Neurotrophins induce death of hippocampal neurons via the p75 receptor - PubMed (original) (raw)

Neurotrophins induce death of hippocampal neurons via the p75 receptor

W J Friedman. J Neurosci. 2000.

Abstract

Nerve growth factor (NGF) and related neurotrophins influence neuronal survival and differentiation via interactions with the trk family of receptors. Recent studies have demonstrated that neurotrophins may also induce cell death via the p75 receptor. The importance and generality of neurotrophin-induced death in the brain have not been defined but may play a critical role during development and in disease-associated neuronal death. Here we demonstrate for the first time that all four members of the neurotrophin family directly elicit the death of hippocampal neurons via the p75 receptor. The hippocampus is a complex structure with many different neuronal subpopulations, and signals that influence neuronal death during development may have a critical impact on the mature function of this structure. In these studies we show that each neurotrophin causes the death of hippocampal neurons expressing p75 but lacking the cognate trk receptor. Neurotrophin-induced neuronal death is mediated by activation of Jun kinase. These studies demonstrate that neurotrophins can regulate death as well as survival of CNS neurons.

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Figures

Fig. 1.

Fig. 1.

Expression of neurotrophin receptors in embryonic hippocampal neurons after 5 d in culture. a, Neurons were treated for 2 hr with vehicle [control (C)] or the different neurotrophins. Lysates were immunoprecipitated with anti-p75 (192 IgG), run on a Western blot, and probed with anti-p75 (9651). PC12 cell lysates were used as a positive control (+). No signal was detected in PC12 lysates in the absence of the immunoprecipitating antibody (−).b, Hippocampal neurons were treated for 5 min with vehicle (C) or the different neurotrophins and analyzed for tyrosine phosphorylation of the trk proteins (arrowhead). Phosphorylation of trk receptors was induced by BDNF and NT4 and by NT3 treatment, indicating the presence of TrkB and TrkC, respectively, in the neuronal cultures (arrowhead). No TrkA phosphorylation was detected after NGF treatment. c, Hippocampal neurons were immunostained with antibodies against p75, TrkC, or TrkB.Arrows indicate positively stained neurons, and_arrowheads_ indicate negatively stained neurons.

Fig. 2.

Fig. 2.

Neurotrophins induce the death of hippocampal neurons. Neurons were cultured for 5 d and treated with vehicle or neurotrophins in triplicates. Survival is reported as the percent of intact nuclei compared with that in untreated controls and is presented as the mean ± SEM. a, Dose–response curve for neurotrophin-induced death of hippocampal neurons after overnight exposure to NGF, BDNF, NT3, or NT4. Data are expressed as the percent of control in triplicate samples from eight independent experiments (n = 24). The mutant tri-NGF that cannot bind p75 did not elicit neuronal death. An asterisk indicates values different from the control value at p < 0.001. b, Time course of neurotrophin-induced death of hippocampal neurons. Neuronal loss was apparent by 6 hr of treatment and was maximal at 24 hr. No additional cell loss was detected after longer treatment times. Data reported are from triplicate samples from three independent experiments (n = 9 cultures per sample). An asterisk indicates values different from control at p < 0.01. The apparent absence of error bars for some samples indicates that the error was smaller than the symbol used.

Fig. 3.

Fig. 3.

p75 mediates neurotrophin-induced death.a, Anti-p75 antiserum prevented neurotrophin-induced death of hippocampal neurons. Cultured neurons were treated overnight with neurotrophins (100 ng/ml) in the presence of normal rabbit serum (1:100 dilution; open bars) or a blocking antibody to p75 (9651; 1:100 dilution; filled bars). Data are expressed as the percent of control in triplicate samples from four independent experiments (n = 12). An_asterisk_ indicates values different from control at_p_ < 0.001. b, Hippocampal neurons from p75 −/− mice (hatched bars) were compared with neurons from wild-type mice (open bars) for the ability of neurotrophins to induce death. Cultured neurons were treated with neurotrophins (100 ng/ml) overnight. Data are expressed as the percent of intact nuclei compared with that of untreated neurons (n = 5 cultures per treatment), and an_asterisk_ indicates values different from control at_p_ < 0.001.

Fig. 4.

Fig. 4.

Neurotrophins induce apoptosis of neurons expressing p75 in the absence of trk receptors. Hippocampal neurons were treated with NGF (a–c), BDNF (d–f), or NT3 (g–i) for 6 hr. All cultures were immunostained with anti-p75 (192 IgG;a, d, g) and labeled with Hoechst 33342 (c, f, i) to identify the nuclei of apoptotic cells. NGF-treated cultures were immunostained with anti-pan-Trk (b), BDNF-treated cultures were labeled with anti-TrkBin(e), and NT3-treated cultures were labeled with anti-TrkCin2 (h). Chromatin condensation, indicative of apoptosis, was detected in neurons that expressed p75 in the absence of the relevant trk receptor (arrowheads). Healthy nuclei in neurons expressing both p75 and the relevant trk are indicated with arrows. Scale bar, 20 μm.

Fig. 5.

Fig. 5.

Blocking the actions of endogenously produced BDNF and/or NT4 with TrkB–IgG enhances neuronal loss in response to NGF. TrkB–IgG (10 μg/ml) was added to the cells 1 hr before the addition of neurotrophins for overnight treatment. Data are expressed as the percent of intact nuclei relative to that in the absence of added neurotrophins. Open bars indicate cells in the presence or absence of the indicated neurotrophins alone. Stippled bars show neurons with the IgG control fragment. Hatched bars indicate cells treated with TrkB–IgG. An_asterisk_ indicates values different from control at_p_ < 0.001; double asterisks_indicate a value different from NGF alone or the NGF + IgG control at_p < 0.01. Data reported are from triplicate samples from two independent experiments (n = 6).

Fig. 6.

Fig. 6.

The JNK-signaling pathway mediates neurotrophin-induced death of hippocampal neurons. a, NGF induces JNK phosphorylation in hippocampal neurons. Neurons were treated with NGF (100 ng/ml) for the times indicated. b, JNK phosphorylation was also induced by BDNF and NT3 (100 ng/ml) treatment of hippocampal neurons, but not by mutant NGF (tri-NGF) that lacks p75 binding. Shown is a 20 min time point. c, Cultures were treated with NGF for 20 min in the presence or absence of CEP-1347/KT7515 (CEP). Incubation of cells with 200 n

m

CEP-1347/KT7515 prevented JNK phosphorylation by NGF, whereas CEP-1347/KT7515 by itself had no effect. d, Phosphorylation of JNK by NGF was prevented by anti-p75 (9651). Cells were pretreated for 1 hr with anti-p75 and then exposed to NGF (100 ng/ml). A 20 min time point is shown. Blots shown in_a–d_ were probed for P-JNK (top panels) and stripped and reprobed for JNK (bottom panels).e, Prevention of JNK activation with CEP-1347/KT7515 blocked neurotrophin-induced death. Cultures were treated with neurotrophins overnight in the absence (open bars) or presence (hatched bars) of 200 n

m

CEP-1347/KT7515. Data are expressed relative to the control number of cells in the absence of added neurotrophins. An _asterisk_indicates values different from control at p < 0.01. Data are from triplicate samples from three independent experiments (n = 9).

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