- Email: [email protected]
STUDIES OF STRUCTURAL CHANGES IN AB PEPTIDE DURING INTERACTION WITH LIPID MEMBRANES AND CHANNEL FORMATION
Poster Presentations: P1 LRP1 as described for Fe65. Methods: Interaction between GULP1 molecules was analysed usingGULP1 constructs carrying two different epitope tags (HA-tagged or GFP-tagged). To confirm GULP1-dimerization we employed a protein fragment complementation assay (PCA) where GULP1 was fused to non-bioluminescent amino-or carboxy-terminal fragments of humanized Gaussia Luciferase (hGLuc) or non-fluorescence amino-or caboxy-terminal fragments of Venus-YFP. Specific GULP1-dimerization reconstitutes luciferase/venus and provides a measurable bioluminescent/ fluorescence signal. Results: Upon co-immunoprecipitation, we observed that the GFP-tagged GULP1 was precipitated with HA-tagged GULP1. To confirm our results we did a protein complementation assay. We observed GULP1 dimerization in living cells and found that co-expression of APP enhances dimerization of GULP1. Furthermore, we analysed localization of GULP1 dimers by confocal microscopy as well as nuclear extraction assays showing that GULP1 dimers can be found both in the cytosol and the nucleus. Complex formation of APP, GULP1 and LRP1 was analysed by triple-immunoprecipitation. We could show that GULP1 functions as a molecular bridge between APP and LRP1. Conclusions: Taken together, these data identify the dimerization properties of GULP1 in vivo. GULP1 forms dimers/oligomers both in the cytosol and the nucleus and the dimerization is further promoted by co-expression of APP. Additionally, GULP1 dimerization enables simultaneous binding of both APP and LRP1.
P1-060
DISTINCT LOCALIZATION OF BETA- AND GAMMA-SECRETASE IN RAT BRAIN SYNAPSES
Jolanta Linnea Lundgren1, Saheeb Ahmed2, Sophia Schedin Weiss1, Bengt Winblad1, Gunnar K. Gouras3, Lars O. Tjernberg4, Susanne Frykman1, 1Karolinska Institutet, Stockholm, Sweden; 2European Neuroscience Institute, G€ ottingen, Germany; 3Lund University, Lund, 4 Sweden; Karolinska Institutet, Huddinge, Sweden. Contact e-mail: jolanta. [email protected] Background: The synaptotoxic amyloid b-peptide (Ab) is critically involved in the pathogenesis of Alzheimer Disease (AD) and is believed to play an important role in synaptic degeneration, which is one of the earliest hallmarks of AD. Ab is produced from its precursor protein, APP, which is sequentially cleaved by b- and g -secretase. However, the cellular localization of these cleavage events has not been fully elucidated. We have previously shown that Ab is constitutively released from synapses. Despite our and others efforts, the mechanism behind this release is still not clear, though it has been linked to endocytosis of synaptic vesicles. Methods: We use rat brain subcellular fractionation, including controlled pored glass purification of synaptic vesicles, followed by western blotting as well as proximity ligation assay to study the cellular localization of b- and g -secretase, APP and its’ cleavage products. Results: We show that active b-secretase, but not the components of g -secretase, accumulates in synaptic vesicles in rat brain. Consequently we also see a profound accumulation of APP-C-terminal fragment, the cleavage product of b-secretase, while APP itself accumulates to a minor extent. Conclusions: We demonstrate that b- and g -secretase normally reside in different cellular compartments at the rat brain synapse.
P1-061
P325
ponents of the membrane, possibly leading to insertion of A b peptide into the plasma membrane and formation of ion conducting pores. The consequence of this events is dysregulation of intracellular Ca 2+ ions homeostasis leading to cellular dysfunction and neurodegeneration. Methods: 1. Ab peptide production and liposomes preparation.2. Lipid overlay assay and western-blot analysis.3. Measurement of Ca2+ Uptake into Liposomes.4.
