The pathogenesis of sporadic cerebellar ataxia remains unknown. In this study, we demonstrate that proinflammatory cytokines, IL-18 and IL-1β, reciprocally regulate kainate-induced cerebellar ataxia in mice. We show that systemic administration of kainate activated IL-1β and IL-18 predominantly in the cerebellum of mice, which was accompanied with ataxia. Mice deficient in caspase-1, IL-1R type I, or MyD88 were resistant to kainate-induced ataxia, while IL-18- or IL-18R α-deficient mice displayed significant delay of recovery from ataxia. A direct intracerebellar injection of IL-1β-induced ataxia and intracerebellar coinjection of IL-18 counteracted the effect of IL-1β. Our data firstly show that IL-18 and IL-1β display differential direct regulation in kainate-induced ataxia in mice. Our results might contribute toward the development of a new therapeutic strategy for cerebellar ataxia in humans.

Kainate, an excitatory amino acid extracted from seaweed, has significantly contributed to understanding epileptogenesis (1). Previously, the effects of kainate on hippocampal neurons have been studied for delineating the mechanism of kainate-induced ataxia (1, 2). It is reported that l-glutamate, the major excitatory neurotransmitter in the brain, acts on three classes of ionotropic glutamate receptors: N-methyl-D-aspartate, α-amino-hydroxy-5-methyl-4-isoxazole propionic acid, and kainate receptors (3, 4). Kainate receptors consist of a set of genes (GluR5–7, KA-1, and KA-2), are widely distributed throughout the brain (5, 6, 7, 8, 9, 10), and are implicated in epileptogenesis and neuronal cell death (11).

IL-1β and IL-18 are proinflammatory cytokines that are produced as a precursor form and proteolytically activated by caspase-1 (12). They are expressed in various tissues including the CNS (13, 14, 15). IL-1β is shown to exert neuroendocrine as well as neurodegenerative effects on animals (14, 15). It has been reported that convulsant stimuli increase the production of IL-1β and its receptor in rodent CNS within hours of seizure induction (16, 17, 18). Recently, Vezzani et al. reported that IL-1β prolongs hippocampal seizures in a N-methyl-D-aspartate receptor-dependent manner, and the action was inhibited by IL-1 receptor antagonist (IL-1ra)3 (19, 20). Concerning IL-18, a crucial role for IL-18 in mediating neuroinflammation and neurodegeneration in the CNS under pathological conditions has been indicated (21).

Cerebellar ataxia, dysfunction of the cerebellum, causes problems such as loss of balance and motor coordination. Some types of cerebellar ataxia can be caused by several genetic mutations, including a group of autosomal dominant spinocerebellar ataxias (22) and autosomal recessive Ataxia telangiectasia (23); however, a large number of patients remain undiagnosed (sporadic cerebellar ataxias). In this study, we examined the roles of IL-1β and IL-18 in kainate-induced ataxia in mice. We demonstrated that IL-1β is activated specifically in the cerebellum by the systemic administration of kainate and is involved in kainate-induced ataxia in mice. Furthermore, we show that IL-18 in the cerebellum is involved in the recovery phase of kainate-induced ataxia by counteracting the function of IL-1β in the cerebellum. Our results show the possible anti-ataxic effect of IL-18 and may suggest new therapeutic strategies for cerebellar ataxia in humans.

Abs to GluR-5, GluR-6, IL-1β, IL-18, IL-1RI, calbindin, and glial fibrillary acidic protein were purchased from Santa Cruz Biotech; Abs to IL-18R and ST2L were purchased from R&D Systems; and an Ab to IL-33 were purchased from Alexa Biochem. Alexa488- or and Alexa564-conjugated anti-IgG were purchased from Molecular Probes.

Six- to 10-wk-old male mice were used in this study. BALB/c mice and C57BL/6 mice were purchased from Sankyo Laboratories. IL-1RI−/− mice with a C57BL/6 × 129 background and IL-18−/− mice with a C57BL/6 background were purchased from The Jackson Laboratory, and caspase-1−/− mice (24) with a BALB/c background, IL-18Rα−/− mice (25) with a C57BL/6 background, and MyD88−/− mice with a C57BL/6 background were provided by Dr. K. Kuida (Vertex Pharmaceuticals, Cambridge, MA) (24), Dr. T. Hoshino (Kurume University School of Medicine, Fukuoka, Japan) (25), and S. Akira (WPI Immunology Frontier Research Center, Osaka, Japan), respectively. These mice were maintained in our animal facility. Mice were housed under controlled temperature (23–25°C) and light (lights on from 08:00 h to 20:00 h) conditions. Food and water were freely available. The procedures for these animal experiments were reviewed and approved by the Committee for Animal Experiments at the University of Toyama.

