- Email: [email protected]
P4-210: Effects of mild cholesterol depletion on APP processing and gamma-secretase
Poster Presentations P4: mation, and this increased baseline activity over two-fold. Proteoliposomes containing cerebrosides or gangliosides significantly increased ␥-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids differentially increased the cleavage of APP-, APLP1- and Notch-like substrates in a dose-dependent manner. Reconstitution of ␥-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared to other membrane compositions, and reconstitution in a brain lipid extract yielded much higher activity than in liver or heart extracts. Conclusions: Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of ␥-secretase and that cholesterol, in particular, plays a major regulatory role. P4-210
EFFECTS OF MILD CHOLESTEROL DEPLETION ON APP PROCESSING AND GAMMA-SECRETASE
Cristina Guardia-Laguarta, Mireia Coma, Jordi Clarimon, Lidia Sereno, Jose Manuel Agullo, Laura Molina, Rafael Blesa, Teresa Gomez-Isla, Alberto Lleo, Laboratory Alzheimer Memory Unit Hospital Sant Pau, Barcelona, Spain. Contact e-mail: [email protected] Background: Epidemiological studies have suggested a link between high serum cholesterol levels and risk for Alzheimer’s disease. A is one of the primary components of amyloid plaques in Alzheimer’s disease (AD). It has been suggested that cellular cholesterol levels can modulate its metabolism by affecting beta- or gamma-secretase activity, although this remains controversial. In this report, we investigate in detail the relation between cholesterol membrane reduction and changes in APP processing and gamma-secretase function. Methods: We developed an assay to induce mild cellular cholesterol depletion by treating CHO or H4 cells with lovastatin, pravastatin or methyl- -cyclodextrin. We measured A 40 and 42 levels by ELISA and the APP C-terminal fragments by Western blot. We also tested the cleavage of Notch receptor by Western blot and a luciferase assay. We applied a Fluorescence Resonance Energy Transfer (FRET)based assay (FLIM) to measure APP-PS1 interactions in intact cells under these conditions. We also developed a novel FRET assay to measure the effect of cholesterol reduction on the distribution of lipid rafts. Results: We found that mild cholesterol depletion led to a cholesterol-dependent decrease on A 40 and 42 production in different cell types. The observed reduction in A levels paralleled the reduction in total cholesterol levels. We did not detect changes on AICD or NICD generation suggesting that cholesterol depletion does not affect gamma-secretase activity. Western blot analyses showed a specific cholesterol-dependent decrease in APP C-terminal fragments. Finally, FRET experiments demonstrated that cholesterol depletion disrupts the APP-PS1 interaction and alter the distribution of lipid rafts. Conclusions: Mild cellular cholesterol depletion affects APP metabolism by reducing APP C-terminal fragments but does not affect gamma-secretase. This effect may be mediated by changes in lipid raft distribution. P4-211
THE FUNCTIONAL ROLES OF TRANSMEMBRANE DOMAINS OF PRESENILIN 1 IN THE FORMATION OF ACTIVE GAMMASECRETASE COMPLEX
Naoto Watanabe, Taisuke Tomita, Takeshi Iwatsubo, The University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected] Background: Genetic and biochemical analysis revealed that gammasecretase is a hetero-tetrameric membrane protein complex, comprised of presenilin (PS), nicastrin, aph-1 and pen-2. PS, the catalytic subunit of gamma-secretase, harbors nine transmembrane domains (TMDs). To identify the individual roles of TMDs of PS1, we swapped each TMD with that of functionally irrelevant proteins preserving the TMD topology and found that PS1 TMD4 bears a direct binding motif to Pen-2 (Watanabe et al., 2005). Intriguingly, all TMD-swap mutants except for TMD3 lost gamma-
T733
secretase activity, while these mutants were able to form hetero-tetrameric gamma-secretase complex. Methods: PS1 mutants were stably expressed in DKO or MEF cells and analyzed by immunoblotting and ELISA. Photoaffinity probes were incubated with the solubilized membranes. The labeled proteins were collected by streptavidin beads after irradiation. Results: Overexpression of PS1 CTF restored the gamma-secretase activity in cells expressing TMD8- or TMD9-swap PS1 as previously described (Laudon et al. 2004). However, coexpression of NTF failed to recover the gamma-secretase activity of TMD1- or TMD2-swap mutant, whereas that of the other mutants (i.e., TMD4-, TMD5- or TMD6-swap) was restored by exogenous PS1 NTF. Overexpression of TMD2-swap PS1 in MEF wt cells caused the accumulation of APP C-terminal stub in a similar manner to that by D385A mutant, which was not caused by TMD1-swap PS1, suggesting that TMD1 and TMD2 play distinct roles in the gamma-secretase mediated intramembrane cleavage. To test the possibility that TMD1 is involved in the formation of enzymatically active sites, photoaffinity labeling experiments using transition-state analogue- or helical peptide-based photoprobe were performed. We found that the gamma-secretase complex containing TMD1-swap PS1 mutant was unable to form either a catalytic center or an initial substrate binding site. Conclusions: These results suggest that each TMD of PS1 plays a specific role in the acquisition of the gamma-secretase activity after the assembly of the complex, e.g., maintaining the conformational stability, serving as the inter-molecular interaction sites, or forming a functional domain required for the proteolytic process. P4-212
