Signal transduction events mediated by the BDNF receptor gp 145trkB in primary hippocampal pyramidal cell culture

Abstract

The trkB gene encodes a tyrosine kinase receptor, gp145trkB, for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). To understand the role of gp145trkB in the nervous system, we have investigated its expression in embryonic rat hippocampal pyramidal cell cultures and examined the effects of BDNF on signal transduction in the primary neurons. The expression of trkB transcripts was established by PCR analysis and in situ hybridization. In addition to gp145trkB, the pyramidal neuronal cultures expressed transcripts specific for the NT-3 receptor gp145trkC, but not for the high-affinity NGF receptor gp140trk or for p75LNGFR, a low-affinity receptor for all known members of the NGF family of neurotrophins including the gp145trkB ligands, BDNF and NT-4. The presence of gp145trkB receptors in the primary neuronal cultures was confirmed by immunocytochemical analysis in which > 90% of the cells stained with affinity-purified polyclonal antibodies to gp145trkB. Immunoblots using this antibody revealed a single approximately 140 kDa protein in both adult hippocampus and pyramidal cultures. Addition of recombinant BDNF to these cultures induced the tyrosine phosphorylation of gp145trkB, as determined by antiphosphotyrosine staining of gp145trkB immunoprecipitates. Moreover, BDNF treatment activated the microtubule-associated protein (MAP) kinases, as determined by an increase in MAP2 phosphorylation in vitro. Both the 41 and 44 kDa forms of MAP kinase were activated by BDNF. BDNF also increased c-fos expression in over 90% of the cells. These results indicate that gp145trkB does not require p75LNGFR to form a functional receptor for BDNF in hippocampal pyramidal neurons.

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways

“…The transient phosphorylation (activation) of ERK1/2 in hippocampal neurons stimulated with BDNF, observed in the present work, is in agreement with previous findings in the same preparation, 52 and with the relatively fast rate of desensitization of TrkB receptors. 57 A downregulation of TrkB receptors under sustained exposure to BDNF may also account for the transient phosphorylation (activation) of Akt, which followed a kinetics similar to that observed for ERK1/2 phosphorylation.…”

“…Our results also show that de novo protein synthesis is required for neuroprotection by BDNF and, accordingly, incubation of hippocampal neurons with the neurotrophin increased Bcl-2 protein levels. The effects of BDNF and NT-3 observed in the present work are in agreement with the reported presence of TrkB and TrkC receptors in the hippocampus from early in development, [50][51][52] in contrast with the lack of TrkA receptors. 26 Accordingly, pre-incubation of hippocampal neurons with NGF did not affect glutamate toxicity.…”

“…56 In the latter study, neurotrophins targeted mainly a subpopulation of neurons that express p75 receptors and lack Trk neurotrophin receptors. Since the majority of hippocampal neurons present in the cultures used in this work express Trk receptors (not shown), in agreement with previously reported results showing that about 90% of cultured hippocampal neurons express these receptors, 52 this may account for the apparent discrepancy between our findings and the results reported by Friedman. 56 This difference in the expression of Trk receptors may be due to developmental changes in the abundance of the receptors, since we have used 7-8 DIV cells and neurotrophin toxicity was observed in five DIV neurons.…”

Small, Nonpeptide p75NTRLigands Induce Survival Signaling and Inhibit proNGF-Induced Death

“…Similar cultures have been reported to express little or no TrkA (Ip et al, 1993;Marsh et al, 1993;Brann et al, 1999), but consideration of differences in species, timing, media, and the presence or absence of a glial feeder layer prompted examination of Trk expression in the cultures used in the present studies (Fig. 3A).…”

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

“…It is well established that many TrkB-mediated cellular responses are accompanied by an increase in c-fos protein levels within 30 ± 60 min following BDNF treatment Marsh et al, 1993). Recent data suggest that induction of c-fos protein synthesis is dependent on ERK1 and ERK2 activation (Karin, 1994;Treisman, 1994).…”