- Email: [email protected]
P1-225 X11L and Alcadein form a tripartite comprex with APP and suppress the γ-cleavage of CTFβ
S160
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Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis COMBINING FUNCTIONAL IMAGING WITH MICROARRAY: A NOVEL
MEMBRANE-TRAFFICKING PATHWAY IMPLICATED IN ALZHEIMER'S DISEASE Scott Small*. Columbia University, New York, NY USA. Contact e-mail: sas68 @columbia.edu
Background: The exploratory power of microarray- the ability to compare the expression levels of thousands of genes between healthy and diseased tissue- is also its main analytic limitation. Here we confront this problem by relying on functional imaging to generate a hypothesis-driven model on how a molecule involved in AD pathogenesis should behave - both spatially and temporally. Objectives: Functional imaging studies have confirmed histological findings, showing that the entorhinal cortex is the hippocampal subregion most sensitive to AD, while the dentate gyms is the subregion most resistant to disease pathogenesis. Accordingly, the first assumption in our model was that when comparing AD and control brains a pathogenic molecule should be differentially expressed in the entorhinal cortex. Imaging studies have further shown that while the function of the entorhinal cortex is uniformly diminished in AD patients, entorhinal function is stable across the age-span among healthy controls. Our model's second assumption, therefore, was that a pathogenic molecule should behave over time in parallel with this pattern. Methods: Both the entorhinal cortex and the dentate gyms were isolated from brains with pathologically proven AD, and from controls that ranged the age-span. A separate microarray analysis was performed on each tissue sample for a total of 24 studies. The microarray dataset was then analyzed statistically in accordance with the spatio-temporal model. All results were confirmed with RT-PCR and immunocytochemistry. Results: An increase in the expression of a trafficking molecule, VPS35, best conformed to the model, discriminating sporadic AD from controls. VPS35 is the core component of a sorting complex that traffÉcs type I membrane-proteins, and acts to increase the concentration of cargo proteins in the endosome and trans-golgi network. To confirm that VPS35 is relevant to AD pathophysiology, we generated small interference RNA (siRNA) directed against human VPS35. When injected into 293 cells expressing human APP695 we observed a dose dependent suppression of A1340 and A[~42 secretion. Conclusions: Our results implicate an unexplored membrane-trafficking pathway associated with sporadic AD. We are currently investigating how VPS35 overexpression increases A[3 production.
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X l l L A N D A L C A D E I N F O R M A TRIPARTITE
COMPREX WITH APP AND SUPPRESS THE y-CLEAVAGE O F CTF[~ Naomi Miyagi .1 , Megumi Sakuma 1, Yoichi Araki 1,2, Toshiharu Suzuld 1.
1Hokkaido university, Sapporo, Japan; 2University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected]
Background: We isolated Xll-like(XllL) as an APP-binding protein. It stabilizes intracellular APP metabolism mad suppresses the secretion of AlL We found that X l l L also interacts with Alcadein (Ale), a novel membrane protein containing extracellular cadherin motifs. X11L needs its PI damain to interact with APP and Ale. Unlikely it is usually documented, X11L could simultaneously associate with APP and Ale through its PI domain and form a tripartite complex. The tripartite complex formation enhanced the XllL-dependent stabilization of intracellular APP metabolism and suppression of A[~ secretion. Objective: To elucidate this mechanism, we investigated whether APP and Ale need the same region in PI domain of X l l L when they interact with it. We also investigated that X l l L and Alemediated suppression of A[3 generation is due to inhibition of y-cleavage of CTF[L Method: We prepared the various constructs of X l l L that result in lower binding-affinity to APP. To analyze the cleavage at y-site, we used APP-C99(CTF[3). HEK293cells were transfected with various combinations of the plasmids. To check the interaction of proteins, we performed coimmunoprecipitation. The immunoprecipitates were then subjected to Western blot analysis. A[~ was quantified with sandwich ELISA by using specific monoclonal antibodies. Result: APP and Ale need different region
in the PI domein of X11L. We also found that C99 formed tripartite complex with X l l L and Ale, as did APP. The expression of X l l L suppressed the secretion of A~ derived from C99. When Ale was also coexpressed, this effect was remarkably enhanced. Moreover, we found that X11L inhibited the interaction of PS with C99, which caused strong suppression of the y-cleavage of C99. This X11L-dependent effect of y-cleavage inhibition was enhanced by Ale. Conclusion: We suggested that APP and Ale recognize the different regions for interaction with the PI domein of X11L, resulting in formation of tripartite complex. X l l L could suppress the y-cleavage of CTF, and Ale enhances this effect. Our finding shed light on molecular mechanism how X11L and Ale regulate APP metabolism in neuron.
