Oral Sessions: O1-02: Biomarkers: Novel Biomarkers in Blood and CSF

cleavage of APP. This increase was abolished by inhibiting g -secretase,

suggesting that the two protease activities are functionally linked. Interestingly, directing cells to different neuronal fates resulted in altered cleavage
patterns of APP. From both control and fAD lines, Ab generated from neurons directed to forebrain cortical fates showed a higher Ab42 to Ab40 ratio
relative to neurons directed to more caudal fates of the hindbrain and spinal
cord. Moreover, consistent with fate-specific biochemical effects on Ab, the
APP V717I mutation led to increased Tau expression in forebrain cultures
but not in more caudally directed neurons. Conclusions: These studies identify previously unappreciated effects of an fAD APP mutation in human
neurons and demonstrate that human iPSC technology provides a powerful
system for analyzing the effects of both genetic alterations and cell types on
processes directly relevant to human brain diseases.
O1-01-06

HUMAN STEM CELL MODELS OF

FRONTOTEMPORAL DEMENTIA CAUSED BY
A NON-CODING HEXANUCLEOTIDE REPEAT
EXPANSION IN C9ORF72

Selina Wray1, Elisavet Preza1, Sara Rollinson2, Mina Ryten1,

Adrian Isaacs1, Henry Houlden1, Martin Rossor3, Adriano Chio4,
Huw Morris5, Stuart Pickering-Brown6, John Hardy3, Rick Livesey7,
Tilo Kunath8, 1UCL Institute of Neurology, London, United Kingdom;
2
Manchester University, Manchester, United Kingdom; 3University College
London, London, United Kingdom; 4University of Torino, Torino, Italy;
5
Cardiff University, Cardiff, United Kingdom; 6University of Cambridge,
Manchester, United Kingdom; 7University of Cambridge, Cambridge,
United Kingdom; 8University of Edinburgh, Edinburgh, United Kingdom.
Contact e-mail: selina.wray@ucl.ac.uk
Background: A non-coding hexanucleotide repeat expansion in C9ORF72
is the most common genetic cause of frontotemporal dementia and motor
neuron disease. In order to generate physiologically-relevant cell models
of C9ORF72 mediated FTD to study disease mechanisms, we have generated iPSC from 3 patient fibroblast lines carrying the C9ORF72 mutation.
Methods: iPSC were generated by retroviral expression of the pluripotency-associated transcription factors Oct3/4, Sox2, Klf4 and cMyc. Fibroblasts expressing the reprogramming factors were maintained on SNL
feeder cells for 25-30 days and emerging iPSC colonies with an embryonic
stem cell-like morphology were picked for further expansion and characterisation. iPSC were differentiated into cortical neurons by dual SMAD inhibition followed by an extended period of in vitro corticogenesis (Shi et al,
2012). Results: 10-15 iPSC lines have been generated per patient, and these
have been characterised by standard methods including immunoreactivity
for the stem cell markers SSEA4, Tra-1-81, Nanog and Oct4, RT-qPCR to
ensure silencing of retroviruses and a pluripotency gene expression signature as determined by pluritest. The presence, size and stability of the repeat
expansion during reprogramming was analysed by repeat-primed PCR and
Southern blot. Interestingly, we found a contraction in repeat size in some
iPSC lines compared to the starting fibroblast line. iPSC were differentiated
into cortical neurons and their identity was confirmed by the expression of
cortical markers including Tbr1 and Ctip2. Conclusions: C9ORF72 iPSC
have been differentiated into cortical neurons and these are currently being
characterised for the presence of RNA foci, TDP-43 pathology and gene expression signatures of disease. Patient-derived C9ORF72 cortical neurons
will provide a powerful model to understand the molecular mechanisms underlying C9FTD/ALS, in addition to establishing a platform for drug screening.
ORAL SESSIONS: O1-02
BIOMARKERS: NOVEL BIOMARKERS IN BLOOD AND CSF
O1-02-01

A CASPASE-GENERATED EPITOPE OF TAU

MEASURED IN SERUM PREDICTS PROGRESSION
OF DISEASE IN EARLY ALZHEIMERS DISEASE
PATIENTS

Kim Henriksen1, Dilek Inekci1, Kevin Duffin2, Bob Dean2,

Morten Karsdal3, 1Nordic Bioscience, Herlev, Denmark; 2Eli Lilly and

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Company, Indianapolis, Indiana, United States; 3Nordic Bioscience A/S,