Mice were lightly anesthetized with ethyl alcohol (wake up time: <20 s). The solution (0.5 μl) was injected into the center of the cerebellum using a 27G needle with a stopper held ∼2 mm from the top of the needle and a microsyringe pump system.

To estimate the effect of kainate and the other reagents on behavioral activity, a rotarod test was performed. To this end, Rota-Rod Treadmill (Ugo Basile) that consists of a gritted plastic rod flanked by two large round plates was used. Before performing the test, mice were trained on the rotarod until they reached a stable performance in this test (>120 s on rotarod) for one or two days before experiments. The test session was performed in accordance with the training session. Mice were brought to the experimental room at least 1 h before the experiment, and then placed on the accelerating rotarod apparatus (Ugo Basile Accelerating Rota-Rod “Jones & Roberts” for Rats 7750) with an initial speed of four rotations per minute; thereafter, the speed gradually increased to 60 rotations per minute. The time that the mouse remained on the rod was measured. A maximum of 120 s was allowed to test each animal. In the rotarod test, mice were pretreated with reagents or the vehicle, and the experiment was started 20 min after treatment.

Cell lysates were prepared by homogenizing the tissues (cerebral cortex, cerebellum, hippocampus, or spinal cord) in lysis buffer, centrifuged at 10,000 × g for 5 min at 4°C, and the supernatant was harvested. After determining the protein concentration, the cell lysates were used to measure the activity of caspase-1 using N-acetyl-Tyr-Val-Ala-Asp-P-nitroanilide as a substrate (Medical and Biological Laboratories). The reaction mixture was incubated for 2 h at 37°C. Caspase-1 activity was monitored with the absorbance at 420 nm, reacting chromophore P-nitroanilide.

Tissue samples (cerebral cortex, cerebellum, hippocampus, spinal cord) were homogenized in cell lysis buffer containing 137 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, and 1 μg/ml leupeptin. The total protein concentration was determined using a Bio-Rad protein assay kit. Proteins were separated by electrophoresis using a 10 or 16% SDS-polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. The membrane was preincubated with a 5% skim milk solution for 1 h at room temperature, then incubated with primary Ab to either IL-1β or IL-18 at 4°C overnight, and subsequently incubated with a HRP-linked Ab against either mouse, rabbit, or goat IgG for 1 h. Membrane-bound HRP-labeled protein bands were reacted with a chemiluminescence detection solution (Amersham Biosciences). Chemiluminescent signals were detected using x-ray film. The amount of active IL-1β and IL-18 was evaluated by measuring the density of the active form cleaved by caspase-1 (17kD and 18 kD, respectively) using the National Institutes of Health Image program. The data were normalized with β-actin.

Mice were anesthetized with ethyl carbamate (1.5g/kg, i.p.) and perfused with 4% paraformaldehyde following PBS (pH 7.4). The mouse cerebellum was then isolated. The tissue was soaked in 4% paraformaldehyde for 4 h and then in 30% sucrose solution overnight at 4°C. A section (30 μm) was prepared using a cryostat. After washing with PBS, the section was soaked in PBS containing 0.3% Triton X-100 for 30 min and then in PBS containing 0.1% FBS for 30 min. The tissue section was incubated with the first Ab to either GluR-5, GluR-6, IL-1, IL-18, IL-1RI, IL-18R, calbindin, or glial fibrillary acidic protein overnight at 4°C, then washed with PBS, and reacted with Alexa488- or Alexa564-conjugated anti-IgG for 2 h. After washing with PBS, the sections were mounted with PBS/glycerol containing 0.05% triethylenediamine. Fluorescent signals were detected using a confocal microscope (Bio-Rad).

All data are represented as the mean ± SEM. Statistical significance was analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons or Student’s t test; p < 0.05 was considered significant.

