Hughes, A. L., Messineo-Jones, D., Lad, S.P., and Neet, K.E. (2001) Distinction Between Differentiation, Cell Cycle, and Apoptosis Signals in PC12 Cells by the Nerve Growth Factor Mutant, D9/13, which is Selective for the p75 Neurotrophin Receptor. J. Neurosci. Res. 63, 10-19.

Department of Biochemistry and Molecular Biology, Finch University of Health Sciences/The Chicago Medical School, N. Chicago, IL 60064

Abstract. The common neurotrophin receptor, p75NTR (low affinity nerve growth factor receptor), participates in the high affinity binding with the TrkA nerve growth factor (NGF) receptor, may mediate apoptosis, and may signal independently in a cell-specific manner. The potential of p75NTR to signal independently of TrkA was investigated with a NGF mutant protein (NGFD9/13) that binds poorly to TrkA (J. Biol. Chem. 270, 6278-6285, 1995). The NGFD9/13 mutant does not activate TrkA autophosphorylation and fails to stimulate the normal NGF-induced growth arrest, demonstrating that TrkA activation is required to arrest PC12 cells at the NGF-activated G1/S cell cycle checkpoint. However, apoptosis is successfully blocked and cell survival is promoted by the NGFD9/13 mutant in naïve PC12 cells after serum withdrawal, suggesting that p75NTR can signal for survival autonomously of TrkA. Annexin V binding, an indication of apoptotic plasma membrane disruption, is inhibited by both NGF and the NGFD9/13 mutant after serum deprivation. Both NGF and the NGFD9/13 mutant inhibit the rapid apoptotic internucleosomal DNA cleavage of PC12 cells upon serum deprivation. Furthermore, the level of caspase3-like activity that is rapidly activated by serum withdrawal from PC12 cells is reduced by both the NGFD9/13 protein and NGF. Finally, upon serum withdrawal both NGF and the NGFD9/13 mutant activate nuclear translocation of the transcriptional factor NF-kB (nuclear factor kB), a process involved in cell survival. These results are consistent with p75NTR inhibition of caspase-mediated apoptosis in PC12 cells. The different physiological responses elicited by NGFD9/13 indicate the potential for individual signaling by the two NGF receptors and also demonstrate the utility of NGF mutants for receptor-selective signal transduction.

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Figure 1. Bioassay of NGF and NGFD9/13 with PC12 cells. Neurite bearing cells were counted after 48 h (defined medium) or 7 days (serum-containing medium) with cells possessing a neurite more than one cell body length were scores as positive.

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Figure 2. Analysis of TrkA autophosphorylation by NGF and by NGFD9/13. PC12 cells were treated with NGF or NGFD9/13 for 15 min at 37 °C. Cell lysates were analyzed for TrkA phosphorylation by immunoprecipitation and anti-phosphotyrosine blots. Lane 1: molecular weight marker. Lane 2: Control untreated cells. Lane 3: 4 nM NGF. Lane 4: 8 nM NGF. Lane 5: 8 nM NGFD9/13. Lane 6: 4 nM tumor necrosis factor (TNF) as negative control. The position of the 140 kDa TrkA band is marked with the arrow.

Figure 3 (MS#5). Immunocytochemistry staining for NF-kB in serum-deprived PC12 cells with NGF or NGFD9/13. Cells were cultured in serum-free DMEM medium for 2 days in the absence (left) or presence of 4 nM NGF (center) or NGFD9/13 (right). Cells were fixed, stained with an antibody directed toward the p65 Rel subunit of NF-kB, visualized with DAB, and counterstained with eosin Y.

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Figure 4 (MS#3). Flow cytometric analysis of BrdU pulse labeled PC12 cells in the presence of NGF or NGFD9/13 in defined medium. Cells were incubated in the absence (left) or presence of 4 nM NGF (center) or NGFD9/13 (right) for 6 h and then labeled with BrdU for 2 h. After fixation, cells were stained with an anti-BrdU fluorescein-conjugated antibody, and counterstained with propidium iodide. BrdU immunostaining (green fluorescence) is presented on the y-axis and DNA content (red fluorescence) is on the x-axis. The boxed region contains percent of cells in S phase (%S). For Figs 4 & 5, each flow analysis was performed on a minimum of 10,000 cells and independently repeated 2 times and the data were quantitated using Coulter Epics Elite Multigraph software.

Figure 5 (MS#6). Flow cytometric analysis of BrdU pulse labeled in serum-deprived PC12 cells with NGF or NGFD9/13. Cells were cultured in serum-free DMEM medium for 8 h in the absence (top right) or presence of 4 nM NGF (bottom left) or NGFD9/13 (bottom right) and then labeled with BrdU for 2 h. Cells were also grown in serum containing medium for comparison and labeled in the same manner (top left). After fixation, cells were stained with an anti-BrdU/fluorescein conjugate antibody, and counterstained with propidium iodide. BrdU immunostaining (green fluorescence) is presented by the y-axis and DNA content (red fluorescence) is on the x-axis. Boxed regions (A) contain cells undergoing DNA fragmentation and, therefore, reflecting a DNA content less than diploid.

Figure 7. Live cell immunocytochemistry staining for annexin V binding in serum-deprived PC12 cells with NGF or NGFD9/13. Cells were cultured in serum-free DMEM medium for 6 h in the absence (A) or presence of 4 nM NGF (B) or NGFD9/13 (C), incubated with annexin V, visualized with DAB, and counterstained with eosin Y. Early apoptotic cells show a black stained plasma membrane (arrowheads); late apoptotic cells have gross membrane changes in permeability, fail to exclude the eosin Y (arrows), and hence have a black plasma membrane with a pink cytoplasm (seen here as dark gray). Cells were photographed at 20X magnification; the bottom row shows 2.5X enlargements of a portion of the fields above.

Figure 8. CPP32-like caspase activity measured in cell lysates from serum- deprived PC12 cells with NGF or NGFD9/13. Cells were cultured in serum medium (serum) or in serum-free DMEM medium for 8 h in the absence (DMEM) or presence of 4 nM NGF or NGFD9/13. CPP32-like protease activity in the PC12 cell lysate was measured by release of free fluorophore from a modified CPP32 consensus sequence peptide, detected at a specific emission wavelength of 460 nm. Relative fluorescence units were normalized by total protein in the lysate as measured by the BCA method.

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