Vesna Kostanjevecki

Phosphorylation of amyloid precursor carboxy-terminal fragments enhances their processing by a gamma-secretase-dependent mechanism

In Alzheimers disease, the complex catabolism of amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta) peptide, the major component of amyloid deposits. APP is cleaved by beta- and alpha-secretases to generate APP carboxy-terminal fragments (CTFs). Abeta peptide and amyloid intracellular domain are resulting from the cleavage of APP-CTFs by the gamma-secretase. In the present study, we hypothesize that post-translational modification of APP-CTFs could modulate their processing by the gamma-secretase. Inhibition of the gamma-secretase was shown to increase the total amount of APP-CTFs. Moreover, we showed that this increase was more marked among the phosphorylated variants and directly related to the activity of the gamma-secretase, as shown by kinetics analyses. Phosphorylated CTFs were shown to associate to presenilin 1, a major protein of the gamma-secretase complex. The phosphorylation of CTFs at the threonine 668 resulting of the c-Jun N-terminal kinase activation was shown to enhance their degradation by the gamma-secretase. Altogether, our results demonstrated that phosphorylated CTFs can be the substrates of the gamma-secretase and that an increase in the phosphorylation of APP-CTFs facilitates their processing by gamma-secretase.

IMPROVEMENTS TO INNOTEST® PHOSPHO-TAU 181P

replicates and technical replicates were analyzed for 3 AD and 3 non-AD over ten consecutive days. For the vast majority of the peptides CVs were <5%. The second, 6 AD and 6 non-AD, indicated slightly increased levels of cystatin C, beta-2-microglobulin, and APP, as well as clearly increased levels of chromogranin A, secretogranin-2, and neurosecretory protein VGF for AD. Moreover, for complex samples the advantage of high resolution instrumentation became evident because of the superior possiblity to avoid signal interferences. Conclusions: The assay is reproducible and consumes little amount per analysis. Preliminary results should be interpreted with caution due to the limited sample material. Data from a larger study, using clinically well characterized samples, is under way and will be presented.

IMPROVEMENTS TO INNOTEST® HTAU AG

properties of these proteins and the interference of matrix components make the establishment of referencematerials very challenging. As a consequence up to now, the trust in the in commercial assays is limited. Since CSF quality control samples were not broadly available. Methods: A large number of individual CSF samples were pooled to establish 5 samples with different A b 1-42 and Ab 1-40 concentrations. Different compounds were added to the pools before lyophilization. The pools underwent one freeze-thaw cycle before aliquotation (250 ml, polypropylene screw-cap tubes). All aliquots were lyophilized and stored at -20 C. The consistency and homogeneity of the prepared panel was evaluated using three different lot numbers of the assays and analysis in 3 different laboratories by different users. In addition, the performance of the proficiency panel was integrated into a multicenter study and compared to performances of neat CSF or run-validation samples (1⁄4 analyte in buffer). Results: Addition of components do not affect analytical behavior of the CSF sample. Lyophilisation of the samples was successful. Stability was considerably increased by that procedure as analytes showed only 5% CV after one week at 37 C. Neat CSF, with and without the additives, as well as samples from the proficiency panel, showed a very good parallelism (1⁄4 absence of matrix interference in the assays) upon dilution for the three analytes evaluated in the study. The production process did not change the intrinsic characteristics of the CSF samples. Overall variability in a multicenter study for the proficiency panel did not exceed > 10%, including the sum of % CVof intra-run, inter-run, inter-operator, and inter-lab. Conclusions: A panel of 5 stabilized and lyophilized CSF samples with target values covering the whole range of the calibration curve of the A b 1-42 , Ab 1-40 and Total-Tau ELISA. The excellent precision data obtained in a multicenter study make this panel an excellent new tool to verify the quality of the biomarker measurements in research settings, but also for routine labs. In the absence of an international reference standard, this commercially available proficiency panel may significantly increase the trust in the values measured by ELISA of CSF diagnostics.

P-064: Measurement of β-amyloid isoforms in human plasma using the INNO-BIA plasma Aβ forms:(Pre)-clinical validation

(p 0 0001), whereas, after adjustment for age and sex, episodic memory and executive function did not. Baseline hippocampal atrophy was associated with increasing atrophy rates (hippocampal volume: p 0 025; MTA-score: p 0 008), in contrast whole brain volume, WMH and lacunes, adjusted for age and sex, were not significantly associated with hippocampal atrophy rates. Conclusions: In MCI, older age, poorer general cognition, hippocampal atrophy and APOE e4 predict subsequent accelerated rates of hippocampal atrophy, suggestive of the accumulation of Alzheimer-type pathology, which may become clinically manifest in the future. These markers may improve identification of MCI subjects at risk for AD.