The effect of kainate on motor coordination in mice was examined with the rotarod test (26), as judged by a decrease in latency before falling from a rotating rod. An i.p. injection of kainate (20 mg/kg), but not saline as a vehicle, induced ataxic gait (Fig. 1,A). The retention time on the rotating rod was decreased at 20 min and the effect was almost abolished by 90 min after injection (Fig. 1,A). Because IL-1β is indicated to play a critical role in seizures (16, 17, 18, 19, 20) and kainate is reported to increase in IL-1β transcripts in several regions of the brain (27), we considered whether kainate-induced ataxia might be mediated by IL-1β. To verify this possibility, we first examined the effect of kainate on caspase-1−/− mice in which IL-1β cannot be activated. Interestingly enough, kainate showed almost no effect on motor coordination in caspase-1−/− mice (Fig. 1,B), suggesting that IL-1β is involved in kainate-induced ataxia. We then examined the effect of kainate injection in IL-1 receptor type I (IL-1RI)−/− mice. We found that kainate showed little effect, if any, on the motor coordination of IL-1RI−/− mice (Fig. 1,C). Because MyD88 is reported to play a central role in IL-1β-mediated signal transduction (28), we then examined the effect of kainate in mice deficient in MyD88. We found that the effect of kainate was limited in MyD88−/− mice as seen in IL-1RI−/− mice (Fig. 1,D). Next, we examined whether the difference of ages and genetic backgrounds showed the different susceptibility to kainate-induced ataxia using various ages (six to 10 wk old) of C57BL/6 (Fig. 1,E) or BALB/c mice (Fig. 1 F), because we used caspase-1−/− mice on a BALB/c background and IL-1RI−/− or MyD88−/− mice on a C57BL/6 background of 6- to 10-wk-old. The result showed that the time courses of ataxia-induction and its recovery after systemic administration of kainate were corresponding in different ages of BALB/c or C57BL/6 mice. Taken together, these results indicate that i.p. injection of kainate induces ataxia gait in mice through the activation of caspase-1 and IL-1β.

FIGURE 1.

Deficiency with caspase-1, IL-1 receptor type I, or MyD88 attenuates the effect of kainate on inducing ataxia, but deficiency with IL-18 or its receptor delay the recovery from kainate-induced ataxia. A, Effect of kainate on locomotion activity in normal BALB/c mice was examined. Before kainate administration, a rotarod test was performed every 10 min (three times) and then kainate (KA) (20 mg/kg: •) or saline (○) was i.p. injected into BALB/c mice at time 0. A rotarod test was performed at 20, 40, 60, 90, and 120 min after kainate injection. ∗, p < 0.05 when compared with average data before kainate injection (n = 5). B–D, G, and H, Effect of kainate on locomotion activity in wild-type mice vs gene-targeted mice was examined. A rotarod test was performed using (B) caspase-1−/− (•) or wild-type (○) mice, (C) IL-1RI−/− (•) or wild-type (○) mice, (D) MyD88−/− (•) or wild-type (○) mice, (G) IL-18−/− (•) or wild-type (○) mice, and (H) IL-18R−/− mice or wild-type (○) mice as mentioned above. E and F, Effect of kainate on locomotion activity in mice with different ages and genetic backgrounds was examined. A rotarod test was performed using either C57BL/6 mice (E) or BALB/c mice (F) 6- to 10-wk old. At time 0, kainate was injected. ∗, p < 0.05 when compared with the average data before kainate injection (n = 5). #, p < 0.05 when compared with wild-type mice (n = 5). Data are represented as the mean ± SEM.

FIGURE 1.

Deficiency with caspase-1, IL-1 receptor type I, or MyD88 attenuates the effect of kainate on inducing ataxia, but deficiency with IL-18 or its receptor delay the recovery from kainate-induced ataxia. A, Effect of kainate on locomotion activity in normal BALB/c mice was examined. Before kainate administration, a rotarod test was performed every 10 min (three times) and then kainate (KA) (20 mg/kg: •) or saline (○) was i.p. injected into BALB/c mice at time 0. A rotarod test was performed at 20, 40, 60, 90, and 120 min after kainate injection. ∗, p < 0.05 when compared with average data before kainate injection (n = 5). B–D, G, and H, Effect of kainate on locomotion activity in wild-type mice vs gene-targeted mice was examined. A rotarod test was performed using (B) caspase-1−/− (•) or wild-type (○) mice, (C) IL-1RI−/− (•) or wild-type (○) mice, (D) MyD88−/− (•) or wild-type (○) mice, (G) IL-18−/− (•) or wild-type (○) mice, and (H) IL-18R−/− mice or wild-type (○) mice as mentioned above. E and F, Effect of kainate on locomotion activity in mice with different ages and genetic backgrounds was examined. A rotarod test was performed using either C57BL/6 mice (E) or BALB/c mice (F) 6- to 10-wk old. At time 0, kainate was injected. ∗, p < 0.05 when compared with the average data before kainate injection (n = 5). #, p < 0.05 when compared with wild-type mice (n = 5). Data are represented as the mean ± SEM.