Fig. 1. Interaction of Ab40 peptide with lipids. A: Protein/lipid overlay assay. The Ab peptide was incubated with SM (sphingomyelin), PS (phosphatidylserine), GM1 (ganglioside), DPPC (dipalmitoylphosphatidylcholine) and cholesterol on nitrocellulose membranes. Ab peptide bound to lipids was detected by chemiluminescence. B: Western blot analysis of Ab peptide binding to liposomes. Ab was incubated with liposomes composed of different lipid compositions. Ab bound to liposomes was detected by Western blot.
Fig. 2. Ab-mediated Ca2+ ions influx into liposomes. Liposomes composed of DPPC/GM1/cholesterol were incubated with Ab40 at different concentrations. The rate of calcium influx was monitored by fluorescence measurement of Fura-2.
STUDIES OF STRUCTURAL CHANGES IN AB PEPTIDE DURING INTERACTION WITH LIPID MEMBRANES AND CHANNEL FORMATION
Kaja Przygonska, Magda Kulma, Krzysztof Tarnowski, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a neurodegenerative disease correlated with aggregation of A b peptide to form insoluble intracellular deposits composed of fibrils in neuronal cells. The growing body of evidence suggests that the plasma membrane of neural cells plays an essential role in the pathophysiology of AD. One of the observed pathological events in AD is disruption of intracellular calcium ions homeostasis. This effect is thought to be mediated by the interaction of A b species with the lipid com-
Fig. 3. The percent of deuteration level along the sequence of A: Ab40 and B: Ab40 bound with liposomes incubated for 10 seconds in D20 solution pH 7.4.
P326
Poster Presentations: P1
Hydrogen/deuterium exchange (HDX) Mass Spectrometry (MS) analysis to verify the structural changes in A b 40 that occur during incorporation into the lipid membranes. Results: In our experiments, we determined appropriate lipid composition for Ab40 interaction with liposomes. Firstly, we found that Ab40 exhibited the highest affinity to negatively charged ganglioside GM1 among various lipids (Fig. 1A). In the subsequent experiments we established proper lipids composition in large unilamellar vesicles. The western-blot analysis showed the most intensive interaction of Ab with liposomes containing DPPC/GM1/cholesterol (molar ratio 5:2:3) (Fig. 1B). Afterwards calcium uptake experiments revealed that incubation of Ab40 in the presence of liposomes leads to Ca2+ influx into to the vesicles, in a concentration dependent manner (Fig. 2).In the structural analysis of Ab40 peptide during interactions with lipids bilayer using HDXMS we observed that the interaction with liposomes causes changes in the middle part of the peptide (residues from 10 thto 35 thamino acid). This region is protected against hydrogen/deuterium exchange what suggests that this region is less dynamic and more stable and structured (Fig. 3B). Conclusions: These experiments suggests that the middle part of Ab40 is involved in lipid-protein interactions and could participate in the ion channel formation what may play a significant and inevitable cytotoxic role in the pathogenesis of AD.