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MAPPING ABETA AMYLOID TOPOLOGY USING
CYSTEINE MUTANTS Shankaramma Shivaprasad*, Ronald Wetzel. UTMedical Center, Knoxville, TN, USA. Contact e-mail: [email protected]
Background: Amyloid fibrils are filamentous aggregates known to be rich in a "cross-[3" structural motif in which [~-strand segments are oriented perpendicular to the long fibril axis, while the hydrogen bonds between the strands are oriented parallel with this axis. To test our working model for how the A~ peptide folds in this amyloid motif, we are inquiring into long-range interactions within A[~ molecules in fibrils. Objective(s): We designed three double cysteine-mutants of A[~(1-40) to determine which residues in the packed fibril structure are in close proximity. Previous work revealed that single cysteine replacements at residues 17, 34 and 36 are not accessible when fibrils are treated with an alkylating reagent, indicating that these residues are buried, possibly in the hydrophobic interior; in contrast, a cysteine replacement at residue 35 is accessible to modification, and hence exposed to solvent. Double cysteine mutants were therefore prepared, wherein one cysteine was introduced at position 17 in all the three mutants, and the other at positions 34, 35 or 36. Methods: In one set of experiments, the mutant fibrils (grown under reducing conditions) were subjected to oxidative cross-linking analysis. In another set, the mutant peptides were oxidized at the monomer level and followed fibril formation reaction, including determining the thermodynamics of fibril formation. To do this, the soluble portion of the peptide was measured after fibril formation reaction came to equilibrium. Results and Conclusions: Of the three mutants studied, only fibrils made from the reduced L17C/34C mutant gave efficient disulfide cross-linking on oxidation. Further, although all three oxidized monomers can make amyloid fibrils, oxidation improved fibril stability, with respect to the reduced peptide, most for the L17C/34C mutant. While more needs to be done to determine whether these cysteine mutant fibrils are wild-type like in their properties, earlier indications are that the side chains of residues 17 and 34 are close in space, and facing each other, in the WT A[3 fibril.
