- Email: [email protected]
P3-331: A novel assay to quantitatively measure gamma-secretase activity against the S3 site of notch
T618 P3-328
Poster Presentations P3: CLEAVAGE OF ENDOGENOUS SUBSTRATES BY ␥-SECRETASE IN RAT BRAIN MEMBRANES
Jenny Frånberg, Birgitta Wiehager, Bengt Winblad, Helena Karlstro¨m, Lars O. Tjernberg, Susanne Frykman, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: ␥-Secretase is the final secretase that cleaves the amyloid precursor protein (APP) to generate the amyloid -peptide (A) and is therefore a target for development of therapeutics for Alzheimer’s disease. In addition to the A-generating cleavage site, ␥-secretase cleaves APP at the ⑀-site generating a C-terminal fragment, the APP intracellular domain (AICD). Apart from APP, ␥-secretase has at present been shown to cleave more than 30 substrates including Notch, N-cadherin, EphrinB, and p75Neurotrophin receptor. All of the substrates are type I transmembrane proteins that are cleaved in a similar way as APP. The ␥-secretase released intracellular domains (ICDs) appear in many cases to play a role in signal transduction. The growing list of reported ␥-secretase substrates makes the development of ␥-secretase inhibitors as potential drugs more difficult. Methods: To study the cleavage of different endogenous substrates by ␥-secretase in brain tissue we isolated membrane fractions from embryonic rat brain (day 17) and adult rat brain (3 months old). The membranes were incubated at 37 °C in the absence or presence of the ␥-secretase inhibitors L-685,458 and DAPT and the ICDs from APP, EphrinB1, N-cadherin, Notch 1 and p75-Neurotrophin receptor were detected by western blot analysis. Results: Cleavage of EphrinB1, N-cadherin and p75-Neurotrophin receptor was detected in the embryonic rat brain membranes but not in the adult rat brain membranes. However, the substrate levels were also higher in the embryonic compared to the adult brain. APP and Notch 1 processing was observed in both embryonic and adult rat brain but to a higher extent in the embryonic rat brain. Conclusions: The ␥-secretase activity was higher in embryonic rat brain compared to adult rat brain. This embryonic rat brain membrane system could be of use when studying the effect of inhibitors or modulators on ␥-secretase cleavage. P3-329
PRESENILIN / ␥-SECRETASE–MEDIATED CLEAVAGE REGULATES TYROSINE PHOSPHATASE LAR SIGNALING VIA CATENIN
Annakaisa Haapasalo, Mari K. Turunen, Mikko Hiltunen, Hilkka Soininen, University of Kuopio, Kuopio, Finland. Contact e-mail: [email protected] Background: Receptor protein tyrosine phosphatase LAR (leukocyte common antigen-related) is a type-I transmembrane protein implicated in the regulation of cell adhesion, neurite outgrowth, and synaptic function. LAR interacts with and dephosphorylates -catenin, which is a structural component in cadherin-mediated cell adhesions, and which also regulates transcription of several genes by forming a complex with transcription factors T-cell factor (TCF) and lymphoid enhancing factor (LEF). We have shown recently that LAR is a new substrate for presenilin (PS)/␥-secretase and undergoes proteolytic processing analogous to that of -amyloid precursor protein (APP). ADAM17 (a disintegrin and metalloproteinase 17), one of the ␣-secretases cleaving APP, sheds LAR ectodomain and generates membrane-attached LAR C-terminal fragments (CTFs). The CTFs are subsequently cleaved by PS/␥-secretase releasing LAR intracellular domains (LICD). Our studies have indicated that LICD localizes in the cytoplasm and nucleus, the same compartments as -catenin, and that overexpression of LICD significantly decreases the mRNA levels of endogenously expressed cyclin D1, one of the target genes regulated by -catenin-TCF-LEF complex. Here, our aim was to elucidate the molecular mechanism how LICD exerts its regulatory function on -catenin-mediated transcription. Methods: To examine whether LICD and -catenin associate with each other and whether this possible association results in altered -catenin phosphorylation or subcellular localization, we performed coimmunoprecipitation, Western blotting and subcellular fractionation experiments in Chinese hamster ovary (CHO) cells stably overexpressing LICD
