EMBRYONIC NEURONAL DEATH DUE TO NEUROTROPHIN AND NEUROTRANSMITTER DEPRIVATION OCCURS INDEPENDENT OF APAF-1 N. HONARPOUR, a K. TABUCHI, b;c J. M. STARK, d R. E. HAMMER, c;e T. C. SU ë DHOF, b;c L. F. PARADA, f X. WANG, c;e J. A. RICHARDSON g and J. HERZ a * a Department of Molecular Genetics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Building, Dallas, TX 75390, USA b Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA c Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA d Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA e Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA f Center for Developmental Biology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA g Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA AbstractöApoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Speci¢cally, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-de¢cient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, di¡er- entiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of di¡erentiated neurons. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: caspase, cytochrome c, apoptosis, cell death, neuron, mouse. Apoptotic cell death occurs throughout the nervous sys- tem, in all cell types. It is estimated that at least half of the original cell population comprising the nervous sys- tem undergoes apoptotic cell death during development (Oppenheim, 1981; Burek and Oppenheim, 1999). Apo- ptosis has been hypothesized to play several roles during neural development such as optimizing the number of synaptic connections, removing unnecessary neurons, and helping in the patterning of the nervous system (Burek and Oppenheim, 1999). A neuron's chance for survival during development is thought to be dependent on the extent of its presynaptic and postsynaptic connec- tions. This suggests that neurons are initially overpro- duced and then compete for target-derived or presynaptic trophic factors, including neurotrophins and neurotransmitters (Cowan et al., 1984; Okado and Oppenheim, 1984; Linden, 1994). Cytochrome c release from the mitochondria (in response to apoptotic stimuli) is a critical step in initiat- ing apoptosis and is tightly regulated by pro- and anti- apoptotic Bcl-2 family members (Adams and Cory, 1998; Tsujimoto and Shimuzu, 2000). In the cytoplasm, cytochrome c is recruited to apoptotic protease-activat- ing factor-1 (Apaf-1), and the complex of Apaf-1, cyto- chrome c, and dATP results in the activation of procaspase-9, which subsequently activates procaspase-3 (Li et al., 1997). The activation of caspase-3 is a terminal step in apoptotic cell death. The apoptotic cascade directed by cytochrome c release has proven to be impor- 263 *Corresponding author. Tel. : +1-214-648-5633 ; fax : +1-214-648- 8804. E-mail address : herz@utsw.swmed.edu (J. Herz). Abbreviations : Apaf-1, apoptotic protease-activating factor-1 ; BDNF, brain-derived neurotrophic factor ; DRG, dorsal root ganglion ; E, embryonic day ; EDTA, ethylenediaminetetraacetic acid ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulfonic acid) ; NT, neurotrophin ; PBS, phosphate-bu¡ered saline ; PCR, polymerase chain reaction; SDS^PAGE, sodium dodecyl sulfate^ polyacrylamide gel electrophoresis ; TrK, tyrosine kinase recep- tor ; TUNEL, terminal (TdT)-mediated dUTP-biotin nick end labeling. NSC 5118 10-9-01 Cyaan Magenta Geel Zwart www.elsevier.com/locate/neuroscience Neuroscience Vol. 106, No. 2, pp. 263^274, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII:S0306-4522(01)00275-5 0306-4522 / 01 $20.00+0.00