c-Jun N-terminal Kinase Mediates Ligand-independent p75NTR Signaling in Mesencephalic Cells Subjected to Oxidative Stress

Abstract

The p75 neurotrophin receptor (p75^NTR) is a multifunctional protein that regulates cellular responses to pathological conditions in specific regions of the nervous system. Activation of p75^NTR in certain neuronal populations induces proteolytic processing of the receptor, thereby generating p75^NTR fragments that facilitate downstream signaling. Expression of p75^NTR has been reported in neurons of the ventral midbrain, but p75^NTR signaling mechanisms in such cells are poorly understood. Here, we used Lund Human Mesencephalic cells, a population of neuronal cells derived from the ventral mesencephalon, to evaluate the effects of oxidative stress on p75^NTR signaling. Subjection of the cells to oxidative stress resulted in decreased cell-surface localization of p75^NTR and intracellular accumulation of p75^NTR fragments. Oxidative stress-induced p75^NTR processing was reduced by pharmacological inhibition of metalloproteases or γ-secretase, but was unaltered by blockade of the ligand-binding domain of p75^NTR. Furthermore, inhibition of c-Jun N-terminal Kinase (JNK) decreased p75^NTR cleavage induced by oxidative damage. Altogether, these results support a mechanism of p75^NTR activation in which oxidative stress stimulates JNK signaling, thereby facilitating p75^NTR processing via a ligand-independent mechanism involving induction of metalloprotease and γ-secretase activity. These findings reveal a novel role for JNK in ligand-independent p75^NTR signaling, and, considering the susceptibility of mesencephalic neurons to oxidative damage associated with Parkinson's disease (PD), merit further investigation into the effects of p75^NTR on PD-related neurodegeneration.

Keywords: JNK; Parkinson’s disease; neurotrophin; p75(NTR); proteolysis; reactive oxygen species.

Figure 1.. Expression of p75^NTR in Neuronal Cells Derived from the Ventral Mesencephalon..

A; phase-contrast images of undifferentiated LUHMES cells (left image) or LUHMES cells that were differentiated for five days in medium containing 1 μg/mL tetracycline, 1 mM db-cAMP, and 2 ng/mL GDNF (right image). B; confocal micrographs revealing p75^NTR expression in differentiated LUHMES cells. Following fixation the cells were permeabilized with 0.1% triton X-100 in PBS, immunolabeled with an antibody specific for the extracellular domain of p75^NTR (green), and subjected to nuclear staining using DAPI (5 μg/mL). The right image depicts an enlargement of the neurite featured in the square inset of the left image. C. Representative confocal micrographs of LUHMES cells that were differentiated for five days, fixed with 4% paraformaldehyde, and immunolabeled using antibodies specific for TH (red) or the p75^NTR-ECD (green). Counterstaining was performed by incubation with the nucleic acid dye DAPI at a concentration of 5 μg/mL. D; Representative confocal micrographs of LUHMES cells that were differentiated for five days and treated with vehicle solution or with the indicated concentrations of 6-OHDA. The cells were fixed after 24 hours of treatment and immunolabeled for the neuronal marker ß-III tubulin (red). Nuclei were labeled by DAPI staining (5 μg/mL) and scored as healthy or apoptotic. Arrowheads indicate apoptotic nuclei. E; quantification of apoptosis of differentiated LUHMES cells that were treated with the indicated concentrations of 6-OHDA and analyzed as described in 1D (n=6). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; TH, tyrosine hydroxylase; n, number of experiments, each featuring an independent cell culture preparation.

Figure 2.. Regulated Intramembrane Proteolysis of p75^NTR is Induced by 6-hydroxydopamine in Mesencephalic Cells.

A; schematic depicting fragments generated by proteolytic processing of p75^NTR. Extracellular cleavage of full-length p75^NTR (p75^NTR-FL) generates a 25 kDa, membrane-bound C-terminal fragment (p75^NTR-CTF). Cleavage of the transmembrane domain of the p75^NTR-CTF releases the 20 kDa intracellular domain of p75^NTR (p75^NTR-ICD). B; representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were exposed to 10 μM 6-OHDA or vehicle solution for 18 hours. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. C - E; densitometric analysis of p75^NTR-CTF (C.), p75^NTR-ICD (D.), or p75^NTR-FL (E.) from immunoblots described in 2B (n = 6; c and d, Student’s t test; e, Mann-Whitney test). F; Confocal micrographs of LUHMES cells that were differentiated for five days and fixed after treatment for 24 hours with vehicle solution or 7.5 μM 6-OHDA. The unpermeabilized cells were immunolabeled with an antibody specific to the extracellular domain of p75^NTR (green), and counterstaining was performed by incubation with the cell-permeable nucleic acid dye DAPI at a concentration of 5 μg/mL. G; Mean fluorescence of LUHMES cells that were treated and immunostained as described in 2F (165 cells from three independent experiments, Mann-Whitney test). H; representative western blot analysis of p75^NTR in lysates of differentiated LUHMES cells that were exposed to vehicle solution or 10 μM 6-OHDA for 18 hours. Blotting was performed using an antibody specific for the p75^NTR-ECD. Immunoblotting for actin was performed as a loading control. I; densitometric analysis of p75^NTR-FL from immunoblots described in 2H (n= 4; Student’s t test). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; 6-OH, 6-hydroxydopamine; HNE, 4-hydroxynonenal; A.U., arbitrary units; *, p< 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; n, number of experiments, each featuring an independent cell culture preparation.

Figure 3.. Proteolytic processing of p75^NTR is Induced by Oxidative Stress in Mesencephalic Cells.

A; Representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were exposed to vehicle solution or 6-OHDA for 18 hours after a 30-minute pretreatment with vehicle solution or 10 mM N-acetyl cysteine. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. B-D; densitometric analysis of p75^NTR-CTF (B.), p75^NTR-ICD (C.), or p75^NTR-FL (D.) from immunoblots described in 3A (n = 5; ANOVA with Tukey’s HSD). E; representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were exposed to vehicle solution, 1 μM HNE, or 2 μM HNE for 18 hours. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. F – H;, densitometric analysis of p75^NTR-CTF (F.), p75^NTR-ICD (G.), or p75^NTR-FL (H.) from immunoblots described in 3E (n = 5, ANOVA with Tukey’s HSD). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; 6-OH, 6-hydroxydopamine; NAC, N-acetyl cysteine; HNE, 4-hydroxynonenal; A.U., arbitrary units; *, p< 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; n, number of experiments, each featuring an independent cell culture preparation.

Figure 4.. Oxidative Stress Induces p75^NTR Processing in Mesencephalic Cells via a Ligand-independent Mechanism.

A; representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were treated for 18 hours with vehicle solution or 10 μM 6-OHDA following 1 hour of pretreatment with vehicle solution or ligand-blocking antibody specific for the p75^NTR-ECD. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. B – D; densitometric analysis of p75^NTR-CTF (B.), p75^NTR-ICD (C.), or p75^NTR-FL (D.) from immunoblots described in 5A. Pretreatment with ligand-blocking antibody caused no significant change in 6-OHDA-induced cleavage of p75^NTR (n = 5, ANOVA with Tukey’s HSD). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; α-p75, ligand-blocking antibody for p75^NTR; A.U., arbitrary units; *, p< 0.05. **, p < 0.01. ***, p < 0.001; ****, p < 0.0001; N.S., not significant; n, number of experiments, each featuring an independent cell culture preparation.

Figure 5.. 6-OHDA Promotes p75^NTR Cleavage in Mesencephalic Cells by Stimulating Metalloprotease and γ-secretase Activity.

A; representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were treated for 18 hours with vehicle solution or 10 μM 6-OHDA following 1 hour of pretreatment with the control solvent dimethyl sulfoxide (DMSO), 10 μM TAPI-1, or 250 nM DAPT. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. B - D; densitometric analysis of p75^NTR-CTF (B.), p75^NTR-ICD (C.), or p75^NTR-FL (D.) from immunoblots using lysates of differentiated LUHMES cells that were exposed to 10 μM 6-OHDA or vehicle solution following a one-hour pretreatment with 10 μM TAPI-1 or DMSO. Pretreatment with TAPI-1 significantly decreased accumulation of p75^NTR fragments, and exposure to 6-OHDA did not cause a significant loss of p75^NTR-FL in cells pretreated with TAPI-1 (n = 7, ANOVA with Tukey’s HSD). E - G; densitometric analysis of p75^NTR-CTF (E.), p75^NTR-ICD (F.), or p75^NTR-FL (G.) from immunoblots using lysates of differentiated LUHMES cells that were exposed to 10 μM 6-OHDA or vehicle solution following a one-hour pretreatment with 250 nM DAPT or DMSO. Pretreatment with DAPT significantly decreased accumulation of the p75^NTR-ICD following exposure to 6-OHDA (n = 7, ANOVA with Tukey’s HSD for 5E and 5F, Kruskal-Wallis with Dunn’s test for 5G). H. representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were treated for 18 hours with vehicle solution or 10 μM 6-OHDA following 1 hour of pretreatment with the control solvent dimethyl sulfoxide (DMSO) or 10 μM BB94. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. I – K; densitometric analysis of p75^NTR-CTF (I.), p75^NTR-ICD (J.), or p75^NTR-FL (K.) from immunoblots described in 5H. (n= 8, ANOVA with Tukey’s HSD for 5I and 5K, Kruskal-Wallis with Dunn’s test for 5J). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; 6-OH, 6-hydroxydopamine; TAPI, TAPI-1; A.U., arbitrary units; *, p< 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; n, number of experiments, each featuring an independent cell culture preparation.

Figure 6.. c-Jun N-terminal Kinase Activity is Required for Oxidative Stress-induced Proteolysis of p75^NTR.

A; representative western blot analysis of p75^NTR fragments in lysates of differentiated LUHMES cells that were treated for 18 hours with vehicle solution or 10 μM 6 OHDA following 1 hour of pretreatment with control solvent DMSO or with the JNK inhibitor SP600125. Fragments of p75^NTR were detected using an antibody specific for the p75^NTR-ICD. Cropped regions indicate different exposure times. Immunoblotting for actin was performed as a loading control. B-D; densitometric analysis of p75^NTR-CTF (B.), p75^NTR-ICD (C.), or p75^NTR-FL (D.) from immunoblots described in 6A. Pretreatment with SP600125 significantly reduced the accumulation of p75^NTR fragments and the loss of p75^NTR-FL associated with exposure to 6-OHDA (n = 6, ANOVA with Tukey’s HSD). E. Confocal micrographs of LUHMES cells that were differentiated for five days, pretreated with vehicle solution or 10 μM SP600125 for 1 hour, and fixed after exposure for 24 hours to vehicle solution or 7.5 μM 6-OHDA. The cells were immunolabeled with an antibody specific for ßIII tubulin (green), and counterstaining was performed by incubation with the nucleic acid dye DAPI at a concentration of 5 μg/mL. F. quantification of apoptosis of differentiated LUHMES cells that were treated as described in 6E (n = 4, ANOVA with Tukey’s HSD). Abbreviations: Veh, vehicle; 6-OHDA, 6-hydroxydopamine; 6-OH, 6-hydroxydopamine; SP6, SP600125; A.U., arbitrary units; *, p< 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant; n, number of experiments, each featuring an independent cell culture preparation.