UNCORRECTED PROOF THE G PROTEIN INHIBITOR, PERTUSSIS TOXIN, INHIBITS THE SECRETION OF BRAIN DERIVED NEUROTROPHIC FACTOR E. C. GUNTHER,* C. S. VON BARTHELD,² L. J. GOODMAN,³§ J. E. JOHNSONk and M. BOTHWELL*¶ *Department of Physiology and Biophysics, Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA ²Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA §Department of Neuroscience, Genentech, Inc., South San Francisco, CA 94080, USA kDepartment of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA Abstract ÐSecretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo- optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain derived neurotrophic factor between embryonic days 13±17 (E13±17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42% whereas injections at E19 had no effect. Co-injection of brain derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125 I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly due to diminished competition from endogenously released brain derived neurotrophic factors for the receptors that mediate retrograde axonal transport. These data suggest that the release of a major fraction of brain derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: Neurotrophin, cell death, retrograde transport, chicken, retina, BDNF. The neurotrophic hypothesis holds that the survival of developing neurons is regulated by a limiting amount of neuro- trophin released by cells within the target region of inner- vation. Neurons compete for this limited amount of trophic factor, thus matching the ®nal neuronal number with the size of their target. 34 Although this simplistic hypothesis does not encompass all of the diverse functions of neurotrophins revealed by recent investigations, it adequately describes the function of neurotrophins in many systems. Mechanisms that regulate the supply of neurotrophins are thus fundamental for the development of the nervous system. Recent studies suggest that in some populations of target cells, a signi®cantly large intracellular reservoir of neurotrophin may exist relative to that which is released to exert trophic effects on innervating neurons. 4 This suggests that mechanisms that regulate the secretion of neurotrophins likely play a critical role in de®n- ing neurotrophic support and in¯uencing the form and func- tion of neural structures. A well-characterized system that behaves in accord with the neurotrophic hypothesis is the developing isthmo-optic nucleus (ION) of the chick visual system. During develop- ment, ION neurons project axons from the midbrain to inner- vate target amacrine cells in the inner nuclear layer of the contralateral retina. From embryonic day 13 (E13) to E17, ION neurons depend on trophic support produced by the amacrine target cells. 7,8 This trophic support appears to be mediated largely by brain derived neurotrophic factor (BDNF), based on gain of function and loss of function studies with BDNF and trkB fusion proteins, respectively. 44,33,43 During this period, 50% of ION neurons normally undergo programmed cell death resulting from limited trophic support. 10,45 This system provides a convenient in vivo oppor- tunity to study mechanisms of neurotrophic signaling: intra- ocular administration of agents that modulate trophic support of the ION results in quanti®ably altered survival of ION neurons. The neurotrophins BDNF and nerve growth factor (NGF) are released in an activity-dependent manner by hippocampal neurons. 5,15,16 The AtT-20 anterior pituitary corticotroph cell line also has been extensively used to characterize secretory mechanisms of neuropeptides, including neurotrophins. 15,20 We have found recently that BDNF is targeted to a novel pool of regulated secretory granules in AtT-20 cells that are released under control of mechanisms that differ from those Pertussis toxin inhibits BNNF release 1 1 Neuroscience Vol. 00, No. 0, pp. 00±00, 2000 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00 PII: S0306-4522(00)00309-2 Pergamon www.elsevier.com/locate/neuroscience ¶To whom correspondence should be addressed. Tel.: 11-206-543-7924; fax: 11-206-543-0934. E-mail address: mab@u.washington.edu (M. Bothwell). ³Present address: Lynx Theraputics, Inc. 25861 Industrial Blvd. Hayward, CA 94545 Abbreviations: ACTH, adrenocorticotrophic hormone; ADP, adenosine diphosphare; BDNF, brain derived neurotrophic factor; BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; CO2, carbon dioxide; DMEM, Dulbecco's modi®ed eagle medium; ECLIA, electro- chemiluminescent immunoassay; ELISA, enzyme linked immunosor- bant asssay; FBS, fetal bovine serum; GCL, ganglion cell layer; HRP, horse raddish peroxidase; INL, inner nuclear layer; ION, isthmo optic nucleus; IPL, inner plexiform layer; KCl, potassium chloride; NaCl, sodium chloride; NGF, nerve growth factor; OPL, outer plexiform layer; PBS, phosphate buffered saline; PKA, protein kinase A; PR, photo receptor; PTX, pertussis toxin; SEM, standard error of the mean; TPA, tripropylamine. Neuroscience A4 280mm deep ± Own style ± AUTOPAGINATION 2 NSC4572 08-08-2000 08:54 NW1239 klh A lden