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Vasoactive intestinal peptide attenuates cytochrome c translocation, and apoptosis, in rat hippocampal stem cells
Neuroscience Letters 325 (2002) 151–154 www.elsevier.com/locate/neulet
Vasoactive intestinal peptide attenuates cytochrome c translocation, and apoptosis, in rat hippocampal stem cells Francis J. Antonawich a,b,*, Sami I. Said b–e a
Department of Neurology, HSC T12-020, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8121, USA b Northport DVA Medical Center, Northport, NY 11768, USA c Department of Medicine, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA d Department of Physiology, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA e Department of Pharmacology, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA Received 29 December 2001; received in revised form 6 February 2002; accepted 7 February 2002
Abstract While widely distributed in the brain, one area with concentrated levels of vasoactive intestinal peptide (VIP) is the hippocampus. In this study, rat hippocampal stem cells were used to examine VIP’s effects on apoptotic cell death induced by withdrawal of trophic support. In the apoptotic cascade, the translocation of cytochrome c from the mitochondria to the cytoplasm activates caspases, resulting in cell death. VIP decreased this translocation of cytochrome c in a dose-dependent manner, and reduced apoptosis. This demonstrates that VIP regulates neuronal apoptosis and may contribute to stem cell homeostasis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progenitor cell; Pituitary adenylate cyclase activating peptide; PACAP Cytochrome c
Originally viewed as a ‘classic gut peptide’, vasoactive intestinal peptide (VIP) has demonstrated a number of neuropeptide effects [18]. These influences range from promoting cell survival and differentiation [14,15], to neuroprotection from chemical [11,22], viral [4] and excitotoxic influences [9]. In this paper, we further demonstrate its modulatory effect on the apoptotic cell death cascade in central nervous system (CNS) progenitor cells. While apoptosis or programmed cell death is evident in normal cell development and homeostasis, it is also prevalent in numerous neurodegenerative conditions. Wyllie originally characterized apoptosis by DNA fragmentation, loss of normal cell contacts, cell blebbing and cell condensation, with subsequent phagocytosis of pyknotic cells [23]. Apoptosis involves the activation of a series of apoptosisregulating genes and their associated proteins. One such group is the B-cell lymphoma (bcl-2) family of anti- and pro-apoptotic proteins. Numerous insults to the brain, such as exposure to excitotoxin or transient global ischemia, lead to an overexpression of the pro-apoptotic member, bax [12]. Bax interacts with the anti-apoptotic proteins, bcl-2 and a * Corresponding author. Tel.: 11-631-444-3646; fax 11-631444-1474. E-mail address: [email protected] (F.J. Antonawich).
homolog bcl-xl, via dimerization. Unfavorable alterations in the ratio of bax to bcl-2 (bcl-xl) accelerate programmed cell death [2]. High bax protein levels result in the formation of mitochondrial pores, causing an increase in the release of cytochrome c [1,20]. Studies have demonstrated that cytochrome c, released into the cytoplasm, activates the caspase family of cysteine proteases [25]. Cytochrome c is a water-soluble protein whose primary function is to transport electrons within the mitochondria, between coenzyme Q and cytochrome oxidase. It has been demonstrated that cytochrome c activates the terminal enzyme, caspase 3. This is accomplished by cytochrome c forming a complex in the cytoplasm with apoptosis protease activating factor-1 and the precursor form of caspase 9 [1,13,21]. This union activates caspase 9, which propagates the activation of various members of the caspase family [24]. Evidence does exist demonstrating that VIP can both increase bcl-2 protein levels [3], as well as inhibit caspase 3 activation [6,8]. The present study was designed to demonstrate its influence on the translocation of cytochrome c from the mitochondria to the cytosol in progenitor cells undergoing apoptosis. Young, adult, female, Fischer 344 rats, weighing approximately 125 g, were used for the isolation and amplification of hippocampal progenitor cells. Their brains were removed
