- Email: [email protected]
P1-285 Phosphorylation of APP at THR668 regulates the function of APP intracellular domain fragment
Poster Session PI : Molecular Mechanisms of Neurodegeneration - Signal Transduction transmembrane sequences (A[3), and a short intracellular fragment (AICD = APP intracellular domain) that may function as a transcriptional activator in a complex with the adaptor protein Fe65 and the nuclear protein Tip60. APP is closely related to APLPl and APLP2, but only APP is known to be cleaved by BACE 1 and to be involved in Alzheimer's disease. We now demonstrate that similar to APP, APLP1 and APLP2 are also cleaved by BACE 1 - but not by ADAM 9, another APP-protease -, and also transactivate nuclear Tip60 in a complex with Fe65. Paradoxically, although BACE 1 cleavage appears to be specific for APP and APLP's, their cleavage sequences exhibit no homology, and a short sequence (7 amino acids) from APE when placed close to the membrane, converts a membrane protein that is normally not cleaved by BACE 1 into a BACE 1 substrate. Our data demonstrate that APLP's and APP are processed similarly to act via the same nuclear target, suggesting that BACE 1 cleavage regulates a common function of APP and APLP's in neurons.
•P-J-•
PHOSPHORYLATION OF APP AT THR668 R E G U L A T E S T H E F U N C T I O N O F APP
S 177
in its regulation, have been implicated in different diseases including neurodegenerative diseases, such as Alzheimer's disease (AD). Importantly, in AD, there is a significant increase of oxidative stress-related lipid peroxidation aldehydic products such as 4-hydroxynonenal (HNE) that are thought to play a key role in disease pathogenesis. While the involvement of lipid peroxidation in neuronal cell death is increasingly appreciated, the impact of subtoxic levels of oxidative stress on neuronal function is largely unknown. To further analyze this, as well as the relationship between oxidative stress and GSK313, in this study we examined the effects of a single exposure of human neuroblastoma IMR-32 cells to HNE on GSK315 phospho-dependent activity and on intracellular signaling cascades that may regulate its phosphorylation state. We provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK313 activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK313 as a mediator of the signaling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes. These findings provide a key liitk between oxidative stress and abnormal phosphorylafion in AD.
INTRACELLULAR DOMAIN FRAGMENT Tadashi Nakaya*, Eiko Kawaguchi, Toshiharu Suzuki. Hokkaido
University, Sapporo, Japan. Contact e-mail: [email protected]
•
I S E S S E N T I A L F O R S U R V I V A L A F T E R TRAUMATIC
Background: Recent observations support that APP intracellular domain cleaved at gamma/epsilon-site (AICD) enters the nucleus and participates in gene transactivation followed its association with FE65. However, it is still unclear how the transactivation of gene by AICD is regulated. Our previous reports showed that Thr668 numbering for APP695 is phosphorylated in adult brain but not non-neuronal tissues. The phosphorylation regulates the interaction between APP and FE65 by altering conformation of APP cytoplasmic domain [J. Biol. Chem. (2001) 276, 40353-40361]. Objective(s) & Methods: Here we focused on the role of phosphorylation of AICD at Thr668 on FE65-dependent gene transactivation. To investigate the regulation of the transactivation function of AICD, we constructed the Gal4-AICD fusion protein with mutaion at Thr668 and analyzed with reporter gene assay. Endogenous protein levels were analyzed using anti-APP or, antiphospho APP antibodies. Results: In mouse, the phosphorylation level of AICD increased in late embryonic and neonatal brain, suggesting that AICD is functionally regulated by phosphorylation in the brain of these developmental stages. To elucidate this possibility, we examined the transactivation assay using the AICD substituted Thr for Ala (AICD-A), which induces an AICD conformation that mimics the one induced by phosphorylation. This construct showed a decreased gene transactivation activity by its poor ability to bind FE65. Conclusions: Because AICD is thought to generate constitutively from APP without any ligand binding and stimulation, the post-generative modification such as phosphorylation may be an important molecular switch for regulation of AICD activity. Although genes, which are subjected to expressional regulation by AICD- and FE65-dependent transactivation, are still unrevealed, our finding contributes to elucidate the regulatory mechanisms that control the gene transactivation of AICD.