Close modal

Next, we examined which region in the brain caspase-1 and IL-1β is activated. Caspase-1 activity was measured in various regions of the brain after kainate administration, and specifically increased in the cerebellum as well as hippocampus, but not in the cerebral cortex or spinal cord (Fig. 2,A). Kinetic study showed that activity in the cerebellum peaked within 20 min and returned to the basal level by 60–90 min, while activity in the hippocampus peaked within 60 min and fell to the basal level by 90 min (Fig. 2,A). Because ataxia was induced within 20 min after kainate administration, it is conceivable that kainate-induced activation of caspase-1 in the cerebellum evoked IL-1β activation and gait disturbance in mice. We then examined the expression of activated IL-1β at the protein level in various regions of the brain by Western blotting (Fig. 2,B). As the precursor form of IL-1β is processed with caspase-1, their active form can be detected by the decrease of their m.w. IL-1β was activated within 20 min after kainate injection in the cerebellum (Fig. 2 B), but was not detected in the cerebral cortex, hippocampus, or spinal cord (data not shown). Although it has been reported that kainate induced the increase in IL-1β mRNA in the cerebral cortex, thalamus, and hypothalamus (27), it remains unclear whether IL-1β is activated at the protein level in these loci. Our results show that systemic administration of kainate activates IL-1β predominantly in the cerebellum and indicates that this activation may play a critical role in kainate-induced ataxia.

FIGURE 2.

Systemic administration of kainate activates caspase-1, IL-1β, and IL-18 predominantly in cerebellum. Kainate (KA, 20 mg/kg) was i.p. injected into normal BALB/c mice and activity of caspase-1, IL-1β, and IL-18 was assessed. A, Activity of caspase-1 in cerebral cortex (○), cerebellum (▵), hippocampus (▪), and spinal cord (♦) was examined at various times after kainate-injection. Y-axis shows the percentage of caspase-1 activity compared with that at time 0. ∗, p < 0.05 when compared with the data at time 0 in each brain sample (n = 4). B, Caspase-1-processed protein levels of IL-1β and IL-18 in cerebellum were analyzed after systemic administration of kainate. Extracts from various parts of brain were separated on SDS-PAGE, and activated IL-1β (○) and IL-18 (•) were detected with immunoblotting. Y-axis shows the relative protein levels of activated IL-1β and IL-18 that are normalized with the protein level of β-actin (n = 3). Because significant levels of activated IL-1β and IL-18 were not detected in the cerebral cortex, hippocampus, and spinal cord, their data are omitted. Data are represented as the mean ± SEM.

FIGURE 2.

Systemic administration of kainate activates caspase-1, IL-1β, and IL-18 predominantly in cerebellum. Kainate (KA, 20 mg/kg) was i.p. injected into normal BALB/c mice and activity of caspase-1, IL-1β, and IL-18 was assessed. A, Activity of caspase-1 in cerebral cortex (○), cerebellum (▵), hippocampus (▪), and spinal cord (♦) was examined at various times after kainate-injection. Y-axis shows the percentage of caspase-1 activity compared with that at time 0. ∗, p < 0.05 when compared with the data at time 0 in each brain sample (n = 4). B, Caspase-1-processed protein levels of IL-1β and IL-18 in cerebellum were analyzed after systemic administration of kainate. Extracts from various parts of brain were separated on SDS-PAGE, and activated IL-1β (○) and IL-18 (•) were detected with immunoblotting. Y-axis shows the relative protein levels of activated IL-1β and IL-18 that are normalized with the protein level of β-actin (n = 3). Because significant levels of activated IL-1β and IL-18 were not detected in the cerebral cortex, hippocampus, and spinal cord, their data are omitted. Data are represented as the mean ± SEM.

Close modal

To verify the above possibility, we directly injected recombinant IL-1β into the cerebellum and examined the motor coordination. To confirm whether the intracerebellar injection was accurate, Evans blue solution (0.5 μl of 1% solution) was injected. As shown in Fig. 3,A, Evans blue solution was spread only in the field of the cerebellum. An intracerebellar injection of IL-1β (0.01–0.1 μg/site) induced ataxia in a dose-dependent manner (Fig. 3,B). As a negative control, we injected IL-6 (0.01 and 0.1 μg/site) intracerebellarly and observed no induction of ataxia (Fig. 3 C), which confirmed the IL-1β-specific ataxia induction.

FIGURE 3.

Intracerebellar injection of IL-1β induces ataxia, but intracerebellar injection of IL-18 together with IL-1β counteracted the effect of IL-1β in normal mice. A, Evans blue (0.5 μl) was intracerebellarly injected and its distribution was examined. B–F, Effect of intracerebellar injection of IL-1β, IL-18, or IL-6 on locomotion activity was examined. Various doses of IL-1β (B), IL-6 (C), IL-18 (D), or IL-18 with IL-1β (0.1 nmol/site) (E) were intracerebellarly injected and rotarod test was performed as mentioned in Fig. 1. F, Five min after intracerebellar injection of various doses of IL-18, kainate (KA, 20 mg/kg) was i.p. administered, and a rotarod test was performed. Y-axis shows the area under the curve (AUC) for 1 h after injection. ∗, p < 0.05 when compared with vehicle (VEH: saline)-treated group (n = 5). Dashed line shows the data from vehicle-treated group (E, without IL-1β; F, without kainate). Data are represented as the mean ± SEM.

