Novel diagnostic cerebrospinal fluid protein biomarkers for pathologic subtypes of frontotemporal dementia identified by proteomics

Abstract

Introduction:
Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotemporal dementia (FTD) and its pathologic subtypes are lacking.

Methods:
Unbiased high-resolution mass spectrometry-based proteomics was applied on CSF of FTD patients with TAR DNA-binding protein 43 (TDP-43, FTD-TDP, n = 12) or tau pathology (FTD-tau, n = 8), and individuals with subjective memory complaints (SMC, n = 10). Validation was performed by applying enzyme-linked immunosorbent assay (ELISA) or enzymatic assays, when available, in a larger cohort (FTLD-TDP, n = 21, FTLD-tau, n = 10, SMC, n = 23) and in Alzheimer's disease (n = 20), dementia with Lewy bodies (DLB, n = 20), and vascular dementia (VaD, n = 18).

Results:
Of 1914 identified CSF proteins, 56 proteins were differentially regulated (fold change >1.2, P < .05) between the different patient groups: either between the two pathologic subtypes (10 proteins), or between at least one of these FTD subtypes and SMC (47 proteins). We confirmed the differential expression of YKL-40 by ELISA in a partly independent cohort. Furthermore, enzyme activity of catalase was decreased in FTD subtypes compared with SMC. Further validation in a larger cohort showed that the level of YKL-40 was twofold increased in both FTD pathologic subtypes compared with SMC and that the levels in FTLD-tau were higher compared to Alzheimer's dementia (AD), DLB, and VaD patients. Clinical validation furthermore showed that the catalase enzyme activity was decreased in the FTD subtypes compared to SMC, AD and DLB.

Discussion:
We identified promising CSF biomarkers for both FTD differential diagnosis and pathologic subtyping. YKL-40 and catalase enzyme activity should be validated further in similar pathology defined patient cohorts for their use for FTD diagnosis or treatment development.

Full text

CSF Biomarkers
Novel diagnostic cerebrospinal fluid biomarkers for pathologic subtypes
of frontotemporal dementia identified by proteomics
Charlotte E. Teunissen
a,
*, Naura Elias
a
, Marleen J. A. Koel-Simmelink
a
, Sisi Durieux-Lu
a
,
Arjan Malekzadeh
a
, Thang V. Pham
b
, Sander R. Piersma
b
, Tommaso Beccari
c
,
Lieke H. H. Meeter
d
, Elise G. P. Dopper
d,e
, John C. van Swieten
d,e
, Connie R. Jimenez
b,
*,
Yolande A. L. Pijnenburg
e,
*
a
Neurochemistry Laboratory and Biobank, Department of Clinical Chemistry, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam,
The Netherlands
b
OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
c
Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
d
Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands
e
Alzheimer Center & Department of Neurology, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
Abstract Introduction: Reliable cerebrospinal fluid (CSF) biomarkers enabling identification of frontotem-
poral dementia (FTD) and its pathologic subtypes are lacking.
Methods: Unbiased high-resolution mass spectrometry–based proteomics was applied on CSF of FTD
patients with TAR DNA-binding protein 43 (TDP-43, FTD-TDP, n 512) or tau pathology (FTD-tau,
n58), and individuals with subjective memory complaints (SMC, n 510). Validation was performed
by applying enzyme-linked immunosorbent assay (ELISA) or enzymatic assays, when available, in a
larger cohort (FTLD-TDP, n 521, FTLD-tau, n 510, SMC, n 523) and in Alzheimer’s disease
(n 520), dementia with Lewy bodies (DLB, n 520), and vascular dementia (VaD, n 518).
Results: Of 1914 identified CSF proteins, 56 proteins were differentially regulated (fold change
.1.2, P,.05) between the different patient groups: either between the two pathologic subtypes
(10 proteins), or between at least one of these FTD subtypes and SMC (47 proteins). We confirmed
the differential expression of YKL-40 by ELISA in a partly independent cohort. Furthermore,
enzyme activity of catalase was decreased in FTD subtypes compared with SMC. Further validation
in a larger cohort showed that the level of YKL-40 was twofold increased in both FTD pathologic
subtypes compared with SMC and that the levels in FTLD-tau were higher compared to Alzheimer’s
dementia (AD), DLB, and VaD patients. Clinical validation furthermore showed that the catalase
enzyme activity was decreased in the FTD subtypes compared to SMC, AD and DLB.