Herlev, Denmark. Contact e-mail: kh@nordicbioscience.com
Background: Biomarkers enabling detection of early neurodegeneration
are needed to identify patients at risk of Alzheimers disease. However, serological biomarkers for AD are hampered by their restricted crossing of the
blood-brain-barrier (BBB). Enzyme-generated protein fragments (neo-epitopes), due to their small size, will pass the BBB. Caspase-mediated cleavage of micro-tubule associated protein Tau is central in the initiation of
neuronal death and thereby AD, hence we focused on developing an ELISA,
which specifically detects a caspase-3 generated fragment of Tau. Methods:
A competive ELISA (Tau-C) specifically recognizing the Asp421 sequence
generated by Caspase-3 was developed and optimized for serum reactivity.
Recombinant Tau and rat brain extracts were cleaved using caspase-3 to validate specificity of the epitope. Brains extracts from Tg4510 tauopathy mice
were used to confirm pathological relevance. Lysates of apoptotic neurons
were evaluated in the ELISA. Baseline serum samples from 100 early AD
patients with both baseline and follow-up ADAS-Cog11 assessments were
used to assess the diagnostic and prognostic potential of the marker.
Results: The Tau-C antibody recognized the cleavage site in tau generated
by Caspase with an IC 50 of 19ng/mL. The antibody also showed a clear separation between intact and Caspase-cleaved Tau and brain extracts. Brain
extracts from Tg4510 mice had 2-fold higher levels of the tau neo-epitope
than wt controls (p<0.05). In apoptotic neurons Tau-C could be detected,
while only background levels were observed in the control cells. The assay
performed well in serum, with robust and reproducible levels of analyte detected. In the AD patients, no correlation with baseline ADAS-Cog11 was
observed; however, a significant correlation to change in ADAS-Cog11
score over both 28 and 64 weeks was observed (R0.4, P<0.05).
Conclusions: We developed a technically robust and sensitive ELISA for
measurement of a brain-specific tau neo-epitope in serum and plasma samples from humans and rodents. Importantly, Tau-C shows a correlation to
change in cognitive function, which clearly indicates that this marker is
the first serum marker, which can predict progression of AD.
O1-02-02

ALTERED BLOOD THIAMINE METABOLISM IN

ALZHEIMERS DISEASE

Chunjiu Zhong, Xiaoli Pan, Guoqiang Fei, Jingwen Lu, Zhongshan

Hospital, Fudan University, Shanghai, China. Contact e-mail: zhongcj@
163.com
Background: Reduced cerebral glucose metabolism is an invariant feature
in Alzheimers disease. A key coenzyme of glucose metabolism and the active form of thiamine, thiamine diphosphate, has been shown to be significantly reduced in Alzheimers disease. Altered blood thiamine metabolism
may represent a usefulbiomarker for the diagnosis of Alzheimers disease.
Methods: Control subjects with normal cognition (n500) and patients
with Alzheimers disease(n110) and vascular dementia (n53) were recruited. Blood thiamine diphosphate, thiamine monophosphate and thiamine levels as well as the activities of blood thiamine pyrophosphokinase
were measured using high performance liquid chromatography. Results:
Bloodthiamine diphosphatelevels in patients with Alzheimers disease
were significantly decreased as compared with control subjects
(82.3162.23 vs. 118.6061.12 nmol/L, P<0.001), whereas bloodthiamine
monophosphateand thiamine levels were increased (5.2760.47 vs.
3.5960.09 nmol/L, P<0.001 and 4.2160.42 vs. 3.4160.11 nmol/L,
P<0.01). Bloodthiamine diphosphatelevel exhibited significantly diagnostic value, with an area under receiver operating characteristic curve
(AUC)of 0.871, a sensitivity of 80.00% and a specificity of 79.60%. The ratio of blood thiamine diphosphate level over seventh square root of blood
thiamine level exhibited an even higher diagnostic value, with an AUC of
0.893, a sensitivity of 80.00% and a specificity of 84.00% when the cutoff point was 85.16. Over two-thirds of patients with vascular dementiawere
above this cut-off point and could be differentiated from patients with Alzheimers disease. In Alzheimers disease patients with APOE 4 alleles, the
ratio of blood thiamine diphosphate level over seventh square root of blood
thiamine level exhibited an excellent diagnostic power with an AUC of
0.906, a sensitivity of 82.69% and a specificity of 84.60%. Of mechanism,

