- Email: [email protected]
4EBP1 and P70S6K signaling controls of translation in neural cells
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
S178
Poster Session P I : Molecular Mechanisms of Neurodegeneration - Signal Transduction
pathways, mRNA translation is altered in the brain of Alzheimer's Disease (AD) patients. We have previously demonstrated that the pro-apoptotic PKR/eIF2 pathway, which attenuates protein translation, is up-regulated in AD degenerating neurons and that A[3 neurotoxicity rapidly increased PKR activation under the control of caspase 8 and calcium (J Biol Chem, 2003, 278,49819-27. J Neurochem, 2002, 83, 1215-25. Neuroreport, 2002,13,2429-32). Very little is known about the involvement of mTOR/4EBP1 and P70S6K pathways that modulate eIF4E translation factor and increase protein translation. A previous report has shown that phosphorylated (activated) P70S6K was up regulated in AD (Am J Pathol, 2003, 163, 591-607). Objective(s): To determine if mTOR/4EBP and P70S6K pathways are altered by A[3 exposure in neural cells Methods: In the present study, human (SH-SY5Y) and murine (neuro-2A) neuroblastoma cells were exposed to A[3 peptides 1-42 and 40-1 from 30 min to 24h. The native and phosphorylated (activated) forms of 4EBP1 controlled by mTOR signaling and of P70S6K were analyzed by western blots. Results: After several hours of AI~ exposure, protein expression of both native forms of 4EBP1 and P70S6K were decreased. At the opposite the expression of both phosphorylated forms were increased as well as the ratio between phosphorylated and native forms of 4EBP1 and P70S6K. Conclusions: These results suggest that A[3 induces a rapid and sustained activation of PKR/elF2 pathway leading to decreased translation and apoptosis, but at the same time, A~ produces an activation of the trophic mTOR/IrTOS6K pathways in a possible attempt for neural ceils to balance PKR activation. Further studies will be needed to decipher the role of the mTOR/P70S6K signaling pathways in neuroprotection.
•
ALTERED SYNAPTIC SIGNALING AND SIGNAL TRANSDUCTION IN ALZHEIMER'S DISEASE
Lina Emilsson*, Peter Saetre, Elena Jazin. Dept. of Evolution, Genomics and Systematics, Uppsala, Sweden. Contact e-mail: lina.emilsson @ebc. uu.se
Background: There is a wealth of evidence for a progressive accumulation of A[3 as the primary cause of synaptic failure, neuronal dysfunction and neuronal death in Alzheimer's disease (AD). However the molecular mechanisms behind these dysfunctions are not well understood. Objective: To identify genes involved in disturbed molecular pathways underlying AD pathology. Methods: More than 20 000 human genes and ESTs were investigated in pooled autopsy brain samples, 61 patients diagnosed with AD (CERAD-positive) and 53 control individuals, on two different sets of cDNA microarrays. Genes were classified after their likelihood of being differentially expressed in AD. Highly ranked genes were then verified with Real-time RT-PCR in each individual separately. Results: The microarray experiments indicated that the expression levels of numerous genes involved in synaptic signaling and signal transduction were altered in AD. The expression levels of 25 genes involved in these molecular pathways or in APP metabolism were further investigated with Real-time RT-PCR. Six genes involved in vesicle transport, three genes involved in glutamate metabolism and signaling, and six genes involved in signal transduction were identified as differentially expressed in AD. G-protein signaling 4 (RGS4) showed the clearest expression difference between AD and control individuals. Conclusions: Our results suggest that disturbed signal vesicle trafficking, glutamate signaling, and deregulation of intracelhilar calcium signaling are all important features, which operate in concert, in AD pathogenesis. We hypothesize that RGS4 play an important role in one or more of these molecular pathways. Experiments are underway to study the functional relevance of RGS4 in AD.
