ProNGF, but Not NGF, Switches from Neurotrophic to Apoptotic Activity in Response to Reductions in TrkA Receptor Levels

Abstract

Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially synthesized as a precursor, proNGF, which is the predominant form in the central nervous system. NGF and proNGF bind to TrkA/p75NTR to mediate cell survival and to sortilin/p75NTR to promote apoptosis. The ratio of TrkA to p75NTR affects whether proNGF and mature NGF signal cell survival or apoptosis. The purpose of this study was to determine whether the loss of TrkA influences p75NTR or sortilin expression levels, and to establish whether proNGF and mature NGF have a similar ability to switch between cell survival and cell death. We systematically altered TrkA receptor levels by priming cells with NGF, using small interfering RNA, and using the mutagenized PC12nnr5 cell line. We found that both NGF and proNGF can support cell survival in cells expressing TrkA, even in the presence of p75NTR and sortilin. However, when TrkA is reduced, proNGF signals cell death, while NGF exhibits no activity. In the absence of TrkA, proNGF-induced cell death occurs, even when p75NTR and sortilin levels are reduced. These results show that proNGF can switch between neurotrophic and apoptotic activity in response to changes in TrkA receptor levels, whereas mature NGF cannot. These results also support the model that proNGF is neurotrophic under normal circumstances, but that a loss in TrkA in the presence of p75NTR and sortilin, as occurs in neurodegenerative disease or injury, shifts proNGF, but not NGF, signalling from cell survival to cell death.

Keywords: PC12 cells; TrkA; apoptosis; neurotrophin; p75NTR; proNGF; sortilin.

Figure 1

ProNGF and nerve growth factor (NGF) are neurotrophic in primed PC12 cells. (A) Representative Western blot of receptor levels in PC12 cells primed for 0 to 4 days with 50 ng/mL 2.5S NGF; (B) PC12 cells were primed for 18 h in 50 ng/mL NGF and incubated for 24 h in 0.5 nM proNGF, WT-NGF, 2.5S NGF, or medium alone. Both proNGF and NGF reduced the percentage of cell death compared to the medium alone. Percentage of cell death is expressed relative to levels in medium-treated cells. Error bars represent standard error of the mean (SEM). *** p < 0.001. n = 8 for proNGF and medium, while n = 7 for WT-NGF and 2.5S NGF. Data pooled from two independent experiments; (C) PC12 cells were primed for 18 h in 50 ng/mL NGF, treated with increasing concentrations of proNGF or NGF for 5 min, and the activation of extracellular signal-regulated kinase (ERK) and Akt was analyzed by Western blot; (D) Quantification of ERK activation. P-ERK is normalized to total ERK and is expressed relative to untreated cells. Error bars represent SEM. n = 4 for proNGF and n = 3 for 2.5S NGF; (E) Quantification of Akt activation. P-Akt is normalized to total Akt and is expressed relative to untreated cells. Error bars represent SEM. n = 4 for proNGF and n = 2 for 2.5S NGF; and (F) Western blot of collected media showing that proNGF remains intact throughout the experiment.

Figure 2

ProNGF causes cell death in primed PC12 cells when TrkA is knocked down, while mature NGF has no effect. (A,B) PC12 cells were treated with 150 nM TrkA siRNA, control siRNA, or vehicle, and primed for 18 h in 50 ng/mL 2.5S NGF. Western blot showing reduced TrkA in PC12 cells treated with TrkA siRNA, compared to control siRNA or vehicle controls. Sortilin and p75NTR levels were unchanged as a result of TrkA knockdown; (C–F) PC12 cells were transfected with 150 nM TrkA siRNA, control siRNA, or vehicle, primed for 18 h in 50 ng/mL 2.5S NGF, and treated for 24 h with 0.5 nM (C) WT-NGF, n = 11 for TrkA siRNA and control siRNA, n = 7 for vehicle (D) 2.5S NGF, n = 11 for TrkA siRNA, n = 12 for control siRNA, and n = 8 for vehicle (E) proNGF, n = 11 for TrkA siRNA, n = 12 for control siRNA, and n = 8 for vehicle; (F) PC12 cells were treated with 150 nM TrkA siRNA, primed for 18 h in 50 ng/mL 2.5S NGF, and treated for 24 h with 0.5 nM proNGF, WT-NGF, 2.5S NGF, or medium alone. n = 11 for proNGF, WT-NGF, and 2.5S NGF, and n = 12 for medium alone. Percentage of cell death is expressed relative to levels in medium-treated cells. For all graphs: Error bars represent SEM. *** p < 0.001. Data pooled from three independent experiments; and (G) Western blot of collected media showing that proNGF remained intact throughout the experiment.

Figure 3

ProNGF is apoptotic in PC12nnr5 cells lacking TrkA. (A) Top panel: Representative Western blot of primed PC12nnr5 and PC12nnr5 B5 cell lysates. Each lane is derived from a separate plate. Bottom panel: quantification of A, where relative amounts of TrkA, sortilin, and p75NTR were normalized to β-actin. Similar results were obtained in a second experiment; (B) PC12nnr5 cells were treated for 48 h with medium alone or 0.5 nM WT-NGF, 2.5S NGF, or proNGF. White arrows show representative dead cells. Scale bars represent 5 μm; (C) ProNGF causes more apoptosis than medium, WT-NGF, or 2.5S NGF in PC12nnr5 cells. n = 10 for all groups. Data pooled from two independent experiments; (D) Re-expression of TrkA in PC12nnr5 B5 cells treated for 48 h with 0.5 nM proNGF, WT-NGF, or medium alone. n = 14 for proNGF and WT-NGF and n = 15 for 2.5S and medium alone. Data pooled from three independent experiments. Percentage of cell death is expressed relative to levels in medium-treated cells. Error bars represent SEM. *** p < 0.005; and (E) Western blot of collected media showing that proNGF remained intact throughout the experiment.