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and hippocampi dissected into cold Dulbecco’s phosphatebuffered saline (PBS). The tissue was finely diced, then resuspended (2–5 ml per lobe of hippocampus) in papain, neutral protease (dispase II), DNAse (PPD) solution. Tissue was incubated at 37 8C with occasional mixing for 1 h. The PPD was washed from the tissue by repeated centrifugation (400 £ g) and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum (FBS). The cell suspension was initially plated onto uncoated dishes in DMEM/F12 (1:1) containing 10% FBS. After 24 h, progenitor cells form neurospheres in the media. This medium was collected, centrifuged (400 £ g) and the progenitor cells, resuspended in DMEM/F12 supplemented with N2 and 20 ng/ml basic fibroblast growth factor (bFGF), were plated on laminin coated dishes. Every other day, 75% of the media was removed and replaced. As the cells began to attach and grow, any remaining debris was gently rinsed away. The isolated cells were ready for passage in about 4 weeks. Progenitors were maintained in DMEM/F12 (1:1) supplemented with neuronal growth support factors N2 and 20 ng/ml human bFGF (fibroblast growth factor-2; FGF-2). Passage of cells required 20–30% conditioned medium. Confirmation of progenitor cell identity was established by immunohistochemical staining, using neuronal (either b-tubulin type III or Neu N antisera), astroglial (glial fibrillary acidic protein antisera) and oligodendrocyte (Gal C antisera)specific markers. In order to determine the neuroprotective effects of VIP (supplied by the late Professor Viktor Mutt, Karolinska Institute, Stockholm, Sweden), it was diluted in PBS 1 0.1% bovine serum albumin and exposed to the progenitor cell cultures at three concentrations, based on previous work [19]. VIP has both a high-affinity and lowaffinity receptor, with most of the neuroprotective influences attributed to the low-affinity member. Since the specific receptor in hippocampal progenitor cells was not known, dosages of 2 nM (test high affinity), 5 mM (test low affinity) and 10 mM (test low affinity) were used. A 25 cm 2 confluent flask was passed into a 75 cm 2 flask using normal media (DMEM/F12 1 N2 1 FGF-2) and allowed to grow overnight. Five flasks were examined for the experiment (control; apoptotic; apoptotic 1 VIP, 2 nM; apoptotic 1 VIP, 5 mM; and apoptotic 1 VIP, 10 mM). The control flask merely had 50% of its conditioned medium removed and fresh normal medium added. The apoptotic flask had 100% of its medium removed and replaced with medium containing DMEM/F12 without N2 or FGF-2. Each of the VIP-treated flasks was treated like the apoptotic flask with the addition of VIP. The cultures were then incubated for 12 h (at 37 8C, 5% CO2), then collected for subcellular fractionation. This trial was conducted in triplicate and duplicated (n ¼ 6). Subcellular fractionation for the cytosolic and mitochondrial preparations was performed based on our published methods [1]. Hippocampal progenitor cells were suspended
in ice cold Lysis buffer containing 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 ( £ 6H2O), 1 mM EDTA ( £ 2H2O), 1 mM EGTA, 1 mM dithiothreitol and protease inhibitors (phenylmethylsulphonyl fluoride, 17 mg/ ml; aprotinin, 8 mg/ml; leupeptin, 2 mg/ml; Sigma). The cells were homogenized with a tissue tearer (VirTir, 50 strokes, high speed). The filtrate was centrifuged at 2100 rpm for 10 min at 4 8C. The pellet was stored at 220 8C and represented the nuclei and unlysed cells. The supernatant was re-centrifuged at 2900 rpm for 15 min at 4 8C. The supernatant, or cytosolic preparation, was removed and stored at 220 8C, while the pellet, or mitochondrial preparation, was resuspended in 200 ml of Lysis buffer and stored at 220 8C [3,6,7]. A bicinchoninic acid protein assay (Pierce, Inc.) was conducted and 50 mg protein samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/immunoblotting (15% gels). Since cytochrome oxidase does not leave the mitochondria, mouse anti-cytochrome oxidase (1:300, Molecular Probes, Inc.) was