FIGURE 3.

Intracerebellar injection of IL-1β induces ataxia, but intracerebellar injection of IL-18 together with IL-1β counteracted the effect of IL-1β in normal mice. A, Evans blue (0.5 μl) was intracerebellarly injected and its distribution was examined. B–F, Effect of intracerebellar injection of IL-1β, IL-18, or IL-6 on locomotion activity was examined. Various doses of IL-1β (B), IL-6 (C), IL-18 (D), or IL-18 with IL-1β (0.1 nmol/site) (E) were intracerebellarly injected and rotarod test was performed as mentioned in Fig. 1. F, Five min after intracerebellar injection of various doses of IL-18, kainate (KA, 20 mg/kg) was i.p. administered, and a rotarod test was performed. Y-axis shows the area under the curve (AUC) for 1 h after injection. ∗, p < 0.05 when compared with vehicle (VEH: saline)-treated group (n = 5). Dashed line shows the data from vehicle-treated group (E, without IL-1β; F, without kainate). Data are represented as the mean ± SEM.

Close modal

Because casapse-1 also activates IL-18 (29), we examined the activation of IL-18 in the cerebellum after kainate i.p. injection. Like IL-1β, IL-18 was also activated in the cerebellum (Fig. 2,B). The level of activated IL-18 in the cerebellum peaked within 40 min and activated IL-18 was still observed until 2 h after kainate injection. We then tested whether kainate induces ataxia in IL-18−/− or IL-18Rα−/− mice (Fig. 1, G and H). Systemic administration of kainate induced ataxia in these mice within 20 min, as seen in wild-type mice. To our surprise, recovery from the kainate-induced ataxia was significantly delayed in these mice. Ataxic gait was still observed after 2 h in IL-18−/− mice, and after 4 h in IL-18Rα−/− mice. The results posed the possibility that IL-18 may enhance the recovery phase of kainate-induced ataxia.

To assess the possibility described above, we examined the effect of intracerebellar injection of IL-18 with or without IL-1β. As shown in Fig. 3,D, an intracerebellar injection of only IL-18 (0.01–0.1 μg/site) did not induce ataxia. Interestingly enough, intracerebellar injection of IL-18 (0.01–0.1 μg/site) together with IL-1β dose-dependently inhibited IL-1β (0.1 μg/site)-induced ataxia (Fig. 3,E). Furthermore, pretreatment with IL-18 (0.01–0.1 μg/site, intracerebellarly, at −10 min) inhibited kainate-induced ataxia in a dose-dependent manner (Fig. 3 F). These results strongly indicate that IL-18 in the cerebellum may play an important role in the recovery phase of kainate-induced ataxia in mice by counteracting with IL-1β.

Finally, we investigated which cells in the cerebellum express IL-1R and IL-18R by immunohistochemistry. Fig. 4 shows that both IL-1R and IL-18R were expressed in Purkinje cells. Their specific expression was confirmed by staining tissue sections of either IL-1RI−/− mice or IL-18Rα−/− mice. We also examined the expression of kainate receptors, IL-1β, and IL-18 in the cerebellum by immunohistochemistry (Fig. 5). GluR5 was expressed in astrocytes (glial fibrillary acid protein-positive) as well as Purkinje cells (calbindin-positive), while GluR6 was mainly expressed in Purkinje cells. IL-1β was expressed mainly in Purkinje cells and IL-18 was expressed in both Purkinje cells and astrocytes. We could not clearly detect the expression of IL-1β or IL-18 in other cells, including microglias or granular cells.

FIGURE 4.

Immunohistochemical analysis of IL-1R and IL-18R in cerebellum is performed after the isolation of cerebellum from either normal mice, IL-1R−/− mice, or IL-18R−/− mice. The tissue section was stained with Ab to either IL-1RI or IL-18R, and then reacted with Alexa564-conjugated anti-rabbit or anti-goat IgG. To identify Purkinje cells, tissue sections were stained with Ab to calbindin, followed by Alexa488-conjugated anti-rabbit or anti-goat IgG. Fluorescent signals were detected using a confocal microscope. Scale bar, 100 μm.

FIGURE 4.