Discussion: We identified promising CSF biomarkers for both FTD differential diagnosis and path-
ologic subtyping. YKL-40 and catalase enzyme activity should be validated further in similar pathol-
ogy defined patient cohorts for their use for FTD diagnosis or treatment development.
Ó2016 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Keywords: Biomarkers; Cerebrospinal fluid; Proteomics; Frontotemporal dementia; Pathology; TDP-43; Tau; Differential
diagnosis
1. Introduction
Frontotemporal dementia (FTD) is the second most prev-
alent dementia of patients aged ,65 years that clinically
presents with either behavior and personality changes or
*Shared senior authors.
*Corresponding author. Tel.: 131-20-4443680; Fax: 131-20-4443895.
E-mail address: c.teunissen@vumc.nl
http://dx.doi.org/10.1016/j.dadm.2015.12.004
2352-8729/ Ó2016 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-94

language disturbance. The disease is often misdiagnosed in
the early stage, either as a psychiatric disorder or as a
different type of dementia such as Alzheimer’s dementia
(AD). The pathology is characterized by two main distinct
subtypes, i.e., tau pathology accounting for roughly one
half of cases and TAR DNA-binding protein 43 (TDP-43)–
pathology for the other half [1,2]. The clinical spectrum of
FTD does not correlate with the distinct pathologies,
except when the underlying pathology of FTD is predicted
by the presence of an autosomal dominant mutation, which
is found in only 20%–30% of the patients [3]. Mutations
in the C9orf72 and GRN genes correspond to TDP-43
pathology, and mutations in the microtubule-associated pro-
tein tau (MAPT) to tau pathology, but an autosomal domi-
nant family history is found in only 20%–30% of the
patients. In addition, FTD with amyotrophic lateral sclerosis
or motor neuron disease is almost always associated with un-
derlying TDP-43 pathology [4]. Correct diagnosis and sub-
typing is very relevant to determine patient management
plans and boost therapy development, especially to develop
treatments targeting either tau or TDP-43 pathologic
mechanisms.
Thus far, no reliable biomarker or set of biomarker with
both high sensitivity and high specificity is available for
FTD, let alone its pathologic subtypes. The cerebrospinal
fluid (CSF) biomarkers for AD, i.e., (p)Tau and amyloid
b-42 (Ab42), appear to be of limited value for the diagnosis
of clinical FTD [5,6], although a prognostic value of tau in
diagnosed FTD patients has been reported [7]. A reduced
CSF P-tau-181-to-tau ratio has recently been found to iden-
tify patients with TDP-43 pathology at a sensitivity and
specificity of each 82% [8], which awaits independent vali-
dation.
A good technology to identify multiple novel bio-
markers in body fluids is mass spectrometry–based prote-
omics [9]. So far, no comprehensive discovery at the
protein level has been performed as previous proteomics
studies used low resolution methods for profiling of a
limited set of abundant CSF peptides or proteins in clini-
cally defined FTD patient groups [10–14]. A recent
immunoassay-based proteomics study focused on an anal-
ysis of 151 biomarkers for different pathologic subtypes
of FTD. This has yielded several proteins that discrimi-
nated FTD-TDP-43 and FTD-tau, including interleukins
(IL-23 and IL-7), which combined had an 86% sensitivity
and 78% specificity [15].
In the present study, we aimed to identify novel
pathology-specific biomarkers for FTD by in-depth protein
profiling of antemortem collected CSF of FTD patients
with known underlying pathology. We applied unbiased
CSF proteomics methods [16] in patients with confirmed
tau or TDP-43 pathology, either by genetic testing or post-
mortem analysis, and controls with subjective memory com-
plaints (SMC). We validated the findings using alternative
assays in a largely independent cohort and in patients with
other dementias.
2. Methods
2.1. Patients
Patients with established FTD subtypes and SMC were
included from the biobanks of the Amsterdam Dementia
Cohort and from the Erasmus MC. The method to define
the pathology to is outlined in Table 1, which shows that
the majority was based on postmortem examination. FTD
pathology was reviewed according to international criteria
[17]. Pathologic examination was performed according to
protocolized procedures by the Dutch brain bank, including
specific immunostaining for TDP-43 pathology and tau pa-
thology. Genetic testing was performed for mutations in
the MAPT and progranulin genes and for the hexanucleotide
repeat at C9orf72. The discovery cohort contained 30 pa-
tients, the validation cohort 53 patients, and 17 of the 53 pa-
tients in the validation cohort overlapped with the discovery
cohort, as outlined in Table 1.
