Tau in platelets as a potential biological marker for Alzheimer's disease

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Poster Presentations P3 BEHAVIORAL DECLINE CORRELATES WITH THE SIGNIFICANT INCREASE OF THE PHOSPHO-TAU IN CEREBROSPINAL FLUID OF THE RAT MODEL OF HUMAN TAUOPATHY

Norbert Zilka1,2, Miroslava Korenova1, Branislav Kovacech1,2, Michal Novak1,2, 1Institute of Neuroimmunology, Bratislava, Slovakia; 2 Axon Neuroscience, Vienna, Austria. Contact e-mail: Norbert.Zilka@ savba.sk Background: Little is known about the cerebrospinal fluid (CSF) biomarkers in the rodent models of human tauopathies. The aim of the present study was to identify the relationship between progressive neurobehavioral decline and the CSF p-tau181 levels in the transgenic rats expressing human truncated tau protein. Methods: CSF was collected from cisterna magna. CSF levels of p-tau181 were measured independently in a longitudinal study. Behavioral changes were quantified using NeuroScale scoring method. Results: In the behavioral study, the transgenic rats fell into two main groups based on the baseline behavioral functioning: 1) transgenic rats suffering from mild neurobehavioral impairment (MNI, score: 3.3-26) and 2) transgenic rats suffering from severe neurobehavioral impairment (SNI, score: 36-44). The analysis of the brain sarkosyl insoluble p-tau181 revealed signifficant increase of the insoluble tau in SNI transgenic rats when compared to MNI counterparts. In order to determine whether CSF phosphotau reflects the behavioral decline and increase of sarcosyl insoluble tau in the brain, p-tau181 were measured in the CSF in the longitudinal study. The study showed significant increase of CSF p-tau181 during the progression of the disease from MNI to SNI. Moreover increased levels of p-tau181 in CSF correlate with significant increase of the sarkosyl insoluble p-tau181 levels in the brain. Conclusions: Increasing p-tau181 level during progressive behavioral decline suggests that p-tau181 may be useful as a surrogate biomarker for preclinical drug development of the disease modifying treatments. P3-196

DETECTION OF ABETA OLIGOMERS IN ALZHEIMER’S DISEASE PATIENT CSF WITH A NOVEL MISFOLDED PROTEIN ASSAY

Carol Man Gao1, Joseph P Fedynyshyn1, Erika Magdangal1, Alice Yam1, Kaj Blennow2, Xuemei Wang1, Cleo M Salisbury1, Sophie Allauzen1, 1 Novartis Diagnostics, Emeryville, CA, USA; 2Sahlgrenska University Hospital, University of Goteborg, Molndal, Sweden. Contact e-mail: man. [email protected] Background: Alzheimer’s Disease (AD) is a progressive neurodegenerative disease that likely starts long before clinical signs of dementia appear. There is a critical need for diagnostics that can detect the disease at these early stages before irreversible cellular and physiological changes (e.g., cell death and loss of synaptic function) have taken place. To accommodate this medical and clinical need, we have developed a novel misfolded protein based assay to detect early stages of the disease from patient cerebral spinal fluid (CSF). While the disease processes which underlie AD are not yet fully understood, it is becoming increasingly clear that soluble Abeta oligomers play an important role. We report the development of a novel assay system which incorporates selective capture of misfolded proteins aggregates and subsequent specific identification of Abeta oligomers from AD patient cerebral spinal fluid (CSF). Methods: Test samples were subjected to a novel misfolded protein capture agent and enrichment technique followed by quantitation of constituent Abeta monomers via multiplexed immunoassay. Soluble extracts from AD patient brain homogenates, synthetic Abeta oligomers, and the CSF of 26 AD patients (12 MCI and early AD, 8 progressive AD, and 2 late AD) and 10 agedmatched controls were examined. Results: The Novartis misfolded protein assay can reliably detect soluble exogenous Abeta aggregates and oligomers spiked into control human CSF. Preliminary data obtained in 36 clinical CSF samples demonstrate diagnostic sensitivity and specificity of >95% and 90% respectively with excellent differentiation between controls and AD patient samples. Conclusions: Circulating Abeta oligo-

mers can be detected in CSF and may provide a useful biomarker for early AD detection. We hope to further develop this assay to ultimately provide a reliable method of both identifying prodromal phase AD and monitoring AD progression. P3-197