Immunohistochemical analysis of IL-1R and IL-18R in cerebellum is performed after the isolation of cerebellum from either normal mice, IL-1R−/− mice, or IL-18R−/− mice. The tissue section was stained with Ab to either IL-1RI or IL-18R, and then reacted with Alexa564-conjugated anti-rabbit or anti-goat IgG. To identify Purkinje cells, tissue sections were stained with Ab to calbindin, followed by Alexa488-conjugated anti-rabbit or anti-goat IgG. Fluorescent signals were detected using a confocal microscope. Scale bar, 100 μm.

Close modal
FIGURE 5.

Immunohistochemical analysis of kainate receptors, IL-1, IL-18, IL-1R, and IL-18R in cerebellum was performed after isolation of cerebellum from normal mice. The tissue section was prepared and stained with Ab to either GluR5, GluR6, IL-1β, IL-18, IL-1RI, IL-18Rα, calbindin, or glial fibrillary acid protein (GFAP), then followed with FITC-conjugated anti-IgG for 1 h. Fluorescent signals were detected using a confocal microscope. Scale bar, 100 μm.

FIGURE 5.

Immunohistochemical analysis of kainate receptors, IL-1, IL-18, IL-1R, and IL-18R in cerebellum was performed after isolation of cerebellum from normal mice. The tissue section was prepared and stained with Ab to either GluR5, GluR6, IL-1β, IL-18, IL-1RI, IL-18Rα, calbindin, or glial fibrillary acid protein (GFAP), then followed with FITC-conjugated anti-IgG for 1 h. Fluorescent signals were detected using a confocal microscope. Scale bar, 100 μm.

Close modal

Kainate receptors are reported to be expressed in various regions in the brain, including the cerebellum, in rodents (5), and it has been demonstrated that kainate induced the expression of IL-1β transcripts in various regions in the brain (27). In the present study, we showed the kainate-induced processing of IL-1β and IL-18 in the cerebellum, and demonstrated that IL-1β plays an important role in inducing kainate-triggered ataxia and that IL-18 has a positive regulatory role in recovery from kainate-induced ataxia.

This effect of kainate was really induced via a kainate receptor in the cerebellum since i.p. administration of 2-amino-5-hydroxy-5-methyl-4-isoxazolepropion acid/kainate-receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (0.3–3 mg/kg), and 6-cyano-7-nitroquinoxaline-2,3-dione (0.3–3 mg/kg), 30 min before systemic administration of kainate (20 mg/kg) injection specifically and dose-dependently inhibited kainate-induced ataxia (data not shown). Furthermore, intracerebellar injection of 6,7-dinitroquinoxaline-2,3-dione (0.25–0.75 μg/site) dose-dependently inhibited kainate-induced ataxia. These results indicate that the ataxia was induced specifically via 2-amino-5-hydroxy-5-methyl-4-isoxazolepropion acid/kainate receptors.

Resistance to kainate-induced ataxia of caspase-1-, IL-1R-, and MyD88-deficient mice (Fig. 1) suggested an important role of IL-1β in kainate-induced ataxia. Time course of caspase-1-activation and the resultant IL-1β-processing in the cerebellum was corresponding to that of kainate-induced ataxia (Fig. 1 and 2), and direct intracerebellar injection of IL-1β elicited the ataxia (Fig. 3). These results indicate the involvement of IL-1β in the cerebellum in kainate-induced ataxia. Regarding the relationship between IL-1β in the cerebellum and ataxia, it was reported that two ataxic mutant mice, staggerer mice with retinoic acid receptor-related orphan receptor (nuclear hormone receptor superfamily) deficiency (30) and lurcher mice with δ2-glutamate receptor deficiency (31) showed abnormal IL-1β expression in the cerebellum (32). In these mice, the neurodegenerative effect of IL-1β was attributed to the cerebellar ataxia. Concerning kainate, it was reported that kainate-induced ataxia was due to its neurodegenerative effect (2, 11). In this study, we examined apoptosis in the cerebellum at various times (30 min to 24 h) after kainate injection using TUNEL methods; however, we could not observe any apoptotic cells in the cerebellum (data not shown). The result indicates that although kainate affects various regions in the brain to induce neurodegenerative effects, the neurodegenerative effect may not be the direct cause in kainate-induced and IL-1β-mediated cerebellar ataxia in mice. The other possible effect of IL-1β on kainate-induced ataxia is its regulatory effect on neurotransmitter systems. IL-1 was reported to be involved in the regulation of inhibitory as well as excitatory neurotransmitter systems (33), while Purkinje cells were reported to express γ-amino-butyric acid, an inhibitory neurotransmitter (34). Of note, γ-amino-butyric acid was indicated to be involved in fast cerebellar oscillation associated with ataxia in a mouse model of Angelman syndrome (35). Taken together, it is assumed that IL-1 might induce the release of γ-amino-butyric acid from Purkinje cells and inhibits neuronal activity, including that of Purkinje cells. In this context, we are electrophysiologically examining the effect of kainate, IL-1β or IL-18, or both on Purkinje cells in the cerebellum.

