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* Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciònes Cientificas/Universidad Autonóma de Madrid, Madrid, Spain;
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Madrid, Spain;
Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain; and
Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain
2Correspondence: Centro de Biología Molecular "Severo Ochoa", CSIC/UAM, Campus UAM de Cantoblanco, 28049 Madrid. Spain. E-mail: jjlucas{at}cbm.uam.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ATP • calcium • apoptosis • polyglutamine • BBG
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Patients suffer from motor dysfunction, cognitive decline, and psychological disturbances over 10 to 15 yr until death. Neuropathology is characterized by brain atrophy and neuronal death predominantly in cortex and striatum and by the presence of neuropil and nuclear neuronal inclusion bodies also in cortex and striatum, as well as in less affected brain regions (2)
.
The precise molecular interactions by which mutant-htt exerts its toxicity remain unknown, although several pathogenic mechanisms have been proposed (3
4
5)
, the relative contributions of which may vary with disease progression. Among these, synaptic alterations (6)
and increased susceptibility to neuronal death (7)
are known contributors to HD symptomatology, in which there is an initial phase without neuronal loss. The initial phase is characterized by neuronal dysfunction associated with decreases in neuronal volume, arborization, and fiber density in the cortex and striatum of HD patients (8
, 9)
. Furthermore, these alterations are mirrored in mouse models of HD (10
, 11)
. While neuronal loss is not initially observed in symptomatic mice, later stages of disease progression exhibit apoptotic (12
, 13)
and nonapoptotic neuronal loss (9
, 14
, 15)
, contributing to brain atrophy. Accordingly, therapeutic strategies aimed at preventing neuronal loss have been proposed for Huntington’s disease (7
, 16)
.
While decreased metabolism has long been associated with HD (17)
, recent findings have surprisingly demonstrated decreased neuronal apoptosis in HD animal models in response to small drugs that inhibit metabolism (18)
. Specifically, in vivo administration of drugs that impair ATP production such as rotenone or oligomycin decreased neuronal death in Caenorhabditis elegans and Drosophila models of HD (18)
. These effects may represent an attenuation of toxic effects elicited by extracellular ATP. In support of this hypothesis, a recent study reported that extracellular ATP induces both apoptotic and nonapoptotic death of SN4741 dopaminergic neurons through stimulation of the P2X7 receptor (19)
.
The P2X7 receptor is an ATP-gated cation channel initially cloned from rat brain (20)
and highly expressed in cells of hematopoietic lineage and in multiple cell types of the immune system (20)
. Within the brain, it was first reported in microglia (21)
and later in other types of glia and in neurons, with prominent expression in synaptic terminals (22)
. A systematic analysis of P2X7 receptors expression throughout the entire rat brain has recently been reported (23)
. This detailed in situ hybridization analysis of P2X7 mRNA expression combined with double labeling with cell type-specific markers confirmed robust expression in neurons throughout the brain, as well as in scattered glial cells such as microglia, oligodendrocytes, and, to a lesser extent, astrocytes (23)
.
In neurons, the P2X7 receptor has been shown to regulate axonal growth and branching (24)
and to modulate neurotransmitter release from presynaptic terminals (22
, 25
, 26)
. Stimulation of P2X7 in the immune system regulates cytokine production and release (27)
. P2X7 receptors have also been shown to mediate apoptosis in response to elevations in intracellular calcium in hematopoietic and immune system cells (28
29
30)
and in neurons (19)
.
In view of the reported role of the P2X7 receptor in mediating ATP induced neuronal death, and the antiapoptotic effect of ATP-synthesis inhibitors in C. elegans and Drosophila models of HD, we hypothesize a potential role of P2X7 receptors in HD pathogenesis. Accordingly, we have explored the status of P2X7 receptors in cellular and mouse models of HD, as well as the therapeutic potential of P2X7 antagonist administration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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32
33)
. Mice were bred and maintained at the Animal facility of Centro de Biología Molecular "Severo Ochoa" (Madrid, Spain). Four to five mice were housed per cage with food and water available ad libitum. Mice were maintained in a temperature-controlled environment on a 12:12-h light-dark cycle with light onset at 7:00 AM. All animal studies were performed with authorization of the Consejo Superior de Investigaciònes Cientificas (CSIC) bioethics committee and were performed following institutional guidelines for animal handling and research.
Synaptosomal preparation
Tet/HD94 and R6/1 mice were cervically dislocated and decapitated, and the forebrain was removed. The synaptosomal isolation was made through a Percoll gradient, as described previously (34)
.
