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NEUROPHARMACOLOGY
-Selective Agonists in Vitro and in VivoPharmacology Research Laboratories (A.I., S.Y., K.M., A.M., N.M.) and Exploratory Research Laboratories (Y.M., T.Y., M.M., Y.K.), Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
Received October 16, 2006; accepted December 12, 2006.
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Abstract |
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subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPAR agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPAR agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPAR agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPAR agonists could be useful tools for understanding the role of PPAR in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.
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, and . The PPAR is expressed in tissues, such as the brain, liver, kidney, spleen, fat skeletal muscle, digestive tube, skin, and placenta (Kliewer et al., 1994
; Braissant et al., 1996; Schoonjans et al., 1996). PPAR functions in adipocyte differentiation (Bastie et al., 1999), cerebral function (Xing et al., 1995). and epidermal differentiation (Matsuura et al., 1999). Approximately 90% of PPAR knockout mice are dead at viviparity, with survivors showing poor growth compared with wild type through viviparity and postdelivery (Barak et al., 2002). Furthermore, their brains are small in proportion to the body sizes, and myelin formation in the corpus callosum is abnormal (Peters et al., 2000). Therefore, it seems that PPAR plays an intimate role in development, lipid metabolism, cerebral myelin formation, and epidermal cell growth. However, PPAR may play a beneficial role in some aspect of tissue damage because it was reported that the PPAR-selective agonist L-165041 is highly protective in ischemic acute renal failure (Letavernier et al., 2005), and PPAR activation promotes renal cell survival following hypertonic stress (Hao et al., 2002).
In the brain, there is considerable expression of PPAR mRNA and protein, with expression at the cellular level in oligodendrocytes and neurons, suggesting a role for PPAR in both myelination and neuronal function within the central nervous system (CNS) (Woods et al., 2003). Although there are no studies that directly addressed the role of the PPAR in cerebral ischemia or neurodegenerative conditions such as Parkinson's disease (PD), some reports show that the PPAR agonist can protect cultured neurons from cell death (Cazevieille et al., 1993; Smith et al., 2004). The role of the individual PPAR subtypes, and any crossover between, in the process of neurodegeneration pathway has not yet been fully elucidated; however, to date, there are a number of studies showing that PPAR agonists may have beneficial therapeutic effects in animal models of cerebral infarction and PD (Breidert et al., 2002; Dehmer et al., 2004). The PPAR agonist, pioglitazone, attenuates the neurodegeneration in a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, although the protective action was suggested to be a consequence of its anti-inflammatory properties rather than any direct suppression of neuronal cell death (Breidert et al., 2002; Dehmer et al., 2004). The PPAR agonists pioglitazone and rosiglitazone also reduce infarct volume in rats following focal ischemia/reperfusion (Shimazu et al., 2005; Zhao et al., 2006); however, this again may not be due to direct suppression of neuronal cell death but rather an indirect protection occurring as a consequence of the anti-inflammatory properties of such PPAR agonists (Luo et al., 2006). Certain compounds with PPAR agonist properties have also been shown to suppress the progression of neurodegeneration in cerebral infarction (Deplanque et al., 2003) and in traumatic brain injury (Besson et al., 2005). Because the individual compounds with PPAR agonist activity also activate PPAR and/or PPAR to varying degrees, the inter-relationship between the agonist activity for each of the PPAR subtypes and the beneficial effects on neurodegeneration disease have not yet been fully clarified.
In this study, to explore the neuroprotective efficacy of PPAR agonists, we initially examined the subtype specificity of the PPAR agonists L-165041 and GW501516 in reporter gene assays and then evaluated their in vitro neuroprotective properties in an experimental model of cell death using human neuroblastoma SH-SY5Y cells. Cell damage was induced by exposure to thapsigargin, an endoplasmic reticular calcium ATPase inhibitor, 1-methyl-4-phenylpyridinium (MPP+), a dopaminergic neurotoxin, or staurosporine, a nonspecific PKC inhibitor. Endoplasmic reticular stress, which can be induced by thapsigargin, is thought to participate in neuronal cell death in diseases such as Alzheimer's disease and PD (Katayama et al., 1999; Imai et al., 2000) as well as stroke (Paschen, 1996). Staurosporine-induced PKC inhibition also reportedly plays an important part of in neurodegenerative processes in cerebral ischemia (Durkin et al., 1997). Therefore, the cell deaths induced by these neurotoxic agents, along with MPP+, are regarded as well established in vitro substitutes to model aspects of cerebral ischemia and PD. Furthermore, the neuroprotective properties of the PPAR agonists, L-165041 and GW501516, were also investigated in vivo using the rat model of transient focal ischemia and MPTP-induced PD model in mice.