All subjects underwent extensive dementia screening at
baseline, including physical and neurologic examination,
mini-mental state examination (MMSE), neuropsychological
investigation, electroencephalogram,magnetic resonance im-
aging, and laboratory tests, including lumbar puncture. De-
mentia diagnoses were made by consensus in a
multidisciplinary meeting according to standard criteria
[18,19]. Probable AD was diagnosed according to the
criteria of the National Institute of Neurological and
Communicative Disorders and Stroke-Alzheimer’s Disease
and Related Disorders association [20], and all patients met
the core clinical National Institute on Aging-Alzheimer’s As-
sociation criteria [21]. Definite FTD was diagnosed using the
criteria of Rascovsky et al. [2]. Control groups of subjects
with SMC consisted of individuals who presented with cogni-
tive complaints, but cognitive and laboratory investigations,
were normal and criteria for mild cognitive impairment, de-
mentia, or any other neurologic or psychiatric disorders
known to cause cognitive complaints were not met. CSF bio-
markers abeta (1–42), tTau, and pTau were not used for the
clinical diagnosis of any of the patients. Groups were matched
for age and gender. Patient characteristics of the discovery and
validation cohorts are presented in Table 1.
The study was performed according to the ethical
principles of the Declaration of Helsinki and was appro-
ved by the local ethics committee. We obtained written
informed consent from all subjects participating in the study.
2.2. CSF biobanking and proteomics analysis
CSF and blood were collected and stored at 280Cin
polypropylene tubes (Sarstedt, N€
umbrecht, Germany) after
centrifugation within 1 hourafter withdrawal according to in-
ternational biobanking consensus guidelines optimized for
CSF proteomics [22].CSFAb42, total tau, and p-tau were
measured with commercially available enzyme-linked immu-
nosorbent assays (ELISAs; INNOTEST Fujirebio, Ghent,
Belgium) on a routine basis as described previously [23].
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-94 87

Proteomics analysis was essentially performed as de-
scribed previously [16] and details are presented in
Supplementary File 1. Because this is a typical proteomics
discovery study, where the candidates are to be validated,
we did not apply a multiple testing correction and applied
aPvalue of .05.
2.3. Independent assay validation
The following assays were used: human adipocyte fatty
acid binding protein (FABP4) ELISA kit was obtained
from BioVendor (Karasek, Czech Republic). The MicroVue
YKL-40 enzyme immunoassay kit was from Quidel Corpo-
ration (San Diego, USA). Complement factor D (CFD)
Quantikine ELISA and interleukin 1 receptor accessory pro-
tein (IL1RAP) DuoSet ELISA were from R&D Systems
(Abingdon, UK). Human apolipoprotein L1 (APOL1)
ELISA was from Proteintech (Manchester, UK). Catalase
enzyme activity was measured using an EnzyChrom
Assay from BioAssay Systems (Hayward, CA, USA). Total
b-hexosaminidase (HexA) activity was measured using
3-mM 4-methylumbelliferyl-2-acetamido-2-deoxy-b-D-
glucopyranoside (4MUGlcNAc) as a substrate in 0.1-M cit-
ric acid/0.2-M disodium phosphate buffer. b-HexA was
measured using 3-mM 4-Methylumbelliferyl 6-Sulfo-2-
acetamido-2-deoxy-b-D-glucopyranoside in 0.1 M citric
acid/0.2 M disodium phosphate buffer. a-Galactosidase A
(GLA) was measured using 4.5-mM 4-methylumbelliferyl-
a-D-galactoside in 0.2-M Na/acetate buffer.
Candidate biomarkers for validation were selected based
on the following criteria: (1) fold change .1.2 and Pvalue
,.05; (2) mean spectral count in all patients in one of the
patients groups .2; and (3) number of identified peptide se-
quences covering at least 20% of the protein. We searched
for availability of ELISAs and selected those with a detailed
validation report (i.e., recovery, linearity, and coefficients of
variation (CV) presented of individual samples). We briefly
tested the analytical performance of all assays for analysis of
CSF as described in Supplementary File 1. We, furthermore,
performed extensive validation of the FABP4 and YKL-40
assays according to the immunoassay validation SOP devel-
oped by the BIOMARKAPD project [24], the results of
which are also presented in Supplementary File 1. Precision
data of all assays are presented in Table 2.