Neuronal primary culture and transfection
Primary cultures of striatal and cortical neurons were prepared according to modifications of established procedures (35
, 36)
. Cortex and striatum were dissected and dissociated individually from each pup, at postnatal day 1, using the Papain Dissociation System (PDS; Worthington Biochemical Corp., Lakewood, NJ, USA). To identify pups of the Tet/HD94 genotype, the remainder of brain tissue was maintained in X-Gal solution, whereas the pups of R6/1 genotype were identified by PCR, extracting DNA from their tails. The P2X7-KO pups did not require genotyping since they are produced from a homozygous colony. Neurons were plated at a density of 100,000 cells/cm2 and maintained in Neurobasal medium (NB; Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 1% B-27, 5% fetal calf serum (FCS), 0.5 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, and grown on 3 µg/ml laminin (Sigma, St. Louis, MO, USA) and 10 µg/ml poly-L-lysine-coated cell culture Chamber Slides (Nunc) or 12 mm coverslip. The cells were incubated in 95% air/5% CO2 in a humidified incubator at 37°C. For calcium-imaging studies, neurons were transfected at 3-4 d in vitro with expression plasmids encoding an N-terminal fragment of Htt fused to GFP (pGW1-Httex1-[Q17 or Q72]-GFP) kindly provided by Drs. Finkbeiner and Arrasate (Gladstone Institute, San Francisco, CA, USA) (37)
. Neuronal transfection was carried using Lipofectamine 2000 (9 µl; Invitrogen, Carlsbad, CA, USA) and 3 µg of pGW1-Httex1-[Q17 or Q72]-GFP vectors. The transfection mix was removed after 3 h, and the neurons were washed and maintained in culture for 3-4 d until functional assay.
Quantitative PCR experiments
Total RNA from mouse cortex and striatum was isolated using the RNeasy plus mini kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. RNA isolation and RT reactions were performed, as described previously (38)
. Quantitative real-time PCR was performed using gene-specific primers and TaqMan MGB probes for mouse P2X7 (exon boundary 5-6) and β-actin (Applied Biosystems, Nutley, NJ, USA). Fast thermal cycling was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) as follows: denaturation, one cycle of 95°C for 20 s, followed by 40 cycles each of 95°C for 1 s and 60°C for 20 s. The results were normalized as indicated by parallel amplification of the endogenous control β-actin.
Semiquantitative PCR experiments were also performed using primers that specifically amplify C-terminal region of full-length P2X7 transcript (sense primer, 5'-GGT GCC AGT GTG GAA ATT G-3'; antisense primer, 5'-TAG GGA TAC TTG AAG CCA CT-3'; 358-bp PCR product). The PCR reaction mixes contained AmpliTaq Gold PCR Master Mix (Applied Biosystems), and 0.6 µM of sense and antisense primers in a total volume of 25 µl. For P2X7, the reaction was conducted for 30 cycles with their respective primers and for 20 cycles with β-actin primers as internal standard (sense primer, 5'-GTA CCA CAG GCA TTG TGA TGG ACT-3'; antisense primer, 5'-TGC CAC AGG ATT CCA TAC CCA AGA-3'; 381-bp PCR product). The number of cycles was predetermined to fall within the linear range of amplification of each PCR product. PCR involved an initial denaturation at 95°C for 10 min; denaturation at 95°C for 45 s; 57°C for 40 s annealing step; and extension at 72°C for 50 s.
Antibodies
The following antibodies were used: polyclonal antibodies against P2X7 receptor from GE Health Care-Pharmacia (Little Chalfont, UK), from Chemicon (Temecula, CA, USA), and from Alomone (Jerusalem, Israel); polyclonal anti-DARPP-32 and monoclonal anti-neuronal nuclei (NeuN) were obtained from Chemicon (Temecula, CA, USA); monoclonal anti-
-tubulin, monoclonal anti-synaptophysin, and monoclonal anti-glial fibrillar acidic protein (GFAP) were obtained from Sigma (St. Louis, MI, USA); monoclonal anti-β-III tubulin was provided by Promega (Madison, WI, USA); polyclonal anti-cleaved caspase-3 was obtained from Cell Signaling (Beverly, MA, USA); and polyclonal anti-Iba-1 was obtained from Wako (Osaka, Japan).
Western blot analysis
Extracts for Western blot analysis were prepared by homogenizing fresh dissected mouse brain regions in ice-cold extraction buffer consisting of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM EDTA, and protease inhibitors (2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The samples were homogenized at 4°C and protein content determined by Bradford. Total protein (20 µg) was electrophoresed on 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The experiments were performed using the following primary antibodies: polyclonal anti-P2X7 (1:1000), monoclonal anti--tubulin (1:10,000), monoclonal anti-synaptophysin (1:2000). The filters were incubated with the antibody at 4°C overnight in 5% nonfat dried milk. A secondary goat anti-mouse (monoclonal) or goat anti-rabbit (polyclonal) antibody (both 1:1000) from DakoCytomation (Glostrup, Denmark) was used followed by ECL detection (Amersham, Piscataway, NJ, USA).
Quantification was performed by using a GS-710 Calibrated Imaging densitometer scanner controlled by Quantity One PC software (Bio-Rad, Hercules, CA, USA). In all cases, the average intensity value of the pixels in a background-selected region was calculated and was subtracted from each pixel in the samples. The densitometry values obtained in the linear range of detection with these antibodies were normalized with respect to the values obtained with an anti--tubulin antibody in order to correct for any deviation in loaded amounts of protein. Statistical analysis was performed using one-way ANOVA followed by Bonferroni test.