or naı¨ve control CHO cells. Results: Our results showed that LICD coimmunoprecipitated with -catenin, indicating the presence of both proteins in the same protein complexes and thus potential interaction. However, LICD overexpression did not cause major differences in -catenin tyrosine phosphorylation status or subcellular localization as compared to control cells. In our current experiments, we are investigating if LICD overexpression regulates formation or function of the transcriptional complex between -catenin and TCF-LEF. Conclusions: Taken together, our data suggest that LICD, produced by PS/␥-secretase activity, directly associates with -catenin and regulates its signaling. Furthermore, our results agree with the notion that PS/␥-secretase plays an important role in the regulation of diverse cellular functions, such as adhesion or gene expression, via proteolytic cleavage of its substrate proteins. P3-330
LUCIFERASE COMPLEMENTATION IMAGING AS A REAL-TIME REPORTER FOR THE GAMMASECRETASE CLEAVAGE OF NOTCH
Ma. Xenia G. Ilagan, Guojun Zhao, Dilip Chandu, Mary Blandford, David Piwnica-Worms, Raphael Kopan, Washington University School of Medicine, Saint Louis, MO, USA. Contact e-mail: [email protected] Background: As a key protease involved in the production of the amyloidogenic A peptides, the gamma-secretase enzyme has been a prime therapeutic target for Alzheimer’s disease. However, gamma-secretase cleaves other Type I membrane protein substrates, several of which play important roles during development and tissue renewal in adults, including the Notch receptors. Notch activation is triggered by ligand binding and depends on gamma-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA-binding protein CSL to activate gene expression. Methods: We report the use of luciferase complementation imaging (LCI) technology to monitor the interactions between the NICD and its nuclear cofactor CSL in real time. In this reporter system, complementary halves of firefly luciferase (NLuc and CLuc), which have no activity on their own, reconstitute luciferase activity when brought in close proximity by specific interactions between NICD and CSL. Results: Our validation studies demonstrate LCI to be a specific, robust and sensitive system for imaging NICD/CSL interactions. Because the luciferase fragments contribute negligible binding energy and the luciferase fusion proteins constitute a complete assay system, they allow measurement of reconstituted luciferase activity in near real time, enabling kinetic analyses of gamma-secretase activity and of Notch inhibition in live cells, from which A production can be simultaneously assessed. We have begun screening for molecules and gene products capable of modulating Notch cleavage and activation and are in the process of validating leads. Conclusions: The LCI system is versatile; it can be adapted for studying the pathway in different cell types and can be modified to monitor interactions of Notch with other proteins, including components of gamma-secretase. We are currently adapting the system to simultaneously monitor interactions of gamma-secretase with two substrates (Notch and APP) and assessing its utility as a screen for modifiers of substrate selection. P3-331
A NOVEL ASSAY TO QUANTITATIVELY MEASURE GAMMA-SECRETASE ACTIVITY AGAINST THE S3 SITE OF NOTCH
Laura A. Ingano1, Ikuo Hayashi1, Gang Yu2, Sanjiv Shah1, Mark Shearman1, 1Merck Research Laboratories, Boston, MA, USA; 2 University of Texas Southwestern Medical Center, Dallas, TX, USA. Contact e-mail: [email protected] Background: Gamma-secretase is an aspartyl protease which cleaves APP to release abeta, and is a popular therapeutic target for the treatment of AD. Progress to pharmacologically inhibit or modulate gamma-secretase activity is hindered by the challenge to block or alter abeta production while maintaining gamma-secretase activity against other substrates. Toxicities
Poster Presentations P3: observed from the potent inhibition of gamma-secretase are likely due to altered Notch processing. Therefore, it is essential to drug discovery efforts to have a high-throughput platform to quantitatively measure Notch cleavage by gamma-secretase. Methods: Here we present a novel in vitro ELISA, employing a biotin flag antibody, a cleaved Notch (V1744) specific antibody and a N100FLAG recombinant protein, to measure the generation of the Notch intracellular domain (NICD). Results: This assay compliments the in vitro APP C100FLAG abeta assay by allowing a direct comparison of compound efficacy on gamma secretase activity against the S3 Notch cleavage site compared to the ␥-cleavage sites of APP. Conclusions: Together, these assays will aid in identifying a Notch-sparing mechanism of inhibiting gamma-secretase activity. P3-332