used to screen the subcellular fractionation preparations. Cytochrome c levels were examined using mouse anti-cytochrome c (1:1250, Pharmingen) antisera. This was followed by 0.15 mg/ml biotinylated goat anti-mouse IgG for 1 h at room temperature, horseradish peroxidase avidin–biotin complex reagent (Elite; Vector Labs.) for 1 h, then visualized by 10 min-exposure to diaminobenzidine. The Scion image analysis system was used to measure protein band intensities. The intensities were adjusted for trial difference by standardizing them to the control culture intensities. The significances of differences between intensity measurements of the cytosolic and mitochondrial preparations were estimated with an analysis of variance followed by a Duncan’s multiple range test to compare individual group means. Statistical analysis was carried out using the Statistical Analysis System (SAS Institute, Cary, NC). Following growth factor withdrawal, hippocampal progenitor cells undergo apoptosis. This is demonstrated by the activation of a number of pro-apoptotic regulatory proteins and the subsequent translocation of the mitochondrial enzyme cytochrome c. A facilitation of cytochrome c into the cytosol becomes evident by 12 h after growth factor withdrawal. The modulation of apoptosis by VIP appears to be influenced by the low-affinity VIP receptor, as no significant effects were observed at 2 nM VIP, while both the 5 and 10 mM dosages significantly decreased cytochrome c’s movement into the cytoplasm (Fig. 1). These findings suggest that the low-affinity VIP receptor may modulate the apoptotic cascade by limiting cytochrome c translocation into the cytoplasm. VIP influences neurogenesis based on the cell type and stage of development. While micromolar concentrations will decrease cortical proliferation both in vivo and in vitro at ,E13.5 days, exposure for .15.5 days was asso-
F.J. Antonawich, S.I. Said / Neuroscience Letters 325 (2002) 151–154
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the trophic and protective actions of the neuropeptide VIP on neurogenic cells. By inhibiting cytochrome c translocation, the apoptotic cascade can be disrupted, and activation of caspases prevented. This influence on hippocampal progenitor cells is particularly interesting since VIP, while widely distributed in the brain, is concentrated in certain areas including the hippocampus, and thus may contribute to normal homeostasis of these germinal cells.
Fig. 1. Cytochrome c protein levels in hippocampal progenitor cells. Hippocampal progenitor cells were induced to undergo apoptosis by the withdrawal of FGF and N2 supplement. Both 5 and 10 mm concentrations of VIP, for 24 h, significantly (P , 0:05) protected the cells from translocation of cytochrome c (n ¼ 3 cultures in triplicate per group).
ciated with an increased progenitor cell proliferation from superior cervical ganglia in culture [16]. These effects both appear to be mediated via the low-affinity (PAC1) VIP receptor, rather than by one of the high-affinity VIP (VPAC1, VPAC2) receptors. The neurotrophic and neuroprotective effects of VIP may be mediated in large part via glial-derived neurotrophism. The release of femtomolar-acting peptides from VIP-stimulated astrocytes prevents cell death from a variety of neurotoxic substances [5,7]. Furthermore, VIP exhibits anti-apoptotic activity following excitotoxic lung injury by preserving bcl-2 expression as well as inhibiting caspase 3 activation after N-methyl-daspartate exposure [3,6]. In the present study, we examined the neuroprotective effects of VIP in neuronal stem cells. CNS proliferative cells were originally viewed as simply glial precursors, until Reynolds and Weiss [17] demonstrated in vitro the derivation of undifferentiated multipotent cells. While the largest populations of neural stem cells reside in the subventricular zone and hippocampal dentate gyrus, additional cells have been isolated from the septum, striatum, cortex, hypothalamus and spinal cord [10,14]. With the proper mitogenic stimuli, these germinal cells are capable of forming a mixed culture of neurons, astrocytes and oligodendrocytes. VIP appears to modulate the apoptotic cascade in neuronal progenitor cell cultures after growth factor withdrawal, as evidenced by decreasing cytochrome c translocation. This effect appears to be mediated by the low-affinity VIP receptor since micromolar concentrations of VIP were necessary to obtain significant reductions in cytosolic cytochrome c levels. The mechanism of this attenuated action remains to be determined. Since our experiments were conducted on mixed cultures, the release of astrocyteinduced femtomolar-peptides may have contributed to the protection. The evidence described in this paper further demonstrates
