Abstract
Platelet-derived growth factor (PDGF) beta receptor activation inhibits N-methyl-d-aspartate (NMDA)-evoked currents in hippocampal and cortical neurons via the activation of phospholipase Cgamma, PKC, the release of intracellular calcium, and a rearrangement of the actin cytoskeleton. In the hippocampus, the majority of NMDA receptors are heteromeric; most are composed of 2 NR1 subunits and 2 NR2A or 2 NR2B subunits. Using NR2B- and NR2A-specific antagonists, we demonstrate that PDGF-BB treatment preferentially inhibits NR2B-containing NMDA receptor currents in CA1 hippocampal neurons and enhances long-term depression in an NR2B subunit-dependent manner. Furthermore, treatment of hippocampal slices or cultures with PDGF-BB decreases the surface localization of NR2B but not of NR2A subunits. PDGFbeta receptors colocalize to a higher degree with NR2B subunits than with NR2A subunits. After neuronal injury, PDGFbeta receptors and PDGF-BB are up-regulated and PDGFbeta receptor activation is neuroprotective against glutamate-induced neuronal damage in cultured neurons. We demonstrate that the neuroprotective effects of PDGF-BB are occluded by the NR2B antagonist, Ro25-6981, and that PDGF-BB promotes NMDA signaling to CREB and ERK1/2. We conclude that PDGFbetaR signaling, by preferentially targeting NR2B receptors, provides an important mechanism for neuroprotection by growth factors in the central nervous system.
Highlights
Trimeric receptors (2NR1/NR2A/NR2B) are likely to be present [1]
When NR2A-containing NMDA receptors were inhibited with NVPAAM077 [28], Platelet-derived growth factor (PDGF)-BB decreased peak NMDA-evoked currents (Fig. 1C)
Taken together these experiments suggest that the inhibition of NMDA currents by PDGF-BB is largely dependent on NR2B- and not NR2A-containing NMDA receptors
Summary
Reagents and Antibodies—PDGF-BB was purchased from R&D Systems (Minneapolis, MN). Ro25-6981 was purchased from Tocris Bioscience (Bristol, UK). A similar protocol was followed to examine the cell surface expression of NR2B and PDGF receptors in cultured hippocampal neurons (15–23 days in vitro) except the 10-min drug treatment was at 37 °C. For total NMDA receptor activation, cells were incubated for 3 min with 100 M NMDA, 10 M glycine, 1 M tetrodotoxin, 40 M 6-cyano-7-nitroquinoxaline-2,3-dione, and 5 M nifedipine after pretreatment with vehicle or 10 ng/ml PDGF-BB for 10. After 24 h, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.4% Triton in PBS, blocked for 30 min with 10% bovine serum albumin, and incubated with anti-MAP2 (1:5000, Sigma) overnight at 4 °C. The following day, the cells were washed in 0.4% Triton/PBS three times and incubated with anti-mouse horseradish peroxidase antibody (Amplex Elisa Development Kit, Molecular Probes).
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