- Email: [email protected]
P1-413
S218
Poster Presentations P1
although the importance of their interaction was still unclear. Our previous and current observations suggest that FE65 is a signaling molecule for the cellular stress, and APP can be a stress acceptor mediated by the phosphorylation at Thr668 of APP. These observations will contribute to the elucidation of the APP-mediated intracellular signaling pathway through FE65. P1-410
THE INTRACELLULAR DOMAIN OF THE AMYLOID-BETA PRECURSOR PROTEIN IS NOT INVOLVED IN NUCLEAR SIGNALING
Sebastien S. Hebert, Lutgarde Serneels, Katleen Craessaerts, Carmen Derks, Bart De Strooper, VIB Vlaams Interuniversitair Instituut voor Biotechnologie, Leuven, Belgium. Contact e-mail: [email protected] Background: The main hallmark of Alzheimer’s disease (AD) is the presence of insoluble amyloid-beta (A-beta) deposits in the brain. A-beta peptides are produced by proteolytic processing of the amyloid-beta precursor protein (APP) by beta- and gamma-secretases. Blocking A-beta generation by inhibiting or modulating gamma-secretase processing of APP is an attractive but problematic drug target for the treatment of AD. Apart from side effects caused by interference with the Notch signaling pathway, recent results suggest that blocking gamma-secretase would also interfere with other signaling pathways. Indeed, proteolytic processing of APP by gamma-secretase releases, concomitant with the A-beta peptides, the APP intracellular domain (AICD) which has been proposed to function in gene transcription regulation. Objectives: Several candidate AICD target genes have been identified over the last years including KAI1, APP, GSK3-beta and Neprilysin. Here, we raised the questions whether gamma-secretase inhibition would affect the expression of these putative AICD target genes and whether AICD has indeed a direct role in gene transcription regulation. Methods and Results: We found no effects of gamma-secretase inhibitors on the protein expression levels of these putative AICD target genes in different cell lines. Also deficiencies of different members of the gamma-secretase complex or the APP-family did not consistently affect the expression levels of these candidate AICD target genes in fibroblasts and in vivo. While AICD overexpression could activate weakly different endogenous promoters, the observed activity was at least one order of magnitude lower than the one obtained with the authentic gene transcription activator NICD (the Notch intracellular domain). We finally demonstrate that Fe65, a major AICD binding protein, transactivates a wide variety of different promoters, including the viral SV40 promoter, independently from AICD co-expression. Conclusions: We demonstrate that the selective inhibition of gamma-secretase and A-beta production does not cause major effects on the levels of genes that were suggested to be regulated by AICD. While this work cannot exclude that other yet unidentified genes are controlled by AICD mediated signaling, we dare to conclude that deregulation of the proposed AICD target genes is not a major additional concern when contemplating gamma-secretase inhibitors as a therapy in AD. P1-411
NUCLEO-CYTOPLASMIC SHUTTLING X11L2/X11GAMMA/MINT3 CONSTITUTES THE TRANSCRIPTIVELY ACTIVE COMPLEX WITH AICD
Akio Sumioka, Toshiharu Suzukil, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected] X11/mint family proteins contain multiple protein-interaction domains; CASK interaction (CI) domain, munc19 interaction (MI) domain, phospho-tyrosine interaction (PI) domain that binds to APP intracellular domain and two postsynaptic density protein, disc-large, zo-1 (PDZ) domains, that allow them to assemble multiple protein complexes. They regulate APP processing through their interaction with APP and are thought to play synaptic functions through the complexes as synaptic receptor tranport and/or synaptic vesicle exocyotosis. Human X11L2/X11-gamma/mint3 is composed of 575 amino acid residues, is a member of the X11 family and physiologically exhibits ubiquitous expression in several tissues. Here we report that X11L2 interacts