CHARACTERIZATION OF THE BACE1B ISOFORM
Joseph L. Johnson, Nathan Bruender, Keerthi Jayasundera, University of Minnesota Duluth, Duluth, MN, USA. Contact e-mail: [email protected] Background: Alzheimer’s Disease (AD) is a devastating neurodegenerative disease that begins with subtle memory loss and proceeds relentlessly, eventually leading to complete incapacitation and death. Amyloid plaques are a characteristic feature of AD brains and are predominantly composed of a short peptide fragment termed ABeta, which is derived from the amyloid precursor protein (APP) by proteolytic cleavage. One prevailing hypothesis proposes that ABeta production is the causal event leading to AD. Two proteases are responsible for the generation of ABeta from APP, beta-secretase (BACE1) and gamma-secretase. BACE1 is an attractive therapeutic target because preventing its cleavage of APP has been shown to decrease ABeta production, which should stop or even reverse the course of AD. Recent studies identified the presence of mRNA for several genetically distinct BACE1 isoforms in vivo, but the activity of these different enzymes and their functions in vivo remain unclear. We will discuss our efforts to produce and characterize the BACE1B isoform. Methods: We will report on the use an of an insect expression system to produce the recombinant BACE1B isoform and its characterization utilizing fluorescence-based assays. Results: Recombinant BACE1B can be expressed and purified. BACE1B has measurable activity, albeit less than the full length isoform, BACE1A. Conclusions: The alternativelyspliced BACE1B isoform may have in vivo roles distinct from BACE1A. P3-333
A NOVEL RECIPROCAL AND BIPHASIC RELATIONSHIP BETWEEN MEMBRANE CHOLESTEROL AND -SECRETASE ACTIVITY IN SH-SY5Y CELLS AND HUMAN PLATELETS
Janet A. Johnston, WeiWei Liu, Stephen Todd, David T. R. Coulson, G. Brent Irvine, Bernadette McGuinness, David Craig, Maeve McConville, A. Peter Passmore, Queen’s University, Belfast, United Kingdom. Contact e-mail: [email protected] Background: Research into Alzheimer’s disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters modulate amyloid precursor protein (APP) processing, altering production of A. APP processing depends on the encounter between APP, and -and ␥-secretases, and is thus influenced by cholesterol-dependent factors including protein trafficking, and distribution between membrane sub-domains. To overcome these confounding factors, we have directly investigated endogenous membrane -secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y cells and human platelets. Methods: SH-SY5Y cells were exposed to either methyl--cyclodextrin (MCD); exogenous cholesterol (complexed with MCD); or cell-permeable -secretase inhibitor (Calbiochem). Blood samples were obtained from human subjects in accordance with local ethical committee procedures, and platelets prepared1,2. SH-SY5Y
T619
cell or platelets were lysed (25 mM HEPES, pH 7.2), homogenised and centrifuged (100,000 g, 1 h). Pellets were suspended in membrane solubilisation buffer (25 mM HEPES, 2% CHAPS), centrifuged (20,000 g, 10 min). The supernatant (membrane fraction) was assayed for -secretase activity as described1,2 using a wild-type APP fluorogenic substrate, MCA-EVKMDAEFK-(DNP)-NH2 (Calbiochem), initial rates calculated over the 8-25 min linear phase, and expressed as pmoles MCA detected/min/g membrane protein. Membrane fraction cholesterol levels were determined by Amplex Red Cholesterol Assay (Invitrogen), and results expressed as pmol cholesterol detected/g membrane protein. BACE1 levels were assayed by ELISA, and results expressed as ng BACE1/g membrane protein. Results: SH-SY5Y membrane cholesterol significantly influenced membrane -secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. The same pattern was identified in human platelets. Interestingly, platelets from cognitively-impaired individuals (AD or milder cognitive impairment) were significantly more likely to lie within the negative correlation zone than controls. Pharmacological inhibition of SH-SY5Y -secretase activity resulted in increased membrane cholesterol levels. Conclusions: Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane -secretase activity, and suggest that this regulatory mechanism is disrupted in the platelets of cognitively-impaired individuals. 1. Johnston, J.A. et al., Neurobiol of Aging, doi:10.1016/j.neurobiolaging.2006.11.003. 2. Liu, W.W. et al., Dement Geriatr Cogn Disord. (2007) 24:464-468. P3-334
