AMYLOID DEPOSITION, TAU AGGREGATION AND MICROGLIAL ACTIVATION CORRELATE WITH VASCULAR BURDEN IN VIVO IN ALZHEIMER'S DISEASE

Poster Presentations: Monday, July 17, 2017 Table 1 Levels of emerging CSF markers by MCI subgroup

Ca3, pg/ml SAP, ng/ml TCC, mAU/ml HFABP, pg/ml

CN N¼15

MCI Low AD likelihood N¼18

MCI SNAP N¼14

MCI IAP N¼18

MCI High AD likelihood N¼27

7257L,S,I,H (4553) 10.7 (8.8) 36.3 (35.5) 1033 (516)H

3116C (2325) 11.1 (17.7) 18.8 (25.0) 905 (325)S,H

4476C (3330) 8.9 (6.4) 28.0 (36.0) 1303 (441)L,I

3064C (1881) 7.5 (6.2) 31.9 (32.6) 963 (340)S,H

4117C (2613) 10.2 (23.2) 62.4 (151.1) 1517 (626)C,L,I

Results are mean (SD). CN ¼ cognitive normal; MCI ¼ mild cognitive impairment; SNAP ¼ suspected non-Alzheimer’s disease pathophysiology; IAP ¼ isolated amyloid pathology SAP ¼ serum amyloid P; TCC ¼ C5b-9 terminal complement complex; HFABP ¼ heart-fatty acid binding protein. C ¼p<0.05 compared to CN; L¼p<0.05 compared to Low AD likelihood group; S¼p<0.05 compared to SNAP group; I¼p<0.05 compared to IAP group; H¼p<0.05 compared to high AD likelihood group.

neurodegeneration within all MCI subgroups. Moreover, HFABP levels are related to overall tau-related neurodegeneration in individuals with MCI.

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DNA METHYLATION STATUS OF AN EXTENDED APOE LOCUS REGION IN ALZHEIMER’S DISEASE

Yvonne Shao1, McKenzie Shaw1, Maria Khrestian1, Giana D’Aleo2, James B. Leverenz3, Lynn M. Bekris1, 1 Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 2 Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA; 3 Lou Ruvo Center for Brain Health, Cleveland Clinic, Cleveland, OH, USA. Contact e-mail: [email protected] Background: In humans and in mouse models, APOE ε2 carriers have

higher apoE levels, compared to ε3 and ε4 (ε2>ε3>ε4) and are protected against Alzheimer’s disease (AD) pathology, such as the toxic Ab protein, suggesting that APOE ε2 plays an important role in the central nervous system. It has been suggested that if apoE2 or apoE3 protein levels are therapeutically increased, without increasing apoE4, this will allow for better clearance and less deposition of the toxic Ab protein in AD. Many questions still remain on how APOE expression is regulated by distal elements at this locus and how they might be exploited to modulate apoE levels in AD. We have described the underlying mechanistic influence by APOE exon 4 regulatory element at the APOE ε2/ε3/ε4 haplotype location. Notably, the activity of this exon 4 regulatory element appears to depend on epigenetic factors that either enhance transcription or decrease expression depending on the APOE ε2/ε3/ε4 haplotype. The central hypothesis of this investigation is that the complex regulatory structure surrounding the APOEgene is regulated by key epigenetic factors depending on tissue type and disease status. Methods: DNA was extracted from human brain (n¼12) and lymphocytes (n¼85) and percent methylation was measured using the EZ DNA MethylationÔ Kit (Zymo Research) and Infinium HumanMethylation450 BeadChip Kit (Illumina). Percent methylation of each cg site was evaluated for an association with disease status and age using a multivariate analyses (SPSS). Results: Extended methylation analysis of the APOE locus suggests that methylation at TOMM40 and APOC1, but not the APOE exon 4-

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3’UTR CpG, are different between AD and controls in the hippocampus. Methylation at the APOE exon 4-3’UTR CpG island, APOC1 and APOC1P1 is different in MCI compared to controls in lymphocytes. Conclusions: This exploratory DNA methylation analysis of an extended region of the APOE locus identified regions outside of the APOE gene that are differentially regulated according to disease status. These results suggest that other regions, in addition to APOE, are differentially methylated in AD and MCI, compared to controls, and identifies regions that may be regulated by methylation at this locus.