Calcium studies: microfluorimetric analysis
Synaptosome preparation pellets containing 0.5 mg/ml protein were placed on polylysine-coated coverslips. Primary neurons were analyzed after at least 3 d in culture. Synaptosomes or primary cultures were then washed with HBM buffer (140 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM NaHCO3, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4), and they were loaded with FURA-2AM solution (5 µM) for 30 min at 37°C to allow for the intracellular hydrolysis of FURA-2AM dye. The coverslips were washed again with HBM Mg2+-free medium and mounted in a superfusion chamber in a Nikon Eclipse TE-2000 microscope (Nikon, Tokyo, Japan). This medium was the same as mentioned above, but the MgCl2 was substituted by the accurate glucose concentration to preserve osmolarity. Neurons were continuously superfused at 1.2 ml/min with Mg2+-free perfusion medium during functional assays. Initially, pulses of Bz-ATP 10 µM for 30 s were applied to synaptosomes in the absence of any other drug. Sequential 50-s pulses of BzATP ranging from 100 nM to 1 mM were assayed on neurons. To check the effect of specific P2X7 receptor antagonists, neurons were incubated for 5 min with Brilliant Blue G (BBG) (from 100 nM to 1 µM) or o-ATP at 100 µM before 100 µM BzATP stimulation. Finally, a pulse of 60 mM K+ was applied at the end of each experiment to confirm the viability of neurons and the functionality of synaptosomes under study. All compounds were dissolved in Mg2+-free HBM medium.
Neurons were visualized using a Nikon microscope with an x40 S Fluor 0.5–1.3 oil lens. The wavelength of the incoming light was filtered to 340 and 380 nm with the aid of a monochromator (10-nm bandwidth, Optoscan monochromator; Cairin, Faversham, UK). The 12-bit images were acquired with an ORCA-ER C 47 42–98 charged-couple device camera from Hamamatsu (Hamamatsu City, Japan) controlled by Metafluor 6.3r6 PC software (Universal Imaging Corp., Cambridge, UK). The exposure time was 250 ms for each wavelength and the changing time <5 ms. The images were acquired continuously and buffered in a fast SCSI disk. The time course data represent the average light intensity in a small elliptical region inside each cell. The background and autofluorescence components were subtracted at each wavelength.
Immunohistochemistry
Brains were fixed in 4% PFA and cryoprotected in 30% sucrose solution. Thirty-micrometer sagital sections were cut on a freezing microtome (Leica Microsystems, Wetzlar, Germany) and collected in 0.1% azide-PBS solution. Next, brain sections were pretreated for 1 h with 1% BSA, 5% FBS, and 0.2% Triton X-100 and then incubated with primary antibodies at the following dilutions: polyclonal anti-P2X7 (1:200), polyclonal anti-cleaved caspase-3 (1:50), polyclonal anti-Iba-1 (1:200), and monoclonal anti-GFAP (1:200). Finally, brain sections were incubated in avidin-biotin complex using the Elite Vectastain kit (Vector Laboratories). Chromogen reactions were performed with diaminobenzidine (Sigma) and 0.003% H2O2 for 10 min. Sections were coverslipped with Fluorosave. Estimation of the number of caspase-3-, Iba-1-, or GFAP-positive cells in cortex of PBS or BBG administered mice was performed by counting the number of positive cells in the motor, somatosensory, and visual cortex areas encompassed in five equidistant 30-µm sagital sections ranging from 1.32 to 2.04 mm lateral coordinates, according to Paxinos mouse brain atlas (39)
. Data are presented as the mean ± SE number of cells extrapolated to the whole mentioned cortical areas between the 1.32 and 2.04 mm lateral coordinates.
Immunofluorescence studies
Sagital mouse brain sections were pretreated with Sudan Black B, 1% BSA, and 1% TX 100 in PBS buffer and incubated with primary antibodies at the following dilutions: polyclonal anti-P2X7 (1:100), polyclonal anti-DARPP-32 (1:500), monoclonal anti-GFAP (1:200), polyclonal anti-Iba-1 (1:100), polyclonal anti-cleaved caspase-3 (1:50), monoclonal anti-NeuN (1:100), and monoclonal anti-synaptophysin (1:200). Subsequently, the brain sections were washed with PBS buffer and incubated with secondary antibodies at the following dilutions: goat anti-rabbit IgG labeled with Texas Red (Molecular Probes, Eugene, OR, USA) (1:400) and goat anti-mouse IgG labeled with Oregon Green 488 from Molecular Probes (1:200). Finally, the brain sections were washed with PBS and mounted following the standard procedures. Controls were performed by following the same procedure but substituting the primary antibodies by PBS in presence of 1% BSA. Similar procedures were followed with synaptosomal preparation, except the Sudan Black B pretreatment. Colocalization of two markers was analyzed by taking successive Oregon Green 488 and Texas Red fluorescent images using an Axioskop 2+ microscope (Zeiss, Oberkochen, Germany) and a CCD camera (Coolsnap FX color; Photometrics, Tucson, AZ, USA). Positive signal was considered for the different antibodies if the mean intensity values were >140 on a 0-255 scale with 0 = white and 255 = black. The cutoff value of 140 was determined from visual analysis of immunolabeling and by comparison with control (maximal level obtained with preabsorbed antibodies).