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GENE EXPRESSION PROFILES OF TRANSCRIPTS IN A M Y L O I D P R E C U R S O R P R O T E I N T R A N S G E N I C MICE: UP-REGULATION OF MITOCHONDRIAL M E T A B O L I S M AND APOPTOTIC GENES IS AN EARLY C E L L U L A R C H A N G E IN A L Z H E I M E R ' S
DISEASE P. Hemachandra Reddy* 1, Shannon McWeeney 2, Byung Park 2, Maria Manczak I , Ramana V. Gutala I , Youngsin Jung 1, Vincent Yau 2, Robert Searles 1, Motomi Mori 2, Quinn Joseph 2. 1Oregon Health and
Science University, Beaverton, OR, USA; 2Oregon Health and Science University, Portland, OR, USA. Contact e-mail: [email protected]
Background: The pathogenesis of Alzheimer's disease (AD) has not been established with certainty, but abnormalities in the processing of the amyloid precursor protein (APP) have been implicated. While abnormal processing of APP is hypothesized to play a role in AD pathogenesis, the early cellular changes related to AD progression are still unknown. Objective: Our objective was to determine genes that are critical for early cellular changes in AD progression. Methods: We investigated an established APP transgenic
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Poster Session P I : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis COMBINING FUNCTIONAL IMAGING WITH MICROARRAY: A NOVEL
MEMBRANE-TRAFFICKING PATHWAY IMPLICATED IN ALZHEIMER'S DISEASE Scott Small*. Columbia University, New York, NY USA. Contact e-mail: sas68 @columbia.edu
Background: The exploratory power of microarray- the ability to compare the expression levels of thousands of genes between healthy and diseased tissue- is also its main analytic limitation. Here we confront this problem by relying on functional imaging to generate a hypothesis-driven model on how a molecule involved in AD pathogenesis should behave - both spatially and temporally. Objectives: Functional imaging studies have confirmed histological findings, showing that the entorhinal cortex is the hippocampal subregion most sensitive to AD, while the dentate gyms is the subregion most resistant to disease pathogenesis. Accordingly, the first assumption in our model was that when comparing AD and control brains a pathogenic molecule should be differentially expressed in the entorhinal cortex. Imaging studies have further shown that while the function of the entorhinal cortex is uniformly diminished in AD patients, entorhinal function is stable across the age-span among healthy controls. Our model's second assumption, therefore, was that a pathogenic molecule should behave over time in parallel with this pattern. Methods: Both the entorhinal cortex and the dentate gyms were isolated from brains with pathologically proven AD, and from controls that ranged the age-span. A separate microarray analysis was performed on each tissue sample for a total of 24 studies. The microarray dataset was then analyzed statistically in accordance with the spatio-temporal model. All results were confirmed with RT-PCR and immunocytochemistry. Results: An increase in the expression of a trafficking molecule, VPS35, best conformed to the model, discriminating sporadic AD from controls. VPS35 is the core component of a sorting complex that traffÉcs type I membrane-proteins, and acts to increase the concentration of cargo proteins in the endosome and trans-golgi network. To confirm that VPS35 is relevant to AD pathophysiology, we generated small interference RNA (siRNA) directed against human VPS35. When injected into 293 cells expressing human APP695 we observed a dose dependent suppression of A1340 and A[~42 secretion. Conclusions: Our results implicate an unexplored membrane-trafficking pathway associated with sporadic AD. We are currently investigating how VPS35 overexpression increases A[3 production.
~
X l l L A N D A L C A D E I N F O R M A TRIPARTITE
COMPREX WITH APP AND SUPPRESS THE y-CLEAVAGE O F CTF[~ Naomi Miyagi .1 , Megumi Sakuma 1, Yoichi Araki 1,2, Toshiharu Suzuld 1.
1Hokkaido university, Sapporo, Japan; 2University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected]
Background: We isolated Xll-like(XllL) as an APP-binding protein. It stabilizes intracellular APP metabolism mad suppresses the secretion of AlL We found that X l l L also interacts with Alcadein (Ale), a novel membrane protein containing extracellular cadherin motifs. X11L needs its PI damain to interact with APP and Ale. Unlikely it is usually documented, X11L could simultaneously associate with APP and Ale through its PI domain and form a tripartite complex. The tripartite complex formation enhanced the XllL-dependent stabilization of intracellular APP metabolism and suppression of A[~ secretion. Objective: To elucidate this mechanism, we investigated whether APP and Ale need the same region in PI domain of X l l L when they interact with it. We also investigated that X l l L and Alemediated suppression of A[3 generation is due to inhibition of y-cleavage of CTF[L Method: We prepared the various constructs of X l l L that result in lower binding-affinity to APP. To analyze the cleavage at y-site, we used APP-C99(CTF[3). HEK293cells were transfected with various combinations of the plasmids. To check the interaction of proteins, we performed coimmunoprecipitation. The immunoprecipitates were then subjected to Western blot analysis. A[~ was quantified with sandwich ELISA by using specific monoclonal antibodies. Result: APP and Ale need different region
in the PI domein of X11L. We also found that C99 formed tripartite complex with X l l L and Ale, as did APP. The expression of X l l L suppressed the secretion of A~ derived from C99. When Ale was also coexpressed, this effect was remarkably enhanced. Moreover, we found that X11L inhibited the interaction of PS with C99, which caused strong suppression of the y-cleavage of C99. This X11L-dependent effect of y-cleavage inhibition was enhanced by Ale. Conclusion: We suggested that APP and Ale recognize the different regions for interaction with the PI domein of X11L, resulting in formation of tripartite complex. X l l L could suppress the y-cleavage of CTF, and Ale enhances this effect. Our finding shed light on molecular mechanism how X11L and Ale regulate APP metabolism in neuron.
