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98 Gene expression of transcription factors in apoptotic cerebellar granule cells
97
98
Signal Transduction in Cell Death.
GENE EXPRESSION OF TRANSCRIPTION FACTORS IN APOI’TOTlC CEREBELLAR GRANULE CELLS
R Perez-Polo, K Werrbach-Perez, L Tong, G. Taghalatela, J Papaconstantinou, D. Sampath, T. Tolliver. IJniversity of Texas Medical Branch, Galveston, TX, IJSA, 77555-0652
D.F. Condorellil, A. Copan?, A. Calogero’, P. Dell’Albti’, F. Nicolett? and A.M. Giuffrida Stella’ Institute of Biochemistry1 (School of Medicine) and Institute of P~~~o~o~~J (School of Pharmacy), University of Catania; Istituto di Neuroscienze, Neuromed’ (Potilli), Italy
Apoptotic death plays a major role in development, trauma qury responses, and age-associated deficits tn the nervous system where neurotrophins (NT) wn spare neurons from death under these condltlons. While theu mechanisms of actlon are diverse and overlappmg, NTs differentially enhance cellular defenses by stimulating antioxidant activity and pyridinenueleotide metabolism, critical to neuronal resistonce to md recuperation from injuq. While causal factors for neuronal death range from the inttinsic (best described for programmed cell death (PCD] in development) to the extrinsic [due to trauma- and ischemia-mediated oxidatlve stress), we hypothesize that initially distinct regulatory pathways for death of NT-responsive neurons converge into common sipnalling pathways at the level of transcription factor activation. Our hypothesis IS that neuronal apoptosis due to oxidatlve stress results from dlsruption of glutathione (CSH) homeostasis and hanscription factors~gnalling that share elements with intrinsic neuronal death due to NT deprivation. We have found that NGF sparing from cell death involves the sctivatlon of the NF-KB and AP-I transcription factors. We found evidence for apoptosis and altered transcription factor signal transduction pathways in aged and oridatively stressed brain. Supported in part by NINDS NS- 18708, NS-33288, NS-3 1998 This is publication 46a from USPHS grant PO1 AG-10514
Cultured cerebellar granule cells undetwent apoptotic degeneration when grown in medium containing 10 instead of 2.5 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured Cfos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/Z68 was differentially regulated by extracellular K’ concentradon at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K’) are committed to degenerate. At this stage, c-fos mRNA levels were only detectable in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or fi-actin mRNA levels were only detectable at 7 DIV, when, in cultures grown in 10 mM K’, the percentage of apoptotic cells had already reached a plateau.
99
100
MECHANISMS OF NEURONAL DEGENERATION: A FINAL COMMON PATHWAY?
REGULATION
OF INJURY RESPONSE IN GLIA
1. de VeIlis, M. Kahn and S. Kumar. Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, CA. 90024-1759 USA
CM. Troy*, V. Singh*. L. Stefanis+. L.A. Greene*, ML. Shelanski* Departments of *Pathology and+Neurology. Taub Center for Alzheimer’s Disease Researchand Center for Neurobiology and Behavior. College of physicians and Surgeons, Columbia University, New York, New York 10032. USA.
During CNS injury and diseasethe levels of a number of cytokines are rapidly increased, e.g. TNFB, IFNB, IL-1 and CNTF. These cytokines can activate astroqtes and micro& which in turn secrete similar injurious signals, resulting in an inflammatory cascade which can involve: blood brain barrier breakdown, adhesion molecules upregulation. astrogliosis, cell death and demyelination. We have investigated the role of cytokines in the early steps of injury response during CNS development, in both in viva and in vitro models. Intracerebral injections of CNTF into developing postnatal rat pups resulted in astrogliosis and activation of microglia. Co-injections of CNTF and TNFa had a synergistic effect with astrogliosis reaching far into the contralateral side. Novel regulatory regions within the GFAP promoter that are important for CNTF induced transcriptional activation were identified by transfection experiments of serially deleted GFAP constructs into CG-4 cells. This model of glial progenitor was found to express TNF& and CNTF receptors at all developmental stages in vitro. CNTF, LIF, PDGF and NT-3 rescued cells from experimentally or developmentally induced cell death. CG4 cells were found to express full length functional TrkC receptors. Members of the IL-6 family of cytokines selectively induced several transcription factors. Differences in the expression of these factors appear to correlate with their efficacy as survival factors. The in vitro data strongly support a direct action of various cytokines on glial cells during an inflammatory response. In viva experiments in progress suggest a much more complex scenario.(Supported by NICHD and the National Multiple Sclerosis Society).
Our previous results indicated that the initiating causes of apoptotic deatb of neumnal cells induced by down-regulation of Cu*-Zn’+ superoxide dismutase (SODl) and withdrawal of trophic support (serumINGF) are different. However, bcl-2 rescues cells in either paradigm suggesting common downstream elements to tie cell death pathway. To determine whether the ICE (interleukin 18 converting enzyme) family of proteases. which is required for apoptosis on tmpbic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their accessto substrates.Blcckadz of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist, rescues neuronal cells from apoptotic death following apoptosis induced by down-regulation of SODI. as well as from tropbii factorMGF&privation. There were suhstantid differences in lbe concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and tmphic factor deprivation. These results suggest the involvement of different members of the IQ family, different subsna(esor both in the two diffeerent initiating causesof cell death
74
98
Signal Transduction in Cell Death.