~ - ~
AMYLOID BETA-PROTEIN IS A BETA-ADRENERGIC RECEPTOR AGONIST
Urule Igbavboa* 1, Ann E. Studniski 1, John Capen 1, Todd M. Malt 1, Leslie N. Johnson-Anuna I , Grace Y. Sun 2, W. Gibson Wood I . 1 Univ. of Minnesota/VA Medical Center, Minneapolis, MN, USA; 2 Univ. of Missouri, Columbia, MO, USA. Contact e-mail: igbavOOl @umn.edu
Background: Amyloid beta-protein (A[~), a protein that is thought to be an important contributor to neurodegeneration that occurs with Alzheimer's disease, alters cellular cholesterol dynamics, particularly cholesterol trafticking in astrocytes and neurons. We have recently reported that A~1-42 increased apoE levels, modified cholesterol distribution within the Golgi complex and reduced cholesterol levels in the plasma membrane of astrocytes (J. Biol. Chem., 278, 17150-17157, 2003). The mechanism for the A[3-induced increase in apoE levels is unclear at this time but there is evidence suggesting that AI3 is acting on apoE transcription that may involve cAMP and the transcription factor, activator protein-2 (AP-2). Formation of cAMP is initiated by stimulation of [~-adrenergic receptors. We propose that extracellullar A[~ may initially act as a [~-adrenergic receptor agonist stimulating cAMP formation, leading to the downstream expression of apoE. Objective: The objective of these experiments was to determine if A~1-42 acted as an agonist on ~-adrenergic receptors. Method: DITNC1 rat astrocytes were used in all experiments. Cells were incubated with fresh A~1-42 and subtype selective [3-adrenergic receptor agonists and antagonists. Levels of cAMP were determined by an enzyme immunoassay and apoE abundance was determined by Western analysis. Results: A[~ stimulated cAMP formation and this stimulation was inhibited by [~-adrenergic receptor antagonists, particularly those acting on receptor subtypes 1 and 2. A[~ enhanced cAMP formation in the presence of agonists for receptor subtypes 1 and 2. ApoE abundance was increased when incubated with dibutyryl-cAMP that is a cell permeable analogue of cAMP. Conclusions: A~ acts a ligand for [3-adrenergic receptors stimulating formation of cAMP that in turn leads to an increase in apoE abundance. In addition to regulation of cAMP levels by [~-adrenergic receptors, involvement of G-stimulatory protein and adenylate cyclase are well-established. AI3 induced formation of cAMP may involve several sites along that pathway.
•
DEFICIENCY OF G-PROTEIN COUPLED RECEPTOR KINASES AT EARLY STAGES OF ALZHEIMER'S DISEASE: AN ASSOCIATION WITH MILD TO MODERATE I3-AMYLOID ACCUMULATION
Zhiming Suo* 1,2, Min Wu 1, Bruce A. Citron 1,2, Gwendolyn T. Wong 3, Barry W. Festoff 1,2. 1Veterans Affairs Medical Center, Kansas City, MO, USA; 2University of Kansas Medical Center, Kansas City, KS, USA," 3Schering-Plough Research Institute, Kenilworth, N J, USA. Contact e-mail: [email protected]
Background: Overwhelming evidence indicates that the effects of 13amyloid (A[5) are dose-dependent both in vitro and in vivo, which implies that A[~ is not directly detrimental to brain cells until it reaches a threshold concentration. Studies in vascular tissues suggested that there are two phases of A[~ effects: one phase involves I~M range of A[~ that is directly toxic to the cells; the other is with sub-threshold nM range of A[3 that has no direct effects but indirectly potentiates vasoconstriction induced by others. Objective(s): To study molecular mechanisms by which subthreshold A[3 induces microglial hyperactivity with a hope to gain insights into the pathogenic mechanisms at early stages of Alzheimer's disease (AD). Methods: We used cultured microglial ceils as an in vitro model to characterize effects of A[3, and then studied the molecular mechanisms underlying the microglial hyperactivity induced by sub-threshold AlL The major finding from the in vitro study was validated in vivo in an AD transgenic mouse model. Results: We found that there were two phases of dose-dependent A[3 effects on microglial cells as well: at the threshold of 5 p,M and above, A~ directly induced tumor necrosis factor-c~ (TNF-c0 release; at sub-threshold doses, A[~ indirectly potentiated TNF-c~ release induced by certain G-protein coupled receptor (GPCR) activators.