Cultured neuron survival assays: propidium iodide–calcein staining
Twenty-four hours after plating, cortical and striatal neurons cultured from Tet/HD94 and R6/1 mice were shifted to medium lacking serum and B27. Cultured neurons were then treated as previously described (24)
. Briefly, neurons were stimulated with Bz-ATP at concentrations from 1 µM to 1 mM or equal volume of PBS solution, and 48 h later, cell viability was assessed by propidium iodide–calcein staining (Molecular Probes). To eliminate the possible effect of adenosine generated by extracellular hydrolysis of Bz-ATP, 0.1 U adenosine deaminase was added to neurons. In some cases, cortical neurons were pretreated with BBG at concentrations from 3 nM to 1 µM for 10 min before 10 or 100 µM Bz-ATP was assayed. After several brief rinses, cells were visualized by fluorescence microscopy using an Axiovert 135 microscope (Zeiss). Nine fields (selected at random) were analyzed per well (
400 cells/field) in two independent experiments.
Drug treatment
BBG and A-438079 were diluted at 3 mg/ml in vehicle solutions. The vehicle solutions were calcium- and magnesium-free PBS plus 0.2% DMSO for BBG and saline solution for A-438079. BBG (45.5 mg/kg) and A-438079 (34.2 mg/kg=100 µmol/kg) in their corresponding vehicles were then administered i.p. to mice, BBG every 48 h, and A-438079 every 24 h. Control littermates were treated with the same relation (volume/body weight) with vehicle solutions. The mice were weighed once a week during drug administration.
Spectrophotometry analysis of BBG levels in plasma and brain
BBG was added at certain concentrations either to PBS, plasma, or cerebral homogenate, and the optimal "signature wavelength" was scanned between 200 and 800 nm using a spectrophotometer (Ultrospec III; Pharmacia). In all cases, the maximal absorbance was obtained at 576 nm. Levels of BBG in plasma and in brain homogenates from BBG-treated mice (wild type and R6/1) were determined according to calibration curves generated with a range of BBG dilutions made either in plasma or in brain homogenate. In mice treated during 1 mo, BBG levels reached 7.08 ± 0.86 µM in plasma and 152.6 ± 2.54 nM in brain, while those treated for 4 mo reached 20.04 ± 7.44 µM in plasma and 226.12 ± 6.8 nM in brain.
Rota-Rod behavioral testing
R6/1 mice were tested for motor coordination at different ages (from 5 to 8 mo old) on an accelerating Rota-Rod apparatus (Ugo Basile, Comerio, Italy). Initially, each mouse was pretrained at a fixed speed (8 rpm) by being repeatedly placed on the Rota-Rod as many times as necessary until it was able to remain on the apparatus for 60 s. After this pretraining, Rota-Rod was set to accelerate from 4 to 40 rpm over 5 min, and mice were tested twice at 3-h intervals on two consecutive days (for a total of 4 trials). During accelerating trials, the latency of each mouse to fall from the Rota-Rod was measured. Data are represented as means ± SE of latency to fall from the Rota-Rod in the four accelerating trials per genotype and treatment condition.
Statistics
All experiments were repeated at least three times, and the results are presented as the mean ± SE. Where indicated, we performed analyses of significance using a one-way ANOVA test followed by Bonferroni test correction or a Student’s t test followed to Mann-Whitney U-test (Origin 7.0 software; OriginLab Corporation, Northampton, MA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and in Supplemental Fig. 1, only the 75-kDa band corresponding to P2X7 receptor was detected by Western blot analysis in both striatal and cortical homogenates. Interestingly, P2X7 levels were increased (P=0.024) in the striatum of 10-mo-old R6/1 mice that ubiquitously express N-terminal mutant htt with a 115 residue PolyQ stretch (32)
, as compared to their nontransgenic littermates (Fig. 1A
). A nonsignificant trend toward increased levels was observed at the age of 5 mo (Fig. 1A
), coinciding with the onset of motor impairment (40)
. Similarly, Tet/HD94 mice that conditionally express exon-1 mutant htt with a shorter PolyQ repeat selectively in forebrain neurons and display a milder and later motor phenotype (13
, 31)
, displayed increased striatal levels of P2X7 receptor beginning at the age of 12 mo (P=0.0025). This occurred right after onset of motor phenotype and persisted in all older Tet/HD94 mice tested (Fig. 1B
). It is worth noting that P2X7 receptor levels progressively decline with age in the striatum of wild-type mice (Fig. 1A, B
), while such decline does not take place in R6/1 mice and is much slower in Tet/HD94 mice. Therefore, it seems that mutant huntingtin expression rather than increasing the total striatal content of P2X7 receptor, attenuates the age-dependent decrease in P2X7 striatal level. Cortical homogenates showed higher levels of P2X7 receptor respect to striatum (2.1 fold, P=0.001), but no differences were found in cortical samples from either HD mouse models as compared to corresponding control littermates (Fig. 1C, D
).
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Using quantitative RT-PCR, we then analyzed the level of P2X7 receptor mRNA in both mouse models with primers that recognize the exon 5-6 boundary of P2X7 mRNA. A higher level of P2X7 mRNA was observed in the cortex than in the striatum (Fig. 1E, F
). This finding is in good agreement with the Western blot results and with recently reported mapping of P2X7 mRNA expression in rat brain by in situ hybridization and by RT-PCR (23)
. Interestingly, increased mRNA levels were observed in the cortex of both mouse models as compared to their wild-type littermates by real-time quantitative PCR (Fig. 1E, F
) and also by semiquantitative RT-PCR with previously reported primers (38)
that amplify sequences on P2X7 exon 13 from wild type but not from P2X7 knockout samples (data not shown). In contrast, no alterations in mRNA were found in striatal samples (Fig. 1E, F
).