EXPLORING CSF BIOMARKER CUTPOINTS WITH PENALIZED SPLINE ANALYSIS AND MAXIMIZED LOG RANK TEST

Leah Burns1, Gilbert L’Italien1, Zhenchoa Guo1, Pablo Lapuerta2, Robert M. Berman1, Stephen Kaplita1, Howard Feldman1, Michael Donohue3, 1Bristol - Myers Squibb, Wallingford, CT, USA; 2 Bristol - Myers Squibb, Princeton, NJ, USA; 3University of California, San Diego, CA, USA. Contact e-mail: [email protected] Background: Previous studies from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) support the hypothesis that cerebral spinal fluid biomarkers (Ab1-42, tau, Ab1-42-to-tau ratio) predict incident dementia. However, there exists no consensus, objective method for determining CSF biomarker cutpoints that discriminate dementia from nondementia. The quantitative relationship between CSF biomarker levels and Alzheimer’s Disease (AD) risk can be used to identify biomarker cutpoints that provide optimal discrimination. This information is important in determining patients appropriate for enrollment into clinical trials aimed at early intervention. The objective of this evaluation was to assess spline analysis and log rank test as a method for establishing CSF biomarker cutpoints that discriminate between patients with mild cognitive impairment (MCI) who progress to AD and those who do not. Methods: A retrospective analysis was conducted on 197 subjects from the ADNI MCI cohort with up to 3 years’ clinical follow-up who had baseline measures of CSF biomarkers. Cox proportional hazards regression was used to assess baseline CSF biomarkers (Ab1-42, tau, Ab1-42:tau ratio [Ab:tau]) predictive of time to AD progression within 2 years. Spline analysis and a log-rank test statistic were used to determine optimal biomarker cutpoints on a continuous scale of the biomarkers. If spline-estimated log hazards were nonlinear with respect to a biomarker, the changepoint method of Contal and O’Quigley was used to determine the CSF cutoff. Results: Spline fits suggested a nonlinear relationship between the log hazards and the CSF measures. An optimal cutpoint in Ab:tau, suggested from proposed statistical methods, was associated with an 8.0-fold (95% CI 2.5 to 25.6) greater risk of developing AD from MCI, while controlling for medial temporal lobe and hippocampal volumes. Conclusions: Spline analysis and log-rank test demonstrates potential as a method for evaluating biomarkers that are nonlinearly related to dementia transition. The method under evaluation identified a cutpoint associated with significant risk of progression from MCI to AD within 2 years. Future research aims to establish the validity and generalizability of these findings applied to other MCI cohorts. P3-198

TAU IN PLATELETS AS A POTENTIAL BIOLOGICAL MARKER FOR ALZHEIMER’S DISEASE

Gonzalo Farias1,2, Karen Neurmann1,2, Andrea Slachevsky2,3, Patricio Perez1,2, Ricardo Maccioni1,2, 1Laboratory of Cellular and Molecular Neurosciences, Faculty of Sciences, University of Chile, Santiago, Chile; 2International Center for Biomedicine (ICC), Santiago, Chile; 3Dept. of Neurological Sciences, Faculty of Medicine, Universidad de Chile, Santiago, Chile, Santiago, Chile. Contact e-mail: [email protected] Background: Platelets are a mayor peripheral reservoir of amyloid precursor peptide (APP), so they have been postulated as a useful peripheral model for the diagnosis and monitoring of therapeutic response in Alzheimer’s disease (AD). Tau is a protein with primary involvement in the pathophysiology of AD, and undergoes changes such as hyperphosphorylation and aggregation since the early stages of the disease. Here we demonstrate that tau protein is also present in platelets and it could serve as a novel biological marker for AD Methods: In a first stage, blood samples

Poster Presentations P3 were obtained from 89 cognitive healthy control subjects, age 30 to 85. Platelets were separated via differential centrifugation, fixed and analyzed with flow cytometry and immunofluorescence with antibodies against tau (Tau-5) and phosphorylated tau (PHF-1). Platelet proteins were immunobloted with the same antibodies, while platelets tau was immunoprecipitated with Tau-5. In a second stage, blood samples were obtained from 30 AD patients and 15 paired age controls and subjected to the same analysis and to platelets APP inmunoblots. Results: Immunofluorescence, blots and immunoprecipitation experiments were positive for the presence of both tau and phosphorylated tau in platelets. Immunoblots showed the presence of high molecular weight fractions that appear to correspond to aggregated forms of tau. The relative amount of tau and phospho-tau were relatively stable at different ages, but the ratio of aggregated species to normal tau was significantly higher in AD patients than controls. A correlation was evidenced between tau species and cognitive impairment. Conclusions: The present is the first description of tau in platelets. The assessment of tau species in platelets provides a tool for establishment of a novel biological marker for AD.