2.4. Statistics
Statistics were performed in SPSS (version 20). None of
the validation results had a normal distribution, as tested by
the Shapiro-Wilk test. Differences in mean values between
clinical groups obtained by ELISA were analyzed with
one-way analysis of variance on the ranked values, correcting
for age as indicated in the results. Spearman correlation
Table 1
Characteristics of included patient populations
Characteristics Age (y)
Sex
(female/
male)
MMSE
score
Disease
duration (y) n
Amsterdam/
Rotterdam Method pathology definition
FTD discovery cohort
SMC 60.9 (15.7) 5/5 10 5/5
TDP-43-FTD 60.9 (5.7) 5/7 12 3/9 PGRN mutation (n 52)/pathology
confirmed (n 510)
Tau-FTD 53.4 (6.7)*3/5 8 2/6 MAPT mutation (n 57)/Pathology
confirmed (n 51)
FTD validation cohort
SMC 60.7 (10.1) 12/9 27.3 (2.9) 23 20/3
TDP-43-FTD 60.5 (5.8) 10/12 26.2 (3.1) 3.3 (2.5) (n 518) 20 12/8 PGRN mutation (n 52)/C9orf72
hexanucleotide repeat (n 52)/
FTD-MND (n 52)/pathology
confirmed (n 514)
Tau-FTD 55.2 (13.6)*6/3 NA 2.9 (1.0) (n 58) 10 4/6 MAPT (n 56)/pathology
confirmed (n 54)
Overlap discovery and validation cohort
SMC 60.1 (19.2) 5/2 7 5/2
FTD-TDP-43 59.9 (4.7) 2/5 7 4/3 GRN (n 52)/pathology
confirmed (n 55)
FTD-Tau 51.5 (6.3) 1/2 3 0/3 MAPT (n 53)
Dementia validation cohort
Alzheimer’s disease (AD) 63.7 (6.9) 8/12 19.7 (6.9) 20
Dementia with Lewy bodies (DLB) 62.9 (5.1) 3/17 23.2 (4.8) 20
Vascular Dementia (VaD) 63.6 (5.4) 3/15 22.3 (4.8) 18
Abbreviations: MMSE, mini-mental state examination; FTD, frontotemporal dementia; TDP-43, TAR DNA-binding protein 43; SMC, subjective memory
complaints; MAPT, microtubule-associated protein tau; NA, not available; SD, standard deviation.
NOTE. Mean values (SD) are given.
*Significantly different between Tau-FTD and the other two groups (P,.05).
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-9488

analyses were performed to study correlations between
markers or demographics. A Pvalue ,.05 was considered
significant.
3. Results
3.1. Identification of discriminatory CSF biomarkers and
functions
To facilitate in-depth coverage of the CSF proteome, CSF
samples were subjected to high-abundant protein depletion
followed by SDS-PAGE (sodium dodecyl sulfate polyacryl-
amide gel electrophoresis) fractionation, in-gel tryptic
digestion, and nanoLC-MS/MS analysis. In total, 1914 pro-
teins were identified in CSF (Supplementary Table 1). The
beta binomial test for comparison of the protein spectral
counts in the three patient groups yielded 56 differentially
regulated proteins (Tables 3–7). Supplementary Fig. 1 shows
a heat map of the relative concentrations of these proteins in
each individual patient sample.
3.1.1. FTD-tau versus FTD-TDP-43
Ten proteins were differentially regulated between FTD-
tau and FTD-TDP-43 patients (Table 3). The largest negative
fold change was observed for KLK7 and F13A1 (,22.0
fold), and the largest positive fold change was observed
for UBL3 (.2.1-fold increase in FTD-TDP-43 compared
with FTD-tau).
3.1.2. FTD-tau versus both SMC and TDP-43
In total, six proteins were differentially regulated be-
tween FTD-tau patients compared with both SMC and
FTD-TDP-43 patients (Table 4). The proteins APOL1 and
N-Myc downstream regulated gene (NDRG)4 had the high-
est positive fold change (.3.5 higher spectral counts in
FTD-tau compared with both SMC and TDP-43).
3.1.3. FTD-tau versus SMC
In total, 25 proteins were differentially regulated between
FTD-tau patients compared with SMC only (Table 5). The
proteins IL1RAP, OLFML1 had the highest negative fold
change (,22.0 times decreased in FTD-tau compared
with SMC). The proteins MAT2A, CA1, CAT, and
S100A7 had the highest positive fold change (.2.4-fold in-
crease in FTD-tau compared with SMC).
