- Email: [email protected]
P3-256
S450 P3-254
Poster P3:: Tuesday Posters SAP97 SCAFFOLDING PROTEIN MEDIATES ALPHA-SECRETASE ADAM10 TRAFFICKING AND DIRECTLY PROMOTES ITS ACTIVITY BOTH IN VITRO AND IN VIVO
Elena Marcello1, Fabrizio Gardoni1, Daniela Mauceri1, Barbara Borroni2, Alessandro Padovani2, Monica Di Luca1, 1University of Milano, Milano, Italy; 2Department of Neurological Sciences, University of Brescia, Brescia, Italy. Contact e-mail: [email protected] Alzheimer disease (AD) is a chronic neurodegenerative disorder caused by a combination of events impairing normal neuronal function. Here we found a molecular bridge between key elements of primary and secondary pathogenic events in AD, namely the elements of the amyloid cascade and synaptic dysfunction associated with the glutamatergic system. In fact, we report that SAP97, a protein involved in dynamic trafficking of proteins to the excitatory synapse, is responsible for driving ADAM10 (the most accredited candidate for a-secretase) to the postsynaptic membrane, by a direct interaction through its SH3 domain. NMDA receptor activation mediates this event and positively modulates a-secretase activity. Furthermore, perturbing ADAM10/SAP97 association in vivo by cell-permeable peptides impairs ADAM10 localization in postsynaptic membranes, and consequently decreases the physiological APP metabolism. Our findings indicate that glutamatergic synapse activation through NMDA receptor promotes the non-amyloidogenic APP cleavage, strengthening the correlation between APP metabolism and synaptic plasticity. P3-255
-SECRETASE ACTIVITY IN HUMAN PLATELETS
Wei Wei Liu, David T.R. Coulson, Stephen A. Todd, Suzanne Murphy, G. Brent Irvine, David Craig, A. Peter Passmore, Janet A. Johnston, Queen’s University Belfast, Belfast, United Kingdom. Contact e-mail: [email protected]
-secretase activity is the rate-limiting step in A peptide production from amyloid precursor protein (APP). Over time, A peptides aggregate and result in the formation of the amyloid plaques that characterise Alzheimer’s disease (AD) neuropathology. Human platelets contain full-length and processed APP. We investigated whether -secretase activity was altered in platelets from individuals with Alzheimer’s disease, compared to controls. Platelets were isolated from blood from 86 AD and 115 age-matched control individuals, lysed (25 mM HEPES, pH 7.2) and fractionated into particulate and soluble fractions by centrifugation at 100,000 g for 1 h. The particulate (membrane) fraction pellet was re-suspended in 25 mM HEPES containing 2% CHAPS. 5 g protein from each platelet membrane sample was assayed for -secretase activity in triplicate using the internally-quenched fluorogenic substrate (MCA)EVKMDAEFK(DNP)-NH2 (Calbiochem) in 50 mM sodium acetate, pH 4.5 at 37°C. Fluorescence was read continuously over 30 min, and initial rates were calculated. We identified a significant 17% increase in platelet membrane fraction -secretase activity in individuals with Alzheimer’s disease compared to controls (p ⬍ 0.0003, unpaired t-test). There was no significant correlation between platelet membrane -secretase activity and Mini Mental State Examination (MMSE) score in the Alzheimer’s disease group (mean MMSE ⫽ 17.7, range 1-23). Elevated -secretase activity in an accessible peripheral tissue is of considerable interest in relation to Alzheimer’s disease pathobiology and diagnosis. The range of -secretase activity detected in different individuals raises important questions about in vivo regulation of this proteolytic activity. The lack of correlation between platelet -secretase activity and MMSE suggests that -secretase activity does not increase as a secondary result of the disease process.