P1-062
ACTIVATION OF THE WNT/B-CATENIN PATHWAY REPRESSES THE TRANSCRIPTION OF THE B-APP CLEAVING ENZYME (BACE1)
Nazanin Mirzaei1, Callum Parr1, Mark Christian2, Magdalena Sastre1, 1 Imperial College London, London, United Kingdom; 2University of Warwick, Coventry, United Kingdom. Contact e-mail: n.mirzaei11@ imperial.ac.uk Background: Alterations in the Wnt signalling pathway have been implicated in Alzheimer’s disease (AD) because of its effects on axonal growth, on synaptogenesis and amyloid-b (Ab) toxicity; however its role in the processing of the amyloid precursor protein (APP) remains unknown. Methods: The canonical Wnt signalling pathway was activated by overexpression of the agonist Wnt3a, or the key regulator of the signalling cascade b-catenin or by inhibition of glycogen kinase synthase-3 in N2a cells. On the other hand, inhibition of the pathway was achieved by transfection of the antagonists secreted Frizzled receptor protein-1 or Dickkopf-1. The effects of both activation and inhibition of the pathway on APP processing were measured by western blotting, quantitative real-time PCR, dual luciferase promoter activity assays and chromatin immunoprecipitation (ChIP). Results: Activation of the Wnt pathway resulted in a reduction in Ab levels and in the activity and expression of b-APP cleaving enzyme (BACE1). Conversely, inhibition of the pathway produced the opposite effects. ChIP analysis demonstrated that b-catenin binds specifically to regions within the promoter of BACE1 containing putative T-cell factor/lymphoid enhancer-binding factor-1 (TCF/LEF) motifs, consistent with canonical Wnt target regulation. Interestingly, we also demonstrate that the transcription factor TCF3 binds preferentially to the same binding region in BACE1 promoter following Wnt3a stimulation, indicating that TCF3 functions as a transcriptional repressor of BACE1 gene transcription. Conclusions: In conclusion, Wnt/b-catenin stimulation may repress BACE1 transcription via binding of TCF3 to BACE1 gene and therefore, activation of the Wnt pathway may hold key to new treatments in AD.
P1-063
THE PPARGAMMA COFACTOR RIP140 REGULATES BACE1 GENE EXPRESSION
Katrin Blondrath, Magdalena Sastre, Imperial College London, London, United Kingdom. Contact e-mail: [email protected] Background: Activation of nuclear receptor Peroxisome Proliferator Activated Receptor gamma (PPARg) has been reported to be protective in Alzheimer’s disease (AD) mouse models and in cell lines, by decreasing
inflammation and Ab levels. Therefore, the role of PPARg cofactors will be critical in order to regulate the activity of PPARg. Our aim was to study the potential role of the PPARg cofactor R eceptor I nteracting P rotein 140 (RIP140) in AD. Methods: Two different kind of SDS pages were used to separate proteins of either whole cell lysates, membrane preparations or Aß peptides. 10% home-made Tris-Glycine polyacrylamide gel were used to separate proteins of whole cell lysates sized between 42 and 140 kD and precast NuPAGEÒ NovexÒ 4-12% Bis-Tris Protein Gels (life Technologies), to separate proteins sized 4 to 100kD. After the transgfer to a PVDF or Nitrocellulose Membrane at 400mV for one hour, Membrane were either blocked in milk or BSA and immunostaining with specific antibodies was performed. Antibodies used were: 6E10, Covance raised against Abeta, BACE1 and GsK3 from cell signaling, IDE and beta-Actin from abcam, ApoE and CD10 (Neprilysin) fom Santa Cruz. The 6D7 antibody against RIP140 was a kind gift provided by Dr. Mark Christian. Promotor activity was performed by using a standard Luciferase kit and a plate reader from Promega. Results: Results obtained in neuroblastoma cells suggest that RIP140 over expression reduced Ab generation and BACE1 expression and mRNA level in a PPARg-dependent manner. Conversely, knockdown of RIP140 increased the level of BACE1 protein, mRNA and promoter activity. Therefore, RIP140 seems to act as a transcriptional co-activator for PPARg leading to reduced BACE1 transcription. In addition, studies in animal models, lacking or over expressing RIP140 indicated that many genes involved in AD and the insulin pathway appeared to be modulated by RIP140. Hippocampal homogenates of RIP140 transgenic mice showed transcriptional induction of growth factors such as growth associated protein 43 (GAP43) and Insulin growth factor2 (IgF2) as well as up-regulation of genes involved in the clearance of Ab, namely insulin degrading enzyme (IDE) and Neprilysin. The frontal cortex of these animals exhibited strongly reduced BACE1 protein expression level, in line with results obtained in vitro. In addition, RIP140 over expressing in this brain area of mice displayed a decrease in microtubule associated proteins 1 and 2, both being involved in the tau pathology of AD. Studies in animals lacking RIP140 revealed that BACE1 mRNA level were increased in the frontal cortex area amongst other regulations on genes which haven been found differently regulated in the RIP140 over expressing background. Conclusions: The results indicate that important enzymes that are implicated in Ab generation and degradation are regulated by RIP140 transcriptional modulation. Therefore, RIP140 could serve as therapeutic target for AD.