GENE EXPRESSION OF TRANSCRIPTION FACTORS IN APOI’TOTlC CEREBELLAR GRANULE CELLS
R Perez-Polo, K Werrbach-Perez, L Tong, G. Taghalatela, J Papaconstantinou, D. Sampath, T. Tolliver. IJniversity of Texas Medical Branch, Galveston, TX, IJSA, 77555-0652
D.F. Condorellil, A. Copan?, A. Calogero’, P. Dell’Albti’, F. Nicolett? and A.M. Giuffrida Stella’ Institute of Biochemistry1 (School of Medicine) and Institute of P~~~o~o~~J (School of Pharmacy), University of Catania; Istituto di Neuroscienze, Neuromed’ (Potilli), Italy
Apoptotic death plays a major role in development, trauma qury responses, and age-associated deficits tn the nervous system where neurotrophins (NT) wn spare neurons from death under these condltlons. While theu mechanisms of actlon are diverse and overlappmg, NTs differentially enhance cellular defenses by stimulating antioxidant activity and pyridinenueleotide metabolism, critical to neuronal resistonce to md recuperation from injuq. While causal factors for neuronal death range from the inttinsic (best described for programmed cell death (PCD] in development) to the extrinsic [due to trauma- and ischemia-mediated oxidatlve stress), we hypothesize that initially distinct regulatory pathways for death of NT-responsive neurons converge into common sipnalling pathways at the level of transcription factor activation. Our hypothesis IS that neuronal apoptosis due to oxidatlve stress results from dlsruption of glutathione (CSH) homeostasis and hanscription factors~gnalling that share elements with intrinsic neuronal death due to NT deprivation. We have found that NGF sparing from cell death involves the sctivatlon of the NF-KB and AP-I transcription factors. We found evidence for apoptosis and altered transcription factor signal transduction pathways in aged and oridatively stressed brain. Supported in part by NINDS NS- 18708, NS-33288, NS-3 1998 This is publication 46a from USPHS grant PO1 AG-10514
Cultured cerebellar granule cells undetwent apoptotic degeneration when grown in medium containing 10 instead of 2.5 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured Cfos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/Z68 was differentially regulated by extracellular K’ concentradon at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K’) are committed to degenerate. At this stage, c-fos mRNA levels were only detectable in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or fi-actin mRNA levels were only detectable at 7 DIV, when, in cultures grown in 10 mM K’, the percentage of apoptotic cells had already reached a plateau.
99
100
MECHANISMS OF NEURONAL DEGENERATION: A FINAL COMMON PATHWAY?
REGULATION
OF INJURY RESPONSE IN GLIA
1. de VeIlis, M. Kahn and S. Kumar. Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, CA. 90024-1759 USA
CM. Troy*, V. Singh*. L. Stefanis+. L.A. Greene*, ML. Shelanski* Departments of *Pathology and+Neurology. Taub Center for Alzheimer’s Disease Researchand Center for Neurobiology and Behavior. College of physicians and Surgeons, Columbia University, New York, New York 10032. USA.
During CNS injury and diseasethe levels of a number of cytokines are rapidly increased, e.g. TNFB, IFNB, IL-1 and CNTF. These cytokines can activate astroqtes and micro& which in turn secrete similar injurious signals, resulting in an inflammatory cascade which can involve: blood brain barrier breakdown, adhesion molecules upregulation. astrogliosis, cell death and demyelination. We have investigated the role of cytokines in the early steps of injury response during CNS development, in both in viva and in vitro models. Intracerebral injections of CNTF into developing postnatal rat pups resulted in astrogliosis and activation of microglia. Co-injections of CNTF and TNFa had a synergistic effect with astrogliosis reaching far into the contralateral side. Novel regulatory regions within the GFAP promoter that are important for CNTF induced transcriptional activation were identified by transfection experiments of serially deleted GFAP constructs into CG-4 cells. This model of glial progenitor was found to express TNF& and CNTF receptors at all developmental stages in vitro. CNTF, LIF, PDGF and NT-3 rescued cells from experimentally or developmentally induced cell death. CG4 cells were found to express full length functional TrkC receptors. Members of the IL-6 family of cytokines selectively induced several transcription factors. Differences in the expression of these factors appear to correlate with their efficacy as survival factors. The in vitro data strongly support a direct action of various cytokines on glial cells during an inflammatory response. In viva experiments in progress suggest a much more complex scenario.(Supported by NICHD and the National Multiple Sclerosis Society).
Our previous results indicated that the initiating causes of apoptotic deatb of neumnal cells induced by down-regulation of Cu*-Zn’+ superoxide dismutase (SODl) and withdrawal of trophic support (serumINGF) are different. However, bcl-2 rescues cells in either paradigm suggesting common downstream elements to tie cell death pathway. To determine whether the ICE (interleukin 18 converting enzyme) family of proteases. which is required for apoptosis on tmpbic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their accessto substrates.Blcckadz of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist, rescues neuronal cells from apoptotic death following apoptosis induced by down-regulation of SODI. as well as from tropbii factorMGF&privation. There were suhstantid differences in lbe concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and tmphic factor deprivation. These results suggest the involvement of different members of the IQ family, different subsna(esor both in the two diffeerent initiating causesof cell death
74