P3-199

RELATIONSHIP BETWEEN CSF BIOMARKERS AND LANGUAGE FLUENCY IN ASYMPTOMATIC MIDDLE-AGED ADULTS AT INCREASED RISK FOR ALZHEIMER’S DISEASE: THE ESPRIT STUDY

Hanna M. Blazel1,2, Jodi Barnet1,2, Carey E. Gleason1,2, N. Maritza Dowling1,2, Bruce P. Hermann1,2, Sterling C. Johnson1,2, Henrik Zetterberg3, Kaj Blennow3, Alyce Marsh1, Luigi Puglielli1,2, Craig Atwood1,2, Mark A. Sager1,2, Sanjay Asthana1,2, Cynthia M. Carlsson1,2, 1University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2Wisconsin Alzheimer’s Disease Research Center, Madison, WI, USA; 3Sahlgrenska Academy at University of Gothenburg, Molndal, Sweden. Contact e-mail: [email protected]

Background: Patients with Alzheimer’s disease (AD) demonstrate characteristic changes in CSF biomarkers, showing reduced CSF beta-amyloid (Ab) and elevated tau levels. However, the onset and course of these changes is yet to be described in preclinical stages. Additionally, the relationship between preclinical CSF biomarkers, genetic risk, and cognitive abilities remains uncertain. Methods: Baseline neuropsychological and CSF data from asymptomatic middle-aged adult children of persons with AD were included in this analysis. Verbal fluency measures included category (animal) and letter (FAS) fluency tasks, collected on the same day as the CSF biomarker data. Spearman correlations were used to identify rela-

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tionships between verbal fluency scores and CSF Ab and tau measures. Results: Participants (n ¼ 100, mean SD age 53.4 6 7.9, 70% women, education 16.2 6 2.9 yrs, 38% ApoE4 positive) were cognitively healthy at baseline (MMSE 29.5/30). In ApoE4 carriers, lower category verbal fluency scores were significantly correlated with reduced CSF Ab 42 levels (n ¼ 32, r ¼ .378, p ¼ .033). An inverse relationship was noted between letter verbal fluency scores and CSF T-Tau levels (n ¼ 32, r ¼ -.344, p ¼ .054) in ApoE4 carriers. These relationships were not noted in ApoE4 non-carriers. Conclusions: In the ESPRIT study, there is evidence to suggest an association between genetic risk, CSF biomarkers and verbal fluency performance in middle-aged, cognitively asymptomatic individuals at-risk for AD. These data support previous findings indicating that disease processes may be occurring decades before the onset of symptoms. Further investigation of these preclinical findings is warranted for continued study of early detection in AD.

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METHIONINE SULFOXIDE, AN INDEX OF OXIDATIVE STRESS, MEASURED IN PLASMA OF PERSONS WITH FAMILIAL ALZHEIMER’S DISEASE MUTATIONS

John M. Ringman1, Andrew T. Fithian2, Karen Gylys1, Jeffrey L. Cummings1, Giovanni Coppola2, Domenico Pratico3, Jackob Moskovitz4, Gal Bitan1, 1Easton Center for Alzheimer’s Disease Research at UCLA, Los Angeles, CA, USA; 2University of California, Los Angeles, Los Angeles, CA, USA; 3Temple University, Philadelphia, PA, USA; 4 University of Kansas, Lawrence, KS, USA. Contact e-mail: jringman@ mednet.ucla.edu Background: There is convergent evidence for increased oxidative stress in persons with Alzheimer’s disease that may play a role in propagating the illness. Various oxidized molecules can be measured in biological fluids reflecting the level of oxidative stress present. We have preliminary evidence that F2-isoprostanes are elevated in CSF and plasma of persons carrying PSEN1 and APP mutations, suggesting a role of oxidative stress in familial AD (FAD). Oxidation of proteins at methionine residues (methionine sulfoxide or MetO) can be measured using a novel polyclonal antibody and has been reported to be increased in late-onset AD. We sought to ascertain if MetO was elevated in the plasma of persons carrying FAD mutations. Methods: Plasma was collected from 30 persons from families harboring PSEN1 or APP mutations. Using a polyclonal antibody raised against an oxidized methionine-rich zein protein (antiMetO-DZS18), MetO levels were measured and compared between FAD mutation carriers (MCs) and their non-mutation carrying (NCs) kin. A subset of subjects (n ¼ 15) had plasma F2-isoprostane levels measured and these were correlated with MetO levels. Results: Of 30 subjects, 18 were MCs (PSEN1 ¼ 13, APP ¼ 5) and 12 were NCs. Among MCs, 3 were demented, 5 had questionable cognitive impairment, and 10 were pre-symptomatic. Using the anti-MetO-DZS18 antibody, four electrophoretic bands were identified with apparent mobilities corresponding to 120, 135, 160, and 200 kilodaltons (kDa). Visual inspection by blinded investigators suggested that the 120 kDa band had the greatest variability among subjects. The percent area of the gel occupied by the 120 kDa band was non-significantly greater in FAD MCs than NCs (mean 11.8 vs. 4.0, Mann-Whitney U test: p ¼ 0.079, t-test: p ¼ 0.019). There was no clear relationship between disease status, age or mutation type and MetO levels. For the 15 subjects for whom both MetO and F2-isoprostane levels were available, the correlation was 0.80 (p < 0.001). Conclusions: We have preliminary evidence for elevated MetO levels in plasma of persons carrying FAD mutations though the relationship to disease status is unclear. MetO levels measured using the anti-MetO-DZS18 antibody correlate well with those of F2-isoprostane levels measured using mass spectrometry.