with the transcription factor and constitutes the transcriptively active complex with AICD. We demonstrate that, in cultured cells, an X11L2 protein continuously shuttles between the cytoplasm and the nucleus, and its localization is regulated by the specific sequence within X11L2, by laser microscopy technique. We demonstrate that X11L2, likely to FE65, fused with Gal4-DNA binding domain exhibits transcriptional activity and X11L2 activates the transcriptional activity mediated by Gal4-DNA binding domain fused AICD by reporter gene assay. We characterise the Gal4BD-X11L2 mediated transcriptional activity and demonstrate that X11L2 interacts with the transcription factor by co-immuno preciptation assay. Our findings show that Nucleocytoplasmic shuttling protein X11L2 plays as a cofactor of the transcription factor mediated gene transactivation and is siginificant for AICD mediated RIP signaling. P1-412
INTRACELLULAR AMYLOID 〉 ENHANCED EXPRESSION AND ACTIVITY OF CYTOSOLIC CALCIUM DEPENDENT PLA2 IN NEURONAL CELLS
Joanna B. Strosznajder, Anna Stolecka, Malgorzata Chalimoniuk, Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Background: Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the release of fatty acids from sn-2 position of phospholipids. These enzymes are responsible for maintaining of membrane phospholipids metabolism and the level of arachidonic acid (AA) that is the substrate for eicosanoids synthesis. Up till now the involvement of specific PLA2 isoforms in Alzheimer’s disease amyloid beta toxicity is not fully understood. Objective(s): The aim of this study was to investigate the effect of elevated intracellular amyloid  level on expression and activity of cytosolic PLA2 via NO/cGMP pathway in neuronal cell line (PC12 cells). Methods: Radio-, immunochemical and spectrofluorimetric methods were used. Results: We used control, transfected with human Amyloid Precursor Protein (APP, APPwt) and bearing the Swedish double mutation (APPsw) PC12 cells. The intracellular level of amyloid  in APPsw cells is 6 and 26 fold higher as compared to APPwt and control PC12 cells, respectively. Our results indicated enhancement of nitric oxide synthase (NOS) activity and cGMP level in APPsw and APPwt PC12 cells. The inhibition of ␥-secreatase by DAPT normalized intracellular A induced NOS activity and cGMP level. The protein level and activity of cPLA2 were higher in APPsw PC12 cells compared to APPwt and control PC12 cells. Incubation of [3H]AA prelabelled all cell types (APPwt, APPsw, PC12) in the presence of NO donors for 30 min stimulated [3H]AA release and free radicals generation. The high NO-induced AA release was observed in control PC12 cells. NO had only negligible effect on AA release in APPsw and APPwt PC12 cells as compared to corresponding nontreated cells. Inhibition of cPLA2 by ACOOF3 significantly decreased these processes and also basal activity of cPLA2. Inhibitor of both constitutive isoforms of NOS, NNLA and inhibitor of ␥-secreatase, DAPT reduced AA release and its metabolism in all investigated cells. Conclusions: Our data indicated that cGMP dependent protein kinase may be involved in basal and NO-induced AA release by cPLA2 activation in PC 12 cells. These results presented that intracellular amyloid  via activation of NO/cGMP pathway modulate expression and activity of cPLA2. This work is supported by PBZ-MIN-001/P05/16. P1-413
SORBS1, C-ABL AND C-CBL; INSULIN SIGNALLING PROTEINS IN ALZHEIMER’S DISEASE
Eirini Meimaridou1, Saliha Afzal1, Petra Nowotny2, Alison Goate2, Richard Killick1, Simon Lovestone1, 1Institute of Psychiatry, London, United Kingdom; 2School of Medicine, St Louis, WA, USA. Contact email: [email protected] Insulin signalling has been implicated in the pathogenesis of Alzheimer’s disease (AD) from many different directions including epidemiology, cel-