•
MAPPING ABETA AMYLOID TOPOLOGY USING
CYSTEINE MUTANTS Shankaramma Shivaprasad*, Ronald Wetzel. UTMedical Center, Knoxville, TN, USA. Contact e-mail: [email protected]
Background: Amyloid fibrils are filamentous aggregates known to be rich in a "cross-[3" structural motif in which [~-strand segments are oriented perpendicular to the long fibril axis, while the hydrogen bonds between the strands are oriented parallel with this axis. To test our working model for how the A~ peptide folds in this amyloid motif, we are inquiring into long-range interactions within A[~ molecules in fibrils. Objective(s): We designed three double cysteine-mutants of A[~(1-40) to determine which residues in the packed fibril structure are in close proximity. Previous work revealed that single cysteine replacements at residues 17, 34 and 36 are not accessible when fibrils are treated with an alkylating reagent, indicating that these residues are buried, possibly in the hydrophobic interior; in contrast, a cysteine replacement at residue 35 is accessible to modification, and hence exposed to solvent. Double cysteine mutants were therefore prepared, wherein one cysteine was introduced at position 17 in all the three mutants, and the other at positions 34, 35 or 36. Methods: In one set of experiments, the mutant fibrils (grown under reducing conditions) were subjected to oxidative cross-linking analysis. In another set, the mutant peptides were oxidized at the monomer level and followed fibril formation reaction, including determining the thermodynamics of fibril formation. To do this, the soluble portion of the peptide was measured after fibril formation reaction came to equilibrium. Results and Conclusions: Of the three mutants studied, only fibrils made from the reduced L17C/34C mutant gave efficient disulfide cross-linking on oxidation. Further, although all three oxidized monomers can make amyloid fibrils, oxidation improved fibril stability, with respect to the reduced peptide, most for the L17C/34C mutant. While more needs to be done to determine whether these cysteine mutant fibrils are wild-type like in their properties, earlier indications are that the side chains of residues 17 and 34 are close in space, and facing each other, in the WT A[3 fibril.
~ - ~
GENE EXPRESSION PROFILES OF TRANSCRIPTS IN A M Y L O I D P R E C U R S O R P R O T E I N T R A N S G E N I C MICE: UP-REGULATION OF MITOCHONDRIAL M E T A B O L I S M AND APOPTOTIC GENES IS AN EARLY C E L L U L A R C H A N G E IN A L Z H E I M E R ' S
DISEASE P. Hemachandra Reddy* 1, Shannon McWeeney 2, Byung Park 2, Maria Manczak I , Ramana V. Gutala I , Youngsin Jung 1, Vincent Yau 2, Robert Searles 1, Motomi Mori 2, Quinn Joseph 2. 1Oregon Health and
Science University, Beaverton, OR, USA; 2Oregon Health and Science University, Portland, OR, USA. Contact e-mail: [email protected]
Background: The pathogenesis of Alzheimer's disease (AD) has not been established with certainty, but abnormalities in the processing of the amyloid precursor protein (APP) have been implicated. While abnormal processing of APP is hypothesized to play a role in AD pathogenesis, the early cellular changes related to AD progression are still unknown. Objective: Our objective was to determine genes that are critical for early cellular changes in AD progression. Methods: We investigated an established APP transgenic