␥-SECRETASE–MEDIATED PROTEOLYSIS OF APP AND NOTCH IS REGULATED BY CAVEOLIN-1
Arun Kapoor1,2, Bo-Jeng Wang1, Yung-Feng Liao1, 1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei,11529, Taiwan; 2Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, 11529, Taiwan. Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan. Contact e-mail: [email protected] Background: The deposition of neurotoxic amyloid- (A) peptides in brain leads to the development of Alzheimer’s disease (AD). These 3943-amino acid A peptides are generated by the sequential proteolysis of amyloid precursor protein (APP) by - and ␥-secretases. Accumulated evidence has suggested that cholesterol- and sphingolipid-rich membrane microdomains, such as lipid rafts, are involved in regulating the secretasemediated proteolysis of APP. Caveolin-1, a cholesterol-binding membrane protein and an essential constituent of caveolae, has been shown to directly interact with APP and -secretase. Its expression in the hippocampus of autopsy-confirmed subjects of AD is upregulated. Furthermore, ␥-secretase components have been shown to be co-localized with cholesterol-rich lipid rafts. We thus hypothesized that caveolin-1 could play an active role in governing ␥-secretase-mediated proteolysis of APP. Methods: We used substrate-specific cell-based assays to measure ␥-secretase activity and RNAi approach to suppress the expression of caveolin-1. We also used confocal microscopy to study the colocalization of caveolin-1 with ␥-secretase subunits presenilin-1 (PS1) and nicastrin (NCT). ␥-Secretase mediated S3 cleavage of Notch was studied using cell lines stably expressing N⌬E and transient transfection of Hes-1 luciferase reporter constructs. Results: We demonstrated that the suppression of caveolin-1 expression by RNAi dramatically augments ␥-secretase-mediated cleavage of APP and production of A40. Consistent with these findings, the down-regulation of caveolin-1 also led to an increased S3 cleavage of Notch by ␥-secretase. The expression and maturation of ␥-secretase constituents, such as presenilin-1 (PS1) and nicastrin (NCT), were not altered by the knock-down of
Poster Presentations P3: CLEAVAGE OF ENDOGENOUS SUBSTRATES BY ␥-SECRETASE IN RAT BRAIN MEMBRANES
Jenny Frånberg, Birgitta Wiehager, Bengt Winblad, Helena Karlstro¨m, Lars O. Tjernberg, Susanne Frykman, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: ␥-Secretase is the final secretase that cleaves the amyloid precursor protein (APP) to generate the amyloid -peptide (A) and is therefore a target for development of therapeutics for Alzheimer’s disease. In addition to the A-generating cleavage site, ␥-secretase cleaves APP at the ⑀-site generating a C-terminal fragment, the APP intracellular domain (AICD). Apart from APP, ␥-secretase has at present been shown to cleave more than 30 substrates including Notch, N-cadherin, EphrinB, and p75Neurotrophin receptor. All of the substrates are type I transmembrane proteins that are cleaved in a similar way as APP. The ␥-secretase released intracellular domains (ICDs) appear in many cases to play a role in signal transduction. The growing list of reported ␥-secretase substrates makes the development of ␥-secretase inhibitors as potential drugs more difficult. Methods: To study the cleavage of different endogenous substrates by ␥-secretase in brain tissue we isolated membrane fractions from embryonic rat brain (day 17) and adult rat brain (3 months old). The membranes were incubated at 37 °C in the absence or presence of the ␥-secretase inhibitors L-685,458 and DAPT and the ICDs from APP, EphrinB1, N-cadherin, Notch 1 and p75-Neurotrophin receptor were detected by western blot analysis. Results: Cleavage of EphrinB1, N-cadherin and p75-Neurotrophin receptor was detected in the embryonic rat brain membranes but not in the adult rat brain membranes. However, the substrate levels were also higher in the embryonic compared to the adult brain. APP and Notch 1 processing was observed in both embryonic and adult rat brain but to a higher extent in the embryonic rat brain. Conclusions: The ␥-secretase activity was higher in embryonic rat brain compared to adult rat brain. This embryonic rat brain membrane system could be of use when studying the effect of inhibitors or modulators on ␥-secretase cleavage. P3-329