Poster Presentations P1 lular and animal studies. As insulin inhibits Glycogen Synthase Kinase-3 (GSK-3), which is both a candidate tau kinase and an APP kinase, it is possible that a relative failure of insulin signalling might directly increase the pathological molecular processes of AD. In order to determine if insulin signalling was altered in AD, we aimed to map the expression and activity of proteins on the insulin signalling pathway from diseased and elderly control post-mortem brain samples. One key participant in the insulin signalling pathway is SORBS1. Polymorphisms of the SORBS1 gene have been associated with obesity and type 2 diabetes [Lin et al., 2001] and the gene encoding SORBS1 is adjacent to a locus strongly associated with AD on chromosome 10 [Grupe et al., 2006]. We therefore determined expression of this protein in superior temporal gyrus in 54 AD cases and 14 control samples. No association between SORBS protein levels and AD or with the SNP in the region associating with AD was found. However, protein levels of downstream targets of SORBS1 protein including c-ABL and c-CBL were found to be significantly increased in AD. Ongoing studies of changes in the expression levels of other proteins involved in the insulin pathways and correlation with plaque and tangle counts will be presented. Lin WH, Chiu KC, Chang HM, Lee KC, Tai TY,Chuang LM. Molecular scanning of the human sorbin and SH3-domain-containing (SORBS1) gene: positive association of the T228A polymorphism with obesity and type 2 diabetes. Human molecular genetics 2001 10: 1753-1760 Chong ZZ, Li F, Maiese K. Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer’s disease. Brain Res Rev 49: 1-21, 2005 Caricasole A, Bakker A, Copani A, Nicoletti F,. Gaviraghi G, Terstappen GC. Two Sides of the Same Coin: Wnt Signaling in Neurodegeneration and Neuro-Oncology. Biosci Rep. 25:309-27, 2005 Grupe A et al. A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease. Am J Hum Genet. 78: 78-88 P1-414
DNA DECOY INTERVENTION OF BACE1 SYNTHESIS IN THE GUINEA PIG BRAIN: A THERAPEUTIC APPROACH
Diana C. Ferrari1, Krystyn Z. Bourne2, J. Regino Perez-Polo1, 1 University of Texas Medical Branch, Galveston, TX, USA; 2The University of Texas Medical School, Houston, TX, USA. Contact e-mail: [email protected] Background: The activity of the -site amyloid precursor protein cleaving enzyme type 1 (BACE1) is required for the formation of the -Amyloid peptide (A). A is the primary constituent of the extracellular amyloid deposits and a neuropathological marker for Alzheimer’s disease (AD). AD is characterized by cognitive deficits that correlate with dysfunctional cholinergic neurons in the basal forebrain, known to require nerve growth factor (NGF) for survival and proper cholinergic expression. The human and rat BACE1, as well as NGF and the NGF receptor p75 display NF-B binding consensus sequences in their promoters. Specifically, we have shown that NF-B stimulates BACE1 promoter activity and transcription in activated astrocytic cells (C6 cells and adult primary guinea pig hippocampus astrocytes) while repressing BACE1 in neuronal cells and inactivated astrocytes. Objective(s): To regulate the expression of BACE1 through NF-B specific decoys. Methods: NF-B decoy injections, Western Blot, Immunohistochemistry. Results: Western blot analyses of TNF␣ adult guinea pig primary astrocytes from hippocampus treated for 8 hours with 10M NGF or A showed an increase in the expression of BACE1 correlated with a decrease in IB␣. There was also a BACE1 increase after NGF treatment of PC12 cells. Twenty-four hour treatment of differentiated PC12 cells with 10M A and 1.6g/l NF-B decoys blocked NF-B repression of BACE1. Injection of 120 nmol. NF-B decoys into young male guinea pig hippocampi resulted in increased BACE1 levels after 6
S219
hours as measured by western blot assay. Thus, the young guinea pig hippocampal neurons and astrocytes, the latter typically not displaying activated features, behave as their in vitro counterparts in that NF-B appears to act as a repressor. Immunocytochemistry showed the presence of fluorescein-labeled NF-B decoys in nuclei of hippocampal neurons but not astrocytes as early as 2 hours and persisting for up to 6 hours after injection. Conclusions: These results would suggest that appropriate treatment with NF-B decoys of the aged or chronically stressed guinea pig brain might reduce the age- or stress-related brain pathology. This work has been supported in part by grants from the Sealy Center for Aging. P1-415
CONNEXIN-43: A POTENTIAL TRANSDUCTION TARGET FOR AMYLOID INTRACELLULAR DOMAIN?
