Interleukin-10 Prevents Glutamate-Mediated Cerebellar Granule Cell Death by Blocking Caspase-3-Like Activity

Abstract

Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.

Genetic Evidence for Adenylyl Cyclase 1 as a Target for Preventing Neuronal Excitotoxicity Mediated by N-Methyl-D-aspartate Receptors

“…Once activated, caspases are responsible for the proteolytic cleavage of several substrates leading to cell death. Accordingly, it has been reported that there is an increase in both caspase-3 expression and activity when cultured neurons are treated with glutamate or NMDA (33,34,(61)(62)(63). We assessed the caspase-3 activity in cultured cortical neurons after glutamate treatment and found that the caspase-3 activity was significantly reduced in the cortical neurons of AC1 KO mice, but not in the cortical neurons from AC8 KO mice, compared with wild-type mice.…”

“…Activation of caspase-3 is a hallmark of apoptotic cell death and precedes changes in nuclear morphology (33,34). To further confirm that glutamate could induce apoptotic neuronal death in our model system, we measured caspase-3 activity at various time points in extracts from cortical neuron cultures during the 8 h after treatment with 250 M glutamate.…”

Gangliosides Activate Trk Receptors by Inducing the Release of Neurotrophins

“…Gangliosides increase neurotrophin release in cerebellar granule cells Cerebellar granule cells were prepared as previously described (31,32). At day 8 in vitro, cells were exposed for 5 or 15 min to either control medium alone or containing LIGA20 (5 M) or GM1 (30 M).…”

“…Cerebellar granule cells were prepared from postnatal day 8 Sprague-Dawley rat pups (Taconic Farms Inc., Germantown, NY) as previously described (31,32). In brief, neurons were plated onto 1% poly-Llysine-precoated 100-mm plastic dishes at a density of 2.5 ϫ 10 6 cells/ml and grown in basal Eagle's medium (Invitrogen) containing glutamine (2 mM), fetal calf serum (10%), KCl (25 mM), gentamycin (100 g/ml), and penicillin/streptomycin (10,000 units/ml).…”

“…Neuronal Survival-Cerebellar granule cell survival was determined by mitochondrial dehydrogenases 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (30,32) and according to the manufacturer's specifications (MTT Kit I, Roche Molecular Biochemicals). Briefly, neurons were cultured on 96-well plates; 10 l of the 5 mg/ml MTT labeling reagent was added to each well containing neurons; and plates were incubated for 4 h in a humidified atmosphere.…”