PRESENILIN / ␥-SECRETASE–MEDIATED CLEAVAGE REGULATES TYROSINE PHOSPHATASE LAR SIGNALING VIA CATENIN
Annakaisa Haapasalo, Mari K. Turunen, Mikko Hiltunen, Hilkka Soininen, University of Kuopio, Kuopio, Finland. Contact e-mail: [email protected] Background: Receptor protein tyrosine phosphatase LAR (leukocyte common antigen-related) is a type-I transmembrane protein implicated in the regulation of cell adhesion, neurite outgrowth, and synaptic function. LAR interacts with and dephosphorylates -catenin, which is a structural component in cadherin-mediated cell adhesions, and which also regulates transcription of several genes by forming a complex with transcription factors T-cell factor (TCF) and lymphoid enhancing factor (LEF). We have shown recently that LAR is a new substrate for presenilin (PS)/␥-secretase and undergoes proteolytic processing analogous to that of -amyloid precursor protein (APP). ADAM17 (a disintegrin and metalloproteinase 17), one of the ␣-secretases cleaving APP, sheds LAR ectodomain and generates membrane-attached LAR C-terminal fragments (CTFs). The CTFs are subsequently cleaved by PS/␥-secretase releasing LAR intracellular domains (LICD). Our studies have indicated that LICD localizes in the cytoplasm and nucleus, the same compartments as -catenin, and that overexpression of LICD significantly decreases the mRNA levels of endogenously expressed cyclin D1, one of the target genes regulated by -catenin-TCF-LEF complex. Here, our aim was to elucidate the molecular mechanism how LICD exerts its regulatory function on -catenin-mediated transcription. Methods: To examine whether LICD and -catenin associate with each other and whether this possible association results in altered -catenin phosphorylation or subcellular localization, we performed coimmunoprecipitation, Western blotting and subcellular fractionation experiments in Chinese hamster ovary (CHO) cells stably overexpressing LICD
or naı¨ve control CHO cells. Results: Our results showed that LICD coimmunoprecipitated with -catenin, indicating the presence of both proteins in the same protein complexes and thus potential interaction. However, LICD overexpression did not cause major differences in -catenin tyrosine phosphorylation status or subcellular localization as compared to control cells. In our current experiments, we are investigating if LICD overexpression regulates formation or function of the transcriptional complex between -catenin and TCF-LEF. Conclusions: Taken together, our data suggest that LICD, produced by PS/␥-secretase activity, directly associates with -catenin and regulates its signaling. Furthermore, our results agree with the notion that PS/␥-secretase plays an important role in the regulation of diverse cellular functions, such as adhesion or gene expression, via proteolytic cleavage of its substrate proteins. P3-330
LUCIFERASE COMPLEMENTATION IMAGING AS A REAL-TIME REPORTER FOR THE GAMMASECRETASE CLEAVAGE OF NOTCH
Ma. Xenia G. Ilagan, Guojun Zhao, Dilip Chandu, Mary Blandford, David Piwnica-Worms, Raphael Kopan, Washington University School of Medicine, Saint Louis, MO, USA. Contact e-mail: [email protected] Background: As a key protease involved in the production of the amyloidogenic A peptides, the gamma-secretase enzyme has been a prime therapeutic target for Alzheimer’s disease. However, gamma-secretase cleaves other Type I membrane protein substrates, several of which play important roles during development and tissue renewal in adults, including the Notch receptors. Notch activation is triggered by ligand binding and depends on gamma-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA-binding protein CSL to activate gene expression. Methods: We report the use of luciferase complementation imaging (LCI) technology to monitor the interactions between the NICD and its nuclear cofactor CSL in real time. In this reporter system, complementary halves of firefly luciferase (NLuc and CLuc), which have no activity on their own, reconstitute luciferase activity when brought in close proximity by specific interactions between NICD and CSL. Results: Our validation studies demonstrate LCI to be a specific, robust and sensitive system for imaging NICD/CSL interactions. Because the luciferase fragments contribute negligible binding energy and the luciferase fusion proteins constitute a complete assay system, they allow measurement of reconstituted luciferase activity in near real