Among the molecules that affect kainate-induced ataxia, caspase-1 deficiency showed the most complete abrogation of kainate-induced ataxia, while deficiency of either IL-1RI or MyD88 showed partial effect (Fig. 1). It is reported that more of the IL-1 cytokine family may be activated with caspase-1 (36). Recently, IL-1-like cytokine, IL-33, that was activated with caspase-1 and its receptor, ST2, was demonstrated to exert its effect on the induction of Th2-associated cytokines (37). With this regard, we detected the expression of IL-33 and ST2 in cerebellum by RT-PCR and immunohistochemical method (data not shown). Further investigation is necessary to clarify a role of ST2/IL-33 signaling including other caspase-1-activated IL-1-like cytokine signaling in kainate-induced ataxia in a future study.

Concerning MyD88-deficient mice, partial resistance to the effect of kainate suggests that signaling molecules other than MyD88 could be involved in kainate-induced caspase-1-dependent ataxia. In this respect, signaling of the IL-1R/TLR family is finely and sophisticatedly tuned with multiple signaling molecules (38). For example, Toll/IL-1R domain-containing adaptor inducing IFN-β-related adaptor molecule was reported to be involved in the TLR 4-mediated MyD88-independent signaling pathway, although MyD88 is involved in TLR 4 signaling (39).

It was reported that human purinergic receptor (P2X7) modulates IL-1β and IL-18 processing and their release in response to ATP in caspase-1-independent fashion (40). To examine a possibility whether P2X7 is involved in kainate-induced ataxia, we examined the effect of oxidized ATP, one of P2X7 receptor antagonists (41), on kainate-induced ataxia. An intracerebellar injection of oxidized ATP did not inhibit the kainate-induced ataxia during at least 60 min after kainate-injection (data not shown). Therefore, the result suggested that the participation of P2X7 is little on the induction of kainate-induced ataxia. In accordance with our results, Nicklas et al. (42) showed that kainate decreased the content (or release) of ATP in cerebellar slices.

The most interesting finding in this study is that IL-18, another caspase-1-activated proinflammatory cytokine (12), counteracted the effect of IL-1β in the induction of ataxia with kainate. IL-18- and IL-18R-deficient mice showed the delay of recovery from kainate-induced ataxia (Fig. 1), and intracerebellar injection of IL-18 together with IL-1β inhibited the IL-1β-induced ataxia (Fig. 3). We assume three possible explanations for these results: firstly, IL-18 may induce the expression of IL-1ra that inhibits the binding of IL-1β to its receptor; secondly, IL-1β and IL-18 may exert their effects on different cells in the neuronal network in the cerebellum; thirdly, IL-1β and IL-18 may exert their effects on the same target cells but induce counteracting signals. As for the first possibility, we examined the effect of intracerebellar injection of IL-18 on IL-1ra mRNA expression. IL-18 did not affect the expression of IL-1ra mRNA expression significantly during 60 min after intracerebellar injection, compared with intracerebellar injection of vehicle only (data not shown). Because intracerebellar injection of IL-18 showed a counteracting effect on kainate-induced or IL-1β-induced ataxia within 60 min (Fig. 3), the result suggests that IL-1ra is not involved in the anti-ataxia effect of IL-18 in cerebellum. Regarding the second possibility, immunohistochemical analysis showed that both IL-1RI and IL-18Rα were expressed on Purkinje cells in the cerebellum (Fig. 4). The analysis indicated the expression of IL-18R but not IL-1RI on astrocytes (Fig. 5). Thus, IL-18 may counteract IL-1β by exerting its effect through different target cells. Concerning the third possibility, it has been so far reported that receptors for IL-1 and IL-18 use the same signaling module, MyD88 and IL-1-receptor associated kinases (43). However, because the receptors for both cytokines are expressed on Purkinje cells, it is possible that the receptors for these cytokines use a different signaling pathway in Purkinje cells. With this possibility, ST2, one of the IL-1 receptor family, has been described as a negative regulator for TLR-IL-1R signaling (44), in line with an earlier report that ST2 was unable to activate the IL-1-activated transcription factor NF-κB (45, 46). The detailed mechanisms on the interaction of IL-1β and IL-18 in the induction and recovery of ataxia remain unknown. The electrophysiological examination might reveal the effect of kainate, IL-1β or IL-18, or both on neural network in the cerebellum.