PRESENILIN 1–RELATED DISTURBANCES OF CALCIUM SIGNALING IN B CELLS ISOLATED FROM POLISH PATIENTS SUFFERING FROM FAMILIAL ALZHEIMER’S DISEASE
Lukasz Bojarski1, Pawel Pomorski2, Jochen Herms3, Jacek Kuznicki1,2, 1 International Institute of Molecular and Cell Biology, Warsaw, Poland; 2 Nencki Institute of Experimental Biology, Warsaw, Poland; 3 Department of Neuropathology, Ludwig-Maximilians Universitat, Munich, Germany. Contact e-mail: [email protected] Background: A few hypotheses have been proposed to explain Alzheimer’s disease (AD) pathogenesis. One of these proposes that the dysregulation of calcium homeostasis may be a key accelerating factor or even a cause of other pathological changes observed in AD. Mutations in gene encoding presenilin 1 result in development of familial Alzheimer’s disease (FAD) and were demonstrated to alter cellular calcium signaling. Increased ER calcium content and attenuated capacitative calcium entry (CCE) were reported for cells bearing PS1 FAD mutations. Methods: We investigated effects of 5 pathogenic FAD mutations (P117R, M139V, H163R, S170F, I213F) and non-pathogenic polymorphism (E318G) in presenilin 1 (PS1) gene on calcium homeostasis of B cells isolated from Polish patients. To elucidate the mechanism of observed disturbances in B cells calcium signaling, we assessed intracellular localization and expression levels of PS1 and STIM1 proteins, the latter being main signaling molecule involved in CCE activation. Results: Ratiometric image analysis revealed increased basal cytosolic concentration as well as enhanced thapsigargin induced release of calcium ions from ER of cells with FAD mutations in comparison to lymphocytes from healthy individuals. Moreover, mean capacitative calcium entry (CCE) was attenuated in analysed cells. Our preliminary experiments revealed that in case of some of analysed PS1 FAD mutations cellular level of STIM1 protein was decreased while the amount of PS1 remained unchanged. To confirm the results of studies on human B cells with neuronal model, we investigated STIM1 protein level in hippocampi of transgenic mice and observed decreased level of STIM1 in tissue of PS1 A246E mice in comparison to wt PS1 animals. Conclusions: Decreased level of STIM1 protein may provide plausible explanation for attenuated CCE in cells bearing PS1 FAD mutations. Moreover, the results of experiments conducted on animal AD model corroborate with observations made on human B cells confirming usefulness of lymphocytes in studies on AD.
EFFECTS OF MILD CHOLESTEROL DEPLETION ON APP PROCESSING AND GAMMA-SECRETASE
Cristina Guardia-Laguarta, Mireia Coma, Jordi Clarimon, Lidia Sereno, Jose Manuel Agullo, Laura Molina, Rafael Blesa, Teresa Gomez-Isla, Alberto Lleo, Laboratory Alzheimer Memory Unit Hospital Sant Pau, Barcelona, Spain. Contact e-mail: [email protected] Background: Epidemiological studies have suggested a link between high serum cholesterol levels and risk for Alzheimer’s disease. A is one of the primary components of amyloid plaques in Alzheimer’s disease (AD). It has been suggested that cellular cholesterol levels can modulate its metabolism by affecting beta- or gamma-secretase activity, although this remains controversial. In this report, we investigate in detail the relation between cholesterol membrane reduction and changes in APP processing and gamma-secretase function. Methods: We developed an assay to induce mild cellular cholesterol depletion by treating CHO or H4 cells with lovastatin, pravastatin or methyl- -cyclodextrin. We measured A 40 and 42 levels by ELISA and the APP C-terminal fragments by Western blot. We also tested the cleavage of Notch receptor by Western blot and a luciferase assay. We applied a Fluorescence Resonance Energy Transfer (FRET)based assay (FLIM) to measure APP-PS1 interactions in intact cells under these conditions. We also developed a novel FRET assay to measure the effect of cholesterol reduction on the distribution of lipid rafts. Results: We found that mild cholesterol depletion led to a cholesterol-dependent decrease on A 40 and 42 production in different cell types. The observed reduction in A levels paralleled the reduction in total cholesterol levels. We did not detect changes on AICD or NICD generation suggesting that cholesterol depletion does not affect gamma-secretase activity. Western blot analyses showed a specific cholesterol-dependent decrease in APP C-terminal fragments. Finally, FRET experiments demonstrated that cholesterol depletion disrupts the APP-PS1 interaction and alter the distribution of lipid rafts. Conclusions: Mild cellular cholesterol depletion affects APP metabolism by reducing APP C-terminal fragments but does not affect gamma-secretase. This effect may be mediated by changes in lipid raft distribution. P4-211