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Materials and Methods |
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, , and were synthesized using polymerase chain reaction. The amplified cDNAs were cloned into the multiple cloning site of the pBIND vector (Promega, Madison, WI), resulting in the human and mouse Gal4-PPAR, , and fusion expression plasmid being constructed. The reporter plasmid pG5luc was also purchased from Promega.
Determination of PPAR Subtype Selectivity in a Reporter Gene Assay
The African green monkey fibroblast cell line CV-1 was obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained in DMEM supplemented with penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum. Cells were seeded at 1.5 x 106 cells in a 175-cm2 culture flask and left overnight before transient transfection (Lipofectamine Plus; Invitrogen Corporation, Carlsbad, CA) as described previously (Kojo et al., 2003), with 1 µg of each reporter gene (pG5luc) and PPAR expression plasmids (pBINDhPPAR, pBINDhPPAR, pBINDhPPAR, pBINDmPPAR, pBINDmPPAR, or pBINDmPPAR) together with the control Renilla luciferase expression plasmid RL-TK (Promega). A coactivator expression plasmid was also included when a transfection was performed for the PPAR assay (Kojo et al., 2003). The cells were harvested 4 h after transfection and replated 1.6 x 104 cells/well in a 96-well plate. The cells were then incubated at 37°C for a further 20 h and then treated with various concentrations of L-165041 or GW501516 for 5 h. Cell extracts were prepared, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) and the ARVOsx 1420 multilabel counter (PerkinElmer Life Science, Boston, MA). Firefly luciferase activity was normalized for transfection efficiency against the activity of the internal Renilla luciferase control.
Neuroprotective Efficacy in SH-SY5Y Cells
Cell Cultures. Human neuroblastoma SH-SY5Y cells were obtained from the European Collection of Animal Cell Cultures (Wiltshire, UK). SH-SY5Y cells were maintained in DMEM supplemented with 10% fetal bovine serum and a 1% (v/v) penicillin-streptomycin antibiotic mixture. Cells were grown in an atmosphere of 95% air and 5% CO2 at 37°C. For all cell death experiments, cells were seeded at a density of 7 x 104 cells/well in 96-well culture plates and allowed to attach overnight.
Drug Treatment. L-165041 or GW501516 were dissolved in 100% dimethyl sulfoxide at a concentration of 0.1 M and then diluted in DMEM without serum. Each solution was added to the culture plate 2 h before thapsigargin, MPP+, or staurosporine exposure.
Determination of Cell Viability. SH-SY5Y cells were plated into 96-well plates at 7 x 104 cells/well. Twenty-four hours after plating the SH-SY5Y cells, the media were replaced with a serum-free solution, and the cells were pretreated (2 h) with the appropriate concentration of each PPAR agonist. The cultures were then challenged with thapsigargin (100 nM), MPP+ (3 mM), or staurosporine (150 nM) for 24 h, and cell viability was measured using the Cell Titer 96 Aqueous One Solution Proliferation Assay kit (Promega). In brief, the culture medium was replaced with 100 µl of fresh medium, 20 µl of Cell Titer 96 Aqueous One Solution Reagent was added to each well, and the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 2 h. The absorbance of each well was measured at 490 nm with a Wallac 1420 ARVOsx (PerkinElmer Life Science). In addition, cytotoxicity was quantified by measuring lactate dehydrogenase (LDH) release using a colorimetric LDH assay kit (Roche Molecular Biochemicals, Indianapolis, IN).
Caspase-3 Activity Assay. SH-SY5Y cells were plated into 96-well plates at 7 x 104 cells/well. The cells were pretreated for 2 h with L-165041 or GW501516. The cultures were then challenged with 100 nM thapsigargin, 3 mM MPP+, or 150 nM staurosporine for 3 h. Caspase-3 activity was measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega). The culture medium was replaced with 25 µl of fresh medium, and 25 µl of homogeneous caspase-3/7 reagent was added. The plates were incubated at room temperature for 2 h. Caspase-3/7 activity was analyzed using a Wallac 1420 ARVOsx with appropriate excitation (485-nm) and emission (535-nm) filters.