3.1.4. FTD-TDP-43 versus SMC
In total, 10 CSF proteins were differentially regulated
(P,.05) between SMC and FTD-TDP-43 patients
(Table 6). Of these, GLA and LAMTOR had the largest
negative fold change (,22.5-fold decrease in FTD-TDP-
43 compared with SMC).
3.1.5. Both FTD-tau and FTD-TDP-43 versus SMC
In total, five proteins were differentially regulated be-
tween SMC and FTD-tau or FTD-TDP-43 patients
(Table 6). Among these, SPTBN5 had the largest negative
fold change (.2.5-fold decrease in both FTD subtypes
compared with SMC).
3.2. Validation by independent assays
Validation was performed in a small subset of biomarker
candidates for which independent assays were available
Table 3
Proteins that were differentially regulated between TDP-43-FTD and Tau-
FTD
Gene
name
tau versus
SMC, fold
change
P
value
TDP-43
versus
SMC
fold change Pvalue
Tau versus
TDP-43,
fold
change
P
value
KLK7 22.064 .029
F13A1 22.003 .026
HSPA8 21.634 .036
FSCN1 21.491 .032
CLIC4 21.481 .030
ABI3BP 21.268 .044
MOG 1.295 .042
HEXA 1.309 .009
CHID1 1.553 .035
UBL3 2.168 .045
Abbreviations: TDP-43, TAR DNA-binding protein 43; FTD, frontotem-
poral dementia; SMC, subjective memory complaints.
Table 4
Proteins that were differentially regulated between FTD-tau and both FTD-
TDP-43 and SMC
Gene
name
SMC
versus tau,
fold
change
P
value
SMC versus
TDP-43,
fold
change
P
value
TDP-43
versus tau,
fold
change
P
value
MYOC 21.817 .011 22.054 .016
SHBG 21.420 .026 21.527 .008
FCGBP 21.335 .002 21.427 .049
IGFALS 1.267 .005 1.299 .011
NDRG4 3.459 .000 2.729 .000
APOL1 5.127 .001 2.595 .009
Abbreviations: FTD, frontotemporal dementia; SMC, subjective memory
complaints; TDP-43, TAR DNA-binding protein 43.
Table 2
Assay precision of all assays included in the validation
Marker
Intra-assay
CV (%) Stdev
Inter-assay
CV (%) Stdev
APOL1 2.55 0.219 1.6–5.1 2.5
Catalase 10.33 0.17 34 0.1
FABP4 1.9–3.0 0.5 5.7–8.8 1.8
CFD 3.2 2.1 4.9 2.8
IL-RAcP 3.82 3.99 4.14 0.017
YKL-40 3.1–3.7 0.3 3.1–10.9 4.4
b-Hexosaminidase-A 3.24 0.73 3.15 0.70
a-Galactosidase A 5.68 2.43 5.73 1.41
Abbreviations: CV, coefficients of variation; FABP4, fatty acid binding
protein; CFD, Complement factor D; Stdev, standard deviation.
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-94 89

commercially or via collaboration as specified in the method
section.
Three of six tested ELISA assays met our validation
criteria for reliable detection in CSF. These were FABP4,
YKL-40, complement factor D, IL1RAP, and APOL1. No
assays to analyze the concentration were available, but
enzyme activity assays were available for catalase, total
HexA, b-HexA, and GLA.
The results in Fig. 1 show that the levels of FABP4 were
comparable between the FTD-patient groups and SMC.
The levels of the YKL-40 were twofold increased in both
FTD pathologic subtypes compared with SMC (Fig. 2),
which confirmed the increase in FTD-TDP-43 and FTD-
tau compared with SMC of the discovery results.
The complement factor D levels were comparable be-
tween the FTD patient groups and SMC (Fig. 3).
The catalase enzyme activities were decreased in both the
FTD-TDP-43 and FTD-tau groups compared with the con-
trols (Fig. 4), which is a reverse relation compared to the in-
crease observed in the protein concentration by proteomics.
The total HexA and GLA activity was not significantly
changed in any of the groups (data now shown).