ANALYTICAL PERFORMANCE OF A FULLY AUTOMATED CHEMILUMINESCENT β-AMYLOID 1-40 ASSAY ON THE LUMIPULSE G SERIES

early diagnosis using blood. Using spectral imaging techniques with conformationally-selective fluorescent amyloid probes, we imaged fluorescence from circulating leukocytes from subjects suspected of AD orMCI, and compared those results to those from Cerebrospinal Fluid (CSF) testing. Results: When dividing subjects into CSF positive and CSF negative (where CSF positive is indicated by phospho-Tau/Ab1-42 < 0.12), our technique is able to

Elevated CSF GAP-43 is Alzheimer's disease specific and associated with tau and amyloid pathology

The level of the presynaptic protein growth‐associated protein 43 (GAP‐43) in cerebrospinal fluid (CSF) has previously been shown to be increased in Alzheimers disease (AD) and thus may serve as an outcome measure in clinical trials and facilitate earlier disease detection.

ANALYTICAL PERFORMANCE OF A FULLY AUTOMATED CHEMILUMINESCENT PTAU 181 ASSAY ON THE LUMIPULSE G SERIES

Institute, Vancouver, BC, Canada; Univ of Penn, Philadelphia, PA, USA; Washington University, St. Louis, MO, USA; Memory and Aging Center, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA; National Alzheimer’s Coordinating Center, University of Washington, Seattle, WA, USA; The Harvard Clinical and Translational Science Center, Boston, MA, USA; The Bluefield Project, San Francisco, CA, USA; Penn FTD Center, University of Pennsylvania, Philadelphia, PA, USA; Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA; Stevens Neuroimaging and Informatics Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; Northwestern University, Chicago, IL, USA. Contact e-mail: John. Kornak@ucsf.edu

VALIDATION OF CSF TUBES FOR USE ON THE LUMIPULSE ® G PLATFORM: IMPACT ON Aβ1-42

clinical data, visually evaluated amyloid-PET scans; amyloid-PET positivity was determined bymajority assessment. Intra-reader reliability was determined based on re-ratings of approximately 10% of randomly selected images from available PET scans. Interreader reliability was also assessed. SUVRs were calculated in a standardized cortical region-of-interest using whole cerebellum as the reference region. CSF samples were analyzed using Elecsys immunoassays for Ab42, tTau, and pTau. Results:Data from both ADNI and BioFINDER cohorts will be presented. For BioFINDER, intra-reader reliability based on average positive agreement (APA) and average negative agreement (ANA) was >90%. Inter-reader reliability gave an APA >80% and ANA >90%. Cut-off values for Ab42, pTau/Ab42, and tTau/Ab42 were defined based on visual-read amyloid-PET positivity. The associated positive (PPA) and negative percentage agreements (NPA) at the cut-off value were 91% and 72% for Ab42, and 91% and 89% for both pTau/Ab42 and tTau/Ab42, respectively. Higher concordance could be achieved with reference SUVR. Conclusions:We present a method to determine cut-off values of Elecsys CSF immunoassays based on visual-read amyloid-PET positivity. The Elecsys CSF immunoassays demonstrated excellent ability to predict amyloid-PET positivity, which was further improved by using tTau/ Ab42 and pTau/Ab42 ratios compared to Ab42 alone.

LUMIPULSE ® G TOTAL TAU: KEY PERFORMANCES OF A FULLY AUTOMATED CHEMILUMINESCENT IMMUNOASSAY

P4-467 LUMIPULSE G TOTALTAU: KEY PERFORMANCES OFA FULLY AUTOMATED CHEMILUMINESCENT IMMUNOASSAY Manu Vandijck, Martine Dauwe, Els Huyck, Nathalie Le Bastard, John Lawson, Christopher Traynham, Zivjena Vucetic, Johan Gobom, Kaj Blennow, Geert Jannes, Vesna Kostanjevecki, Fujirebio Europe N.V., Gent, Belgium; Fujirebio US Inc, Malvern, PA, USA; Fujirebio Diagnostics Inc, Malvern, PA, USA; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden. Contact e-mail: manu. vandijck@fujirebio.com

VERIFICATION OF THE ANALYTICAL PERFORMANCE OF THE FULLY AUTOMATED LUMIPULSE G β-AMYLOID 1-42 AND LUMIPULSE G TOTAL TAU ASSAYS

using “conversion to dementia” as a gold standard for diagnosis (in the lack of pathology data). The resulting 5 sequential phases were: 1) pilot studies, 2) clinical assay development for clinical disease, 3) prospective longitudinal repository studies, 4) prospective diagnostic studies, and 5) disease control studies (Table). Because of the required adaptations, biomarkers for AD can be used for biomarker-based diagnoses and not yet for screening purposes. Conclusions:The adaptation of the oncology framework to AD aims to systematize the validation of AD biomarkers. The important limitations restrict the generalizability of results to the general population and the use of such biomarkers for screening purposes. This initiative should be considered as a first, although necessary, step to the definition of a systematic validation of biomarkers for AD.