- Email: [email protected]
BINDING OF THE AB43 PEPTIDE TO APOLIPOPROTEIN E AND ITS ROLE IN CLEARANCE
P346
Poster Presentations: P1
previously associated with AD including tau, apolipoprotein E, complement C1q, neuroligin and heat shock proteins. Studies are ongoing to determine which proteins have altered expression in AD tissue from patients at early versus late stages of disease and tissue from normal age-matched controls. Conclusions: The combined use of laser capture microdissection and LCMS to characterize proteins in specific brain structures or cell types has great promise for future studies. Future results using this technique will complement those from GWAS studies, potentially identifying protein pathways uniquely expressed or altered in AD. The function of these identified proteins and consequences of expression in AD can then be pursued in further research and could provide novel therapeutic targets. P1-124
BINDING OF THE AB43 PEPTIDE TO APOLIPOPROTEIN E AND ITS ROLE IN CLEARANCE
Elisanne A.L.M. Biemans1, H. Bea Kuiperij1, Rob M.W. De Waal2, Marcel M. Verbeek1, 1Department of Neurology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; 2Department of Pathology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands. Contact e-mail: elisanne.biemans@ radboudumc.nl Background: A range of Ab peptides that vary in length have been found in the human brain with Ab40 and Ab42 being the most studied peptides. However, recently the Ab43 peptide was found to be more abundant than previously thought, and might play a role in AD development. Interestingly, it has also been described that Ab43 does not accumulate in CAA. We hypothesize this is due to an altered blood-brain barrier clearance compared to the shorter Ab peptides. Also, the role of apolipoprotein E in this clearance process has been debated for many years. Therefore, in this study, we aim to investigate the binding capacity of the Ab43 peptide to the apolipoprotein E isoforms and its possible role in clearance compared with the shorter Ab peptides. Methods: ApoE2, ApoE3 and ApoE4 were incubated with Ab40, Ab42 and Ab43 to form complexes. The formation of complexes was analyzed with techniques such as SDS-PAGE and Western Blot. In addition, the interaction between complexes and cerebrovascular cells will be determined using several viability assays. Results: First results, based on immunoblot quantification, show an Ab peptide dependent binding affinity for ApoE2 and ApoE3 with Ab40 > Ab42 >> Ab43. For ApoE4, complex formation was low for all Ab peptides. Conclusions: We have found indications that Ab43 has a lower binding affinity to ApoE when compared to shorter Ab peptides. Given that Ab43 does not accumulate in CAA, future functional studies will have to be performed to confirm the hypothesis that this reduced binding leads to altered clearance. We are currently validating our binding capacity experiments with other techniques such as iSPR. P1-125
DETERMINING THE OLIGOMERIC STATE OF THE BETA-SITE APP-CLEAVING ENZYME 1 (BACE1) IN NATURAL MEMBRANES AND DETERGENTS
Filip Liebsch, Gerhard Multhaup, McGill University, Montreal, Quebec, Canada. Contact e-mail: [email protected] Background: The beta-site APP-cleaving enzyme 1 (BACE1) has a transmembrane sequence (TMS), which is necessary for effective BACE1 cleavage of the amyloid precursor protein (APP). An uncommon sulfur-rich motif, MxxxCxxxMxxxCxMxC, spans the entire TMS of BACE1. The sequence is highly conserved among homologues and is reminiscent of a high-affinity binding site for Cu(I) found in other copper-transporting proteins. Methods: We designed model peptides of the BACE1-TMS to investigate metal-ion binding and oligomerization uncoupled from the cytoplasmic and the ectodomain. A set of biophysical and colorimetric methods was used to investigate peptide-metal ion complex formation. The role of the metal-ion binding motif in potentially pre-existing oligomers of full-length BACE1 was assessed by F€orster resonance energy transfer, automated single-molecule fluorescence counting in living cells, as well