Rania Y. Hallaq, Richard Killick, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected] Background: In 1995, the Nagy and Marotta group demonstrated that the stable expression of the C99 -cleaved stub of the -amyloid precursor proteins (APP) in the PC12 rat neuronal like cell line, lead to increased expression of Connexin 43 (Cx43), a gap junction subunit and concomitant increase in gap junctional communication. C99 is consitutively cleaved by ␥-secretase into the -amyloid (A) peptide(s) and the APP intracellular domain (AICD) which can vary in length from 49 to 59 amino acids. At the time of this report a signalling function for AICD, akin to that of the Notch ICDs, was not contemplated. To date this issue remains controversial. Results: We have repeated their study substituting PC12s for the human neuroblastoma cell line, SH-SY5Y, which expresses undetectable amounts of Cx43 by immunoblotting. Several clonal lines have been established expressing C99 or the empty vector. A dramatic increase in Cx43 was detected in only the C99 clones and a close positive correlation between C99 levels and Cx43 was observed. Method: Given that C99 is the precursor of both A and AICD we are now generating new cell lines expressing either AICD-␥59, C99, full length APP (wild type and mutant forms) or APP which lacks the ICD and treating SH-SY5Y cells with soluble A peptides. The cells will then be examined for Cx43 expression levels. In addition we are generating a Luciferase reporter gene containing the human Cx43 gene promoter sequence. Conclusion: These studies will inform us as to whether A or the AICD is responsible for the induction of Cx43 expression and whether either has direct transcriptional effects on the Cx43 promoter. P1-416
APP PROCESSING AND CELL CYCLE REENTRY SIGNALING PATHWAYS ARE INVOLVED IN P25/ CDK5-MEDIATED NEURODEGENERATION
Dohoon Kim, Jonathan C. Cruz, Lily Y. Moy, Xiaoyan Sun, Roderick T. Bronson, Jie Shen, Li-Huei Tsai, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Aberrant activation of Cyclin dependent kinase 5 (cdk5) by its regulatory subunit p25, the cleaved form of p35, has been implicated in a variety of neurodegenerative conditions including Alzheimer’s Disease. We recently demonstrated that aberrant activation of cdk5 by inducible overexpression of p25 results in massive neurodegeneration and brain atrophy in a transgenic model (p25Tg) after 5-6 weeks of induction. Objective(s): To investigate the mechanisms which precede and account for p25/cdk5-mediated neurotoxicity. Methods: We systematically examined p25Tg mouse brains at 2-3 weeks of p25 induction, which is prior to the onset of neurodegeneration, and at later timepoints following the onset of neurodegeneration. Various markers for AD-related pathology, as well as microarray analyses, were utilized. Results: We found that tau hyperphosphorylation was not detected at such an early time of induction. At later timepoints following the onset of neuronal degeneration, tau hyperphosphorylation was observed but was not closely correlated with dying neurons at the cellular level. On the other hand, robust increases in A levels were observed prior to the onset of neurodegeneration, while at later
Poster Presentations P1
although the importance of their interaction was still unclear. Our previous and current observations suggest that FE65 is a signaling molecule for the cellular stress, and APP can be a stress acceptor mediated by the phosphorylation at Thr668 of APP. These observations will contribute to the elucidation of the APP-mediated intracellular signaling pathway through FE65. P1-410
THE INTRACELLULAR DOMAIN OF THE AMYLOID-BETA PRECURSOR PROTEIN IS NOT INVOLVED IN NUCLEAR SIGNALING
Sebastien S. Hebert, Lutgarde Serneels, Katleen Craessaerts, Carmen Derks, Bart De Strooper, VIB Vlaams Interuniversitair Instituut voor Biotechnologie, Leuven, Belgium. Contact e-mail: [email protected] Background: The main hallmark of Alzheimer’s disease (AD) is the presence of insoluble amyloid-beta (A-beta) deposits in the brain. A-beta peptides are produced by proteolytic processing of the amyloid-beta precursor protein (APP) by beta- and gamma-secretases. Blocking A-beta generation by inhibiting or modulating gamma-secretase processing of APP is an attractive but problematic drug target for the treatment of AD. Apart from side effects caused by interference with the Notch signaling pathway, recent results suggest that blocking gamma-secretase would also interfere with other signaling pathways. Indeed, proteolytic processing of APP by gamma-secretase releases, concomitant with the A-beta peptides, the APP intracellular domain (AICD) which has been proposed to function in gene transcription regulation. Objectives: Several candidate AICD target genes have been identified over the last years including KAI1, APP, GSK3-beta and Neprilysin. Here, we raised the questions whether gamma-secretase inhibition would affect the expression of these putative AICD target genes and whether AICD has indeed a direct role in gene transcription regulation. Methods and Results: We found no effects of gamma-secretase inhibitors on the protein expression levels of these putative AICD target genes in different cell lines. Also deficiencies of different members of the gamma-secretase complex or the APP-family did not consistently affect the expression levels of these candidate AICD target genes in fibroblasts and in vivo. While AICD overexpression could activate weakly different endogenous promoters, the observed activity was at least one order of magnitude lower than the one obtained with the authentic gene transcription activator NICD (the Notch intracellular domain). We finally demonstrate that Fe65, a major AICD binding protein, transactivates a wide variety of different promoters, including the viral SV40 promoter, independently from AICD co-expression. Conclusions: We demonstrate that the selective inhibition of gamma-secretase and A-beta production does not cause major effects on the levels of genes that were suggested to be regulated by AICD. While this work cannot exclude that other yet unidentified genes are controlled by AICD mediated signaling, we dare to conclude that deregulation of the proposed AICD target genes is not a major additional concern when contemplating gamma-secretase inhibitors as a therapy in AD. P1-411
NUCLEO-CYTOPLASMIC SHUTTLING X11L2/X11GAMMA/MINT3 CONSTITUTES THE TRANSCRIPTIVELY ACTIVE COMPLEX WITH AICD
Akio Sumioka, Toshiharu Suzukil, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected] X11/mint family proteins contain multiple protein-interaction domains; CASK interaction (CI) domain, munc19 interaction (MI) domain, phospho-tyrosine interaction (PI) domain that binds to APP intracellular domain and two postsynaptic density protein, disc-large, zo-1 (PDZ) domains, that allow them to assemble multiple protein complexes. They regulate APP processing through their interaction with APP and are thought to play synaptic functions through the complexes as synaptic receptor tranport and/or synaptic vesicle exocyotosis. Human X11L2/X11-gamma/mint3 is composed of 575 amino acid residues, is a member of the X11 family and physiologically exhibits ubiquitous expression in several tissues. Here we report that X11L2 interacts
with the transcription factor and constitutes the transcriptively active complex with AICD. We demonstrate that, in cultured cells, an X11L2 protein continuously shuttles between the cytoplasm and the nucleus, and its localization is regulated by the specific sequence within X11L2, by laser microscopy technique. We demonstrate that X11L2, likely to FE65, fused with Gal4-DNA binding domain exhibits transcriptional activity and X11L2 activates the transcriptional activity mediated by Gal4-DNA binding domain fused AICD by reporter gene assay. We characterise the Gal4BD-X11L2 mediated transcriptional activity and demonstrate that X11L2 interacts with the transcription factor by co-immuno preciptation assay. Our findings show that Nucleocytoplasmic shuttling protein X11L2 plays as a cofactor of the transcription factor mediated gene transactivation and is siginificant for AICD mediated RIP signaling. P1-412
INTRACELLULAR AMYLOID 〉 ENHANCED EXPRESSION AND ACTIVITY OF CYTOSOLIC CALCIUM DEPENDENT PLA2 IN NEURONAL CELLS
Joanna B. Strosznajder, Anna Stolecka, Malgorzata Chalimoniuk, Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Background: Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the release of fatty acids from sn-2 position of phospholipids. These enzymes are responsible for maintaining of membrane phospholipids metabolism and the level of arachidonic acid (AA) that is the substrate for eicosanoids synthesis. Up till now the involvement of specific PLA2 isoforms in Alzheimer’s disease amyloid beta toxicity is not fully understood. Objective(s): The aim of this study was to investigate the effect of elevated intracellular amyloid  level on expression and activity of cytosolic PLA2 via NO/cGMP pathway in neuronal cell line (PC12 cells). Methods: Radio-, immunochemical and spectrofluorimetric methods were used. Results: We used control, transfected with human Amyloid Precursor Protein (APP, APPwt) and bearing the Swedish double mutation (APPsw) PC12 cells. The intracellular level of amyloid  in APPsw cells is 6 and 26 fold higher as compared to APPwt and control PC12 cells, respectively. Our results indicated enhancement of nitric oxide synthase (NOS) activity