In view of these results, the previously reported axonal localization of P2X7 receptor (22
, 24
, 25)
and the fact that cortex is the major source of axonal afference into the striatum, we reasoned that the striatal increase in P2X7 receptor level most likely occurs in axons of cortical neurons projecting to the striatum. Immunofluorescence studies were performed on brain sections from wild-type, R6/1, and Tet/HD94 mice to analyze brain cell subpopulations expressing the P2X7 receptor and to explore its subcellular localization in cortex and striatum. The pattern of P2X7 immunostaining was similar in wild-type and HD mice. P2X7-immunopositive cell bodies were found in the cortex (Fig. 2A-C
) but rarely in the striatum (Fig. 2E-H
). In good agreement with P2X7 mRNA expression in rat brain (23)
, double labeling with the neuronal marker NeuN revealed that the vast majority of cortical cells showing P2X7 immunostaining in their somata were neurons (Fig. 2A
). Also supporting findings by Yu et al. (23)
, a smaller fraction of cells with P2X7-immunopositive somata corresponded to microglia, as evidenced by double immunofluorescence with the Iba-1 marker (Fig. 2B
), and virtually no P2X7 labeling could be detected in astrocytes as evidenced by double labeling with the astrocyte marker GFAP (data not shown). In both cortex and striatum, with the exception of the limited P2X7 immunostaining in cell bodies, the bulk of P2X7 immunostaining is located in the neuropil, where it can be found in projections (see asterisks in Fig. 2A-E
) and in buttons (see arrowheads in 2A–E and double labeling with synaptophysin in 2F–H). Double labeling with the neuronal cytoskeleton marker βIII-tubulin confirmed that those projections were axonal processes (Fig. 2C
). Such processes were also found traversing the corpus callosum (Fig. 2D
) and in the striatum (Fig. 2E
). In the latter, however, the punctate pattern is predominant, and double immunofluorescence with markers of striatal neurons (DARPP-32, Fig. 2E
) and synaptic terminals (synaptophysin Fig. 2F-H
) revealed that these buttons are axon terminals, many of which can be found lining the somata of striatal neurons.
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In summary, the analysis of P2X7 receptor level and distribution in the HD mouse models demonstrated that P2X7 receptor mRNA levels are increased in the cortex of symptomatic R6/1 and Tet/HD94 mice, while protein levels are found increased only in the striatum. Because cortical neurons project to the striatum and P2X7 receptor is located in axonal projections, the cortical increase in P2X7 mRNA results in increased P2X7 receptor protein in the striatum where it can be found at the level of synaptic terminals.
Altered P2X7 receptor function in synaptic terminals from Tet/HD94 mice
We next investigated whether the increased level of P2X7 receptor in the striatum of HD mouse models would result in altered calcium permeability in synaptic terminals in response to stimulation of P2X7 receptors. To this end, we prepared synaptosomes from striatum of 10-mo-old R6/1 mice and of 16-mo-old Tet/HD94 mice. In agreement with the Western blot and immunofluorescence data, the percentage of synaptosomes immunolabeled for P2X7 receptor was higher in the synaptosomal preparations of HD mice as compared to control littermates. As shown in Fig. 3A-C
, P2X7/synaptophysin double immunofluorescence revealed that 27.1 ± 3.5% of Tet/HD94 synaptosomes were immunoreactive for P2X7 receptor vs. 18.3 ± 3.2% of wild-type synaptosomes (P=0.032). Furthermore, Western blot of synaptosomal preparation samples revealed an increase in total P2X7 receptor content in the Tet/HD94 samples (Fig. 3D
) comparable to that observed in whole tissue homogenates. Similar results were found in the R6/1 mouse samples (data not shown). Together with the double immunofluorescence data on whole tissue, these data confirm that increased levels of P2X7 in synaptic terminals account for the demonstrated increase in whole striatal homogenates.
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We next analyzed calcium permeability in individual synaptosomes in response to a 30-s exposure to 10 µM concentration of the P2X7 agonist 2',3'-O-(4-benzoyl-4-benzoyl)-ATP (BzATP). In good agreement with the higher percentage of P2X7/synaptophysin double-labeled synaptosomes shown in Fig. 3C
, the percentage of synaptosomes that responded to the agonist was significantly higher in the synaptosomal preparation from HD mice (27.3±2.0 vs. 21.6±1.8%, P=0.01; Fig. 3E
). Interestingly, the calcium imaging recordings in response to BzATP exposure rendered two types of kinetics (Fig. 3F
). In one case, individual synaptosomes displayed an initial peak that remained elevated throughout the exposure to BzATP and progressively declined during BzATP washout (we termed this kinetic as declining profile). The second kinetic profile observed was characterized by multiple upscaling peaks of fluorescence that persisted for at least 25 s after BzATP removal (what we termed upscaling profile). In synaptosomal preparations from wild-type mice, both profiles were similarly represented (55.6±3.2% declining, 44.4±4.1% upscaling). However, in HD mouse synaptosomal preparations, the percentage shifted in favor of the latter (24.5±3.1% declining, 76.5±6.1% upscaling; Fig. 3E
). These data suggest that, in addition to increased P2X7 receptor levels in the striatum of HD mice, an alteration occurs in the functional state of the receptor in the synaptic terminal.