as by blue-native and SDS-gel electrophoresis. Results: We found that the sulfur-rich core motif MxxxCxxxM is involved in metal-ion coordination and oligomerization of BACE1. Addition of Cu(II) facilitated the formation of dimers and trimers of the BACE1 TMS. Importantly, the peptide undergoes a redox reaction with copper ions, resulting in a disulfide bridge involving Cys466 in the center of the conserved MxxxCxxxM motif as the key amino acid. Further, we find peptide trimerization to depend on (i) the presence of monovalent copper ions and (ii) the sulfhydryl group of Cys466.For the full-length protein, FLIM-FRET experiments revealed that BACE1 oligomers are naturally present in living cells, as the oligomeric state of BACE1 remained unchanged in the absence or presence of metalions.We determined a stable trimeric assembly of BACE1 in the plasma membrane by accurate single-molecule fluorescence counting. Although the oligomeric state of full-length BACE1 was not altered by the addition of copper in living cells, the addition of monovalent metal-ions was required to visualize di- and oligomers by Western blot analysis. Conclusions: We propose that BACE1 acts as a bona fide metalloprotein in an oligomeric form in vivo. Additionally, our results demonstrate a novel metal-ion controlled stabilization mediated by the TMS of the BACE1. P1-126
L-NAME REVERSES MORPHINE-INDUCED SPATIAL MEMORY CONSOLIDATION IMPAIRMENT
Golnaz Yadollahi Khales, Abolfazl Alipoor, Maryam Moosavi, Shiraz University of Medical Sciences, Shiraz, Iran. Contact e-mail: [email protected] Background: Memory impairment is among the early and debilitating features of Alzheimer’s disease. Memory is considered to have different phases: acquisition, consolidation and retrieval. The major structure involving in learning and memory is the hippocampus. Morris water maze is a test assessing spatial memory as a hippocampal function. Morphine is reported to disrupt spatial memory consolidation phase. It has been shown that there is a relationship between morphine and nitric oxide (NO). NO is synthesized when L-Arginine is transformed to L-citrullin via nitric oxide synthase (NOS) activity. Morphine is assumed to activate nNOS in CNS by affecting m receptors; which leads to production of NO. This study was aimed to assess if NO is involved in spatial memory consolidation impairment. Methods: Adult male Sprague-Dawely rats weighing 230-270 g were trained in a single session consisting of 8 trials. The probe test was done 24 hours later to assess memory retention. To assess the effect of LNAME (3mg/kg/ip) on memory consolidation impairment induced by morphine (10mg/kg/SQ), Morphine was injected immediately after training. In the groups receiving L-NAME, it was administered simultaneously with morphine. Results: The results showed that post-training administration of L-NAME reversed the deteriorating effects of morphine on memory consolidation. This was due to the observation that trained rats spent more percentage of time in target zone at probe trial compared with the rats given morphine alone. As co-administration of L-NAME and morphine prevents morphine effect on memory consolidation, it seems that NO plays a role in morphine inducing memory consolidation defect. Conclusions: This study might indicate that morphine disrupts memory consolidation process in a NO dependent manner. This helps us in knowing more about neuromodulators and their roles in learning and memory. P1-127
THE INFLUENCE OF CEREBROSPINAL FLUID (CSF) BIOMARKERS ON CLINICAL DEMENTIA EVALUATIONS
Jonathan Gooblar1, Brian D. Carpenter1, Mary A. Coats1, John C. Morris1, B. Joy Snider2, 1Washington University in St. Louis, St. Louis, Missouri, United States; 2Washington University School of Medicine, Saint Louis, Missouri, United States. Contact e-mail: [email protected] Background: Cerebrospinal fluid (CSF) protein values of amyloid-b (Ab 42), tau, and phosphorylated tau 181 (ptau 181) are becoming accepted biomarkers for Alzheimer’s disease (AD) pathology in research settings. The extent of their use and perceived utility in clinical settings, however, is less well studied, and it is unclear how these biomarkers influence clinicians
