Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons

Science

Volumn 286, Issue 5448, 1999, Pages 2358-2361

  Riccio, Antonella ^a   Ahn, Sohyun ^a   Davenport, Christopher M ^a   Blendy, Julie A ^b   Ginty, David D ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b HOSPITAL OF THE UNIVERSITY OF PENNSYLVANIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIC AMP RESPONSIVE ELEMENT BINDING PROTEIN; NERVE GROWTH FACTOR; PROTEIN BCL 2; TRANSCRIPTION FACTOR;

ANIMAL CELL; ARTICLE; CELL SURVIVAL; CONTROLLED STUDY; GENE EXPRESSION; NONHUMAN; PRIORITY JOURNAL; SYMPATHETIC NERVE CELL;

ANIMALS; APOPTOSIS; AXONS; BRAIN-DERIVED NEUROTROPHIC FACTOR; CELL NUCLEUS; CELL SURVIVAL; CELLS, CULTURED; CYCLIC AMP RESPONSE ELEMENT-BINDING PROTEIN; GENE EXPRESSION REGULATION; GENES, BCL-2; GENETIC VECTORS; NERVE GROWTH FACTOR; NEURONS; PC12 CELLS; PROMOTER REGIONS (GENETICS); PROTO-ONCOGENE PROTEINS C-BCL-2; RATS; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; SYMPATHETIC NERVOUS SYSTEM; TRANSFECTION;

ANIMALIA;

EID: 0033579301     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5448.2358     Document Type: Article

References (27)