Focal Cerebral Ischemia Models in Rats
Animals. Male Wistar rats, 9 to 10 weeks old, weighing 250 to 280 g, from Charles River (Hino, Japan) were used. All animals were housed in a room maintained at 23 ± 2°C with 55 ± 5% humidity and a 12-h light/dark cycle (lights on at 7:00 AM). The rats were housed three per cage, allowed free access to food and water, and left at least 1 week before experimentation. All experiments were performed under the guidelines of the Experimental Laboratory Animal Committee of Astellas Pharma Inc. and were in strict accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the number of animals used and the stress to the animals during experimental procedures.
Transient Focal Ischemia. Transient occlusion of the right middle cerebral artery (MCA) was induced by insertion of a nylon suture through the right internal carotid artery to the origin of the MCA as described previously (Iwashita et al., 2004a). In brief, rats were anesthetized with a mixture of 4.0% halothane and oxygen-nitrogen (30% oxygen and 70% nitrogen), with anesthesia maintained during the procedure with a mixture of 1.5% halothane and oxygen-nitrogen. Each animal was placed in the supine position, and a midline incision was made through the skin of the neck. The right common carotid artery (CCA) was exposed with careful protection of the vagus nerve. The external carotid artery, internal carotid artery (ICA), and CCA were carefully isolated and maintained in a "Y" shape using silk sutures. After ligation of the external carotid artery and CCA, an incision to insert a monofilament was made at the bifurcation. The monofilament was a 19-mm-long, 4-0 nylon surgical suture (Nicchou, Tokyo, Japan) coated with silicone (Xantopren L; Heraeus Kulzer, Dormagen, Germany) to thicken the distal 5 mm to approximately 0.4 mm in diameter. The proximal tip of the monofilament was heated to create a globular stopper for ease of removal. The monofilament was introduced into the lumen of the ICA. As a consequence, the monofilament passed through the origin of the MCA and thereby occluded it. A silk suture was tied around the ICA to immobilize the monofilament, the neck wound was closed, and animals were allowed to recover from anesthesia. Ninety minutes after MCA occlusion, each animal was reanesthetized, the neck wound was reopened, the monofilament was removed, and blood flow to the MCA resumed before closure of the neck wound.
Drug Administration. Rats were chronically implanted with a stainless steel cannula inserted into the right lateral cerebral ventricle to facilitate i.c.v. administration at the following coordinates: 0.8 mm posterior to the bregma, 1.5 mm lateral to the midsagittal suture, and 4.0 mm ventral to the skull. L-165041 or GW501516 were dissolved in polyethylene glycol 300 to concentrations of 1 or 10 mg/ml and administered by i.c.v. infusion into the right lateral ventricle using an Alzet micro-osmotic pump (model 1003D) 24 h before MCA occlusion. The infusion volume was adjusted to 1 µl/h; thus, the drugs were administered at either 24 or 240 µg/day over a 2-day period.
Measurement of Brain Damage. Twenty-four hours after reperfusion, each rat was anesthetized with i.p. administration of sodium pentobarbital (50 mg/kg), and the brain was removed after transcardial perfusion with heparinized saline, then cooled in ice-cold saline. The brain was then cut into 2-mm coronal slices using a brain microslicer at +4, +2, 0, -2, -4, and -6 mm from bregma. Brain sections were then freshly stained with a 2% triphenyltetrazolium chloride solution at 37°C for 30 min. To correct for swelling due to brain edema or atrophy caused by tissue damage, the damaged area was quantified by comparing the ratio of the whole area of the left cerebral hemisphere with that of the right cerebral hemisphere. The area of ischemic damage was quantified using an image analyzer system. The total area of damage was determined by summing up the damaged area from six sections and then presented as a percentage of the damaged area relative to the whole coronal area.