Altered P2X7 receptor function in terminals and somata of primary neurons expressing mutant htt
The alteration in calcium homeostasis in synaptic terminals from HD mouse models described above fits well with the proposed role of synaptic alterations in mediating HD symptomatology (6)
. However, as mentioned above, altered calcium permeability through P2X7 receptors might also contribute to neuronal apoptosis (19
, 30
, 41)
. To this end, we investigated alterations in P2X7-mediated calcium permeability in response to expression of mutant-htt in both terminals and somata of primary cultured neurons. To compare mutant-htt-expressing and –nonexpressing neurons in the same culture, we transfected wild-type mouse primary neurons with plasmids driving expression of GFP-fused exon 1 htt with either 17Q or 72 Q repeats. FURA-2 imaging in response to BzATP was then performed both in somata and terminals of neurons expressing either version of htt (identified by their expression of GFP) and of control nontransfected neurons from the same plate (Fig. 4
). In good agreement with the data obtained in the synaptosomal preparations, the same two types of kinetics (declining and upscaling) were observed in response to BzATP in terminals of cultured neurons (Fig. 4C
). Moreover, in accordance with the findings in synaptosomal preparations from wild-type mice, approximately half of the terminals of control (nontransfected) and of 17 Q (transfected with exon 1 17Q-GFP) neurons displayed one or the other kinetic (52.3±3.1% declining and 47.7±4.5% upscaling in control terminals; 50.2±1.9% declining and 49.8±3.5% upscaling in 17Q terminals). As expected, the percentage of terminals of neurons transfected with exon 1 72Q-GFP displaying the upscaling profile was much higher (78.2±9.5 vs. 21.8±7.5% of terminals showing a declining profile; P=0.0012), recapitulating the observation in synaptosomes from HD transgenic mice.
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Regarding the effect of BzATP in neuronal somata, where endoplasmic reticulum also contributes to the net concentration of intracellular calcium, only one type of calcium imaging profile was observed. This was very similar to the declining kinetic that was observed in terminals but with an additional and short lasting initial peak (Fig. 4D, F
). Interestingly, when we performed a dose response curve of the effect of BzATP in somata of 17Q- and 72Q-transfected neurons, we observed a sensitization induced by mutant Htt. More precisely, the EC50 of the effect of BzATP on calcium influx was 37.9 ± 1.21 µM in somata of 17Q-transfected neurons and 4.91 ± 1.86 µM in somata of 72Q-transfected neurons (Fig. 4D, E
). To confirm that the increased in calcium influx was due to sensitization of the P2X7 receptor, we performed similar experiments in primary neurons from P2X7-knockout (P2x7-KO) mice (33)
. In good agreement with previous reports (42)
, the calcium influx in response of BzATP was much smaller in primary neurons from P2X7-KO mice and, more important, the sensitization induced by 72Q-Htt in wild-type neurons did not take place in P2X7-KO neurons (Fig. 4E
). More precisely, the EC50 of the effect of BzATP on calcium influx was 29.82.9 ± 1.14 µM in somata of 17Q-transfected P2X7-KO neurons and 21.03 ± 1.32 µM in somata of 72Q-transfected P2X7-KO neurons (Fig. 4D, E
). In summary, the increased sensitivity to P2X7 agonist-induced calcium influx found in 72Q-transfected wild-type neurons demonstrates that P2X7 receptor function is also altered in neuronal somata as a consequence of N-terminal mutant-htt expression.
HD mouse primary neurons show increased vulnerability to P2X7-mediated apoptosis
To test whether increased P2X7 receptor function would sensitize mutant-htt-expressing neurons to apoptosis triggered by extracellular ATP, we performed survival assays on primary cortical and striatal neuronal cultures from Tet/HD94 mice using the propidium iodine-calcein staining method (Fig. 5
). As previously reported (10)
, viability of untreated primary neurons from Tet/HD94 mice was indistinguishable from that of wild-type mice (Fig. 5C, D
). In the case of Tet/HD94 striatal neurons, a trend toward decreased viability was observed in response to exposure to 10 µM BzATP, though this did not reach statistical significance (Fig. 5D
). However, in cortical neurons, such exposure to the ATP analog that did not affect survival of wild-type neurons dramatically reduced viability of Tet/HD94 neurons by 46.1 ± 7.7% (P=0.0017, Fig. 5A-C
). To further confirm that this apoptotic effect was due to stimulation of P2X7 receptors, cells were preincubated with different doses (0.3 nM to 1 µM) of the P2X7 antagonist BBG starting 10 min before the exposure to Bz-ATP. As shown in Fig. 5C, E
, 1 µM BBG fully prevented the deleterious effect of Bz-ATP on neuron viability. The IC50 of this inhibitory effect of BBG was 170.07 ± 1.38 nM (Fig. 5E
), thus fitting with the reported efficacy of BBG at antagonizing the P2X7 receptor (43)
. We then performed primary neuronal cultures from cortex of R6/1 mice and of their wild-type littermates and analyzed the effect of Bz-ATP in neuronal survival. In good agreement with the results obtained from Tet/HD94 neurons, Bz-ATP-induced death of R6/1 neurons at doses that did not affect survival of wild-type neurons (Fig. 5F
) Taken together, these findings indicate that expression of mutant-htt renders neurons more vulnerable to P2X7 mediated apoptosis.
