AMYLOID-BETA (Aβ) ISOFORMS AND PTMS OF SOLUBLE Aβ OLIGOMERS FROM HUMAN BRAIN

Poster Presentations: Sunday, July 24, 2016

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Ab OLIGOMER ELIMINATING COMPOUNDS IMPEDE NEURODEGENERATION IN VIVO

Dieter Willbold, Forschungszentrum J€ulich, J€ulich, Germany; Heinrich-Heine-Universit€ at, D€usseldorf, Germany. Contact e-mail: d. [email protected] Background: Several lines of evidence suggest a central role of amyloid-b-peptide (Ab) in the pathogenesis of Alzheimer’s disease (AD). More than Ab fibrils, small soluble and prion-like Ab oligomers are suspected to be the major toxic species responsible for disease development and progression. Therefore, eradication of these Ab oligomers is our principal objective for therapy of AD. Previously, we have identified the fully D-enantiomeric peptide D3 by mirror image phage display selection [1] and showed that it was able to specifically eliminate Ab oligomers and convert them into non-toxic species. D3 was able to reduce plaque load in transgenic AD mouse models, and improved cognition even after oral application [2]. More recently, we developed derivatives of D3 with improved properties. Methods: Using our newly developed AbQIAD (quantitative determination of interference with Ab aggregate size distribution) assay we showed that increased Ab oligomer elimination efficiency correlates with increased activity to slow down neurodegeneration in the TBA2.1 mouse model [3]. Results: Moreover, these compounds were also able to improve cognition in the Tg-SwDI mouse model. As expected from D-peptides, D3 and its derivatives showed superior pharmacokinetic properties, such as long half-lives and high oral bioavailability [4, 5]. I will summarize our newest data on highly efficient D3 derivatives on their way towards the first in man, first in class, clinical phase I study. This will include unpublished data. Conclusions: Compounds that specifically and efficiently eliminate Ab oligomers are able to enhance cognition and impede neurodegeneration in vivo. [1] vanGroen et al., ChemMedChem 3, 1848-1852 (2008). [2] Funke et al., ACS Chem. Neurosci. 1, 639-648 (2010). [3] Brener et al., Sci. Rep. 5, 13222 (2015). [4] Jiang et al., PLoS One 10, e0128553 (2015). [5] Leithold et al., Pharm Res. 33, 33(2):328-336 (2015).

P1-099

PURIFICATION AND QUANTITATIVE CHARACTERIZATION OF AMYLOID-BETA OLIGOMERS FROM ALZHEIMER’S DISEASE BRAIN LYSATES

Thomas J. Esparza1, Norelle C. Wildburger1, Nigel J. Cairns1,2, Robyn Roth1, David L. Brody1,3, 1Washington University School of Medicine, St. Louis, MO, USA; 2Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA; 3Hope Center for Neurological Disorders, St. Louis, MO, USA. Contact e-mail: [email protected] Background: Previous efforts to identify toxic amyloid-beta (Ab)

species involved in Alzheimer’s disease (AD) utilized synthetic peptide preparations, over-expressing cell lines, and/or transgenic animal models. Studies that used human patient-derived Ab have demonstrated a greater toxicity than synthetic preparations; thus supporting the need for a clear identification of the soluble Ab constituents. To our knowledge there has not been a successful isolation and mass spectrometric characterization of the soluble Ab oligomer species from AD post-mortem tissue. Methods: We developed a quantitative biochemical extraction protocol that permits scalable isolation and immunopurification of Ab oligomer species. We used clinically demented (CDR ¼ 3) and neuropathologically well-characterized (NA-AA high level of AD neuropathologic

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change) postmortem brain tissue (frontal or parietal lobe). We dissected neocortex from underlying white matter and samples were dounce homogenized in PBS containing protease inhibitors and sub critical micelle concentrations of CHAPS detergent. Oligomer loss was reduced by blocking with albumin during all purification steps. Homogenates are further processed by differential ultracentrifugation which separates Ab oligomers from monomers. Oligomers are further immuno-purified and subjected to mass spectrometric characterization. Results: Using 2-3 grams of frontal or parietal cortex from AD cases, we consistently isolated >70% of the input oligomeric Ab. Our method has provided sufficient purification for the initial mass spectrometric characterization of the soluble Ab oligomer isoforms. We find that our method does not induce artificial oligomers when AD derived Ab monomer is spiked into negative-pathology age-matched tissue and purified. In addition, we have performed initial size and morphological characterization of the Ab oligomers using size-exclusion and immunoelectron microscopy. Conclusions: We have successfully isolated and initially characterized AD patient-derived Ab oligomers. This method will allow the future identification of oligomeric Ab that arises presymptomatically (CDR0 + plaque pathology), and that may drive progression to dementia. Human brain Ab oligomers may be logical targets for preventative interventions.