3.3. Correlations with demographics and clinical
characteristics and between validated biomarkers
CSF YKL-40 was positively correlated with age. We,
therefore, included age as covariate in further analyses of
Table 6
Proteins that were differentially regulated between TDP-43-FTD and SMC
Gene name
SMC
versus tau,
fold
change
P
value
SMC versus
TDP-43,
fold
change
P
value
TDP-43
versus
tau, fold
change
P
value
GLA 25.512 .026
LAMTOR2 22.547 .035
MPZ 21.813 .043
TMEM132B 21.792 .017
IMPA1 21.480 .024
DLD 21.469 .045
GDA 21.205 .034
CPVL 21.351 .013
CTSL1 1.215 .014
ST6GAL2 1.611 .042
Abbreviations: FTD, frontotemporal dementia; SMC, subjective memory
complaints; TDP-43, TAR DNA-binding protein 43.
Table 7
Proteins that were differentially regulated between FTD and SMC (no
specific pathologic subtype)
Gene
name
SMC
versus
tau, fold
change
P
value
SMC versus
TDP-43,
fold
change
P
value
TDP-43
versus
tau, fold
change
P
value
SPTBN5 22.482 .040 22.947 .045
RAD23B 21.901 .016 21.539 .044
AP2B1 21.371 .031 21.306 .039
CFD 21.284 .033 21.223 .037
YKL-40 1.243 .020 1.276 .009
Abbreviations: FTD, frontotemporal dementia; SMC, subjective memory
complaints; TDP-43, TAR DNA-binding protein 43.
SMC TDP43 Tau
0
1
2
3
4
FABP4 (ng/mL)
Fig. 1. FABP4 levels in CSF of different patient groups as measured by
ELISA. Abbreviations: FABP4, fatty acid binding protein; CSF, cerebrospi-
nal fluid; ELISA, enzyme-linked immunosorbent assay; SMC, subjective
memory complaints; TDP-43, TAR DNA-binding protein 43.
Table 5
Proteins that were differentially regulated between Tau-FTD and SMC
Gene
name
SMC
versus
tau, fold
change
P
value
SMC versus
TDP-43,
fold
change
P
value
TDP-43
versus tau,
fold
change
P
value
IL1RAP 22.533 .034
OLFML1 22.013 .023
ISLR2 21.969 .014
CNTNAP3 21.850 .039
TENM2 21.655 .012
GALNS 21.462 .041
F5 21.424 .037
QDPR 21.368 .028
PCMT1 21.307 .049
CTSH 21.307 .018
OLFML3 21.277 .027
NOV 21.266 .011
LCAT 1.347 .032
FSTL5 1.406 .045
CDH15 1.519 .019
CASP14 1.660 .043
ACAN 1.671 .026
FABP4 1.710 .047
FAH 1.731 .018
VCL 1.856 .036
LRP8 1.901 .013
MAT2A 2.471 .022
CA1 3.283 .036
CAT 4.473 .031
S100A7 4.591 .007
Abbreviations: FTD, frontotemporal dementia; SMC, subjective memory
complaint; TDP-43, TAR DNA-binding protein 43; FABP4, fatty acid bind-
ing protein.
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-9490

this marker. CSF YKL-40 was positively correlated with
t-tau and negatively to catalase enzyme activity levels
(data not shown, Table 8). There were no correlations with
MMSE or difference in levels between females and males.
3.4. Validation in other dementia types
We next continued validation of the proteins for which a
differential expression was confirmed, i.e., YKL-40 and
catalase, by analysis of the levels in other common dementia
subtypes, AD, DLB, and vascular dementia (VaD).
The levels of YKL-40 (Fig. 2) were higher in FTD-tau
compared with AD (P,.001), dementia with Lewy bodies
(DLB, P,.001), and VaD (P5.001).
The levels of catalase (Fig. 4) were lower in FTD-tau or
FTD-TDP-43 compared with AD (P,.001), DLB
(P,.05), and in the FTD-tau group compared with VaD
(P5.05).
3.5. Serum analysis
We analyzed YKL-40 concentrations in paired serum of
the FTD patients available in the Amsterdam cohort (12
FTD-TDP-43, 4 FTD-Tau, and 18 SMC). There was no cor-
relation between serum and CSF levels of YKL-40 and no
differences in serum YKL-40 levels between the patient
groups (data not shown).