P1-064
A COMBINATION APPROACH IN A CANINE MODEL OF AGING: IMMUNOTHERAPY WITH BEHAVIORAL ENRICHMENT
Paulina R. Davis1, Tina Beckett1, Ginevra Giannini2, Nathanial Calloway1, M. Paul Murphy1, Edward G. Barrett2, Elizabeth Head1, 1Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United
P1-060
DISTINCT LOCALIZATION OF BETA- AND GAMMA-SECRETASE IN RAT BRAIN SYNAPSES
Jolanta Linnea Lundgren1, Saheeb Ahmed2, Sophia Schedin Weiss1, Bengt Winblad1, Gunnar K. Gouras3, Lars O. Tjernberg4, Susanne Frykman1, 1Karolinska Institutet, Stockholm, Sweden; 2European Neuroscience Institute, G€ ottingen, Germany; 3Lund University, Lund, 4 Sweden; Karolinska Institutet, Huddinge, Sweden. Contact e-mail: jolanta. [email protected] Background: The synaptotoxic amyloid b-peptide (Ab) is critically involved in the pathogenesis of Alzheimer Disease (AD) and is believed to play an important role in synaptic degeneration, which is one of the earliest hallmarks of AD. Ab is produced from its precursor protein, APP, which is sequentially cleaved by b- and g -secretase. However, the cellular localization of these cleavage events has not been fully elucidated. We have previously shown that Ab is constitutively released from synapses. Despite our and others efforts, the mechanism behind this release is still not clear, though it has been linked to endocytosis of synaptic vesicles. Methods: We use rat brain subcellular fractionation, including controlled pored glass purification of synaptic vesicles, followed by western blotting as well as proximity ligation assay to study the cellular localization of b- and g -secretase, APP and its’ cleavage products. Results: We show that active b-secretase, but not the components of g -secretase, accumulates in synaptic vesicles in rat brain. Consequently we also see a profound accumulation of APP-C-terminal fragment, the cleavage product of b-secretase, while APP itself accumulates to a minor extent. Conclusions: We demonstrate that b- and g -secretase normally reside in different cellular compartments at the rat brain synapse.
P1-061
P325
ponents of the membrane, possibly leading to insertion of A b peptide into the plasma membrane and formation of ion conducting pores. The consequence of this events is dysregulation of intracellular Ca 2+ ions homeostasis leading to cellular dysfunction and neurodegeneration. Methods: 1. Ab peptide production and liposomes preparation.2. Lipid overlay assay and western-blot analysis.3. Measurement of Ca2+ Uptake into Liposomes.4.
Fig. 1. Interaction of Ab40 peptide with lipids. A: Protein/lipid overlay assay. The Ab peptide was incubated with SM (sphingomyelin), PS (phosphatidylserine), GM1 (ganglioside), DPPC (dipalmitoylphosphatidylcholine) and cholesterol on nitrocellulose membranes. Ab peptide bound to lipids was detected by chemiluminescence. B: Western blot analysis of Ab peptide binding to liposomes. Ab was incubated with liposomes composed of different lipid compositions. Ab bound to liposomes was detected by Western blot.
Fig. 2. Ab-mediated Ca2+ ions influx into liposomes. Liposomes composed of DPPC/GM1/cholesterol were incubated with Ab40 at different concentrations. The rate of calcium influx was monitored by fluorescence measurement of Fura-2.