1. L. F. Reichardt and I. Farinas, in Molecular and Cellular Approaches to Neural Development, W. M. Cowan, T. M. Jessell, S. L. Zipursky, Eds. (Oxford Univ. Press, New York, 1997), pp. 220-263.
2. A. Bonni, D. D. Ginty, H. Dudek, M. E. Greenberg, Mol. Cell. Neurosci. 6, 168 (1995).
3. D. D. Ginty, A. Bonni, M. E. Greenberg, Cell 77, 713 (1994).
4. S. Ahn et al., Mol. Cell. Biol. 18, 967 (1998).
5. S. Finkbeiner et al., Neuron 19, 1031 (1997).
6. A. Riccio, B. Pierchala, C. L. Ciarallo, D. D. Ginty, Science 277, 1097 (1997).
7. G. A. Gonzalez and M. R. Montminy, Cell 59, 675 (1989).
8. M. Deshmukh and E. M. Johnson Jr., Neuron 21, 695 (1998).
9. A. Riccio, S. Ahn, D. D. Ginty, unpublished observations.
10. D. Rudolph et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4481 (1998).
11. R. S. Struthers, W. W. Vale, C. Arias, P. E. Sawchenko, M. R. Montminy, Nature 350, 622 (1991).
12. X. Tao, S. Finkbeiner, D. B. Arnold, A. J. Shaywitz, M. E. Greenberg, Neuron 20, 709 (1998).
13. I. Garcia, I. Martinou, Y. Tsujimoto, J.-C. Martinou, Science 258, 302 (1992).
14. L. J. Greenlund, S. J. Korsmeyer, E. M. Johnson Jr., Neuron 15, 649 (1995).
15. T. M. Michaelidis et al., Neuron 17, 75 (1996).
16. B. E. Wilson, E. Mochon, L. M. Boxer, Mol. Cell. Biol. 16, 5546 (1996).
17. A. Bonni et a., Science 286, 1358 (1999): J. P. Somers, J. A. DeLoia, A. J. Zeleznik, Mol. Endocrinol. 13, 1364 (1999).
18. A. Bonni et a., Science 286, 1358 (1999): J. P. Somers, J. A. DeLoia, A. J. Zeleznik, Mol. Endocrinol. 13, 1364 (1999).
19. Y.-Z. Liu, L. M. Boxer, D. S. Latchman, Nucleic Acids Res. 27, 2086 (1999).
20. R. E. Mains and P. H. Patterson, J. Cell Biol. 59, 329 (1973).
21. E. H.-Y. Cheng et al., Science 278, 1966 (1997).
22. R. B. Campenot, Proc. Natl. Acad. Sci. U.S.A. 74, 4516 (1977).
23. A. Lanahan, G. Lyford, G. S. Stevenson, P. F. Worley, C. A. Barnes, J. Neurosci. 17, 2876 (1997).
24. 4 (pH 7.2)] containing pEGFP (50 μg/ml), an expression vector encoding enhanced green fluorescent protein (GFP; Clontech), Rhodamine-dextran (4 mg/ml; Sigma; this was excluded in experiments done for cytochrome c immunocytochemistry), and the indicated expression vectors. The concentrations of all other DNAs in the microinjection solutions were 50 μg/ml, except for HYC-Bcl-2 or the empty HYC vector (20), which were 700 μg/ml. Expression of CFP reached a maximum within 3 or 4 days after microinjection. A-CREB potently and specifically inhibits the DNA binding activity of CREB and closely related family members ATF-1 and CREM (4). Even at very high concentrations, A-CREB does not block the DNA binding activity of any other B-ZIP transcription factor tested, including ATF-2, c-Jun, Fos, VBP, JunD, C/EBP, and VBP (4). CREB is the most abundant member of the CREB subfamily of B-ZIP transcription factors in sympathetic neurons as determined by electrophoretic mobility shift assays (9). Thus, the effects of A-CREB are probably through inhibition of DNA binding activity of CREB, rather than that of ATF-1 or CREM. For NGF withdrawal experiments, neurons were placed in a medium containing antibody to NGF (1:1000, Sigma) 2 days after microinjection. Neurons were stained with Hoechst 33258 (Molecular Probes), microinjected neurons were identified by CFP fluorescence microscopy, and injected and noninjected neurons were scored for apoptotic nuclei as described by S. B. Maggirwar et a/. [J. Neurosci. 18, 10356 (1998)]. Cytochrome c immunocytochemistry was done essentially as described (8).
25. Cortical neurons were dissociated from E1B rat embryos and grown in culture (3). PC12 cells were grown in Dulbecco's modified Eagle's medium in the presence of fetal bovine serum (10%) and horse serum (5%). Cultures were treated with medium containing BDNF or NGF, and total RNA was collected and analyzed by Northern (RNA) blot analysis (2). For reverse Northern experiments, sympathetic neurons were grown in 1-mm barrier compartmentalized cultures, which allows for separation of neuronal cell bodies and proximal axons from their distal axons, as described (6, 21). After 7 days of growth to develop compartmentalization of cell bodies and distal axons. growth medium in the cell body compartments was replaced with a medium containing antibody to NGF for 6 days. Then, growth medium bathing distal axons was replaced with medium containing a low concentration of NGF (2 ng/ml) for 2 days. Distal axons were then exposed to a high concentration of NGF (100 ng/ml) for 3.5 hours. Messenger RNA was isolated with Microfast Track Kit (Invitrogen), and double-strand cDNA was obtained with the Superscript Choice System (Gibco). Reverse Northern analysis of Bcl-2 and GAPDH was performed as described (22). Protein immunoblot analysis was performed as described (3). Protein extract (25 μg) prepared from forebrains of mouse embryos was resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with a Bcl-2 monoclonal antibody (1 μg/ml; Santa Cruz Biotechnology), a CREB polyclonal antibody (1:500; NEB), and an α-tubulin monoclonal antibody (1:10,000; Sigma).
26. PC12 cells were transfected with plasmids with Lipofectamine Plus Reagent (LTI), and reporter gene activity was assessed 2 days later with the Dual Luciferase Assay System (Promega). Plasmids amounts used per 60 mm plate of cells were as follows: Bcl-2 reporter constructs, 2 μg; A-CREB and CREBm1 expression plasmids, 4 μg; and GFP, 0.5 μg. Luciferase expression values were normalized for transfection efficiency, which was determined by the expression of the TK-renilla luciferase expression vector (Promega), 0.05 μg of which was included in all transfections. About 30% of PC12 and 80% of HEK 293T cells were transfected as determined by counting the number of GFP-positive cells.
27. We thank A. Kolodkin, R. Misra, F. Rupp, and members of the Ginty laboratory for helpful discussions and comments on this manuscript; A. Lanahan and P. Worley for advice with the reverse Northern experiment; B. Lonze for help with figures; A. Shaywitz and M. Greenberg for the CREB-VP16 constructs; M. Hardwick for discussions and Bcl-2 expression vectors; B. A. Tsui-Pierchala for advice with cytochrome c immunocytochemistry; and L Boxer for Bcl-2-luciferase constructs. CREB mutant mice were provided by G. Schutz and the Deutsches Krebforschungzentrum. Supported by a Pew Scholars Award and NIH grant NS34814-04 (D.D.G.).