and cGMP level in APPsw and APPwt PC12 cells. The inhibition of ␥-secreatase by DAPT normalized intracellular A induced NOS activity and cGMP level. The protein level and activity of cPLA2 were higher in APPsw PC12 cells compared to APPwt and control PC12 cells. Incubation of [3H]AA prelabelled all cell types (APPwt, APPsw, PC12) in the presence of NO donors for 30 min stimulated [3H]AA release and free radicals generation. The high NO-induced AA release was observed in control PC12 cells. NO had only negligible effect on AA release in APPsw and APPwt PC12 cells as compared to corresponding nontreated cells. Inhibition of cPLA2 by ACOOF3 significantly decreased these processes and also basal activity of cPLA2. Inhibitor of both constitutive isoforms of NOS, NNLA and inhibitor of ␥-secreatase, DAPT reduced AA release and its metabolism in all investigated cells. Conclusions: Our data indicated that cGMP dependent protein kinase may be involved in basal and NO-induced AA release by cPLA2 activation in PC 12 cells. These results presented that intracellular amyloid  via activation of NO/cGMP pathway modulate expression and activity of cPLA2. This work is supported by PBZ-MIN-001/P05/16. P1-413
SORBS1, C-ABL AND C-CBL; INSULIN SIGNALLING PROTEINS IN ALZHEIMER’S DISEASE
Eirini Meimaridou1, Saliha Afzal1, Petra Nowotny2, Alison Goate2, Richard Killick1, Simon Lovestone1, 1Institute of Psychiatry, London, United Kingdom; 2School of Medicine, St Louis, WA, USA. Contact email: [email protected] Insulin signalling has been implicated in the pathogenesis of Alzheimer’s disease (AD) from many different directions including epidemiology, cel-
Poster Presentations P1 lular and animal studies. As insulin inhibits Glycogen Synthase Kinase-3 (GSK-3), which is both a candidate tau kinase and an APP kinase, it is possible that a relative failure of insulin signalling might directly increase the pathological molecular processes of AD. In order to determine if insulin signalling was altered in AD, we aimed to map the expression and activity of proteins on the insulin signalling pathway from diseased and elderly control post-mortem brain samples. One key participant in the insulin signalling pathway is SORBS1. Polymorphisms of the SORBS1 gene have been associated with obesity and type 2 diabetes [Lin et al., 2001] and the gene encoding SORBS1 is adjacent to a locus strongly associated with AD on chromosome 10 [Grupe et al., 2006]. We therefore determined expression of this protein in superior temporal gyrus in 54 AD cases and 14 control samples. No association between SORBS protein levels and AD or with the SNP in the region associating with AD was found. However, protein levels of downstream targets of SORBS1 protein including c-ABL and c-CBL were found to be significantly increased in AD. Ongoing studies of changes in the expression levels of other proteins involved in the insulin pathways and correlation with plaque and tangle counts will be presented. Lin WH, Chiu KC, Chang HM, Lee KC, Tai TY,Chuang LM. Molecular scanning of the human sorbin and SH3-domain-containing (SORBS1) gene: positive association of the T228A polymorphism with obesity and type 2 diabetes. Human molecular genetics 2001 10: 1753-1760 Chong ZZ, Li F, Maiese K. Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer’s disease. Brain Res Rev 49: 1-21, 2005 Caricasole A, Bakker A, Copani A, Nicoletti F,. Gaviraghi G, Terstappen GC. Two Sides of the Same Coin: Wnt Signaling in Neurodegeneration and Neuro-Oncology. Biosci Rep. 25:309-27, 2005 Grupe A et al. A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease. Am J Hum Genet. 78: 78-88 P1-414
DNA DECOY INTERVENTION OF BACE1 SYNTHESIS IN THE GUINEA PIG BRAIN: A THERAPEUTIC APPROACH
Diana C. Ferrari1, Krystyn Z. Bourne2, J. Regino Perez-Polo1, 1 University of Texas Medical Branch, Galveston, TX, USA; 2The University of Texas Medical School, Houston, TX, USA. Contact e-mail: [email protected] Background: The activity of the -site amyloid precursor protein cleaving enzyme type 1 (BACE1) is required for the formation of the -Amyloid peptide (A). A is the primary constituent of the extracellular amyloid deposits and a neuropathological marker for Alzheimer’s disease (AD). AD is characterized by cognitive deficits that correlate with dysfunctional cholinergic neurons in the basal forebrain, known to require nerve growth factor (NGF) for survival and proper cholinergic expression. The human and rat BACE1, as well as NGF and the NGF receptor p75 display NF-B binding consensus sequences in their promoters. Specifically, we have shown that NF-B stimulates BACE1 promoter activity and transcription in activated astrocytic cells (C6 cells and adult primary guinea pig hippocampus astrocytes) while