Article Figures & Data
Figures
- Figure 1.
Generation of knock-in alleles of p75NTR lacking the DD or TM cysteine Cys259. A, Schematic of the p75ntr locus (not to scale) with strategy for generation of the ΔDD mutant allele. B, Schematic of the p75ntr locus (not to scale) with strategy for generation of the C259A mutant allele. C, Schematic of C-terminal sequences in wild-type, ΔDD, and C259A p75NTR proteins. Black lines outline the boundaries of exons 4, 5, and 6, denoted by their respective numbers. Sequences corresponding to the extracellular, TM, juxtamembrane (Jux), and DDs are colored in blue, purple, green, and orange, respectively. The C-terminal tail containing a putative PDZ-binding motif is in gray. The ΔDD protein ends in QGDTATSPV, where QGD corresponds to the end of the Jux domain and TATSPV to the C-terminal tail. For the C259A protein, the TGC (Cys) codon was changed to GCA (Ala). Black vertical lines mark the boundaries of exons 4, 5, and 6.
- Figure 2.
Expression of knock-in alleles of p75NTR lacking the DD or TM cysteine Cys259. A, Expression of p75ntr mRNA in cortex (Ctx), hippocampus (Hc), and basal forebrain (BF) of p75NTR wild-type and ΔDD adult mice as assessed by quantitative PCR. For each brain region, expression was normalized to actin mRNA levels. Error bars indicate average (relative to wild-type levels) ± SD of triplicate determinations (n = 3 different animals from each genotype). B, Expression of p75ntr mRNA in Ctx, Hc, and BF of p75NTR wild-type and C259A adult mice as assessed by quantitative PCR. For each brain region, expression was normalized to actin mRNA levels. Error bars indicate average (relative to wild-type levels) ± SD of triplicate determinations (n = 3 different animals from each genotype). C, Expression of p75NTR in cerebral cortex of 3-month-old wild-type (WT) knock-out (KO), ΔDD and C259A mice analyzed by Western blotting (n = 3). ECD, Antibody against extracellular domain; DD, antibody against DD. Ten micrograms of total protein extract was loaded in each lane. Reprobing with β-actin antibodies is shown as a loading control.
- Figure 3.
Induction of caspase-3 activation by proneurotrophins requires the p75NTR DD and TM Cys259. A, Activation of caspase-3 by 12 h treatment with proNGF or proBDNF in cultured embryonic hippocampal (Hc) neurons identified by MAP-2 staining, from p75NTR wild-type, knock-out (KO), ΔDD, and C259A mice. Error bars indicate average ± SD of three independent determinations. **p < 0.01 versus control. B, Activation of caspase-3 by 12 h treatment with proNGF or proBDNF in cultured embryonic cortical (Ctx) neurons identified by MAP-2 staining, from p75NTR wild-type, KO, ΔDD, and C259A mice. Error bars indicate average ± SD of three independent determinations. **p < 0.01 versus control. C, Representative photomicrographs of immunofluorescence staining for cleaved caspase-3 (red) and MAP-2 (green) in cultured cortical neurons. Scale bar, 50 μm.
- Figure 4.
Neuronal death induced by proNGF requires p75NTR DD and TM Cys259. A, Pyknotic nuclei identified by propidium iodide staining after 24 h treatment with proNGF in cultured embryonic hippocampal neurons identified by Tuj1 staining and counterstained with DAPI, from p75NTR wild-type and ΔDD mice. Error bars indicate average percentage of control ± SEM. **p < 0.01 versus control (n = 6). B, Pyknotic nuclei identified by propidium iodide staining after 24 h treatment with proNGF in cultured embryonic hippocampal neurons identified by Tuj1 staining and counterstained with DAPI from p75NTR wild-type and C259A mice. Error bars indicate average percentage of control ± SEM. **p < 0.01 versus control (n = 3). C, Representative photomicrographs of pyknotic nuclei (red), denoted by arrowheads, and Tuj1 staining (green) in cultured hippocampal neurons counterstained with DAPI (blue). Scale bar, 50 μm.
- Figure 5.
NGF and proNGF induce conformational changes in p75NTR that are dependent on the conserved TM cysteine. A, Representative experiment showing traces of average anisotropy change after addition of NGF or vehicle in cells expressing wild-type or C257A rat p75NTR. Addition of NGF, but not vehicle, induced positive anisotropy oscillations above baseline (horizontal axis at 0) that were abolished in the C257A mutant. B, Net anisotropy change over 15 min after addition of NGF or vehicle in cells expression wild-type or C257A p75NTR. Results are expressed as average ± SD (n = 3 experiments; n = 15–17 cells examined per experiment). AUC, Area under curve. **p < 0.001 versus vehicle. C, Representative experiment showing traces of average anisotropy change after addition of proNGF or vehicle in cells expressing wild-type or C257A rat p75NTR. Addition of proNGF induced positive anisotropy oscillations above baseline (horizontal axis at 0) that were abolished in the C257A mutant. D, Net anisotropy change over 15 min after addition of proNGF or vehicle in cells of wild-type or C257A p75NTR. Results are expressed as average ± SD (n = 3 experiments; n = 15–17 cells examined per experiment). **p < 0.001 versus vehicle.
- Figure 6.
Essential role of the p75NTR DD and TM cysteine Cys259 in cell death induced by pilocarpine-mediated seizures. A, TUNEL staining (green) appears mainly on neurons identified by NeuN staining (red) in the hippocampal hilar region of adult wild-type mice 24 h after pilocarpine-induced seizures. Scale bar, 50 μm. B, Representative photomicrographs of TUNEL staining (green) in hippocampus, somatosensory cortex, piriform cortex, and entorhinal cortex of wild-type, KO, ΔDD, and C259A mice. Scale bars, 100 μm, hippocampus; 50 μm, cortices (5 μm, insets). C, TUNEL-positive cells in hippocampus (HC), somatosensory cortex (SC), piriform cortex (PC), and entorhinal cortex (EC) of wild-type and p75NTR KO mice. Error bars indicate average ± SEM. The number of animals used in each group is indicated in brackets. **p < 0.01 versus wild-type. D, TUNEL-positive cells in HC, SC, PC, and EC of wild-type and p75NTR ΔDD mice. Error bars indicate average ± SEM. The number of animals used in each group is indicated in brackets. *p < 0.05 versus wild-type. E, TUNEL-positive cells in HC, SC, PC, and EC of wild-type and p75NTR C259A mice. Error bars indicate average ± SEM. The number of animals used in each group is indicated in brackets. **p < 0.01 versus wild-type.