Pharmacokinetic Study in Rats
Measurement of the concentration of L-165041 and GW501516 in plasma and brain was performed in rats following i.p. administration of a 10 mg/kg dose in a volume of 2 ml/kg (dissolved in polyethylene glycol 300). At 0.5, 1, 3, and 6 h after dosing, blood was collected from the ventral aorta. After removal of the blood from the brain vessel performed by carotid brain perfusion with saline, the brain was collected, and homogenate samples were prepared. A 200-µl plasma sample or a 500-µl brain sample was homogenized with 5.5 ml of saline and further diluted/homogenized in 100 µl of methanol and 5 ml of diethyl ether solution. Each supernatant was subsequently evaporated under a gentle stream of nitrogen, and the dried residue was reconstituted in 100 µl of methanol. A 20-µl volume of reconstitute was injected onto a column (Cadenza CD-C18, 2 x 50 mm) and detected using an Alliance 2790 HPLC system (Waters, Milford, MA) coupled to a single-quadrupole Waters ZMD mass spectrometer (liquid chromatography-mass spectrometry).
MPTP-Induced Parkinson's Disease Model in Mice
Animals. For the MPTP model, 9- to 10-week-old male C57/BL6 mice weighing 19 to 22 g from Charles River were used. Mice were housed in a room maintained at 23 ± 2°C with 55 ± 5% humidity and with a 12-h light/dark cycle (lights on at 7:00 AM). The minimum quarantine period was at least 2 weeks before the experiment. Animals were housed five per cage and allowed free access to food and water, with all experiments again performed in accordance with the Astellas Pharma Inc. guidelines described above. Again, all efforts were made to minimize both the number of animals used and any stress to the animals caused during the experimental procedures.
Administration of MPTP and PPAR Agonists. We used the experimental paradigm as described by Iwashita et al. (2004b) with some minor modifications. In this study, the mice received two 20 mg/kg MPTP i.p. injections with a 2-h interval. Before MPTP treatment, all mice were implanted with an L-shaped cannula, which was inserted into the left lateral cerebral ventricle according to the following coordinates: 0.0 mm posterior to the bregma, 1.2 mm lateral to the midsagittal suture, and 2.5 mm ventral to the skull. L-165041 or GW501516, which was dissolved with 30% dimethyl sulfoxide/saline at concentrations of 1 or 10 mg/ml, was administered by i.c.v. infusion into the right lateral ventricle using an Alzet micro-osmotic pump (model 1007D) at 48 h before MPTP treatment. The infusion volume was adjusted to 0.5 µl/h, with the drugs thus administered at 12 or 120 µg/day over a 6-day period.
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Statistical Analysis
All values are expressed as mean ± S.E.M. Statistical analysis was performed out using a Student's t test comparing two groups and by using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test for the multiple drug-treated groups versus the control group. P < 0.05 was considered to be significant.
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Results |
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Agonists in the Reporter Gene Assay agonists L-165041 and GW501516 exhibited subtype selectivity for mouse and human PPAR.In the mouse PPAR reporter gene assay, L-165041 and GW501516 increased reporter activity at concentrations higher than 5 x 10-6 and 10-8 M, respectively. However, for PPAR and PPAR, L-165041 and GW501516 did not induce the activation at a concentration up to 10-5 and 10-6 M, respectively (Fig. 2, A and B). In human PPAR reporter gene assay, L-165041 and GW501516 increased reporter activity at higher concentrations than 5 x 10-7 M and 10-9 M, respectively, but did not induce PPAR or PPAR activity at similar concentrations (Fig. 2, C and D). Thus, L-165041 was >100-fold selective, and GW501516 was >1000-fold selective for both mouse and human PPAR over other subtypes (Fig. 2).
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Agonists. The effects of the PPAR agonists L-165041 and GW501516 on SH-SY5Y cell death induced by three cytotoxic (thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane; MPP+, a dopaminergic neurotoxin; and the protein kinase inhibitor, staurosporine) agents were examined. In the present) experimental conditions, thapsigargin (100 nM), MPP+) (3 mM), and staurosporine (150 nM) all induced marked cell death 24 h after exposure (Figs. 3, 4, 5). In the thapsigargin-induced cell death studies, both L-165041 and GW501516 produced a concentration-dependent attenuation of cell damage in both the MTT and LDH assays (Fig. 3, A to D). Moreover, GW501516 significantly and concentration-dependently attenuated the cell death induced by MPP+ (Fig. 4, A to D) or staurosporine (Fig. 5, A to D) in both the MTT and LDH assays. L-165041 similarly attenuated MPP+-induced cell damage, whereas the dose-response curve was bell-shaped (Fig. 4, A and C). Finally, L-165041 significantly attenuated staurosporine-induced cell damage in the MTT assay (Fig. 5A), although there was no significant effect in the LDH assay (Fig. 5C).