•
GLYCOGEN SYNTHASE KINASE-3O ACTIVITY IS REGULATED BY PRODUCTS OF LIPID PEROXIDATION: R E L E V A N C E T O A L Z H E 1 M E R
AMYLOID P R E C U R S O R P R O T E I N BRAIN INJURY
Maarten Leyssen*, Derya Ayaz, Simon Reeve, Bart De Strooper, Bassem Hassan. V.I.B./K.U. Leuven, Leuven, Belgium. Contact e-mail:
maarten.leyssen @med.kuleuven.ac.be Background: Amyloid Precursor Protein (APP), a type I membrane spanning protein, is the precursor of A[~-peptides. These peptides, generated by two sequential cleavages of APP, form the main constituents of amyloid plaques in the brains of patients with Alzheimer's disease (AD). Despite years of intensive investigation, surprisingly little is known about the physiological function of APP. A huge body of in vitro and cell culture studies implicate APP in neurite outgrowth, gene transcription and neuronal survival and link its cytoplasmic tall to several signalling cascades. The in vivo relevance of these findings and therefore the physiological function of APP remains unknown. Knockout models for APP in Drosophila melanogaster and mus musculus have failed to assign a significant role to this protein in brain development. Only subtle deficits in synapse formation, locomotion and axonal transport were reported. Even the lethal combination of APP, APLP2 or APLP1, APLP2 knockout in mice causes no obvious brain abnormalities. After Traumatic Brain Injury (TBI) levels of APP are increased both in mammalian model systems and in humans. Objectives: In our work we use Drosophila melanogaster to address the putative role of APP in the central nervous system after TBL Methods: We developed an assay to study Traumatic Brain Injury (TBI) in Drosophila. Next we studied the effects of APP upregulation on specific subsets of neurons in the adult Drosophila brain. Finally we used genetic interaction studies to identify essential components of the APP-pathway. Results and conclusions: We identified APP as an essential molecule for survival after TBI for the first time. Furthermore we show that APP acts by induction of axonal sprouting in the Drosophila brain, possibly to repair lost axonal connections. Genetic interaction studies show that APP transduces its effects via interaction with components which signal to the actin cytoskeleton.
DISEASE Paola Strocchi* 1, Mark A. Smith z, George Perry 2, Massimo Tabaton 3 , Barbara Dozza I . 1University of Bologna, Bologna, Italy," 2Case Western
Reserve University, Cleveland, OH, USA; 3University of Genoa, Genoa, Italy. Contact e-mail: [email protected] Glycogen synthase kinase-3~ (GSK3[3), a constitutively active serine/threonine ldnase initially described as a key enzyme involved in glycogen metabofism, is now known to regulate a diverse array of cell functions. GSK3~ activity is regulated by phosphorylation and is a critical downstream element of the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinases (MAPKs) signaling pathways. Deregulation of GSK313 activity, as well as the up-stream signaling pathways involved
~ A [ ~
MODULATES PKR, MTOR/4EBP1 AND P70S6K SIGNALING C O N T R O L S O F T R A N S L A T I O N IN NEURAL CELLS
Claire Lafay ~, Guylene Page 1 Marie Christine Peranlt 1,2, Raymond C. Chang 3, Jacques Hugon* 1,2.1 University of Poitiers, Poitiers, France;
2CHU Poitiers, Poitiers, France, SDepartment of Anatomy, University of [long Kong, Hong Kong Special Administrative Region of China. Contact e-mail: jacques, hugon @univ-poitiers.fr Background: The initiation of translation control is modulated by different factors including first the PKR/eIF2 and then the mTOR/P70S6K/eW4E
•P-J-•
PHOSPHORYLATION OF APP AT THR668 R E G U L A T E S T H E F U N C T I O N O F APP
S 177
in its regulation, have been implicated in different diseases including neurodegenerative diseases, such as Alzheimer's disease (AD). Importantly, in AD, there is a significant increase of oxidative stress-related lipid peroxidation aldehydic products such as 4-hydroxynonenal (HNE) that are thought to play a key role in disease pathogenesis. While the involvement of lipid peroxidation in neuronal cell death is increasingly appreciated, the impact of subtoxic levels of oxidative stress on neuronal function is largely unknown. To further analyze this, as well as the relationship between oxidative stress and GSK313, in this study we examined the effects of a single exposure of human neuroblastoma IMR-32 cells to HNE on GSK315 phospho-dependent activity and on intracellular signaling cascades that may regulate its phosphorylation state. We provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK313 activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK313 as a mediator of the signaling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes. These findings provide a key liitk between oxidative stress and abnormal phosphorylafion in AD.