Poster Presentations: P1
previously associated with AD including tau, apolipoprotein E, complement C1q, neuroligin and heat shock proteins. Studies are ongoing to determine which proteins have altered expression in AD tissue from patients at early versus late stages of disease and tissue from normal age-matched controls. Conclusions: The combined use of laser capture microdissection and LCMS to characterize proteins in specific brain structures or cell types has great promise for future studies. Future results using this technique will complement those from GWAS studies, potentially identifying protein pathways uniquely expressed or altered in AD. The function of these identified proteins and consequences of expression in AD can then be pursued in further research and could provide novel therapeutic targets. P1-124
BINDING OF THE AB43 PEPTIDE TO APOLIPOPROTEIN E AND ITS ROLE IN CLEARANCE
Elisanne A.L.M. Biemans1, H. Bea Kuiperij1, Rob M.W. De Waal2, Marcel M. Verbeek1, 1Department of Neurology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands; 2Department of Pathology, Donders Centre for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands. Contact e-mail: elisanne.biemans@ radboudumc.nl Background: A range of Ab peptides that vary in length have been found in the human brain with Ab40 and Ab42 being the most studied peptides. However, recently the Ab43 peptide was found to be more abundant than previously thought, and might play a role in AD development. Interestingly, it has also been described that Ab43 does not accumulate in CAA. We hypothesize this is due to an altered blood-brain barrier clearance compared to the shorter Ab peptides. Also, the role of apolipoprotein E in this clearance process has been debated for many years. Therefore, in this study, we aim to investigate the binding capacity of the Ab43 peptide to the apolipoprotein E isoforms and its possible role in clearance compared with the shorter Ab peptides. Methods: ApoE2, ApoE3 and ApoE4 were incubated with Ab40, Ab42 and Ab43 to form complexes. The formation of complexes was analyzed with techniques such as SDS-PAGE and Western Blot. In addition, the interaction between complexes and cerebrovascular cells will be determined using several viability assays. Results: First results, based on immunoblot quantification, show an Ab peptide dependent binding affinity for ApoE2 and ApoE3 with Ab40 > Ab42 >> Ab43. For ApoE4, complex formation was low for all Ab peptides. Conclusions: We have found indications that Ab43 has a lower binding affinity to ApoE when compared to shorter Ab peptides. Given that Ab43 does not accumulate in CAA, future functional studies will have to be performed to confirm the hypothesis that this reduced binding leads to altered clearance. We are currently validating our binding capacity experiments with other techniques such as iSPR. P1-125
DETERMINING THE OLIGOMERIC STATE OF THE BETA-SITE APP-CLEAVING ENZYME 1 (BACE1) IN NATURAL MEMBRANES AND DETERGENTS
Filip Liebsch, Gerhard Multhaup, McGill University, Montreal, Quebec, Canada. Contact e-mail: [email protected] Background: The beta-site APP-cleaving enzyme 1 (BACE1) has a transmembrane sequence (TMS), which is necessary for effective BACE1 cleavage of the amyloid precursor protein (APP). An uncommon sulfur-rich motif, MxxxCxxxMxxxCxMxC, spans the entire TMS of BACE1. The sequence is highly conserved among homologues and is reminiscent of a high-affinity binding site for Cu(I) found in other copper-transporting proteins. Methods: We designed model peptides of the BACE1-TMS to investigate metal-ion binding and oligomerization uncoupled from the cytoplasmic and the ectodomain. A set of biophysical and colorimetric methods was used to investigate peptide-metal ion complex formation. The role of the metal-ion binding motif in potentially pre-existing oligomers of full-length BACE1 was assessed by F€orster resonance energy transfer, automated single-molecule fluorescence counting in living cells, as well