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Agonists against Caspase Activation. To investigate the basis of the antiapoptotic effects of PPAR agonists, caspase-3/-7 activity was assessed in SH-SY5Y cells. Caspase-3/-7 was significantly activated by thapsigargin (Fig. 3, E and F), MPP+ (Fig. 4, E and F), and staurosporine (Fig. 5, E and F). L-165041 and GW501516 treatment significantly and concentration-dependently suppressed the caspase-3/-7 activation after thapsigargin or staurosporine (Figs. 3, E and F, and 5, E and F). L-165041 treatment showed concentration-dependent attenuation of caspase-3/-7 activation after MPP+ (Fig. 4E), whereas GW501516 treatment suppressed in a bell-shaped fashion (Fig. 4F).
Pharmacokinetic Study
The plasma and brain concentrations of L-165041 and GW501516 in rats were determined at 0.5, 1, 3, and 6 h following i.p. administration at 10 mg/kg. Mean plasma concentrations of Cmax were 11.9 and 10.7 µg/ml, respectively, and the plasma concentrations were still high 6 h after dosing (1.64 and 2.35 µg/ml, respectively) (Table 1). However, the highest concentration of these compounds was less than 0.1 µg/g tissue in the brain; thus, Kp values were less than 0.01. Therefore, to ensure that sufficient concentrations of PPAR agonists are attained to assess the neuroprotective properties of the compounds in vivo, both agonists were administered by i.c.v. infusion using Alzet miniosmotic pumps in subsequent in vivo studies.
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Neuroprotective Action of PPAR Agonists in a Rat Model of Focal Cerebral Ischemia
Regional cerebral blood flow (rCBF) decreased to 21.3 ± 3.7% (n = 9) of the baseline level immediately after MCA occlusion, and this decrease was sustained during 90 min of ischemia in the control group. After reperfusion, rCBF increased to 95 to 
100% of baseline within 5 min. In the control group, the dorsolateral cortex and basal ganglia showed extensive damage that could be clearly differentiated from normally perfused area (data not shown). The volume of ischemic brain infarction in the cerebral cortex and subcortex in the control group was 179.60 ± 13.19 and 134.21 ± 11.02 mm3, respectively. Both L-165041 and GW501516 dose-dependently reduced the size of the infarcted cortical area when chronically infused using an Alzet osmotic pump from 24 h before MCA occlusion to 24 h after reperfusion (Fig. 6). Cortical damage was reduced by 27 and 48% at the doses of 240 µg/day L-165041 and GW501516, respectively, and the changes were statistically significant (P < 0.05 by one-way ANOVA followed by Dunnett's multiple comparison test). The protection by GW501516 at the dose of 240 µg/day was also statistically significant in total infarct size (36% reduction) compared with the control group (P < 0.05 by one-way ANOVA followed by Dunnett's multiple comparison test).
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Agonists in a Mouse MPTP Model agonists exert any neuroprotective effects in the mouse MPTP model, L-165041 and GW501516 were evaluated using a two-dose paradigm of MPTP delivery, as indicated in Fig. 7A. Administration of MPTP in this model (2 x 20 mg/kg i.p., 2 h apart) resulted in moderate reduction (approximately 40% of normal levels) of striatal DA content (Fig. 7B). L-165041 or GW501516 were administered by i.c.v. infusion from 48 h before the first injection of MPTP, and after 4 days, each striatum was dissected to quantify the level of DA and its metabolites. Both L-165041 and GW501516 dose-dependently attenuated the depletion of striatal DA, DOPAC, and HVA contents (Fig. 7B). In normal mice, L-165041 and GW501516 had no effects on DA and its metabolites (data not shown).
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Discussion |
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agonists on cytotoxin-induced neuronal damage in vitro and in vivo on brain injury associated with transient focal ischemia in rats and on MPTP-induced dopaminergic neuronal damage in mice. We first confirmed that L-165041 was a selective human and mouse PPAR agonist, with little/no effect on PPAR or PPAR (>100-fold higher selectivity). It has been reported that L-165041 binds to both PPAR and PPAR; however, its affinity for PPAR (Ki = 730 nM) is much weaker than that for PPAR (Ki = 6 nM) (Leibowitz et al., 2000), which is consistent with the present data. GW501516 is also known to be a high-affinity (Ki = 1.1 nM) and selective (> 1000-fold) PPAR agonist, which exerts a high expression induction activity when the agonist activity is assayed by the GAL4-responsive reporter gene (EC50 = 1.2 nM; Oliver et al., 2001). Therefore, both compounds seem to be highly selective PPAR agonists that can be used to investigate the functional roles of this receptor subtype both in vitro and in vivo.