P3-256
A STRUCTURAL MODEL OF A LMP2 POLYMORPHISM, STUDIED IN ALZHEIMER’S DISEASE AND OTHER NEURODEGENERATIVE DISEASES: EXPRESSION, ACTIVITY, INTRACELLULAR LOCALIZATION AND INSIGHT INTO THE REGULATORY MECHANISMS OF 20S IMMUNOPROTEASOME
Michele Mishto, C.I.G., Unibo, Bologna, Italy. Contact e-mail: [email protected] Background: The LMP2 codon 60 polymorphism has been associated with autoimmune diseases and demonstrated to influence susceptibility to TNF-␣ induced apoptosis in blood, and proteasome activity in human aged and Alzheimer’s disease brains. Objective(s): To further investigate in an ad hoc in vitro model the effect of LMP2 codon 60 polymorphism on immunoproteasome. Methods: An in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit and the experimental investigation of immunoproteasome expression, activity and intracellular localization in lymphoblastoid cell lines model was performed. Results: A significant difference in antibody affinity while no major variations in functionality and amount of immunoproteasomes emerged. These data have been integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism, in order to develop a general hypothesis on LMP2 polymorphism effects. Conclusions: We identified three prospective mechanisms, that could explain the biological data on the polymorphism, such as the modulation of the binding affinity of a putative non-catalytic modifier site in the external surface of the immunoproteasome core or the modification of any channel between alpha and beta rings. Because proteasome activity is inhibited in Alzheimer’s disease, further investigation on such a topic can allow an identifying of a possible target for AD (or other neurodegenerative disease) therapy. P3-257
BACE1 CLEAVAGE AND RELEASE OF NEUREGULIN-1 DETERMINES THE ENSHEATHMENT FATE OF AXONS
Michael Willem1, Alistair Garratt2, Linne´a Rabe1, Paul Saftig3, Bart DeStrooper4, Carmen Birchmeier2, Chrsitian Haass1, 1LMU Munich, Munich, Germany; 2Max-Delbru¨ck-Center, Berlin, Germany; 3 University Kiel, Kiel, Germany; 4KULeuven, Leuven, Belgium. Contact e-mail: [email protected] Background: The generation of amyloid peptides (A) from the amyloid precursor protein (APP) depends on sequential cleavage by -secretase and ␥-secretase enzyme activities. Cleavage by ␥-secretase (previously identified as presenilin-1) produces A. BACE1 (-site APP cleaving enzyme), an aspartylprotease, was recently shown to be the major -secretase in the brain, based on the loss of -cleavage in BACE1 KO mice and reduced A levels in mouse brains upon treatment with a BACE1 inhibitor. APLP1, APLP2, PSGL-1, ST6Gal, VGSC subunits and LRP were identified as BACE1 substrates, but the the functional consequence of their processing by BACE1 remains elusive. Recently it has been shown that the EGF-like ligand neuregulin-1 (NRG-1), and its receptors, members of the ErbB receptor tyrosine kinase family are also substrates for regulated intramembrane proteolysis mediated by ␥-secretase. Cleavage of these molecules by BACE1 however has not been previously demonstrated. Genetic studies in mice have demonstrated the multiple essential roles of this signalling system in glial cell and cardiac development. In particular, NRG-1 signalling through ErbB2/ErbB3 receptors is required for early development of neural crest cells, in expansion of the Schwann cell precursor pool and in myelination. Objective: The role of BACE1 in cleavage of the ␥-secretase substrate NRG-1 was studied to determine if BACE1 can influence the release of the active EGF domain. Methods: NRG-1 cleavage was studied in HEK 293 cells and in BACE -/- mice, and myelination of peripheral nerves was analysed by electron microscopy. Results and Conclusion: Biochemical analysis revealed a preferential cleavage of certain NRG-1
Poster P3:: Tuesday Posters SAP97 SCAFFOLDING PROTEIN MEDIATES ALPHA-SECRETASE ADAM10 TRAFFICKING AND DIRECTLY PROMOTES ITS ACTIVITY BOTH IN VITRO AND IN VIVO
Elena Marcello1, Fabrizio Gardoni1, Daniela Mauceri1, Barbara Borroni2, Alessandro Padovani2, Monica Di Luca1, 1University of Milano, Milano, Italy; 2Department of Neurological Sciences, University of Brescia, Brescia, Italy. Contact e-mail: [email protected] Alzheimer disease (AD) is a chronic neurodegenerative disorder caused by a combination of events impairing normal neuronal function. Here we found a molecular bridge between key elements of primary and secondary pathogenic events in AD, namely the elements of the amyloid cascade and synaptic dysfunction associated with the glutamatergic system. In fact, we report that SAP97, a protein involved in dynamic trafficking of proteins to the excitatory synapse, is responsible for driving ADAM10 (the most accredited candidate for a-secretase) to the postsynaptic membrane, by a direct interaction through its SH3 domain. NMDA receptor activation mediates this event and positively modulates a-secretase activity. Furthermore, perturbing ADAM10/SAP97 association in vivo by cell-permeable peptides impairs ADAM10 localization in postsynaptic membranes, and consequently decreases the physiological APP metabolism. Our findings indicate that glutamatergic synapse activation through NMDA receptor promotes the non-amyloidogenic APP cleavage, strengthening the correlation between APP metabolism and synaptic plasticity. P3-255
-SECRETASE ACTIVITY IN HUMAN PLATELETS
Wei Wei Liu, David T.R. Coulson, Stephen A. Todd, Suzanne Murphy, G. Brent Irvine, David Craig, A. Peter Passmore, Janet A. Johnston, Queen’s University Belfast, Belfast, United Kingdom. Contact e-mail: [email protected]
-secretase activity is the rate-limiting step in A peptide production from amyloid precursor protein (APP). Over time, A peptides aggregate and result in the formation of the amyloid plaques that characterise Alzheimer’s disease (AD) neuropathology. Human platelets contain full-length and processed APP. We investigated whether -secretase activity was altered in platelets from individuals with Alzheimer’s disease, compared to controls. Platelets were isolated from blood from 86 AD and 115 age-matched control individuals, lysed (25 mM HEPES, pH 7.2) and fractionated into particulate and soluble fractions by centrifugation at 100,000 g for 1 h. The particulate (membrane) fraction pellet was re-suspended in 25 mM HEPES containing 2% CHAPS. 5 g protein from each platelet membrane sample was assayed for -secretase activity in triplicate using the internally-quenched fluorogenic substrate (MCA)EVKMDAEFK(DNP)-NH2 (Calbiochem) in 50 mM sodium acetate, pH 4.5 at 37°C. Fluorescence was read continuously over 30 min, and initial rates were calculated. We identified a significant 17% increase in platelet membrane fraction -secretase activity in individuals with Alzheimer’s disease compared to controls (p ⬍ 0.0003, unpaired t-test). There was no significant correlation between platelet membrane -secretase activity and Mini Mental State Examination (MMSE) score in the Alzheimer’s disease group (mean MMSE ⫽ 17.7, range 1-23). Elevated -secretase activity in an accessible peripheral tissue is of considerable interest in relation to Alzheimer’s disease pathobiology and diagnosis. The range of -secretase activity detected in different individuals raises important questions about in vivo regulation of this proteolytic activity. The lack of correlation between platelet -secretase activity and MMSE suggests that -secretase activity does not increase as a secondary result of the disease process.