P1-100

AMYLOID-BETA (Ab) ISOFORMS AND PTMS OF SOLUBLE Ab OLIGOMERS FROM HUMAN BRAIN

Norelle C. Wildburger1, Thomas J. Esparza1, Nigel J. Cairns1,2, Randall Bateman1,2,3, David L. Brody1,3, 1Washington University School of Medicine, St. Louis, MO, USA; 2Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA; 3Hope Center for Neurological Disorders, St. Louis, MO, USA. Contact e-mail: [email protected] Background: N-terminally truncated amyloid-beta (Ab) peptides in plaques and cerebral amyloid angiopathy were established as a feature of AD three decades ago. Some of the earliest characterized N-truncated Ab peptides include Ab4-42, pyroglutamate-modified Ab3(pE), and Ab11(pE). In addition to N-truncation, Ab3(pE) was one of the first described Ab PTMs in AD brain. The relative importance of N-truncated and post-translationally modified Ab peptides in the pathogenesis of AD is not completely understood. However, to the best of our knowledge, Ab size variants have not been examined in water-soluble Ab oligomer species from AD brain. Few studies have examined PTMs present in soluble Ab oligomers from either human or transgenic animal models. Examination of PTMs on soluble Ab oligomers to-date includes glutamine deamidation and di-tyrosine crosslinking of synthetic Ab and recombinantly expressed Ab from CHO cells. One study identified phosphorylation of serine 8 in transgenic mice and human AD tissue. Methods: Affinity-purified Ab oligomers from our scalable biochemical extraction protocol were dried in vacuo and resuspended in 1%/10%/5% FA/ACN/MeOH (v/v). Each sample was analyzed in a block-randomized fashion by nLC-MS/MS on a hybrid mass spectrometer (Q-LTQ-Orbitrap Fusion) in an untargeted top-down discovery approach for the unambiguous characterization of Ab isoforms in oligomers from human AD brain. Results: Using 1-2 grams of frontal or parietal cortex from clinically demented (CDR ¼ 3) and pathologically confirmed cases of AD (NIA-AA: high level of AD neuropathologic change) we have 1) identified up to 73 Ab isoforms from oligomeric fractions. 2)

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Poster Presentations: Sunday, July 24, 2016

Identified a number of post-translational modifications (PTMs) including those related to protein aging (e.g., carbamylation). 3) Identified potential single amino acid variants (SAV) in the Ab peptides from human AD brain. Conclusions: We demonstrate for the first time multiple N- and C-terminal truncated Ab isoforms from water-soluble Ab oligomers from human AD brain as well as PTMs and SAVs. These “signatures” will allow future determination of the Ab isoforms that drive neurotoxicity and potential therapeutic targets and biomarkers.

P1-101

AMYLOID-BETA 1-42 (Ab1-42) LEVELS IN THE CEREBROSPINAL FLUID ASSOCIATE WITH SPATIAL MEMORY PERFORMANCE IN AGED BUT NOT IN ADULT MCGILL-R-THY1-APP RATS

Eduardo R. Zimmer1,2, Maxime J. Parent3, Monica Shin4, Min Su Kang4, Antonio Aliaga5, Sonia Do Carmo6, Serge Gauthier4, A. Claudio Cuello5, Pedro Rosa-Neto4, 1Brain Institute of Rio Grande do Sul, Porto Alegre, Brazil; 2Federal University of Rio Grande dos Sul, Porto Alegre, Brazil; 3 Yale School of Medicine, New Haven, CT, USA; 4McGill University Research Centre for Studies in Aging, Verdun, QC, Canada; 5McGill University, Montreal, QC, Canada; 6Department of Pharmacology- McGill University, Montreal, QC, Canada. Contact e-mail: eduardo.zimmer@ ufrgs.br