INTRACELLULAR DOMAIN FRAGMENT Tadashi Nakaya*, Eiko Kawaguchi, Toshiharu Suzuki. Hokkaido
University, Sapporo, Japan. Contact e-mail: [email protected]
•
I S E S S E N T I A L F O R S U R V I V A L A F T E R TRAUMATIC
Background: Recent observations support that APP intracellular domain cleaved at gamma/epsilon-site (AICD) enters the nucleus and participates in gene transactivation followed its association with FE65. However, it is still unclear how the transactivation of gene by AICD is regulated. Our previous reports showed that Thr668 numbering for APP695 is phosphorylated in adult brain but not non-neuronal tissues. The phosphorylation regulates the interaction between APP and FE65 by altering conformation of APP cytoplasmic domain [J. Biol. Chem. (2001) 276, 40353-40361]. Objective(s) & Methods: Here we focused on the role of phosphorylation of AICD at Thr668 on FE65-dependent gene transactivation. To investigate the regulation of the transactivation function of AICD, we constructed the Gal4-AICD fusion protein with mutaion at Thr668 and analyzed with reporter gene assay. Endogenous protein levels were analyzed using anti-APP or, antiphospho APP antibodies. Results: In mouse, the phosphorylation level of AICD increased in late embryonic and neonatal brain, suggesting that AICD is functionally regulated by phosphorylation in the brain of these developmental stages. To elucidate this possibility, we examined the transactivation assay using the AICD substituted Thr for Ala (AICD-A), which induces an AICD conformation that mimics the one induced by phosphorylation. This construct showed a decreased gene transactivation activity by its poor ability to bind FE65. Conclusions: Because AICD is thought to generate constitutively from APP without any ligand binding and stimulation, the post-generative modification such as phosphorylation may be an important molecular switch for regulation of AICD activity. Although genes, which are subjected to expressional regulation by AICD- and FE65-dependent transactivation, are still unrevealed, our finding contributes to elucidate the regulatory mechanisms that control the gene transactivation of AICD.
•
GLYCOGEN SYNTHASE KINASE-3O ACTIVITY IS REGULATED BY PRODUCTS OF LIPID PEROXIDATION: R E L E V A N C E T O A L Z H E 1 M E R
AMYLOID P R E C U R S O R P R O T E I N BRAIN INJURY
Maarten Leyssen*, Derya Ayaz, Simon Reeve, Bart De Strooper, Bassem Hassan. V.I.B./K.U. Leuven, Leuven, Belgium. Contact e-mail:
maarten.leyssen @med.kuleuven.ac.be Background: Amyloid Precursor Protein (APP), a type I membrane spanning protein, is the precursor of A[~-peptides. These peptides, generated by two sequential cleavages of APP, form the main constituents of amyloid plaques in the brains of patients with Alzheimer's disease (AD). Despite years of intensive investigation, surprisingly little is known about the physiological function of APP. A huge body of in vitro and cell culture studies implicate APP in neurite outgrowth, gene transcription and neuronal survival and link its cytoplasmic tall to several signalling cascades. The in vivo relevance of these findings and therefore the physiological function of APP remains unknown. Knockout models for APP in Drosophila melanogaster and mus musculus have failed to assign a significant role to this protein in brain development. Only subtle deficits in synapse formation, locomotion and axonal transport were reported. Even the lethal combination of APP, APLP2 or APLP1, APLP2 knockout in mice causes no obvious brain abnormalities. After Traumatic Brain Injury (TBI) levels of APP are increased both in mammalian model systems and in humans. Objectives: In our work we use Drosophila melanogaster to address the putative role of APP in the central nervous system after TBL Methods: We developed an assay to study Traumatic Brain Injury (TBI) in Drosophila. Next we studied the effects of APP upregulation on specific subsets of neurons in the adult Drosophila brain. Finally we used genetic interaction studies to identify essential components of the APP-pathway. Results and conclusions: We identified APP as an essential molecule for survival after TBI for the first time. Furthermore we show that APP acts by induction of axonal sprouting in the Drosophila brain, possibly to repair lost axonal connections. Genetic interaction studies show that APP transduces its effects via interaction with components which signal to the actin cytoskeleton.
DISEASE Paola Strocchi* 1, Mark A. Smith z, George Perry 2, Massimo Tabaton 3 , Barbara Dozza I . 1University of Bologna, Bologna, Italy," 2Case Western
Reserve University, Cleveland, OH, USA; 3University of Genoa, Genoa, Italy. Contact e-mail: [email protected] Glycogen synthase kinase-3~ (GSK3[3), a constitutively active serine/threonine ldnase initially described as a key enzyme involved in glycogen metabofism, is now known to regulate a diverse array of cell functions. GSK3~ activity is regulated by phosphorylation and is a critical downstream element of the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinases (MAPKs) signaling pathways. Deregulation of GSK313 activity, as well as the up-stream signaling pathways involved
~ A [ ~
MODULATES PKR, MTOR/4EBP1 AND P70S6K SIGNALING C O N T R O L S O F T R A N S L A T I O N IN NEURAL CELLS
Claire Lafay ~, Guylene Page 1 Marie Christine Peranlt 1,2, Raymond C. Chang 3, Jacques Hugon* 1,2.1 University of Poitiers, Poitiers, France;
2CHU Poitiers, Poitiers, France, SDepartment of Anatomy, University of [long Kong, Hong Kong Special Administrative Region of China. Contact e-mail: jacques, hugon @univ-poitiers.fr Background: The initiation of translation control is modulated by different factors including first the PKR/eIF2 and then the mTOR/P70S6K/eW4E