The authors would like to thank Mrs Susan Fiore-Marasa for her excellent technical assistance. This work was supported by research funds from the Department of Veterans Affairs. F.J.A. is a recipient of a Career Development Award from the VA. [1] Antonawich, F.J., Translocation of cytochrome c following transient global ischemia, Neurosci. Lett., 273 (1999) 1–4. [2] Antonawich, F.J., Krajewski, S., Reed, J.C. and Davis, J.N., Bcl-x/Bax interaction after transient global ischemia, J. Cereb. Blood Metab., 18 (1998) 882–886. [3] Bandyopadhyay, A., Dickman, K., Mathew, S.M. and Said, S.I., Vasoactive intestinal peptide (VIP) as an anti-apoptotic agent in the lung, Am. J. Respir. Crit. Care Med., 157 (1998) A690. [4] Brenneman, D.E., Westbrook, G.L., Fitzgerald, S.P., Ennist, D.L., Elkins, K.L., Ruff, M.R. and Pert, C.B., Neuronal cell killing by the envelope protein of HIV and its prevention by VIP, Nature, 335 (1988) 639–642. [5] Brenneman, D.E., Spong, C.Y. and Gozes, I., Protective peptides derived from novel glial proteins, Biochem. Soc. Trans., 28 (2000) 452–455. [6] Dickman, K., Mathew, S., Berisha, H.I., Bratut, M. and Said, S.I., Vasoactive intestinal peptide (VIP) inhibits caspase-3 activation: a basis for anti-apoptotic activity, Am. J. Respir. Crit. Care Med., 161 (2000) A97. [7] Filippatos, G.S., Lalude, O., Parameswaran, N., Said, S.I., Spielman, W. and Uhal, B.D., Regulation of apoptosis by vasoactive peptides, Am. J. Physiol., 281 (2001) L749–L761. [8] Gozes, I. and Brenneman, D.E., A new concept in the pharmacology of neuroprotection, J. Mol. Neurosci., 14 (2000) 61–68. [9] Gressens, P., Marret, S., Hill, J.M., Brenneman, D.E., Gozes, I. and Fridkin, M., Vasoactive intestinal peptide prevents excitotoxic cell death in the murine developing brain, J. Clin. Invest., 100 (1999) 390–397. [10] Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U. and Frisen, J., Identification of a neural stem cell in the adult mammalian central nervous system, Cell, 96 (1999) 25–34. [11] Kaiser, P.K. and Lipton, S.A., VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro, Neuron, 5 (1990) 373–381. [12] Krajewski, S., Mai, J.K., Krajewska, M., Sikorska, M., Mossakowski, M. and Reed, J.C., Upregulation of bax protein levels in neurons following cerebral ischemia, J. Neurosci., 15 (1995) 6364–6376. [13] Li, P., Nijhawan, D., Budiharardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, M., Alnemri, E.S. and Wang, X., Cytochrome c and dATP dependent formation of Apaf-1/ caspase-9 complex initiates an apoptotic protease cascade, Cell, 91 (1997) 479–489. [14] Palmer, T.D., Takahashi, J. and Gage, F.H., The adult rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci., 8 (1997) 389–404.
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[15] Pence, J.C. and Shorter, N.A., In vitro differentiation of human neuroblastoma cells caused by vasoactive intestinal peptide, Cancer Res., 50 (1990) 5177–5183. [16] Pincus, D.W., Di Cicco-Bloom, E.M. and Black, I.B., Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts, Nature, 343 (1990) 564–567. [17] Reynolds, B.A. and Weiss, S., Generation of neurons and astrocytes from isolated cells of the adult mammalian nervous system, Science, 255 (1992) 1707–1710. [18] Said, S., The Viktor Mutt Memorial Lecture: protection by VIP and related peptides against cell death and tissue injury, Ann. N. Y. Acad. Sci., 921 (2000) 264–274. [19] Said, S., Dickman, K., Dey, R.D., Bandyopadhyay, A., DeStefanis, P., Raz, S., Pakbaz, H. and Berisha, H.I., Glutamate toxicity in the lung and neuronal cells: prevention or attenuation by VIP and PACAP, Ann. N. Y. Acad. Sci., 865 (1998) 226–237.