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SYNAPTOLOGY OF LAYER II OF THE TRANSENTORHINAL CORTEX IN ALZHEIMER’S DISEASE

Marta Dominguez, Center of Biomedical Technology (CTB), Politecnica University of Madrid, Madrid, Spain. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is the main cause of demen-

tia, accounting for 60-80% of cases. It is characterized by a progressive and persistent decline of superior cerebral functions, such as memory. During the course of the disease, three main histopathological alterations occur: cerebral atrophy, intracellular neurofibrillary tangles and amyloid plaques. Early loss of episodic memory in AD patients is closely associated with the progressive degeneration of the medial temporal lobe structures, with the transentorhinal cortex (TEC) being one of the first affected areas. This area is considered as a transitional zone between the entorhinal cortex (EC) and the temporal cortex. The main cytoarchitectonic feature of the TEC is that layers III and V merge and sweep obliquely to invade layer II of the EC. Methods: In this study, we performed a three-dimensional ultrastructural analysis of the neuropil from layer II of the TEC, using human brain tissue from 5 patients with AD and from 5 subjects with no apparent neurological diseases. We used an instrument that combines a high-resolution field-emission SEM column with a focused gallium ion beam (FIB), which mills the sample surface on a nanometer scale. The sequential and automated use of FIB milling and SEM imaging allows us to obtain large images stacks that represent a three-dimensional sample. Customized analysis software was used for the reconstruction of synapses, which allowed their number, morphology and spatial distribution to be calculated (EspINA software, v.2.1.9). Results: Our preliminary results show that the total number of synapses per volume in layer II of the TEC in AD patients was significantly lower than in nondemented subjects. We have not found differences in the morphology of the synapses in AD patients compared with nondemented subjects. In addition, the spatial organization of synapses in the neuropil of layer II of the TEC is random, regardless of the subject group. Conclusions: The synaptic density of the TEC is affected in Alzheimer’s disease. There are no changes in morphology or spatial distribution.

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AMYLOID DEPOSITION, TAU AGGREGATION AND MICROGLIAL ACTIVATION CORRELATE WITH VASCULAR BURDEN IN VIVO IN ALZHEIMER’S DISEASE

Melanie Dani1, Zhen Fan1, Melanie Wood1, Ruth Mizoguchi1, Richard Morgan2, Zuzana Walker3, Grazia Daniela Femminella1, Valeria Calsolaro1, David J. Brooks1,4,5, Paul Edison1, 1Imperial College London, London, United Kingdom; 2Chelsea and Westminster Hospital,

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Poster Presentations: Monday, July 17, 2017

London, United Kingdom; 3North Essex Partnership University NHS Foundation Trust, Chelmsford, United Kingdom; 4Aarhus University, Aarhus, Denmark; 5Newcastle University, Newcastle, United Kingdom. Contact e-mail: [email protected] Background: The role of vascular disease in Alzheimer’s disease

(AD) is still debated. White matter hypointensity (WMH) volume is an imaging measure of vascular burden. Using MRI and Positron Emission Tomography (PET) we examined the voxel-level relationships between WMH and 3 key processes in AD: amyloid deposition, tau aggregation and microglial activation. Methods: 15 participants with a clinical diagnosis of AD had T1-weighted MRI, 18F-flutemetamol, 18F-AV1451 and 11C-PBR28 PET scans. 18 healthy controls also had 18F-flutemetamol and 11C-PBR28 scans, and 8 of these also had 18F-AV1451 PET. Z-score maps for each tracer (80-100 minute ratio image for 18F-AV1451, 90-120 minute ratio image for 18F-flutemetamol and logan Vd parametric map for 11C-PBR28 ) were calculated for the AD subjects compared to the controls for each tracer. WMH volumes from the MRI were calculated from Freesurfer software. Multiple regression in SPM8 was used to calculate voxel wise correlations between WMH volume and tracer uptake. A pvalue of p<0.05 was considered significant. Results: Amyloid deposition: there were correlations between WMH volume and amyloid deposition in the 1) frontal lobe (right medial and anterior orbital gyri, left posterior orbital gyrus, and right superior frontal gyri) 2) parietal lobe (left superior parietal gyrus) and 3) corpus callosum. Tau aggregation: there were correlations between WMH burden and tau aggregation in the left and right posterior temporal lobe Microglial activation :there were correlations between WMH volume and microglial activation in the 1) frontal lobe (right superior/middle frontal gyrus, right subgenual frontal cortex, right medial orbital gyrus) 2) parietal lobe (postcentral / precentral gyrus, left inferolateral part of parietal lobe and 3) occipital lobe (left lateral part of occipital lobe). Conclusions: White matter hypointensity volume correlates at voxel-level with amyloid deposition, tau aggregation and microglial activation. These findings emphasise the complex disease pathogenesis occurring in AD, and the need for a multi-targeted treatment.