To examine the neuroprotective properties of L-165041 and GW501516 in cultured cells, the endoplasmic reticular calcium ATPase inhibitor, thapsigargin, the dopaminergic neurotoxin, MPP+, and the PKC inhibitor, staurosporine, were used because these are well established in vitro substitutes that model some of the aspects of cerebral ischemia and PD. In our SH-SY5Y cell assay, GW501516 more potently attentuated the cell damage than L-165041 for all three neurotoxic conditions. The level of neuroprotective potency seemed to correlate with the compound PPAR agonist activity in the human PPAR reporter gene assay. Furthermore, we have recently identified and synthesized some novel PPAR agonists with their potency in activating PPAR agonist activity correlated with their neuroprotective properties in the cell death assays (A. Iwashita, unpublished data). This suggests that GW501516 and L-165041 possess superior neuroprotective properties as a consequence of their PPAR agonist activity and adequate cell membrane permeability.
In our in vitro cell death model, the neuroprotective effects of the selective PPAR agonists were closely correlated with caspase-3/7 inhibitory activity, suggesting that the PPAR agonists possess potent antiapoptotic properties. It was recently reported that PPAR activation stimulates promoter activity of the gene coding the 14-3-3 protein (Liou et al., 2006). Up-regulation of the 14-3-3 protein amplifies phosphorylated Bad binding, sequesters Bad in the cytosol, and results in reduced Bad translocation to the mitochondria, ultimately inhibiting cytochrome c release, caspase-3 activation, and the apoptosis (Liou et al., 2006). Furthermore, the 14-3-3 protein is most abundantly expressed in neurons of the central nervous system, and its binding to Bad is known to inhibit the apoptotic process (Berg et al., 2002). As shown in our present study, the cell death induced by MPP+, thapsigargin, and staurosporine seemed to occur as a result of caspase-3 activation. Considering that apoptotic cell death is associated with phosphorylated Bad, we speculate that the 14-3-3 protein is up-regulated by PPAR activation and that this could be one possible mechanism underlying the antiapoptotic properties of PPAR-selective agonists.
In addition to in vitro neuroprotection, L-165041 and GW501516 produced significant cerebroprotection following focal cerebral ischemia. The neuroprotective properties of GW501516 and L-165041 in the focal ischemia model are consistent with their PPAR agonist activity in the models of in vitro cell death. The neuroprotective effect of the PPAR agonists was more prominent in the cortex than in the striatum, in accordance with previous reports evaluating other neuroprotectants in transient ischemia models (Lo et al., 1998; Iwashita et al., 2003, 2004a). Although the putative mechanism of neuroprotection by L-165041 and GW501516 presumably involves activation of PPAR, infarct volume can be often influenced by other nonspecific effects like improving rCBF or hypothermia. However, the lack of changes in rCBF and in body temperature in PPAR agonist-treated rats (data not shown) suggest that it is unlikely that any hemodynamic actions or hypothermic actions of PPAR agonists contributed to their beneficial neuroprotective actions. Therefore, the present data indicate that PPAR plays an important role in brain damage after transient ischemia.
In the final set of studies, PPAR-selective agonists also seemed to be neuroprotective in the MPTP-induced model of dopaminergic neurodegeneration. In our previous study, we confirmed that the reduction of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta induced by MPTP treatment is well correlated with the decrease in DA and metabolite content in the striatum (Iwashita et al., 2004b). These results support the notion that the quantification of DA content is a suitable substitute to quantifying the dopaminergic cell death by using tyrosine hydroxylase immunonostaining. In the present study, treatment with L-165041 or GW501516 dose-dependently ameliorated the depletion of DA and its metabolites. These data indicate that the PPAR agonists rescued dopaminergic neurons in the substantia nigra par compacta from MPTP neurotoxicity, which is compatible with our in vitro results showing that L-165041 and GW501516 were neuroprotective in the MPP+-induced model of cell death.