STUDIES OF STRUCTURAL CHANGES IN AB PEPTIDE DURING INTERACTION WITH LIPID MEMBRANES AND CHANNEL FORMATION
Kaja Przygonska, Magda Kulma, Krzysztof Tarnowski, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a neurodegenerative disease correlated with aggregation of A b peptide to form insoluble intracellular deposits composed of fibrils in neuronal cells. The growing body of evidence suggests that the plasma membrane of neural cells plays an essential role in the pathophysiology of AD. One of the observed pathological events in AD is disruption of intracellular calcium ions homeostasis. This effect is thought to be mediated by the interaction of A b species with the lipid com-
Fig. 3. The percent of deuteration level along the sequence of A: Ab40 and B: Ab40 bound with liposomes incubated for 10 seconds in D20 solution pH 7.4.
P326
Poster Presentations: P1
Hydrogen/deuterium exchange (HDX) Mass Spectrometry (MS) analysis to verify the structural changes in A b 40 that occur during incorporation into the lipid membranes. Results: In our experiments, we determined appropriate lipid composition for Ab40 interaction with liposomes. Firstly, we found that Ab40 exhibited the highest affinity to negatively charged ganglioside GM1 among various lipids (Fig. 1A). In the subsequent experiments we established proper lipids composition in large unilamellar vesicles. The western-blot analysis showed the most intensive interaction of Ab with liposomes containing DPPC/GM1/cholesterol (molar ratio 5:2:3) (Fig. 1B). Afterwards calcium uptake experiments revealed that incubation of Ab40 in the presence of liposomes leads to Ca2+ influx into to the vesicles, in a concentration dependent manner (Fig. 2).In the structural analysis of Ab40 peptide during interactions with lipids bilayer using HDXMS we observed that the interaction with liposomes causes changes in the middle part of the peptide (residues from 10 thto 35 thamino acid). This region is protected against hydrogen/deuterium exchange what suggests that this region is less dynamic and more stable and structured (Fig. 3B). Conclusions: These experiments suggests that the middle part of Ab40 is involved in lipid-protein interactions and could participate in the ion channel formation what may play a significant and inevitable cytotoxic role in the pathogenesis of AD.
P1-062
ACTIVATION OF THE WNT/B-CATENIN PATHWAY REPRESSES THE TRANSCRIPTION OF THE B-APP CLEAVING ENZYME (BACE1)
Nazanin Mirzaei1, Callum Parr1, Mark Christian2, Magdalena Sastre1, 1 Imperial College London, London, United Kingdom; 2University of Warwick, Coventry, United Kingdom. Contact e-mail: n.mirzaei11@ imperial.ac.uk Background: Alterations in the Wnt signalling pathway have been implicated in Alzheimer’s disease (AD) because of its effects on axonal growth, on synaptogenesis and amyloid-b (Ab) toxicity; however its role in the processing of the amyloid precursor protein (APP) remains unknown. Methods: The canonical Wnt signalling pathway was activated by overexpression of the agonist Wnt3a, or the key regulator of the signalling cascade b-catenin or by inhibition of glycogen kinase synthase-3 in N2a cells. On the other hand, inhibition of the pathway was achieved by transfection of the antagonists secreted Frizzled receptor protein-1 or Dickkopf-1. The effects of both activation and inhibition of the pathway on APP processing were measured by western blotting, quantitative real-time PCR, dual luciferase promoter activity assays and chromatin immunoprecipitation (ChIP). Results: Activation of the Wnt pathway resulted in a reduction in Ab levels and in the activity and expression of b-APP cleaving enzyme (BACE1). Conversely, inhibition of the pathway produced the opposite effects. ChIP analysis demonstrated that b-catenin binds specifically to regions within the promoter of BACE1 containing putative T-cell factor/lymphoid enhancer-binding factor-1 (TCF/LEF) motifs, consistent with canonical Wnt target regulation. Interestingly, we also demonstrate that the transcription factor TCF3 binds preferentially to the same binding region in BACE1 promoter following Wnt3a stimulation, indicating that TCF3 functions as a transcriptional repressor of BACE1 gene transcription. Conclusions: In conclusion, Wnt/b-catenin stimulation may repress BACE1 transcription via binding of TCF3 to BACE1 gene and therefore, activation of the Wnt pathway may hold key to new treatments in AD.