4. Discussion
In this study, we identified 56 candidate biomarkers that
were differentially regulated between the pathologic sub-
types of FTD or between FTD and SMC, in CSF of homoge-
neous patient groups. The result could be confirmed for one
of five biomarkers for which a robust ELISA was available,
***
***
***
***
***
**
*
**
*
Fig. 2. YKL-40 levels in CSF of different patient groups as measured by
ELISA. ***P,.001. Abbreviations: CSF, cerebrospinal fluid; ELISA,
enzyme-linked immunosorbent assay; SMC, subjective memory complaint;
TDP-43, TAR DNA-binding protein 43; AD, Alzheimer’s dementia; DLB,
dementia with Lewy bodies; VaD, vascular dementia.
SMC TDP43 Tau
50
0
100
150
CFD (ng/mL)
Fig. 3. Complement factor D levels in CSF of different patient groups as
measured by ELISA. Abbreviations: CSF, cerebrospinal fluid; ELISA,
enzyme-linked immunosorbent assay; SMC, subjective memory com-
plaints; TDP-43, TAR DNA-binding protein 43.
**
**
***
*
*
***
*
**
*
*
*
Fig. 4. Catalase enzyme activity in CSF of different patient groups.
*P,.05, **P,.01, ***P,.001. Abbreviations: CSF, cerebrospinal fluid;
SMC, subjective memory complaints; TDP-43, TAR DNA-binding protein
43; AD, Alzheimer’s dementia; DLB, dementia with Lewy bodies; VaD,
vascular dementia.
Table 8
Correlation between CSF biomarkers and demographics in all groups*
Demographics
CSF biomarker
YKL-40 Catalase
Age 0.300** 0.093
Disease duration*20.133 20.222
Abeta (1–42) 20.100 20.058
t-Tau 0.397*** 0.044
p-Tau 0.197* 0.084
YKL-40 x 20.240*
Abbreviation: CSF, cerebrospinal fluid.
NOTE. Bivariate Spearman correlation coefficients were calculated
(n 588–101). *P,.05; **P,.01; ***P,.001.
*n 526 (YKL-40) and 19 (catalase).
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-94 91

i.e., YKL-40. Moreover, enzyme activity of one of three
tested proteins (catalase) was confirmed to be differentially
regulated between the FTD subtypes and controls.
The strongest increase in the proteomics results (fold
change .3) was observed for S100A7, GLA, APOL1,
NDRG4, CAT, and CA1 (Tables 3–7). Each of these
specific proteins have not yet been related to FTD, and we
here provide some more background information on these
proteins.
S100A7, also called psoriasin, is increased in several can-
cers and has multiple functions, including inflammatory
roles [25].
GLA and APOL1 are lysosomal proteins. They have not
yet been related to FTD pathology but can be very promising
as they confirm the previously revealed important role of the
autophagy/lysosome system in the etiology of FTD. For
instance, the GRN and CHMP2B genes that are directly
related to FTD are associated with the autophagy/lysosomal
pathway [26,27]. Similarly, the discovered genetic risk
factors for FTD, TMEM106b [28,29],RAB38, and CTCS
[30] are all associated with lysosomal pathways. These
data support further research into the role of the identified
CSF proteins in the FTD pathology.
Catalase and CA1 are relevant proteins for oxidative
stress, but it remains to be determined if the increase is
real and not an artifact, as a recent proteomics study sug-
gested these two, besides hemoglobin and peroxiredoxin,
as suitable biomarkers for blood contaminated CSF [31].
NDRG4 levels were increased specifically in the tau
group. The NDRG4 protein expression is upregulated in
aggressive meningioma [32]. In contrast, the levels were
decreased in glioblastoma or colorectal cancer [33].
NDRG4 is specifically expressed in the brain and heart
and plays a possible role in neuronal differentiation. Interest-
ingly, the NDRG4 messenger RNA expression was
decreased in Alzheimer brain tissue [34].
We validated a subset of biomarkers by immunoassays
as independent methods. YKL-40 has already been proven
to have a role in dementia, i.e., in Alzheimer’s dementia
[35,36], and also in early stages of multiple sclerosis
[37]. Our data expand findings in a recent report showing
increased levels in clinically defined FTD patients and a
positive correlation with CSF t-tau [38]. The 1.2-fold in-
crease in CSF levels in AD is of similar magnitude as
earlier reports, and lack of significant difference in our
cohort is likely due to the smaller number of AD patients
included in our study [39]. The elevation observed in other
diseases and the correlation with t-tau, albeit moderate,
indicate that YKL-40 cannot function as a single
pathology-specific biomarker and is probably a sensitive
biomarker for astrogliosis [37]. Interestingly, neuroinflam-
mation is one of the explanatory mechanisms through
which the progranulin haploinsufficiency can cause FTD
[40]. Further studies should define the role of neuroinflam-
mation and, in particular, astrogliosis, in FTD in more
depth [40–43].