Because we have observed that both GW501516 and L-165041 have very poor ability in terms of brain penetration and no other brain-penetrable PPAR-selective agonists have been reported, we have employed direct infusion of drugs in both animal studies to validate the functional role of PPAR in neurodegeneration in vivo. The question was whether the effective drug concentrations in in vivo animal models were relevant to those observed in in vitro neuroprotective assay, and one assumption could be made as follows. If i.c.v. infused drug solution diffused widespread the brain parenchyma in rats, the drug concentration of 240 µg/day is approximately equal to 240 µg/ml in the brain, and the concentration is calculated to be 5 x 10-4 M considering the brain volume. There is a report that the protein binding of GW501516 was >99% (Pelton, 2006). On the assumption that the ratio of protein binding in GW501516 is approximately 99% both in rats and mice, the free unbound drug concentration in the brain could be approximately 5 x 10-6 M. Therefore, it is reasonable to assume that the effective concentration of the drug in vitro and in vivo could be comparable in the present study. Nevertheless, further studies are warranted in which we need to address efficacy following systemic administration, as well as examining cell permeability and pharmacokinetic properties of other PPAR agonists that have better brain penetration. Another limitation of the present methodology due to the drug's poor brain penetration was that we were able to validate only the preventive efficacy of PPAR agonists. Thus, future studies should be also directed to clarify the therapeutic time window of PPAR agonists in stroke and PD animal models when better CNS PPAR agonists will be available and will be treated after the insults.
To date, several other subtypes of PPAR agonists have been reported, with the PPAR agonist pioglitazone and rosiglitazone reportedly able to decrease ischemic injury in a model of MCA occlusion (Shimazu et al., 2005; Luo et al., 2006; Zhao et al., 2006). Pioglitazone was also shown to be neuroprotective against MPTP neurotoxicity (Breidert et al., 2002; Dehmer et al., 2004). Previous studies indicate that in the CNS, PPAR is expressed at appreciable levels in microglia and astrocytes, both of which regulate inflammatory responses in the brain (Bernardo et al., 2000; Cristiano et al., 2001). These results suggest that the activation of PPAR may indirectly affect neuronal cells, perhaps via an anti-inflammatory mechanism. In contrast, PPAR is relatively highly expressed on neurons compared with the other PPAR subtypes (Lemberger et al., 1996). Because the present in vitro studies show that PPAR agonists directly interact with neuronal cell to suppress cell death, we suggest that PPAR agonists may be efficacious in a wide range of neurodegenerative diseases compared with PPAR agonists.
It has been reported that PPAR agonists such as fenofibrate were able to decrease ischemic injury in a rat model of MCA occlusion (Deplanque et al., 2003). In preliminary studies, we have compared the potency and neuroprotective efficacy of another reference PPAR agonist, Wy14643 (pirinixic acid), with those of PPAR agonists in both the in vitro cell death assay and the rat transient focal ischemia model. GW501516 was the most potent PPAR agonist among the PPAR ligands examined, with this agonist also again the most potent neuroprotective compound in the thapsigargin-induced cell death model (A. Iwashita, unpublished data). In the in vivo ischemia model, GW501516 treatment robustly reduced infarct volume, with the efficacy of this compound greater than that of the PPAR agonists examined (data not shown). Although it would be simple to conclude that PPAR activation will therefore produce neuroprotection, extensive investigations will be required to address whether these properties are dependent upon PPAR activation alone and how PPAR activation ameliorates cell death.
In conclusion, the present studies elucidate for the first time that PPAR-selective agonists are neuroprotective in vivo in models of cerebral infarction and Parkinson's disease. The present results further implicate that blood-brain barrier-penetrable PPAR agonists are useful tools for investigating the role of PPAR in other neurodegenerative disorders and that such compounds are attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; CNS, central nervous system; PD, Parkinson's disease; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; L-165041, 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid; GW501516, [2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid]; MPP+, 1-methyl-4-phenylpyridinium; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; LDH, lactate dehydrogenase; MCA, middle cerebral artery; CCA, common carotid artery; ICA, internal carotid artery; HPLC, high-performance liquid chromatography; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; ANOVA, analysis of variance; rCBF, regional cerebral blood flow; Wy14643, 4-chloro-6-(2,3-xylidine)-2-pyrimidinylthioacetic acid.
Address correspondence to: Dr. Akinori Iwashita, Neuroscience Discovery Research, Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. E-mail: akinori.iwashita{at}jp.astellas.com
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