and follow-up showed a significant learning curve in the MWM acquisition phase (p<0.05). However, the McGill-R-Thy1-APP group presented an inferior performance than the WT group (p<0.05). The WT group presented undetectable levels of CSF Ab1-42. By contrast, McGill-R-Thy1-APP rats presented detectable levels of Ab1-42 in the CSF (baseline (mean6SD): 20026453 pg/mL; follow-up: 14336273 pg/mL). The CSF Ab142 concentration in aged McGill-R-Thy1-APP rats decreased around w30% compared with the baseline (adult). Finally, no correlations were found between Ab1-42 CSF levels and the MWM acquisition phase. However, Ab1-42CSF concentration correlates with the performance in the MWM retention phase at follow-up but not at baseline (table 1). Conclusions: The longitudinal assessment of the McGill-R-Thy1-APP rat demonstrated an age-dependent decline in spatial memory and in the Ab1-42 CSF levels. Interestingly, Ab1-42 CSF levels were only significantly correlated with MWM performance in aged McGill-R-Thy1-APP rats but not in adult rats. Our findings suggest that Ab1-42 CSF concentration does not correlate well in early stages of amyloid deposition but it seems to indicate cognitive decline as amyloid load progresses.

P1-102

Background: Low levels of amyloid-beta 1-42 (Ab1-42) peptides in

the cerebrospinal fluid (CSF) are typically found in Alzheimer’s disease (AD) patients. However, whether Ab1-42 CSF levels are indicators of cognitive decline remains controversial. In this context, animal models expressing mutations in the amyloid precursor protein (APP) are ideally suited for advancing in the understanding of the relationship between levels of Ab1-42 and cognitive decline. Here, we submitted McGill-R-Thy1-APP rats to the Morris water maze test (MWM) and collected CSF longitudinally at two time points. We hypothesize age-dependent cognitive decline and reduction in the Ab1-42 CSF levels with these variables potentially being associated. Methods: McGill-RThy1-APP and wild-type (WT) rats were used for analyzing spatial memory performance (MWM) and collecting CSF. Animals were analyzed in both procedures in a longitudinal fashion: at baseline (9-11 months) and follow-up (16-19 months). In the MWM, the latency to escape and time in the target quadrant were used as outcome measures. The CSF was collected by direct puncture in the cisterna magna and measured using a multiplex specific for Ab1-42. Results: Both groups at baseline

BUILDING A TESTABLE MATHEMATICAL MODEL FOR ALZHEIMER’S DISEASE USING GENE EXPRESSION CHANGES AND BIOCHEMICAL SYSTEMS THEORY (BST)

Randolph A. Coleman1, Ceyda Durmaz1, Alec A. Weech2, Frank J. Castora3, 1College of William and Mary, Williamsburg, VA, USA; 2 National Institutes of Health, Bethesda, MD, USA; 3Eastern Virginia Medical School, Norfolk, VA, USA. Contact e-mail: [email protected] Background: Abnormal mitochondrial function has become recog-

nized as a critical component in the pathogenesis of a variety of neurodegenerative diseases, including AD. We have recently found abnormal expression of several genes critical to mitochondrial energy production and biogenesis in AD brains. Using this subset of mitochondrial genes, we have begun to build a mathematical model of AD using Biochemical System Theory (BST). Through the development and application of appropriate differential equations, the flux of various metabolites and small molecules will be simulated and used to generate a testable model of mitochondrial involvement in AD pathogenesis. Methods: Human Mitochondrial Biogenesis and Energy Metabolism Plus RT2 Profiler PCR Arrays were used to assess expression of 168 mitochondrial function and energy genes

Table 1 Pearson’s correlation analyses between spatial memory performance and cerebrospinal fluid (CSF) amyloid beta 1-42 (Ab1-42) levels Morris water maze (MWM) vs CSF Ab1-42 levels Acquisition Session 1 vs CSF Ab1-42 levels Acquisition Session 2 vs CSF Ab1-42 levels Acquisition Session 3 vs CSF Ab1-42 levels Acquisition Session 4 vs CSF Ab1-42 levels Retention Session vs Ab1-42 levels Acquisition Session 1 vs CSF Ab1-42 levels Acquisition Session 2 vs CSF Ab1-42 levels Acquisition Session 3 vs CSF Ab1-42 levels Acquisition Session 4 vs CSF Ab1-42 levels Retention Session vs Ab1-42 levels

Time-point

Pearson’s r

P-value

Baseline (Adult)

-0.4095 -0.0955 -0.3052 -0.0343 -0.0066 0.3075 0.4844 0.2906 0.1386 0.7889

0.2737 0.8068 0.4246 0.9301 0.9865 0.4209 0.1863 0.4481 0.7221 0.0115*

Follow-up (Aged)