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Oral Sessions: O1-02: Biomarkers: Novel Biomarkers in Blood and CSF

altered blood thiamine metabolism was associated with reduced activities of

thiamine pyrophosphokinase in patients with Alzheiemrs disease as compared with control subjects (1.24 6 0.06 vs. 1.44 6 0.07, P<0.05).
Conclusions: The patients with Alzheimers disease manifest significant
abnormality in blood thiamine metabolism as compared with normal cognitive subjects and patients with vascular dementia. Altered blood thiamine
metabolism represents an ideal diagnostic biomarker for Alzheimers disease with the reliable, non-invasive, simple to perform and inexpensive
merits, and is associated with decreased thiamine pyrophosphokinase activities.
O1-02-03

ESTABLISHING APOLIPOPROTEIN J AS
A POTENTIAL CANDIDATE FOR ALZHEIMERS
DISEASE BIOMARKER PANEL: AUSTRALIAN
IMAGING, BIOMARKER AND LIFESTYLE (AIBL)
FLAGSHIP STUDY OF AGING

Veer Bala Gupta1, Christopher Rowe2, Cassandra Szoeke3, David Ames4,

Lynne Cobiac3, Ashley Bush5, Kathryn Ellis6, Lance Macaulay7,
Colin Masters8, Ralph Martins1, 1Edith Cowan University, Perth, Australia;
2
Austin Hospital, Melbourne, Australia; 3CSIRO, Melbourne, Australia;
4
National Ageing Research Institute Inc. (NARI), Parkville, Australia;
5
Florey Institute of Neuroscience & Mental Health, Parkville, Australia; 6St
Georges Hospital, Kew, Australia; 7CSIRO, Parkville, Australia;
8
University of Melbourne, Melbourne, Australia. Contact e-mail: v.gupta@
ecu.edu.au
Background: Apolipoprotein J (apoJ) is a multifunctional protein, functions as a chaperone of misfolded extracellular proteins. It participates in
functions like cell proliferation and apoptosis. Its levels are increased in
the CSF of patients with AD. The plasma levels have been correlated to
brain atrophy, baseline disease severity and clinical progression in AD.
The present study includes longitudinal plasma measurement of apoJ in order to establish it as a potential analyte to be included in AD biomarker panel
of AIBL. Methods: ApoJ protein levels were measured in the baseline and
18-month follow-up plasma samples from 833 individuals participating in
the Australian Imaging, Biomarkers and Lifestyle (AIBL) Longitudinal
Study of Ageing. It was assayed by a commercially available ELISA kit
(Human Clusterin ELISA, R and D systems). apoJ plasma levels were
then compared between healthy controls (HC), mild cognitively impaired
(MCI) individuals and Alzheimers disease (AD) patients. The data was further compared against other collected demographics including but not limited to cerebral amyloid load as measured by positron emission tomography
(PET), hippocampal volume, APOE genotype and neuropsychological
scores. Results: MCI and AD had significantly higher levels of apoJ at b aseline and 18-month data. MCI group had highest percentage change between
the two time points viz. 2% (HC), 7% (MCI), 0.5% (AD). Significant correlation was observed with PiB-PET SUVR and hippocampal volumes. Increased levels of plasma apoJ correlate with cognitive decline at both
baseline and 18 months. a poJ levels were significantly higher in apoE4 carriers, as compared to the non apoE4 carriers at both the time points. Further
analysis will determine the relationship between plasma apoJ and other potential biomarkers such as apoE. Conclusions: The results of the present
study show that apoJ is a potential candidate to be included in AIBL "AD
biomarker panel" for the future development of an AD diagnostic assay.
Completed measurements of further time point plasma samples from the
same individuals will add great value to this study, by analysing the changes
in plasma apoJ over time.
O1-02-04