•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
S178
Poster Session P I : Molecular Mechanisms of Neurodegeneration - Signal Transduction
pathways, mRNA translation is altered in the brain of Alzheimer's Disease (AD) patients. We have previously demonstrated that the pro-apoptotic PKR/eIF2 pathway, which attenuates protein translation, is up-regulated in AD degenerating neurons and that A[3 neurotoxicity rapidly increased PKR activation under the control of caspase 8 and calcium (J Biol Chem, 2003, 278,49819-27. J Neurochem, 2002, 83, 1215-25. Neuroreport, 2002,13,2429-32). Very little is known about the involvement of mTOR/4EBP1 and P70S6K pathways that modulate eIF4E translation factor and increase protein translation. A previous report has shown that phosphorylated (activated) P70S6K was up regulated in AD (Am J Pathol, 2003, 163, 591-607). Objective(s): To determine if mTOR/4EBP and P70S6K pathways are altered by A[3 exposure in neural cells Methods: In the present study, human (SH-SY5Y) and murine (neuro-2A) neuroblastoma cells were exposed to A[3 peptides 1-42 and 40-1 from 30 min to 24h. The native and phosphorylated (activated) forms of 4EBP1 controlled by mTOR signaling and of P70S6K were analyzed by western blots. Results: After several hours of AI~ exposure, protein expression of both native forms of 4EBP1 and P70S6K were decreased. At the opposite the expression of both phosphorylated forms were increased as well as the ratio between phosphorylated and native forms of 4EBP1 and P70S6K. Conclusions: These results suggest that A[3 induces a rapid and sustained activation of PKR/elF2 pathway leading to decreased translation and apoptosis, but at the same time, A~ produces an activation of the trophic mTOR/IrTOS6K pathways in a possible attempt for neural ceils to balance PKR activation. Further studies will be needed to decipher the role of the mTOR/P70S6K signaling pathways in neuroprotection.
•
ALTERED SYNAPTIC SIGNALING AND SIGNAL TRANSDUCTION IN ALZHEIMER'S DISEASE
Lina Emilsson*, Peter Saetre, Elena Jazin. Dept. of Evolution, Genomics and Systematics, Uppsala, Sweden. Contact e-mail: lina.emilsson @ebc. uu.se
Background: There is a wealth of evidence for a progressive accumulation of A[3 as the primary cause of synaptic failure, neuronal dysfunction and neuronal death in Alzheimer's disease (AD). However the molecular mechanisms behind these dysfunctions are not well understood. Objective: To identify genes involved in disturbed molecular pathways underlying AD pathology. Methods: More than 20 000 human genes and ESTs were investigated in pooled autopsy brain samples, 61 patients diagnosed with AD (CERAD-positive) and 53 control individuals, on two different sets of cDNA microarrays. Genes were classified after their likelihood of being differentially expressed in AD. Highly ranked genes were then verified with Real-time RT-PCR in each individual separately. Results: The microarray experiments indicated that the expression levels of numerous genes involved in synaptic signaling and signal transduction were altered in AD. The expression levels of 25 genes involved in these molecular pathways or in APP metabolism were further investigated with Real-time RT-PCR. Six genes involved in vesicle transport, three genes involved in glutamate metabolism and signaling, and six genes involved in signal transduction were identified as differentially expressed in AD. G-protein signaling 4 (RGS4) showed the clearest expression difference between AD and control individuals. Conclusions: Our results suggest that disturbed signal vesicle trafficking, glutamate signaling, and deregulation of intracelhilar calcium signaling are all important features, which operate in concert, in AD pathogenesis. We hypothesize that RGS4 play an important role in one or more of these molecular pathways. Experiments are underway to study the functional relevance of RGS4 in AD.