Concerning the genetic background of knockout mice, we had used knockout mice of the same genetic backgrounds, i.e., C57BL/6 background for IL-1RI−/−, MyD88−/−, IL-18−/−, and IL-18R−/− mice, but used a BALB/c background for caspase-I−/− mice. Regarding the differences in the development of inflammatory disease of the IL-1ra−/− mice, Horai et al. (47) showed that the IL-1ra-deficient mice on a BALB/c background, but not those on a C57BL/6J background, spontaneously developed chronic inflammatory polyarthropathy. Their results suggested that genes other than IL-1ra are involved in the development of spontaneously developed arthritis. They also observed that IL-1ra−/− mice on the C57BL/6 background developed arthritis at a high incidence when these mice were immunized with type II collagen. Their result demonstrated that IL-1ra−/− mice on C57BL/6 background as well as BALB/c background showed the corresponding susceptibility to experimentally induced arthritis. In our study, we observed the effect of kainate on the acutely induced ataxia (induction within 20 min after the kainate injection and the following subsidence within 60–120 min). Our data showed that the mice deficient with IL-1RI, or MyD88 on the same C57BL/6 background were resistant to kainate-induced ataxia, while IL-18- or IL-18Rα-deficient mice on the same C57BL/6 background displayed significant delay of the recovery from ataxia. In these experiments, we had compared the responses with the wild-type mice on the same C57BL/6 background. We have also compared the locomotion time of normal BALB/c mice and C57BL/6 mice after systemic administration of kainate, and observed the similar responses in normal BALB/c mice and C57BL/6 mice (Fig. 1, E and F). Our data suggest that the effect of the genetic background of BALB/c mice and C57BL/6 mice on kainate-induced ataxia in the acute phase is less important. Taken together, our data clearly showed that IL-1RI or MyD88 was involved in the induction of ataxia, and IL-18 and its receptor in the recovery of the kainate-induced ataxia in the C57BL/6 genetic background.

Recently Zhang et al. (48) showed that IL-18-deficient mice were more sensitive to kainate administration in the induction of neurodegeneration compared with the normal animals. The observation that IL-18 deficiency aggravated kainate-induced hippocampal neurodegeneration seems similar to our observation that the mice with IL-18 deficiency showed a delay of the recovery from kainate-induced ataxia. However, they showed that the exogenous administration of IL-18 aggravated the kainate-induced neurodegeneration. They concluded that IL-18 had a disease-promoting role in kainate-induced excitotoxicity but that the roles of IL-18 in excitotoxic injury in IL-18-deficient mice might be overcompensated by an increase of other microglia-derived disease-promoting factors, such as IL-12. In contrast, we demonstrated that the exogenous administration of IL-18 showed the counteracting effect on kainate-induced ataxia (Fig. 3 F). Our results suggest that although IL-18 and IL-1β are both proinflammatory cytokines, IL-18 showed the counteracting effect in kainate-induced ataxia.

In this study, we provided evidence that caspase-1 and IL-1RI in the cerebellum are essential for kainate-induced ataxia and that IL-18 and IL-18R are positive regulators for the recovery phase of kainate-induced ataxia in mice. Earlier reports on the effect of kainate have demonstrated its effect mainly in the hippocampus, and kainate is reported to show a neurodegenerative effect (2, 49). In this regard, the present study showed that caspase-1 was activated not only in the cerebellum but also in the hippocampus (Fig. 2). The activation of caspase-1 in the hippocampus peaked at 60 min after kainate administration, although ataxia was induced within 20 min and then gradually subsided within 60 to 90 min after kainate stimulation. In our studies, we could not detect either activated IL-1β or activated IL-18 at the protein level in the hippocampus, while we could clearly detect them in the cerebellum. Our result indicates that the activation of caspase-1 in the hippocampus may not be directly attributed to kainate-induced ataxia, but may lead to a neurodegenerative effect of kainate. Further analysis of the molecular as well as physiological basis of IL-1β-induced ataxia and its inhibition by IL-18 in the cerebellum may contribute to clarify the mechanism of kainate-induced ataxia, and might contribute toward the development of a new strategy for the therapy of cerebellar ataxia in humans.

We thank K. Kuida, K. Hoshino, and S. Akira for providing us with caspase-1−/−, IL-18Rα−/−, and MyD88−/− mice, respectively. We are also grateful to S. Hirota for taking care of the knock out mice.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

3

Abbreviations used in this paper: IL-1ra, IL-1 receptor antagonist; IL-1RI, IL-1 receptor type I.

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