THE BETA-AMYLOID AGGREGATE COUNT IN

CSF IS A BIOMARKER FOR ALZHEIMERS
DISEASE

Dieter Willbold1, Susanne Funke1, Lei Wang-Dietrich1, Kun Wang1,

Oliver Bannach2, Eva Birkmann1, 1Forschungszentrum Julich, Julich,
Germany; 2Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany.
Contact e-mail: d.willbold@fz-juelich.de
Background: Alzheimers disease (AD) is a fatal neurodegenerative and
progressive disorder. Currently, no reliable biomarker for pre-symptomatic

diagnosis or therapy monitoring is available. Recent studies support that especially soluble Ab oligomers are the major toxic species during development and progression of AD. Therefore, we suggest that the number of
Ab oligomers in body fluids can be used as the most direct biomarker for
AD. Methods: Here, we present an optimized version of the single aggregate sensitive surface-based fluorescence intensity distribution analysis
(sFIDA) assay. For the first time, it allowed the determination of the Ab oligomer count in cerebrospinal fluid (CSF). Results: We challenged the assay
with CSF samples of 14 AD patients and 12 age-matched control subjects.
The Ab oligomer count allowed a surprisingly clear distinction between
both groups. All samples of the control group showed homogenously low
numbers of Ab oligomers. The samples of the AD group had comparably
high levels of Ab oligomers and displayed high variability. The Ab oligomer
levels clearly correlated with the patients mini-mental state examination
(MMSE) scores. Conclusions: Our results support the idea that Ab oligomers play a decisive role in AD pathology and in turn can be used as a diagnostic biomarker. The sFIDA assay is able to reliably quantify the Ab
oligomers in human CSF. The correlation between MMSE and Ab oligomer
count suggests that the quantitity of Ab oligomers in CSF even reflects the
severity of the disease. Further studies will show whether anti-Ab targeted
therapies can affect the Ab oligomer counts in treated individuals.
O1-02-05

IDENTIFYING MULTI-ANALYTE CSF

BIOMARKERS FOR ALZHEIMERS DISEASE IN
A MULTI-COHORT STUDY

Yuk Yee Leung1, Juan Toledo2, Alexey Nefedov1, Robi Polikar3,

Nandini Raghavan4, Sharon Xie2, Michael Farnum5, Tim Schultz5,
Young Baek2, Victor Lobanov5, Allitia DiBernardo6, Vivianna Van
Deerlin1, Mitchel Kling7, Alice Chen-Plotkin1, Matthew Mailman8,
William Hu9, Richard Perrin10, Anne Fagan14, Murray Grossman1,
David Holtzman10, Holly Soares11, John Morris10, David Baker8,
Steven Arnold2, Vaibhav Narayan6, Virginia Lee1, Leslie Shaw12,
Gayle Wittenberg6, Li-San Wang2, John Trojanowski2, ALZHEIMERS_DISEASE
NEUROIMAGING_INITIATIVE13,1University of Pennsylvania, Philadelphia,
Pennsylvania, United States; 2University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania, United States; 3Rowan University,
Glassboro, New Jersey, United States; 4Janssen Pharmaceuticals, Raritan,
New Jersey, United States; 5Janssen Pharmaceuticals, Spring House,
Pennsylvania, United States; 6Janssen Pharmaceuticals, Titusville, New
Jersey, United States; 7Perelman School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania, United States; 8Janssen R&D,
Titusville, New Jersey, United States; 9Emory University, Atlanta, Georgia,
United States; 10Washington University, St. Louis, St. Louis, Missouri,
United States; 11Bristol Myers Squibb, Wallingford, Connecticut, United
States; 12University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania, United States; 13Boston University, Boston, Massachusetts,
United States. Contact e-mail: yyee@mail.med.upenn.edu
Background: Ab 42 and tau cerebrospinal fluid (CSF) values measured by
ELISA and xMAP technologies are currently used to differentiate between
Alzheimers disease (AD) against cognitively normal (CN) subjects, yet the
standardization of these tests are still underway. It seems likely that a combined set of biomarkers will define a patient-specific signature to diagnose
AD in the future. The utilization of a discovery-based multi-analyte platform (MAP) by Rules Based Medicine allows for the exploration of
a broader set of protein analytes, with the aim of improving the accuracy
of AD diagnosis upon current classical CSF biomarkers. Yet special pre-processing steps are required for proper removal of any experimental biases
present inside these multiplex data. Methods: CSF samples from AD and
CN populations were obtained from three independent cohorts: Alzheimers
disease Neuroimaging Initiative, University of Pennsylvania and Washington University. We first performed quality control steps for data from the
MAP panel, including 1) removal and imputation of analytes with missing
and low values; 2) adjustment of sample outliers by flooring/ceiling their respective protein levels; 3) logarithmic transformation to ensure analytes are
of normal distribution; and 4) z-score transformation to account for batch
effects across cohorts. 54 common analytes were left across these cohorts.