P1-063
THE PPARGAMMA COFACTOR RIP140 REGULATES BACE1 GENE EXPRESSION
Katrin Blondrath, Magdalena Sastre, Imperial College London, London, United Kingdom. Contact e-mail: [email protected] Background: Activation of nuclear receptor Peroxisome Proliferator Activated Receptor gamma (PPARg) has been reported to be protective in Alzheimer’s disease (AD) mouse models and in cell lines, by decreasing
inflammation and Ab levels. Therefore, the role of PPARg cofactors will be critical in order to regulate the activity of PPARg. Our aim was to study the potential role of the PPARg cofactor R eceptor I nteracting P rotein 140 (RIP140) in AD. Methods: Two different kind of SDS pages were used to separate proteins of either whole cell lysates, membrane preparations or Aß peptides. 10% home-made Tris-Glycine polyacrylamide gel were used to separate proteins of whole cell lysates sized between 42 and 140 kD and precast NuPAGEÒ NovexÒ 4-12% Bis-Tris Protein Gels (life Technologies), to separate proteins sized 4 to 100kD. After the transgfer to a PVDF or Nitrocellulose Membrane at 400mV for one hour, Membrane were either blocked in milk or BSA and immunostaining with specific antibodies was performed. Antibodies used were: 6E10, Covance raised against Abeta, BACE1 and GsK3 from cell signaling, IDE and beta-Actin from abcam, ApoE and CD10 (Neprilysin) fom Santa Cruz. The 6D7 antibody against RIP140 was a kind gift provided by Dr. Mark Christian. Promotor activity was performed by using a standard Luciferase kit and a plate reader from Promega. Results: Results obtained in neuroblastoma cells suggest that RIP140 over expression reduced Ab generation and BACE1 expression and mRNA level in a PPARg-dependent manner. Conversely, knockdown of RIP140 increased the level of BACE1 protein, mRNA and promoter activity. Therefore, RIP140 seems to act as a transcriptional co-activator for PPARg leading to reduced BACE1 transcription. In addition, studies in animal models, lacking or over expressing RIP140 indicated that many genes involved in AD and the insulin pathway appeared to be modulated by RIP140. Hippocampal homogenates of RIP140 transgenic mice showed transcriptional induction of growth factors such as growth associated protein 43 (GAP43) and Insulin growth factor2 (IgF2) as well as up-regulation of genes involved in the clearance of Ab, namely insulin degrading enzyme (IDE) and Neprilysin. The frontal cortex of these animals exhibited strongly reduced BACE1 protein expression level, in line with results obtained in vitro. In addition, RIP140 over expressing in this brain area of mice displayed a decrease in microtubule associated proteins 1 and 2, both being involved in the tau pathology of AD. Studies in animals lacking RIP140 revealed that BACE1 mRNA level were increased in the frontal cortex area amongst other regulations on genes which haven been found differently regulated in the RIP140 over expressing background. Conclusions: The results indicate that important enzymes that are implicated in Ab generation and degradation are regulated by RIP140 transcriptional modulation. Therefore, RIP140 could serve as therapeutic target for AD.
P1-064
A COMBINATION APPROACH IN A CANINE MODEL OF AGING: IMMUNOTHERAPY WITH BEHAVIORAL ENRICHMENT
Paulina R. Davis1, Tina Beckett1, Ginevra Giannini2, Nathanial Calloway1, M. Paul Murphy1, Edward G. Barrett2, Elizabeth Head1, 1Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United