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Abstract

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor–dependent expression of genes encoding prosurvival factors.

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REFERENCES AND NOTES

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Sympathetic neurons were isolated from superior cervical ganglia of neonatal rats (19) and grown in normal growth medium (6). Nuclei of neurons grown for 6 to 9 days in vitro (DIV) were microinjected with plasmids with an Eppendorf Transjector and Micromanipulator System. Before microinjection, neurons were placed in serum-free Hanks' balanced salt solution containing bovine serum albumin (1%), penicillin, and streptomycin. Neurons were injected with microinjection solution [48 mM K2HPO4, 14 mM NaH2PO4, and 0.45 mM KH2PO4(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 GFP 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 GFP fluorescence microscopy, and injected and noninjected neurons were scored for apoptotic nuclei as described by S. B. Maggirwar et al. [J. Neurosci. 18, 10356 (1998)]. Cytochrome c immunocytochemistry was done essentially as described (8).
24
Cortical neurons were dissociated from E18 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).
25
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.
26
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.).

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Published In

Science
Volume 286 | Issue 5448
17 December 1999

Submission history

Received: 4 June 1999
Accepted: 16 November 1999
Published in print: 17 December 1999

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Authors

Affiliations

Antonella Riccio
Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205–2185, USA.
Sohyun Ahn
Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205–2185, USA.
Christopher M. Davenport
Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205–2185, USA.
Julie A. Blendy
Department of Pharmacology, University of Pennsylvania Medical Center, Philadelphia, PA 19104–6084, USA.
David D. Ginty*
Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205–2185, USA.

Notes

*
To whom correspondence should be addressed. E-mail: [email protected]

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