluR5 activation of ERK in the hippocampus (Mao et al., 2005; Tappe et al., 2006; Ronesi and Huber, 2008). Thus, in addition to increased Ca2+ release, Homer may also be contributing to increased DHPG-stimulated ERK1/2 phosphorylation in HdhQ111/Q111 striatal neurons.

It has been shown that basal levels of AKT phosphorylation are increased in an immortalized HdhQ111/Q111 striatal cell line (Gines et al., 2003). The increase in basal AKT phosphorylation in HdhQ111/Q111 striatal cells has been shown to be NMDA receptor (NMDAR)-dependent, as the NMDAR antagonist MK801 decreases HdhQ111/Q111 AKT phosphorylation to control levels (Gines et al., 2003). We show here that the mGluR5 selective antagonist MPEP also suppresses basal AKT phosphorylation in neurons derived from HdhQ111/Q111 mice. Therefore, in addition to NMDAR activation, altered mGluR5 signaling may also underlie increased AKT signaling in HdhQ111/Q111 knock-in mice and may represent a neuroprotective signaling response. The activation of AKT by mGluR5 involves phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1) (Rong et al., 2003; Hou and Klann, 2004). mGluR1 activation leads to formation of the functional complex mGluR1-Homer-PIKE (PI3K enhancer), allowing PI3 kinase activation by PIKE, which results in reduced apoptosis (Rong et al., 2003). The observation that the increase in basal AKT activation in HdhQ111/Q111 striatal neurons can be blocked using a mGluR5-specific antagonist suggests that mGluR5 signaling via AKT is selectively enhanced in the absence of basal alterations in ERK and InsP signaling. Phosphorylation of AKT by glutamate receptors is particularly important because it has been shown that AKT activation can protect against neuronal death (Datta et al., 1999; Kandel and Hay, 1999). Furthermore, AKT can also promote phosphorylation of mutated Htt protein, which functions to reduce Htt aggregate formation and neuronal cell death, providing a protective pathway in HD (Humbert et al., 2002; Warby et al., 2009).

The glutamatergic system plays a substantial role in neuronal cell death and there are consistent data implicating ionotropic glutamate receptor activation with the excitotoxic neuronal loss in HD (Zeron et al., 2002; Schiefer et al., 2004). Some studies suggest that the sensitization of the NMDAR containing the subunit NR1/NR2B by the mutated Htt protein is responsible for causing the selective cell death of the medium sized spiny neurons present in the striatum, since these neurons express high level of this NMDAR subtype (Chen et al., 1999; Zeron et al., 2001). Group I mGluR receptors have also been implicated in neuronal cell death. However, there are mixed reports with respect to the role of mGluRs. Treatment of an HD transgenic mouse model with mGluR5 antagonists increases survival (Schiefer et al., 2004). The disruption of calcium signaling and increased neuronal apoptosis that are observed in medium sized spiny neurons of an HD mouse model are also attributed to activation of mGluR1/5 and NMDAR containing the NR2B subunit (Tang et al., 2005). However, other studies have provided evidence that mGluR signaling may be protective. For example, when cortical neuronal cultures are consecutively incubated two times with DHPG, NMDAR-mediated excitotoxicity is attenuated (Bruno et al., 2001; Baskys et al., 2005). The results presented here demonstrate that DHPG-mediated InsP formation is reduced in HdhQ111/Q111 mice due to an increase in PKC-mediated desensitization of mGluR1/5 in HdhQ111/Q111 mice. It is possible that the PKC-mediated mGluR1/5 desensitization is protective, avoiding further increases in calcium release that may result in increased cell death. We find that basal levels of cell death are the same in neuronal primary cultures obtained from HdhQ20/Q20 and HdhQ111/Q111 embryos. However, when PKC-mediated mGluR1/5 desensitization is blocked with the PKC inhibitor Bis I, DHPG stimulation causes an increase in HdhQ111/Q111 neuronal death, although it does not enhance HdhQ20/Q20 neuronal death.

PKC-mediated mGluR1/5 desensitization is present only in the asymptomatic phase of the disease, disappearing in mice older than 11 months. Furthermore, the data presented here demonstrate that mGluR1/5-mediated ERK and AKT activation, which can be protective against neuronal cell death, was higher in HdhQ111/Q111 neurons than in HdhQ20/Q20 neurons. These data might shed some light on why HD symptoms only appear later in life, although the mutated Htt protein is expressed since the beginning of patient's life. It is possible that mGluR1/5-mediated signaling pathways that could lead to cell death (increase in intracellular calcium) are desensitized and that pathways that could lead to neuronal protection (activation of ERK and AKT) are sensitized in the asymptomatic phase of the disease. These alterations might be lost later in life, causing an increase in neuronal death and the development of HD symptoms.

Footnotes

F.M.R. is the recipient of a Heart and Stroke Foundation of Canada (HSFC) Postdoctoral Fellowship. M.P. is the recipient of a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship award. S.S.G.F. holds a Tier I Canada Research Chair in Molecular Neurobiology and is a Career Investigator of the Heart and Stroke Foundation of Ontario. This work was supported by CIHR Grant MA-15506 to S.S.G.F. We also thank Dr. Douglas Sheffler and Dr. Jeffrey Conn for helping us to establish a protocol for InsP formation assay in brain slices, as well as Dr. Stephen M. Sims and Tom Chrones for helping us to perform the calcium experiments.

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