|
Decreased incidence of neuronal apoptosis and phenotypic improvement by in vivo administration of P2X7 antagonist to HD mice
In view of the neuroprotective effect of BBG on P2X7-mediated neuronal death in culture, we investigated the potential therapeutic efficacy of this antagonist in vivo in a mouse model of HD. Because a significant increase in P2X7 level in the striatum of R6/1 mice is first observed at the age of 8 mo (data not shown) BBG (45.5 mg/kg, i.p.) or vehicle (PBS) were administered every 48 h during 4-wk to 8-mo-old R6/1 mice and to their corresponding wild-type littermates. This dosage paradigm was chosen because it results in a 152.6 ± 2.56 nM concentration in the brain of treated mice (see Materials and Methods), thus in the range of the IC50 of BBG to antagonize P2X7 (10-200 nM) (43)
and one order of magnitude below the DL50 of Brilliant Blue-CFC (44)
.
As shown in Fig. 6A
, the progressive decline in body weight that normally occurs in untreated R6/1 mice also occurred in vehicle (PBS)-administered R6/1 mice. However, BBG administration fully prevented this body weight loss in R6/1 mice (Fig. 6A
) with no change evident in the body weight in wild-type mice (data not shown). More important, we also monitored the effect of BBG on motor coordination in the Rota-Rod apparatus. As shown in Fig. 6B, C
, vehicle-treated-R6/1 mice displayed a deterioration in motor performance over the 4-wk testing period (P=0.009). However, BBG-treated mice displayed no significant deterioration over the 4 wk and by the end of the testing period performed significantly better than vehicle-treated mice (P=0.02; Fig. 6B, C
). In contrast, BBG treatment did not affect the motor performance of wild-type mice since both PBS- and BBG-treated wild-type mice showed the typical progressive improvement in motor performance (P=0.01 and P=0.03) with repeated testing over the 4-wk period (Fig. 6C
). In view of the behavioral improvement of R6/1 mice with the BBG antagonist, we decided to test also the recently developed P2X7 antagonist A-438079 that has been reported to modulate pathological nociception after acute administration to rats (45
, 46)
. Since 76 µmol/kg (i.p.) is the reported ED50 of A-438079 in acute reversal of allodynia in the rat model (45)
, we decided to administer 100 µmol/kg i.p. once daily to 8-mo-old R6/1 mice during 4 wk. As shown in Fig. 6A
, A-438079 administration resulted in a marked tendency to prevent the decline in body weight that takes place in vehicle-administered mice (P=0.072). However, in the Rota-Rod test, A-438079-administered mice were indistinguishable from vehicle-administered mice except for the last time point (after 28 d of administration) at which a moderate tendency toward improvement was detected (Fig. 6B
). These minor effects of A-438079 compared to those of BBG probably reflect the reported very short half-life (1.02 h) and limited bioavailability (19%) of this compound (45)
.
|
We then explored whether the efficacy of BBG in preventing additional weight loss and deterioration in motor coordination also correlated with a reduction in the rate of neuronal death observed in R6/1 mice. As shown in Fig. 7A-F
, neuronal apoptosis can be detected in the cortex of 8-mo-old R6/1 mice by immunohistochemistry or immunofluorescence against cleaved caspase-3. Interestingly, and in good agreement with the protective effect of BBG on HD primary cultured neurons, the increased number of cleaved caspase-3-immunoreactive neurons seen in vehicle-administered R6/1 mice was fully prevented in BBG-administered R6/1 mice (Fig. 7A-C
). Double immunofluorescence labeling experiments with NeuN and P2X7 antibodies confirmed the neuronal identity of the caspase-3-positive cells and the fact that they express P2X7 receptor (Fig. 7D-F
). In summary, these results demonstrate a beneficial effect of the P2X7 antagonist in mutant-htt-induced neuronal apoptosis in vivo, that recapitulates the protective effect observed on primary neuronal cultures and which parallels the in vivo positive effect of the antagonist in the body weight and motor coordination phenotypes.
|
In view of these in vivo results and of those obtained in primary HD cultured neurons, the protective effect of P2X7 antagonist administration seems to be clearly exerted in neurons. However, since the P2X7 receptor is also found in glial cells, we analyzed whether BBG administration also affected the gliosis that takes place in HD mice. Immunohistochemical detection of the microglial marker Iba-1 revealed a tendency for an increased number of reactive microglial cells in the cortex (Fig. 7G-I
) and striatum (not shown) of R6/1 mice with respect to their wild-type littermates that did not reach statistical significance and, more important, that was not affected by the BBG treatment (Fig G–I). Similarly, although analysis of GFAP immunohistochemistry did reveal a clear reactive astrocytosis in the cortex (Fig. 7J-L
) and striatum (not shown) of R6/1 mice, this was also unaffected by the BBG administration paradigm (Fig. 7J-L
) that prevented neuronal apoptosis.
