Amyloidogenic properties of isoAsp7-containing beta-amyloid peptide

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Sergey Kozin1, Ivan Cheglakov1, Armen Ovsepyan2, George Telegin2,. Philippe .... Stephen Newhouse15, Richard Dobson3, Simon Lovestone3, 1Institute of.
Poster Presentations: P4 affinity of fibrinogen for Abeta and formed tighter Abeta-altered fibrin clots. In vitro fibrinolysis assays revealed that these Abeta-altered fibrin clots showed an even greater delay in fibrinolysis compared with control Abeta-altered fibrin clots. Furthermore, an AD mouse model with diet-induced HCC had more significant impairments in learning and memory as well as severe cerebral amyloid angiopathy (CAA) and parenchymal Abeta deposits in the olfactory bulb, cortex and hippocampus compared to control groups. Conclusions: Therefore, we propose that effect of HC/HCTL on the interaction between Abeta and fibrin(ogen) may be associated with CAA development and cognitive decline and could be risk factor for AD associated with vascular dysfunction.

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AMYLOIDOGENIC PROPERTIES OF ISOASP7CONTAINING BETA-AMYLOID PEPTIDE

Sergey Kozin1, Ivan Cheglakov1, Armen Ovsepyan2, George Telegin2, Philippe Tsvetkov3, Andrey Lisitsa4, Alexander Makarov3, 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; 2Branch of Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia; 3Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; 4Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia. Contact e-mail: [email protected] Background: Region 1-16 constitutes the metal-binding domain of human Ab, and amino acids 6-14 form the minimal zinc-binding site of the domain. Zinc-dependent Ab dimerization is mediated by a tetrapeptide stretch 1114, which acts as the primary zinc recognition site. It has been found that isomerization of Aspartate 7, the most abundant aging-associated spontaneous chemical modification of Ab, leads to serious structure and function changes in the region 1-16. Namely, isoAsp7-containing Ab (isoAb) significantly differs from the intact Ab by better hydrolysis by the angiotensin converting enzyme, changes in zinc and copper ion chelation, and higher susceptibility to zinc-dependent oligomerization. Since each of these molecular processes is closely related to the aggregation ability of Ab, we assumed that isoAb would be a pathogenic agent of AD. Methods: We have used APP/PS1 doubly tg mice which manifest characteristic cognitive features of AD-like pathology and possess significant amounts of dense core congophilic amyloid plaques starting from 4-6 month age. The experimental groups included male animals which were grown under specific pathogenfree conditions and were subjected to intravenous injections of isoAb starting from 2 month age. Results: It has been shown in experiments on transgenic mice that intravenous injections of isoAb cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. Conclusions: The obtained data suggest that isoAb is a molecular trigger of Alzheimer’s disease and therefore can be used as a promising target for drug therapy of this disease.

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A NATURALLY OCCURRING HUMAN IGG SPECIFIC FOR OLIGOMERIC AND FIBRILLAR BETA-AMYLOID: IN VITRO AND IN VIVO STUDIES

Rama Devudu Puligedda1, Yona Levites2, Brian O’Nuallain3, Tomas Ondrejcak4, Pedro Cruz5, Thomas Ladd5, Michael Rowan6, Scott Dessain1, 1Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, United States; 2University of Florida, Gainesville, Florida, United States; 3Harvard Medical School-Brigham and Womens Hospital, Boston, Massachusetts, United States; 4Trinity College Dublin, Dublin, Ireland; 5University of Florida, Gainesville, Florida, United States; 6Trinity College Dublin, Dublin, Ireland. Contact e-mail: ramdev.puligedda@ gmail.com Background: The toxic effects of b-amyloid protein (Ab) in the brains of Alzheimer’s disease patients have been attributed to soluble oligomeric forms of Ab, which contain amyloid-specific, conformational epitopes. Intravenous immunoglobulin (IVIG) contains IgGs specific for oligomeric and fibrillar Ab forms. These have anti-fibrillar functions and have shown activity in improving cognitive function in a transgenic mouse of Alzheimer’s