time, enabling kinetic analyses of gamma-secretase activity and of Notch inhibition in live cells, from which A production can be simultaneously assessed. We have begun screening for molecules and gene products capable of modulating Notch cleavage and activation and are in the process of validating leads. Conclusions: The LCI system is versatile; it can be adapted for studying the pathway in different cell types and can be modified to monitor interactions of Notch with other proteins, including components of gamma-secretase. We are currently adapting the system to simultaneously monitor interactions of gamma-secretase with two substrates (Notch and APP) and assessing its utility as a screen for modifiers of substrate selection. P3-331
A NOVEL ASSAY TO QUANTITATIVELY MEASURE GAMMA-SECRETASE ACTIVITY AGAINST THE S3 SITE OF NOTCH
Laura A. Ingano1, Ikuo Hayashi1, Gang Yu2, Sanjiv Shah1, Mark Shearman1, 1Merck Research Laboratories, Boston, MA, USA; 2 University of Texas Southwestern Medical Center, Dallas, TX, USA. Contact e-mail: [email protected] Background: Gamma-secretase is an aspartyl protease which cleaves APP to release abeta, and is a popular therapeutic target for the treatment of AD. Progress to pharmacologically inhibit or modulate gamma-secretase activity is hindered by the challenge to block or alter abeta production while maintaining gamma-secretase activity against other substrates. Toxicities
Poster Presentations P3: observed from the potent inhibition of gamma-secretase are likely due to altered Notch processing. Therefore, it is essential to drug discovery efforts to have a high-throughput platform to quantitatively measure Notch cleavage by gamma-secretase. Methods: Here we present a novel in vitro ELISA, employing a biotin flag antibody, a cleaved Notch (V1744) specific antibody and a N100FLAG recombinant protein, to measure the generation of the Notch intracellular domain (NICD). Results: This assay compliments the in vitro APP C100FLAG abeta assay by allowing a direct comparison of compound efficacy on gamma secretase activity against the S3 Notch cleavage site compared to the ␥-cleavage sites of APP. Conclusions: Together, these assays will aid in identifying a Notch-sparing mechanism of inhibiting gamma-secretase activity. P3-332
CHARACTERIZATION OF THE BACE1B ISOFORM
Joseph L. Johnson, Nathan Bruender, Keerthi Jayasundera, University of Minnesota Duluth, Duluth, MN, USA. Contact e-mail: [email protected] Background: Alzheimer’s Disease (AD) is a devastating neurodegenerative disease that begins with subtle memory loss and proceeds relentlessly, eventually leading to complete incapacitation and death. Amyloid plaques are a characteristic feature of AD brains and are predominantly composed of a short peptide fragment termed ABeta, which is derived from the amyloid precursor protein (APP) by proteolytic cleavage. One prevailing hypothesis proposes that ABeta production is the causal event leading to AD. Two proteases are responsible for the generation of ABeta from APP, beta-secretase (BACE1) and gamma-secretase. BACE1 is an attractive therapeutic target because preventing its cleavage of APP has been shown to decrease ABeta production, which should stop or even reverse the course of AD. Recent studies identified the presence of mRNA for several genetically distinct BACE1 isoforms in vivo, but the activity of these different enzymes and their functions in vivo remain unclear. We will discuss our efforts to produce and characterize the BACE1B isoform. Methods: We will report on the use an of an insect expression system to produce the recombinant BACE1B isoform and its characterization utilizing fluorescence-based assays. Results: Recombinant BACE1B can be expressed and purified. BACE1B has measurable activity, albeit less than the full length isoform, BACE1A. Conclusions: The alternativelyspliced BACE1B isoform may have in vivo roles distinct from BACE1A. P3-333
A NOVEL RECIPROCAL AND BIPHASIC RELATIONSHIP BETWEEN MEMBRANE CHOLESTEROL AND -SECRETASE ACTIVITY IN SH-SY5Y CELLS AND HUMAN PLATELETS