as by blue-native and SDS-gel electrophoresis. Results: We found that the sulfur-rich core motif MxxxCxxxM is involved in metal-ion coordination and oligomerization of BACE1. Addition of Cu(II) facilitated the formation of dimers and trimers of the BACE1 TMS. Importantly, the peptide undergoes a redox reaction with copper ions, resulting in a disulfide bridge involving Cys466 in the center of the conserved MxxxCxxxM motif as the key amino acid. Further, we find peptide trimerization to depend on (i) the presence of monovalent copper ions and (ii) the sulfhydryl group of Cys466.For the full-length protein, FLIM-FRET experiments revealed that BACE1 oligomers are naturally present in living cells, as the oligomeric state of BACE1 remained unchanged in the absence or presence of metalions.We determined a stable trimeric assembly of BACE1 in the plasma membrane by accurate single-molecule fluorescence counting. Although the oligomeric state of full-length BACE1 was not altered by the addition of copper in living cells, the addition of monovalent metal-ions was required to visualize di- and oligomers by Western blot analysis. Conclusions: We propose that BACE1 acts as a bona fide metalloprotein in an oligomeric form in vivo. Additionally, our results demonstrate a novel metal-ion controlled stabilization mediated by the TMS of the BACE1. P1-126
L-NAME REVERSES MORPHINE-INDUCED SPATIAL MEMORY CONSOLIDATION IMPAIRMENT
Golnaz Yadollahi Khales, Abolfazl Alipoor, Maryam Moosavi, Shiraz University of Medical Sciences, Shiraz, Iran. Contact e-mail: [email protected] Background: Memory impairment is among the early and debilitating features of Alzheimer’s disease. Memory is considered to have different phases: acquisition, consolidation and retrieval. The major structure involving in learning and memory is the hippocampus. Morris water maze is a test assessing spatial memory as a hippocampal function. Morphine is reported to disrupt spatial memory consolidation phase. It has been shown that there is a relationship between morphine and nitric oxide (NO). NO is synthesized when L-Arginine is transformed to L-citrullin via nitric oxide synthase (NOS) activity. Morphine is assumed to activate nNOS in CNS by affecting m receptors; which leads to production of NO. This study was aimed to assess if NO is involved in spatial memory consolidation impairment. Methods: Adult male Sprague-Dawely rats weighing 230-270 g were trained in a single session consisting of 8 trials. The probe test was done 24 hours later to assess memory retention. To assess the effect of LNAME (3mg/kg/ip) on memory consolidation impairment induced by morphine (10mg/kg/SQ), Morphine was injected immediately after training. In the groups receiving L-NAME, it was administered simultaneously with morphine. Results: The results showed that post-training administration of L-NAME reversed the deteriorating effects of morphine on memory consolidation. This was due to the observation that trained rats spent more percentage of time in target zone at probe trial compared with the rats given morphine alone. As co-administration of L-NAME and morphine prevents morphine effect on memory consolidation, it seems that NO plays a role in morphine inducing memory consolidation defect. Conclusions: This study might indicate that morphine disrupts memory consolidation process in a NO dependent manner. This helps us in knowing more about neuromodulators and their roles in learning and memory. P1-127
THE INFLUENCE OF CEREBROSPINAL FLUID (CSF) BIOMARKERS ON CLINICAL DEMENTIA EVALUATIONS
Jonathan Gooblar1, Brian D. Carpenter1, Mary A. Coats1, John C. Morris1, B. Joy Snider2, 1Washington University in St. Louis, St. Louis, Missouri, United States; 2Washington University School of Medicine, Saint Louis, Missouri, United States. Contact e-mail: [email protected] Background: Cerebrospinal fluid (CSF) protein values of amyloid-b (Ab 42), tau, and phosphorylated tau 181 (ptau 181) are becoming accepted biomarkers for Alzheimer’s disease (AD) pathology in research settings. The extent of their use and perceived utility in clinical settings, however, is less well studied, and it is unclear how these biomarkers influence clinicians