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disease (AD). Methods: We used a novel means for cloning human antibodies in their native configurations, creating stable, antibody-secreting hybridomas using primary human B-cells as starting material. We created hybridomas using B-cells from a healthy young woman and screened for human IgGs that bound to a lambda immunoglobulin light chain amyloid protein and to Ab fibrils. We identified a monoclonal IgG (mAb) and assessed its binding in vitro using ELISA and competition binding assays. We tested the mAb in the anesthetized rat model of long-term potentiation (LTP) in the hippocampus. We generated a single chain variable fragment (scFv) and tested its binding specificity in vitro and its activity when expressed from an adenovirus vector the brains of neonatal CRND8 mice. Results: The 3H3 human mAb binds preferentially to oligomeric and fibrillar Ab, compared to monomeric Ab. It also binds to kappa and lambda immunoglobulin light chain amyloids. DNA sequence analysis of the 3H3 gene coding sequences indicates that the 3H3 is an affinity matured mAb. Amyloid-specific binding was observed with both the intact IgG and the scFv. In the anesthetized rat, 3H3 inhibited the toxic effects of soluble AD brain extract on LTP in the hippocampus. 3H3 scFv brain expression attenuated development of amyloid pathology resulting in lower levels of extractable Ab 40 and a change in the morphology of amyloid plaques from cored to diffuse. Conclusions: These results suggest that naturally occurring human antibodies exist that specifically bind to toxic forms of Ab. These may contribute to the anti-amyloid activity of IVIG and may be suitable for use as AD therapeutics.

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CANDIDATE BLOOD PROTEOME MARKERS OF ALZHEIMER’S DISEASE ONSET AND PROGRESSION: A SYSTEMATIC REVIEW AND REPLICATION STUDY

Chantal Bazenet1, Steven Kiddle2, Martina Sattlecker3, Petroula Proitsi4, Andrew Simmons5, Eric Westman6, Sally Nelson7, David Sterling7, Steven Williams8, Angela Hodges9, Caroline Johnston1, Hilkka Soininen10, Iwona Kloszewska11, Patrizia Mecocci12, Magda Tsolaki13, Bruno Vellas14, Stephen Newhouse15, Richard Dobson3, Simon Lovestone3, 1Institute of Psychiatry, London, United Kingdom; 2King’s College London, London, United Kingdom; 3King’s College London, London, United Kingdom; 4 King’s College London, Institute of Psychiatry, London, United Kingdom; 5 King’s College London, London, United Kingdom; 6Karolinska Institute, Stockholm, Sweden; 7SomaLogics Inc., Boulder, Colorado, United States; 8 Somalogics Inc., Boulder, Colorado, United States; 9King’s College London Institute of Psychiatry, London, United Kingdom; 10Kuopio University and University Hospital, Kuopio, Finland; 11Medical University of Lodz, Lodz, Poland; 12Institute of Gerontology and Geriatrics, Perugia, Italy; 13Aristotle University of Thessaloniki, Thessaloniki, Greece; 14Clinic of Internal Medicine and Gerontology, Toulouse, France; 15KCL, London, United Kingdom. Contact e-mail: [email protected] Background: A blood-based protein biomarker, or set of protein biomarkers, that could predict onset and progression of AD would have great utility; potentially clinically, but also for clinical trials and especially in the selection of subjects for preventative trials. Numerous discovery studies for blood-based protein markers of AD have been undertaken during the last decade, ranging from proteomics technologies using both gel and non-gel based mass spectrometry (MS) to antibody capture. The failure to replicate discoveries has been a fundamental issue regarding the development of a useful diagnostic panel to date. The problems of non-replicability may be the result of heterogeneity of proteomic platforms or research cohorts, over-fitting of data, technical issues, or non-standardized sample collection protocols Methods: In order to explore the degree of concordance amongst the various discovery efforts, we performed a systematic review of 21 published discovery or panel-based (>100 proteins) blood proteomics studies of AD, which had identified a total of 163 candidate biomarkers. We subsequently applied a modified aptamer-based array (SomaLogic) to a large cohort of 687 subjects (AddNeuroMed and KHP/BRC cohorts) in a substantial replication experiment, encompassing 94 out of the 163 previously discovered candidates. Results: Our systematic review reveals that two thirds of