~ - ~
AMYLOID BETA-PROTEIN IS A BETA-ADRENERGIC RECEPTOR AGONIST
Urule Igbavboa* 1, Ann E. Studniski 1, John Capen 1, Todd M. Malt 1, Leslie N. Johnson-Anuna I , Grace Y. Sun 2, W. Gibson Wood I . 1 Univ. of Minnesota/VA Medical Center, Minneapolis, MN, USA; 2 Univ. of Missouri, Columbia, MO, USA. Contact e-mail: igbavOOl @umn.edu
Background: Amyloid beta-protein (A[~), a protein that is thought to be an important contributor to neurodegeneration that occurs with Alzheimer's disease, alters cellular cholesterol dynamics, particularly cholesterol trafticking in astrocytes and neurons. We have recently reported that A~1-42 increased apoE levels, modified cholesterol distribution within the Golgi complex and reduced cholesterol levels in the plasma membrane of astrocytes (J. Biol. Chem., 278, 17150-17157, 2003). The mechanism for the A[3-induced increase in apoE levels is unclear at this time but there is evidence suggesting that AI3 is acting on apoE transcription that may involve cAMP and the transcription factor, activator protein-2 (AP-2). Formation of cAMP is initiated by stimulation of [~-adrenergic receptors. We propose that extracellullar A[~ may initially act as a [~-adrenergic receptor agonist stimulating cAMP formation, leading to the downstream expression of apoE. Objective: The objective of these experiments was to determine if A~1-42 acted as an agonist on ~-adrenergic receptors. Method: DITNC1 rat astrocytes were used in all experiments. Cells were incubated with fresh A~1-42 and subtype selective [3-adrenergic receptor agonists and antagonists. Levels of cAMP were determined by an enzyme immunoassay and apoE abundance was determined by Western analysis. Results: A[~ stimulated cAMP formation and this stimulation was inhibited by [~-adrenergic receptor antagonists, particularly those acting on receptor subtypes 1 and 2. A[~ enhanced cAMP formation in the presence of agonists for receptor subtypes 1 and 2. ApoE abundance was increased when incubated with dibutyryl-cAMP that is a cell permeable analogue of cAMP. Conclusions: A~ acts a ligand for [3-adrenergic receptors stimulating formation of cAMP that in turn leads to an increase in apoE abundance. In addition to regulation of cAMP levels by [~-adrenergic receptors, involvement of G-stimulatory protein and adenylate cyclase are well-established. AI3 induced formation of cAMP may involve several sites along that pathway.
•
DEFICIENCY OF G-PROTEIN COUPLED RECEPTOR KINASES AT EARLY STAGES OF ALZHEIMER'S DISEASE: AN ASSOCIATION WITH MILD TO MODERATE I3-AMYLOID ACCUMULATION
Zhiming Suo* 1,2, Min Wu 1, Bruce A. Citron 1,2, Gwendolyn T. Wong 3, Barry W. Festoff 1,2. 1Veterans Affairs Medical Center, Kansas City, MO, USA; 2University of Kansas Medical Center, Kansas City, KS, USA," 3Schering-Plough Research Institute, Kenilworth, N J, USA. Contact e-mail: [email protected]
Background: Overwhelming evidence indicates that the effects of 13amyloid (A[5) are dose-dependent both in vitro and in vivo, which implies that A[~ is not directly detrimental to brain cells until it reaches a threshold concentration. Studies in vascular tissues suggested that there are two phases of A[~ effects: one phase involves I~M range of A[~ that is directly toxic to the cells; the other is with sub-threshold nM range of A[3 that has no direct effects but indirectly potentiates vasoconstriction induced by others. Objective(s): To study molecular mechanisms by which subthreshold A[3 induces microglial hyperactivity with a hope to gain insights into the pathogenic mechanisms at early stages of Alzheimer's disease (AD). Methods: We used cultured microglial ceils as an in vitro model to characterize effects of A[3, and then studied the molecular mechanisms underlying the microglial hyperactivity induced by sub-threshold AlL The major finding from the in vitro study was validated in vivo in an AD transgenic mouse model. Results: We found that there were two phases of dose-dependent A[3 effects on microglial cells as well: at the threshold of 5 p,M and above, A~ directly induced tumor necrosis factor-c~ (TNF-c0 release; at sub-threshold doses, A[~ indirectly potentiated TNF-c~ release induced by certain G-protein coupled receptor (GPCR) activators.