repressing BACE1 in neuronal cells and inactivated astrocytes. Objective(s): To regulate the expression of BACE1 through NF-B specific decoys. Methods: NF-B decoy injections, Western Blot, Immunohistochemistry. Results: Western blot analyses of TNF␣ adult guinea pig primary astrocytes from hippocampus treated for 8 hours with 10M NGF or A showed an increase in the expression of BACE1 correlated with a decrease in IB␣. There was also a BACE1 increase after NGF treatment of PC12 cells. Twenty-four hour treatment of differentiated PC12 cells with 10M A and 1.6g/l NF-B decoys blocked NF-B repression of BACE1. Injection of 120 nmol. NF-B decoys into young male guinea pig hippocampi resulted in increased BACE1 levels after 6
S219
hours as measured by western blot assay. Thus, the young guinea pig hippocampal neurons and astrocytes, the latter typically not displaying activated features, behave as their in vitro counterparts in that NF-B appears to act as a repressor. Immunocytochemistry showed the presence of fluorescein-labeled NF-B decoys in nuclei of hippocampal neurons but not astrocytes as early as 2 hours and persisting for up to 6 hours after injection. Conclusions: These results would suggest that appropriate treatment with NF-B decoys of the aged or chronically stressed guinea pig brain might reduce the age- or stress-related brain pathology. This work has been supported in part by grants from the Sealy Center for Aging. P1-415
CONNEXIN-43: A POTENTIAL TRANSDUCTION TARGET FOR AMYLOID INTRACELLULAR DOMAIN?
Rania Y. Hallaq, Richard Killick, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected] Background: In 1995, the Nagy and Marotta group demonstrated that the stable expression of the C99 -cleaved stub of the -amyloid precursor proteins (APP) in the PC12 rat neuronal like cell line, lead to increased expression of Connexin 43 (Cx43), a gap junction subunit and concomitant increase in gap junctional communication. C99 is consitutively cleaved by ␥-secretase into the -amyloid (A) peptide(s) and the APP intracellular domain (AICD) which can vary in length from 49 to 59 amino acids. At the time of this report a signalling function for AICD, akin to that of the Notch ICDs, was not contemplated. To date this issue remains controversial. Results: We have repeated their study substituting PC12s for the human neuroblastoma cell line, SH-SY5Y, which expresses undetectable amounts of Cx43 by immunoblotting. Several clonal lines have been established expressing C99 or the empty vector. A dramatic increase in Cx43 was detected in only the C99 clones and a close positive correlation between C99 levels and Cx43 was observed. Method: Given that C99 is the precursor of both A and AICD we are now generating new cell lines expressing either AICD-␥59, C99, full length APP (wild type and mutant forms) or APP which lacks the ICD and treating SH-SY5Y cells with soluble A peptides. The cells will then be examined for Cx43 expression levels. In addition we are generating a Luciferase reporter gene containing the human Cx43 gene promoter sequence. Conclusion: These studies will inform us as to whether A or the AICD is responsible for the induction of Cx43 expression and whether either has direct transcriptional effects on the Cx43 promoter. P1-416
APP PROCESSING AND CELL CYCLE REENTRY SIGNALING PATHWAYS ARE INVOLVED IN P25/ CDK5-MEDIATED NEURODEGENERATION
Dohoon Kim, Jonathan C. Cruz, Lily Y. Moy, Xiaoyan Sun, Roderick T. Bronson, Jie Shen, Li-Huei Tsai, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Aberrant activation of Cyclin dependent kinase 5 (cdk5) by its regulatory subunit p25, the cleaved form of p35, has been implicated in a variety of neurodegenerative conditions including Alzheimer’s Disease. We recently demonstrated that aberrant activation of cdk5 by inducible overexpression of p25 results in massive neurodegeneration and brain atrophy in a transgenic model (p25Tg) after 5-6 weeks of induction. Objective(s): To investigate the mechanisms which precede and account for p25/cdk5-mediated neurotoxicity. Methods: We systematically examined p25Tg mouse brains at 2-3 weeks of p25 induction, which is prior to the onset of neurodegeneration, and at later timepoints following the onset of neurodegeneration. Various markers for AD-related pathology, as well as microarray analyses, were utilized. Results: We found that tau hyperphosphorylation was not detected at such an early time of induction. At later timepoints following the onset of neuronal degeneration, tau hyperphosphorylation was observed but was not closely correlated with dying neurons at the cellular level. On the other hand, robust increases in A levels were observed prior to the onset of neurodegeneration, while at later