In view of the protective effect of BBG administration to 8-mo-old R6/1 mice, we decided to explore potential beneficial effects of BBG in younger mice. We performed a similar experiment with 5-mo-old mice that do not yet display significant increases in P2X7 receptor level (Fig. 1A
) nor increased incidence of cleaved caspase-3 immunoreactivity (data not shown). In these younger R6/1 mice, body weight and Rota-Rod performance were not affected by the 4-wk administration paradigm that was found beneficial in 8-mo-old mice. For this reason, mice were maintained on the BBG or vehicle regimen and were tested every 4 wk until the age of 8 mo. However, despite the longer administration paradigm, antagonist administration did not affect body weight nor Rota-Rod performance in these younger mice, suggesting that P2X7 antagonist therapy may be beneficial only in more advanced stages of disease progression when both increase in P2X7 level and neuronal death occur.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Levels of P2X7 receptor are found increased in the striatum of two different mouse models. We also found a concomitant increase in P2X7 receptor transcription in the cortex and a prominent subcellular localization of the protein in cortical axonal projections and in striatal axon terminals. It thus appears that increased levels in the striatum are due to increased levels in corticostriatal projections, which, in turn, are secondary to increased transcription in cortical neurons. Transcriptional deregulation triggered by the expression of mutant-htt has been extensively postulated as a key mediator in HD pathogenesis (47
, 48)
. We now add P2X7 to the list of genes whose expression is altered in HD, and it would be possible that its increased transcription underlies the multiple P2X7-related alterations that we report here. While many P2X7 gene polymorphisms have been reported, including several in the promoter region (49)
, none of these alter receptor abundance or activity under the tested conditions. With the exception of the above-mentioned study and to the best of our knowledge, no other detailed studies of the promoter region have been reported. Thus, further investigation is required to elucidate the mechanism by which mutant htt expression elicits increased P2X7 transcription. It would be of interest to explore whether any of the above-mentioned P2X7 gene polymorphisms correlate with those currently being investigated as potential mediators of the variability in severity and in age of onset in HD patients with the same CAG repeat mutation length (50)
.
In synaptosomes and axon terminals of mutant-htt expressing neurons, we have found a prevalence of the so-called upscaling calcium imaging profile in response to a P2X7 agonist. This might simply be a consequence of higher levels of the receptor in the terminals as indicated by the Western blot data and the quantification of the P2X7 immunofluorescent synaptosomes. An alternative explanation would be the existence of two different functional states of the P2X7 receptor, depending on mutant-htt-induced post-translational changes. Interestingly, recordings of P2X7-operated single-channel currents have demonstrated that this receptor can mediate two different types of currents characterized by short-lived and long-lived openings, respectively (51)
. Because this was observed in single channel recordings, a difference in receptor density cannot account for the two different kinetics, and the authors suggest post-translational modifications such as phosphorylation. In this regard, changes in kinase-dependent signaling cascades have been previously reported in mutant-htt-expressing cells (52)
.
Regarding the cell type in which P2X7 function is altered and at which BBG exerts its beneficial effect, it is obvious that neurons are affected and that they are involved in the protective effect of the antagonist. This is based on the alteration in P2X7-mediated calcium permeability observed in HD-cultured neurons and in synaptosomes from HD mice, on the fact that in vivo administration of BBG prevents the neuronal apoptosis seen in untreated HD mice, and on the fact that BBG also prevented the increased vulnerability to P2X7-mediated apoptosis observed in HD neuronal cultures devoid of glial cells. However, the possibility of the beneficial action of BBG being in part mediated also by glial cells can not be excluded. In this regard, P2X7 antagonists have been shown to exert anti-inflammatory effect in microglia (27
, 30)
and anti-inflammatory drugs, such as minocycline have also been postulated for treatment of HD (53)
, though concerns remain regarding its efficacy in animal models (54
, 55)
and its safety in humans (56)
. Therefore, apart from the mentioned neuronal effects, in vivo administration of BBG might also have produced an anti-inflammatory effect. However, this does not seem to be the case based on the fact that we did not observe decreased microgliosis or astrocytosis in BBG-administered HD mice.
Finally, the herein-reported therapeutic efficacy of the P2X7 antagonist BBG in HD mice strongly suggest potential of P2X7 antagonists for treatment of HD patients. BBG is slightly less potent at antagonizing human P2X7 receptor than at rodent receptors (43)
. However, other P2X7 antagonists such as KN62 are more potent at human P2X7 receptors (57)
. Regarding the more recent selective antagonist A-438079, its pharmacodynamics and bioavailability do not seem appropriate for chronic i.p. administration protocols. However, given the current research on its applicability for the treatment of other conditions such as chronic pain (58)
, it is conceivable that other routes of administration or close derivatives will be explored soon for chronic administration. Therefore, in view of our data and of the current state of research on new P2X7 antagonist, BBG probably has the best potential for translating into clinical studies. Notably, BBG might have the advantage of predicted low toxicity based on the safety of its analog Brilliant Blue-CFC that has been used as a food additive dye for decades (44)
. Preclinical studies are currently being conducted with BBG itself for ophthalmologic applications (59)
.
In summary, the biochemical, histological, calcium-imaging, and in vivo data presented here support the hypothesis that altered level and function of P2X7 receptor may contribute to HD pathogenesis and strongly suggest a therapeutic potential of P2X7 receptor antagonists for HD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 18, 2008. Accepted for publication January 8, 2009.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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