[20] Schendel, S.L., Montal, M. and Reed, J.C., Bcl-2 family of proteins as ion-channels, Cell Death Differ., 5 (1998) 372–380. [21] Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T. and Alnemri, E.S., Autoactivation of procaspase-9 by Apaf-1 mediated oligomerization, Mol. Cell, 1 (1998) 949–957. [22] Tanaka, S. and Koike, T., VIP suppresses neuronal cell death induced by nerve growth factor deprivation in rat sympathetic ganglion cells in vitro, Neuropeptides, 26 (1994) 103– 111. [23] Wyllie, A.H., Apoptosis: an overview, Br. Med. Bull., 53 (1997) 451–465. [24] Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., Penninger, J.M. and Mak, T.W., Apaf-1 is required for mitochondrial pathways of apoptosis and brain development, Cell, 94 (1999) 739–750. [25] Zhivotovsky, B., Hanson, K.P. and Orrenius, S., Back to the future: the role of cytochrome c in cell death, Cell Death Differ., 5 (1998) 459–460.
Vasoactive intestinal peptide attenuates cytochrome c translocation, and apoptosis, in rat hippocampal stem cells Francis J. Antonawich a,b,*, Sami I. Said b–e a
Department of Neurology, HSC T12-020, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8121, USA b Northport DVA Medical Center, Northport, NY 11768, USA c Department of Medicine, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA d Department of Physiology, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA e Department of Pharmacology, S.U.N.Y. at Stony Brook, Stony Brook, NY 11794-8172, USA Received 29 December 2001; received in revised form 6 February 2002; accepted 7 February 2002
Abstract While widely distributed in the brain, one area with concentrated levels of vasoactive intestinal peptide (VIP) is the hippocampus. In this study, rat hippocampal stem cells were used to examine VIP’s effects on apoptotic cell death induced by withdrawal of trophic support. In the apoptotic cascade, the translocation of cytochrome c from the mitochondria to the cytoplasm activates caspases, resulting in cell death. VIP decreased this translocation of cytochrome c in a dose-dependent manner, and reduced apoptosis. This demonstrates that VIP regulates neuronal apoptosis and may contribute to stem cell homeostasis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progenitor cell; Pituitary adenylate cyclase activating peptide; PACAP Cytochrome c
Originally viewed as a ‘classic gut peptide’, vasoactive intestinal peptide (VIP) has demonstrated a number of neuropeptide effects [18]. These influences range from promoting cell survival and differentiation [14,15], to neuroprotection from chemical [11,22], viral [4] and excitotoxic influences [9]. In this paper, we further demonstrate its modulatory effect on the apoptotic cell death cascade in central nervous system (CNS) progenitor cells. While apoptosis or programmed cell death is evident in normal cell development and homeostasis, it is also prevalent in numerous neurodegenerative conditions. Wyllie originally characterized apoptosis by DNA fragmentation, loss of normal cell contacts, cell blebbing and cell condensation, with subsequent phagocytosis of pyknotic cells [23]. Apoptosis involves the activation of a series of apoptosisregulating genes and their associated proteins. One such group is the B-cell lymphoma (bcl-2) family of anti- and pro-apoptotic proteins. Numerous insults to the brain, such as exposure to excitotoxin or transient global ischemia, lead to an overexpression of the pro-apoptotic member, bax [12]. Bax interacts with the anti-apoptotic proteins, bcl-2 and a * Corresponding author. Tel.: 11-631-444-3646; fax 11-631444-1474. E-mail address: [email protected] (F.J. Antonawich).