Lumbar puncture may be perceived as inconvenient but
gets common practice in the dementia field and risk compli-
cations as severe headache of 0.9%, typical postlumbar
puncture headache of 9%, and back pain of 17% is generally
accepted in this population [44]. Unfortunately, blood levels
of YKL-40 were similar in FTD groups and SMC. This was,
however, not surprising, as blood levels of these biomarkers
have been related to inflammation in several other diseases,
including cardiovascular disease and diabetes, which could
mask brain-related alterations [45–47].
The discovery results on catalase were validated by an ac-
tivity assay, due to the lack of assays to analyze the concen-
trations. It remains to be determined if the activity of this
enzyme correlates to its concentrations, but the decreased
activity observed in FTD is at least encouraging to perform
future validation studies, including studies on the effects of
blood contamination on the activity.
A major strength of the study was the study design, i.e.,
the start with pathologically homogeneous patient groups
that were defined either by postmortem evaluation or genetic
subtyping and the use of an unbiased, well-validated method
for CSF proteomics [48]. Because clinical FTD is heteroge-
neous, which does not correlate strongly to its pathologic
subtypes [49], cohorts with known pathologic subtypes are
very relevant to increase knowledge on pathways, reflected
in biomarkers changes, and therapy development. Another
strength was the stringent screening of ELISA assays as in-
dependent methods and validation in independent cohort
including other dementia types. The validation was success-
ful for 20% of the biomarkers for which a CSF-validated
assay was available, which may forecast a similar success
rate for the remainder-identified proteins. The commercial
availability and shown quality of these assays have impor-
tant implications. First, these can be easily applied for inde-
pendent replication of our study by other research groups,
and second, implementation in clinical practice will be
feasible.
A weakness of this study is that we did not further differ-
entiate the pathologic subtypes, as the TDP-43 can be subdi-
vided into at least four distinct pathologic subtypes,
depending on the cellular location of the TDP-43 inclusions
(e.g., cytoplasmic or axonal) or stage of the pathology
[1,50]. Further subtyping would have led to lower number
of patients per subgroup, due to limited availability.
Nevertheless, the here identified biomarkers can be further
studied in relation to these different pathologies.
So far, there has been a lack of differential diagnostic bio-
markers of the pathologic subtypes of FTD. An interesting
recent report showed that a reduced ratio (,0.37) of p-tau
to t-tau could be a biomarker to identify FTD-TDP-43
from FTD-tau, AD, and healthy seniors with 82% sensitivity
and 82% specificity in cohorts of similar size as ours [8].An
imaging study showed that white matter volume could
discriminate between these pathologic FTD subtypes [51].
Thus, studies focusing on pathologic subtypes of FTD are
just emerging, and it will be interesting to evaluate the
C.E. Teunissen et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 2 (2016) 86-9492

additional value combining different CSF biomarkers and
different biomarker modalities for accurate diagnosis.
Taken together, YKL-40 should be studied further for its
use in diagnosis of FTD, as it discriminated both FTD sub-
types from SMC. Our study presented promising new candi-
date biomarkers for a correct and differential diagnosis of the
FTD subtypes, which is important for inclusion of specific
patients for either TDP-43 or tau-targeted treatments.
Acknowledgments
The Association for Frontotemporal Degeneration and
Alzheimer’s Drug Discovery Foundation, and the ZonMw
Memorabel program project “PRODIA,” as part of the
Deltaplan Dementie, are acknowledged for their grant sup-
port of this study.
Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.dadm.2015.12.004.
RESEARCH IN CONTEXT
1. Systematic review: The authors reviewed the litera-
ture using traditional (e.g., PubMed) sources and
citations within articles. Key words were frontotem-
poral dementia (FTD) and biomarkers and cerebro-
spinal fluid (CSF).
2. Interpretation: Our findings led to the discovery and
validation of several novel CSF biomarkers for the
different pathologic subtypes of FTD. Validation of
inflammatory biomarkers underscores the involve-
ment of these pathways in FTD.
3. Future directions: Future studies should aim at (1) in-
dependent multicenter validation of the biomarkers;
(2) establish the pathways role of these identified
key biomarkers for lysosomal and inflammatory
pathways in FTD pathology; and (3) validation of
biomarkers for which methods are to be developed.
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