Janet A. Johnston, WeiWei Liu, Stephen Todd, David T. R. Coulson, G. Brent Irvine, Bernadette McGuinness, David Craig, Maeve McConville, A. Peter Passmore, Queen’s University, Belfast, United Kingdom. Contact e-mail: [email protected] Background: Research into Alzheimer’s disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters modulate amyloid precursor protein (APP) processing, altering production of A. APP processing depends on the encounter between APP, and -and ␥-secretases, and is thus influenced by cholesterol-dependent factors including protein trafficking, and distribution between membrane sub-domains. To overcome these confounding factors, we have directly investigated endogenous membrane -secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y cells and human platelets. Methods: SH-SY5Y cells were exposed to either methyl--cyclodextrin (MCD); exogenous cholesterol (complexed with MCD); or cell-permeable -secretase inhibitor (Calbiochem). Blood samples were obtained from human subjects in accordance with local ethical committee procedures, and platelets prepared1,2. SH-SY5Y
T619
cell or platelets were lysed (25 mM HEPES, pH 7.2), homogenised and centrifuged (100,000 g, 1 h). Pellets were suspended in membrane solubilisation buffer (25 mM HEPES, 2% CHAPS), centrifuged (20,000 g, 10 min). The supernatant (membrane fraction) was assayed for -secretase activity as described1,2 using a wild-type APP fluorogenic substrate, MCA-EVKMDAEFK-(DNP)-NH2 (Calbiochem), initial rates calculated over the 8-25 min linear phase, and expressed as pmoles MCA detected/min/g membrane protein. Membrane fraction cholesterol levels were determined by Amplex Red Cholesterol Assay (Invitrogen), and results expressed as pmol cholesterol detected/g membrane protein. BACE1 levels were assayed by ELISA, and results expressed as ng BACE1/g membrane protein. Results: SH-SY5Y membrane cholesterol significantly influenced membrane -secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. The same pattern was identified in human platelets. Interestingly, platelets from cognitively-impaired individuals (AD or milder cognitive impairment) were significantly more likely to lie within the negative correlation zone than controls. Pharmacological inhibition of SH-SY5Y -secretase activity resulted in increased membrane cholesterol levels. Conclusions: Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane -secretase activity, and suggest that this regulatory mechanism is disrupted in the platelets of cognitively-impaired individuals. 1. Johnston, J.A. et al., Neurobiol of Aging, doi:10.1016/j.neurobiolaging.2006.11.003. 2. Liu, W.W. et al., Dement Geriatr Cogn Disord. (2007) 24:464-468. P3-334
␥-SECRETASE–MEDIATED PROTEOLYSIS OF APP AND NOTCH IS REGULATED BY CAVEOLIN-1
Arun Kapoor1,2, Bo-Jeng Wang1, Yung-Feng Liao1, 1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei,11529, Taiwan; 2Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, 11529, Taiwan. Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan. Contact e-mail: [email protected] Background: The deposition of neurotoxic amyloid- (A) peptides in brain leads to the development of Alzheimer’s disease (AD). These 3943-amino acid A peptides are generated by the sequential proteolysis of amyloid precursor protein (APP) by - and ␥-secretases. Accumulated evidence has suggested that cholesterol- and sphingolipid-rich membrane microdomains, such as lipid rafts, are involved in regulating the secretasemediated proteolysis of APP. Caveolin-1, a cholesterol-binding membrane protein and an essential constituent of caveolae, has been shown to directly interact with APP and -secretase. Its expression in the hippocampus of autopsy-confirmed subjects of AD is upregulated. Furthermore, ␥-secretase components have been shown to be co-localized with cholesterol-rich lipid rafts. We thus hypothesized that caveolin-1 could play an active role in governing ␥-secretase-mediated proteolysis of APP. Methods: We used substrate-specific cell-based assays to measure ␥-secretase activity and RNAi approach to suppress the expression of caveolin-1. We also used confocal microscopy to study the colocalization of caveolin-1 with ␥-secretase subunits presenilin-1 (PS1) and nicastrin (NCT). ␥-Secretase mediated S3 cleavage of Notch was studied using cell lines stably expressing N⌬E and transient transfection of Hes-1 luciferase reporter constructs. Results: We demonstrated that the suppression of caveolin-1 expression by RNAi dramatically augments ␥-secretase-mediated cleavage of APP and production of A40. Consistent with these findings, the down-regulation of caveolin-1 also led to an increased S3 cleavage of Notch by ␥-secretase. The expression and maturation of ␥-secretase constituents, such as presenilin-1 (PS1) and nicastrin (NCT), were not altered by the knock-down of