homolog bcl-xl, via dimerization. Unfavorable alterations in the ratio of bax to bcl-2 (bcl-xl) accelerate programmed cell death [2]. High bax protein levels result in the formation of mitochondrial pores, causing an increase in the release of cytochrome c [1,20]. Studies have demonstrated that cytochrome c, released into the cytoplasm, activates the caspase family of cysteine proteases [25]. Cytochrome c is a water-soluble protein whose primary function is to transport electrons within the mitochondria, between coenzyme Q and cytochrome oxidase. It has been demonstrated that cytochrome c activates the terminal enzyme, caspase 3. This is accomplished by cytochrome c forming a complex in the cytoplasm with apoptosis protease activating factor-1 and the precursor form of caspase 9 [1,13,21]. This union activates caspase 9, which propagates the activation of various members of the caspase family [24]. Evidence does exist demonstrating that VIP can both increase bcl-2 protein levels [3], as well as inhibit caspase 3 activation [6,8]. The present study was designed to demonstrate its influence on the translocation of cytochrome c from the mitochondria to the cytosol in progenitor cells undergoing apoptosis. Young, adult, female, Fischer 344 rats, weighing approximately 125 g, were used for the isolation and amplification of hippocampal progenitor cells. Their brains were removed
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and hippocampi dissected into cold Dulbecco’s phosphatebuffered saline (PBS). The tissue was finely diced, then resuspended (2–5 ml per lobe of hippocampus) in papain, neutral protease (dispase II), DNAse (PPD) solution. Tissue was incubated at 37 8C with occasional mixing for 1 h. The PPD was washed from the tissue by repeated centrifugation (400 £ g) and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum (FBS). The cell suspension was initially plated onto uncoated dishes in DMEM/F12 (1:1) containing 10% FBS. After 24 h, progenitor cells form neurospheres in the media. This medium was collected, centrifuged (400 £ g) and the progenitor cells, resuspended in DMEM/F12 supplemented with N2 and 20 ng/ml basic fibroblast growth factor (bFGF), were plated on laminin coated dishes. Every other day, 75% of the media was removed and replaced. As the cells began to attach and grow, any remaining debris was gently rinsed away. The isolated cells were ready for passage in about 4 weeks. Progenitors were maintained in DMEM/F12 (1:1) supplemented with neuronal growth support factors N2 and 20 ng/ml human bFGF (fibroblast growth factor-2; FGF-2). Passage of cells required 20–30% conditioned medium. Confirmation of progenitor cell identity was established by immunohistochemical staining, using neuronal (either b-tubulin type III or Neu N antisera), astroglial (glial fibrillary acidic protein antisera) and oligodendrocyte (Gal C antisera)specific markers. In order to determine the neuroprotective effects of VIP (supplied by the late Professor Viktor Mutt, Karolinska Institute, Stockholm, Sweden), it was diluted in PBS 1 0.1% bovine serum albumin and exposed to the progenitor cell cultures at three concentrations, based on previous work [19]. VIP has both a high-affinity and lowaffinity receptor, with most of the neuroprotective influences attributed to the low-affinity member. Since the specific receptor in hippocampal progenitor cells was not known, dosages of 2 nM (test high affinity), 5 mM (test low affinity) and 10 mM (test low affinity) were used. A 25 cm 2 confluent flask was passed into a 75 cm 2 flask using normal media (DMEM/F12 1 N2 1 FGF-2) and allowed to grow overnight. Five flasks were examined for the experiment (control; apoptotic; apoptotic 1 VIP, 2 nM; apoptotic 1 VIP, 5 mM; and apoptotic 1 VIP, 10 mM). The control flask merely had 50% of its conditioned medium removed and fresh normal medium added. The apoptotic flask had 100% of its medium removed and replaced with medium containing DMEM/F12 without N2 or FGF-2. Each of the VIP-treated flasks was treated like the apoptotic flask with the addition of VIP. The cultures were then incubated for 12 h (at 37 8C, 5% CO2), then collected for subcellular fractionation. This trial was conducted in triplicate and duplicated (n ¼ 6). Subcellular fractionation for the cytosolic and mitochondrial preparations was performed based on our published methods [1]. Hippocampal progenitor cells were suspended