THE FUNCTIONAL ROLES OF TRANSMEMBRANE DOMAINS OF PRESENILIN 1 IN THE FORMATION OF ACTIVE GAMMASECRETASE COMPLEX
Naoto Watanabe, Taisuke Tomita, Takeshi Iwatsubo, The University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected] Background: Genetic and biochemical analysis revealed that gammasecretase is a hetero-tetrameric membrane protein complex, comprised of presenilin (PS), nicastrin, aph-1 and pen-2. PS, the catalytic subunit of gamma-secretase, harbors nine transmembrane domains (TMDs). To identify the individual roles of TMDs of PS1, we swapped each TMD with that of functionally irrelevant proteins preserving the TMD topology and found that PS1 TMD4 bears a direct binding motif to Pen-2 (Watanabe et al., 2005). Intriguingly, all TMD-swap mutants except for TMD3 lost gamma-
T733
secretase activity, while these mutants were able to form hetero-tetrameric gamma-secretase complex. Methods: PS1 mutants were stably expressed in DKO or MEF cells and analyzed by immunoblotting and ELISA. Photoaffinity probes were incubated with the solubilized membranes. The labeled proteins were collected by streptavidin beads after irradiation. Results: Overexpression of PS1 CTF restored the gamma-secretase activity in cells expressing TMD8- or TMD9-swap PS1 as previously described (Laudon et al. 2004). However, coexpression of NTF failed to recover the gamma-secretase activity of TMD1- or TMD2-swap mutant, whereas that of the other mutants (i.e., TMD4-, TMD5- or TMD6-swap) was restored by exogenous PS1 NTF. Overexpression of TMD2-swap PS1 in MEF wt cells caused the accumulation of APP C-terminal stub in a similar manner to that by D385A mutant, which was not caused by TMD1-swap PS1, suggesting that TMD1 and TMD2 play distinct roles in the gamma-secretase mediated intramembrane cleavage. To test the possibility that TMD1 is involved in the formation of enzymatically active sites, photoaffinity labeling experiments using transition-state analogue- or helical peptide-based photoprobe were performed. We found that the gamma-secretase complex containing TMD1-swap PS1 mutant was unable to form either a catalytic center or an initial substrate binding site. Conclusions: These results suggest that each TMD of PS1 plays a specific role in the acquisition of the gamma-secretase activity after the assembly of the complex, e.g., maintaining the conformational stability, serving as the inter-molecular interaction sites, or forming a functional domain required for the proteolytic process. P4-212
PRESENILIN 1–RELATED DISTURBANCES OF CALCIUM SIGNALING IN B CELLS ISOLATED FROM POLISH PATIENTS SUFFERING FROM FAMILIAL ALZHEIMER’S DISEASE
Lukasz Bojarski1, Pawel Pomorski2, Jochen Herms3, Jacek Kuznicki1,2, 1 International Institute of Molecular and Cell Biology, Warsaw, Poland; 2 Nencki Institute of Experimental Biology, Warsaw, Poland; 3 Department of Neuropathology, Ludwig-Maximilians Universitat, Munich, Germany. Contact e-mail: [email protected] Background: A few hypotheses have been proposed to explain Alzheimer’s disease (AD) pathogenesis. One of these proposes that the dysregulation of calcium homeostasis may be a key accelerating factor or even a cause of other pathological changes observed in AD. Mutations in gene encoding presenilin 1 result in development of familial Alzheimer’s disease (FAD) and were demonstrated to alter cellular calcium signaling. Increased ER calcium content and attenuated capacitative calcium entry (CCE) were reported for cells bearing PS1 FAD mutations. Methods: We investigated effects of 5 pathogenic FAD mutations (P117R, M139V, H163R, S170F, I213F) and non-pathogenic polymorphism (E318G) in presenilin 1 (PS1) gene on calcium homeostasis of B cells isolated from Polish patients. To elucidate the mechanism of observed disturbances in B cells calcium signaling, we assessed intracellular localization and expression levels of PS1 and STIM1 proteins, the latter being main signaling molecule involved in CCE activation. Results: Ratiometric image analysis revealed increased basal cytosolic concentration as well as enhanced thapsigargin induced release of calcium ions from ER of cells with FAD mutations in comparison to lymphocytes from healthy individuals. Moreover, mean capacitative calcium entry (CCE) was attenuated in analysed cells. Our preliminary experiments revealed that in case of some of analysed PS1 FAD mutations cellular level of STIM1 protein was decreased while the amount of PS1 remained unchanged. To confirm the results of studies on human B cells with neuronal model, we investigated STIM1 protein level in hippocampi of transgenic mice and observed decreased level of STIM1 in tissue of PS1 A246E mice in comparison to wt PS1 animals. Conclusions: Decreased level of STIM1 protein may provide plausible explanation for attenuated CCE in cells bearing PS1 FAD mutations. Moreover, the results of experiments conducted on animal AD model corroborate with observations made on human B cells confirming usefulness of lymphocytes in studies on AD.