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INTRANEURONAL AMYLOID b-PEPTIDE LEVELS ARE AFFECTED BY MONOAMINE OXIDASE B

Bengt Winblad1, Lenka Hromadkova2, Susanne Frykman1, Lars O. Tjernberg1, Sophia Schedin Weiss1, 1Karolinska Institutet, Center for Alzheimer Research, Div. of Neurogeriatrics, Huddinge, Sweden; 2Charles University in Prague, Prague, Czech Republic. Contact e-mail: [email protected] Background: The amyloid b-peptide (Ab), a key molecule in Alzheimer disease (AD) pathogenesis, is formed after proteolytic processing of the amyloid precursor protein by BACE1 followed by g-secretase. Increasing evidence suggest that intracellular Ab is neurotoxic and correlate with AD pathogenesis. Regrettably, direct inhibition of g-secretase has been associated with toxic side effects in clinical trials and alternative strategies should be considered. Here we have identified monoamine oxidase B (MAO-B) as a g-secretase associated protein that regulates Ab production but not Notch processing, and suggest that the g-secretase/MAO-B interaction may be a potential target for pharmacological intervention of AD. Methods: We developed a method to quantify intracellular Ab42 and MAO-B levels in situ in single primary neurons, using immunocytochemistry and confocal microscopy. In contrast to quantification

by biochemical methods such as ELISA, this approach has the advantage that data is obtained from individual cells, making it possible to calculate statistics on a single cell basis. Ab42 was fluorescently labeled by a monoclonal antibody followed by an Alexa Fluor 488-conjugated secondary antibody, whereas MAO-B was fluorescently labeled by a single-protein PLA approach. MAO-B was silenced in primary cortex neurons by siRNA treatment and the effect on intracellular MAO-B and Ab42 levels was determined by image analyses using the software Image J. Results: Quantitative immunofluorescence data based on single cell analysis revealed that intracellular levels of MAO-B protein and Ab42 peptide correlate in nontreated primary cortex neurons. Furthermore, siRNA silencing of MAO-B demonstrated that decreased MAO-B expression led to reduced levels of intracellular Ab42, providing further evidence that MAO-B affects intraneuronal Ab42 levels. Conclusions: Since intracellular Ab42 is a neurotoxic agent in AD, it is important to be able to quantify Ab levels in individual neurons. Our technique enabling such measurements allowed us to find a correlation between Ab42 and MAO-B on an individual cell basis. These findings together with previous studies showing increased g-secretase processing of the immediate substrate C99 in the presence of enhanced levels of MAO-B, indicate that MAO-B affects Ab production via gsecretase. We have therefore developed a screen to identify compounds that inhibit the g-secretase/MAO-B interaction.

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MAPPING PROTEIN-PROTEIN INTERACTIONS BY PEPTIDE ARRAY FOR DISRUPTOR PEPTIDE DEVELOPMENT AND TRANSLATIONAL IMPACT!

Christina Elliott1, George S. Baillie2, Richard Killick1, 1King’s College London, London, United Kingdom; 2University of Glasgow, Glasgow, United Kingdom. Contact e-mail: [email protected] Background: Aberrant cellular signalling is central to the pathogen-

esis of a number of neurodegenerative disorders. Our group focuses in particular on the role of Wnt signalling in b-amyloid toxicity, and have demonstrated not only that aberrant Wnt activation, APP proteolysis and synapse loss are intimately linked, but have identified the key protein-protein interactions responsible. Methods: A fundamental aspect of our pathway-driven approach is the mapping and disruption of protein-protein interactions using peptide array technology. Peptide arrays are created from peptide libraries consisting of overlapping peptides spanning the entire sequence of the protein of interest and spotted onto cellulose coated slides. These peptide arrays can be overlaid with putative binding partners to identify potential binding regions or novel post-translational modification sites. Key amino acid residues can be rapidly identified by alanine/ single substitution scanning. Crucially, we can delineate the minimal “core” binding regions, which can be synthesised and used as cell permeable “disruptor peptides” capable of selectively blocking specific protein-protein interactions. Furthermore, disruptor peptides can be used as the basis of high throughput biochemical and cell based assays for drug discovery. Results: We wish to showcase the power of this technology, using specific examples from our own work on Alzheimer’s disease, and demonstrate its capabilities, which are readily amenable across a broad range of applications, to provide useful research tools in dissecting specific signalling events. Conclusions: Mapping and disrupting protein-protein interactions by peptide array has significant potential to provide translational outcomes for neurodegenerative disease.