in ice cold Lysis buffer containing 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 ( £ 6H2O), 1 mM EDTA ( £ 2H2O), 1 mM EGTA, 1 mM dithiothreitol and protease inhibitors (phenylmethylsulphonyl fluoride, 17 mg/ ml; aprotinin, 8 mg/ml; leupeptin, 2 mg/ml; Sigma). The cells were homogenized with a tissue tearer (VirTir, 50 strokes, high speed). The filtrate was centrifuged at 2100 rpm for 10 min at 4 8C. The pellet was stored at 220 8C and represented the nuclei and unlysed cells. The supernatant was re-centrifuged at 2900 rpm for 15 min at 4 8C. The supernatant, or cytosolic preparation, was removed and stored at 220 8C, while the pellet, or mitochondrial preparation, was resuspended in 200 ml of Lysis buffer and stored at 220 8C [3,6,7]. A bicinchoninic acid protein assay (Pierce, Inc.) was conducted and 50 mg protein samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/immunoblotting (15% gels). Since cytochrome oxidase does not leave the mitochondria, mouse anti-cytochrome oxidase (1:300, Molecular Probes, Inc.) was used to screen the subcellular fractionation preparations. Cytochrome c levels were examined using mouse anti-cytochrome c (1:1250, Pharmingen) antisera. This was followed by 0.15 mg/ml biotinylated goat anti-mouse IgG for 1 h at room temperature, horseradish peroxidase avidin–biotin complex reagent (Elite; Vector Labs.) for 1 h, then visualized by 10 min-exposure to diaminobenzidine. The Scion image analysis system was used to measure protein band intensities. The intensities were adjusted for trial difference by standardizing them to the control culture intensities. The significances of differences between intensity measurements of the cytosolic and mitochondrial preparations were estimated with an analysis of variance followed by a Duncan’s multiple range test to compare individual group means. Statistical analysis was carried out using the Statistical Analysis System (SAS Institute, Cary, NC). Following growth factor withdrawal, hippocampal progenitor cells undergo apoptosis. This is demonstrated by the activation of a number of pro-apoptotic regulatory proteins and the subsequent translocation of the mitochondrial enzyme cytochrome c. A facilitation of cytochrome c into the cytosol becomes evident by 12 h after growth factor withdrawal. The modulation of apoptosis by VIP appears to be influenced by the low-affinity VIP receptor, as no significant effects were observed at 2 nM VIP, while both the 5 and 10 mM dosages significantly decreased cytochrome c’s movement into the cytoplasm (Fig. 1). These findings suggest that the low-affinity VIP receptor may modulate the apoptotic cascade by limiting cytochrome c translocation into the cytoplasm. VIP influences neurogenesis based on the cell type and stage of development. While micromolar concentrations will decrease cortical proliferation both in vivo and in vitro at ,E13.5 days, exposure for .15.5 days was asso-
F.J. Antonawich, S.I. Said / Neuroscience Letters 325 (2002) 151–154
153
the trophic and protective actions of the neuropeptide VIP on neurogenic cells. By inhibiting cytochrome c translocation, the apoptotic cascade can be disrupted, and activation of caspases prevented. This influence on hippocampal progenitor cells is particularly interesting since VIP, while widely distributed in the brain, is concentrated in certain areas including the hippocampus, and thus may contribute to normal homeostasis of these germinal cells.
Fig. 1. Cytochrome c protein levels in hippocampal progenitor cells. Hippocampal progenitor cells were induced to undergo apoptosis by the withdrawal of FGF and N2 supplement. Both 5 and 10 mm concentrations of VIP, for 24 h, significantly (P , 0:05) protected the cells from translocation of cytochrome c (n ¼ 3 cultures in triplicate per group).
ciated with an increased progenitor cell proliferation from superior cervical ganglia in culture [16]. These effects both appear to be mediated via the low-affinity (PAC1) VIP receptor, rather than by one of the high-affinity VIP (VPAC1, VPAC2) receptors. The neurotrophic and neuroprotective effects of VIP may be mediated in large part via glial-derived neurotrophism. The release of femtomolar-acting peptides from VIP-stimulated astrocytes prevents cell death from a variety of neurotoxic substances [5,7]. Furthermore, VIP exhibits anti-apoptotic activity following excitotoxic lung injury by preserving bcl-2 expression as well as inhibiting caspase 3 activation after N-methyl-daspartate exposure [3,6]. In the present study, we examined the neuroprotective effects of VIP in neuronal stem cells. CNS proliferative cells were originally viewed as simply glial precursors, until Reynolds and Weiss [17] demonstrated in vitro the derivation of undifferentiated multipotent cells. While the largest populations of neural stem cells reside in the subventricular zone and hippocampal dentate gyrus, additional cells have been isolated from the septum, striatum, cortex, hypothalamus and spinal cord [10,14]. With the proper mitogenic stimuli, these germinal cells are capable of forming a mixed culture of neurons, astrocytes and oligodendrocytes. VIP appears to modulate the apoptotic cascade in neuronal progenitor cell cultures after growth factor withdrawal, as evidenced by decreasing cytochrome c translocation. This effect appears to be mediated by the low-affinity VIP receptor since micromolar concentrations of VIP were necessary to obtain significant reductions in cytosolic cytochrome c levels. The mechanism of this attenuated action remains to be determined. Since our experiments were conducted on mixed cultures, the release of astrocyteinduced femtomolar-peptides may have contributed to the protection. The evidence described in this paper further demonstrates
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