P3-256
A STRUCTURAL MODEL OF A LMP2 POLYMORPHISM, STUDIED IN ALZHEIMER’S DISEASE AND OTHER NEURODEGENERATIVE DISEASES: EXPRESSION, ACTIVITY, INTRACELLULAR LOCALIZATION AND INSIGHT INTO THE REGULATORY MECHANISMS OF 20S IMMUNOPROTEASOME
Michele Mishto, C.I.G., Unibo, Bologna, Italy. Contact e-mail: [email protected] Background: The LMP2 codon 60 polymorphism has been associated with autoimmune diseases and demonstrated to influence susceptibility to TNF-␣ induced apoptosis in blood, and proteasome activity in human aged and Alzheimer’s disease brains. Objective(s): To further investigate in an ad hoc in vitro model the effect of LMP2 codon 60 polymorphism on immunoproteasome. Methods: An in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit and the experimental investigation of immunoproteasome expression, activity and intracellular localization in lymphoblastoid cell lines model was performed. Results: A significant difference in antibody affinity while no major variations in functionality and amount of immunoproteasomes emerged. These data have been integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism, in order to develop a general hypothesis on LMP2 polymorphism effects. Conclusions: We identified three prospective mechanisms, that could explain the biological data on the polymorphism, such as the modulation of the binding affinity of a putative non-catalytic modifier site in the external surface of the immunoproteasome core or the modification of any channel between alpha and beta rings. Because proteasome activity is inhibited in Alzheimer’s disease, further investigation on such a topic can allow an identifying of a possible target for AD (or other neurodegenerative disease) therapy. P3-257
BACE1 CLEAVAGE AND RELEASE OF NEUREGULIN-1 DETERMINES THE ENSHEATHMENT FATE OF AXONS
Michael Willem1, Alistair Garratt2, Linne´a Rabe1, Paul Saftig3, Bart DeStrooper4, Carmen Birchmeier2, Chrsitian Haass1, 1LMU Munich, Munich, Germany; 2Max-Delbru¨ck-Center, Berlin, Germany; 3 University Kiel, Kiel, Germany; 4KULeuven, Leuven, Belgium. Contact e-mail: [email protected] Background: The generation of amyloid peptides (A) from the amyloid precursor protein (APP) depends on sequential cleavage by -secretase and ␥-secretase enzyme activities. Cleavage by ␥-secretase (previously identified as presenilin-1) produces A. BACE1 (-site APP cleaving enzyme), an aspartylprotease, was recently shown to be the major -secretase in the brain, based on the loss of -cleavage in BACE1 KO mice and reduced A levels in mouse brains upon treatment with a BACE1 inhibitor. APLP1, APLP2, PSGL-1, ST6Gal, VGSC subunits and LRP were identified as BACE1 substrates, but the the functional consequence of their processing by BACE1 remains elusive. Recently it has been shown that the EGF-like ligand neuregulin-1 (NRG-1), and its receptors, members of the ErbB receptor tyrosine kinase family are also substrates for regulated intramembrane proteolysis mediated by ␥-secretase. Cleavage of these molecules by BACE1 however has not been previously demonstrated. Genetic studies in mice have demonstrated the multiple essential roles of this signalling system in glial cell and cardiac development. In particular, NRG-1 signalling through ErbB2/ErbB3 receptors is required for early development of neural crest cells, in expansion of the Schwann cell precursor pool and in myelination. Objective: The role of BACE1 in cleavage of the ␥-secretase substrate NRG-1 was studied to determine if BACE1 can influence the release of the active EGF domain. Methods: NRG-1 cleavage was studied in HEK 293 cells and in BACE -/- mice, and myelination of peripheral nerves was analysed by electron microscopy. Results and Conclusion: Biochemical analysis revealed a preferential cleavage of certain NRG-1