N-methyl-D-aspartate and TrkB receptor activation in cerebellar granule cells: an in vitro model of preconditioning to stimulate intrinsic survival pathways in neurons

Abstract

Delineating the mechanisms of survival pathways that exist in neurons will provide important insight into how neurons utilize intracellular proteins as neuroprotectants against the causes of acute neurodegeneration. We have employed cultured rat cerebellar granule cells as a model for determining the mechanisms of these intraneuronal survival pathways. Glutamate has long been known to kill neurons by an N-methyl-d-aspartate (NMDA) receptor-mediated mechanism. Paradoxically, subtoxic concentrations of NMDA protect neurons against glutamate-mediated excitotoxicity. Because NMDA protects neurons in physiologic concentrations of glucose and oxygen, we refer to this phenomenon as physiologic preconditioning. One of the major mechanisms of NMDA neuroprotection involves the activation of NMDA receptors leading to the rapid release of brain-derived neurotrophic factor (BDNF). BDNF then binds to and activates its cognate receptor, receptor tyrosine kinase B (TrkB). The efficient utilization of these two receptors confers remarkable resistance against millimolar concentrations of glutamate that kill more than eighty percent of the neurons in the absence of preconditioning the neurons with a subtoxic concentration of NMDA. Exactly how the neurons mediate neuroprotection by activation of both receptors is just beginning to be understood. Both NMDA and TrkB receptors activate nuclear factor kappaB (NF-kappaB), a transcription factor known to be involved in protecting neurons against many different kinds of toxic insults. By converging on survival transcription factors, such as NF-kappaB, NMDA and TrkB receptors protect neurons. Thus, crosstalk between these very different receptors provides a rapid means of neuronal communication to upregulate survival proteins through release and transcriptional activation of messenger RNA.

FIGURE 1.

N-Methyl-d-aspartate activates NF-kB in nuclear extracts prepared from rat cerebellar granule cells. Autoradiograph shows that NMDA (100 μM) activates a specific NF-kB DNA-protein binding complex within 40 minutes in cultured rat cerebellar granule cells on day eight in vitro. No specific complex was observed in untreated neurons (no treatment). The addition of an unlabeled competitor DNA resulted in the disappearance of the specific band (NMDA + cold). No bands were detected in the probe (probe only).

FIGURE 2.

Brain-derived neurotrophic factor activates NF-kB in nuclear extracts prepared from rat cerebellar neurons. Two specific DNA-protein binding complexes were induced in neurons by BDNF (100 ng/mL), a concentration that protects about 30% of the neurons against glutamate excitotoxicity (Marini et al., 1998). The NMDA receptor antagonist, MK-801, was added to the neurons to block NMDA receptors, thereby ensuring that the activation of NF-kB was mediated solely through the activation of TrkB receptors.

FIGURE 3.

NF-kB DNA oligonucleotide decoy derived from the 5'- flanking region of Exon 3 of the bdnf gene blocks NMDA receptor-mediated neuroprotection. A double-stranded DNA target sequence based upon the 5'- flanking region of exon 3 of the bdnf gene (see Lipsky et al., 2001) was added to granule cell cultures for 24 h (NF-kB oligo). Neurons were also pretreated with a random sequence of the NF-kB oligonucleotide (scramble oligo). On the following day some culture dishes were treated with a maximum neuroprotective concentration of NMDA (100 μM) for six hours followed by the addition of an excitotoxic concentration of glutamate (100 μM) for 24 hours. Neuronal viability was determined by the fluorescein diacetate staining method (Marini and Paul, 1992). Note that pretreatment of the cultured neurons with NMDA for six hours followed by the addition of glutamate protects all of the vulnerable neurons against glutamate. Glutamate alone kills about 70% of the neurons. Pretreatment with the specific NF-kB oligo in the presence of a maximum neuroprotective concentration of NMDA completely blocked NMDA neuroprotection. Data are expressed as mean ± SE, where n = 6; ^*p < 0.001 compared with untreated neurons; ^**p < 0.003 compared with neurons treated with NMDA + glutamate.

FIGURE 4.

A maximum neuroprotective concentration of NMDA (100 μM) induces Bcl-2 mRNA. A marked decrease in bcl-2 mRNA was observed at twenty-four hours in glutamate-treated neurons (hatched bars). In sharp contrast, preincubation with NMDA (100 μM) followed by the addition of glutamate (100 μM, light hatch) blocked the glutamate-induced decrease in bcl-2 mRNA. This supports the hypothesis that another mechanism exists where NMDA promotes neuronal survival through the anti-apoptotic bcl family of proteins. p < 0.05 versus ^‡glutamate, ^*untreated, or ^§MK-801.

FIGURE 5.

Overview of the neuroprotective activity of N-methyl-d-aspartate. Activation of NMDA receptors results in the influx of extracellular calcium intracellularly and the immediate release (within two minutes) of brain-derived neurotrophic factor (BDNF). Extracellular BDNF then binds to and activates its cognate receptor, TrkB. Thus, coactivation of NMDA and TrkB receptors occurs within ten minutes (see Marini et al., 1998). The influx of calcium and possibly other second messenger systems leads to the phosphorylation of I-kB, the inhibitor of NF-kB. Once phosphorylated, I-kB is rapidly degraded by the ubiquitin-proteosome-mediated pathway resulting in the release of NF-kB. Activated NF-kB translocates to the nucleus where it binds to the 5'- flanking region of exon 3 of the bdnf gene and likely to the promoter of the bcl-2 gene to activate gene transcription to protect vulnerable neurons against the excitotoxic effects of glutamate acting on NMDA receptors. The question marks indicate gaps in knowledge of the interaction between NMDA and TrkB receptors and the signal transduction pathways that mediate the survival effects of coactivation of NMDA and TrkB receptors.