Platelet Phosphorylated TDP-43

22 Pages·2017·0.83 MB·English

by  Abdulbaki Agbas

bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
Platelet phosphorylated TDP- 43: An exploratory study for a peripheral surrogate
biomarker development for Alzheimer’s disease
Rodger Wilhite, Jessica Sage, Abdurrahman Bouzid, Tyler Primavera,
Abdulbaki Agbas (*)
Division of Basic Sciences, College of Osteopathic Medicine, Kansas City University of
Medicine and Biosciences
1750 Independence Avenue, Kansas City, MO64106
Running title: Platelet TDP-43, a dynamic biomarker for Alzheimer’s disease
(*) Lead author: Abdulbaki Agbas, [email protected], [phone]
Keywords: TDP-43, human platelet, Alzheimer’s disease, hippocampus, surrogate marker

ABSTRACT
Alzheimer’s disease (AD) is the sixth leading cause of death in the United States. The World
Health Organization predicted that the world population with AD will rise to about 75 million by
2030. Therefore, AD and other forms of dementia create a non-curable disease population, and
a socioeconomic burden in the world’s societies. It is imperative to diagnose AD and other
neurodegenerative diseases at their early stage. Consequently, it is important to develop a
blood-based biomarker so that the remedial or disease-altering therapeutical interventions for
AD patients would be available at the early stages of the disease. We have identified an easy,
feasible, cost-effective, and less invasive assay method that measures a cellular protein that
may be a potential biomarker candidate for the neurodegenerative diseases; platelet
phosphorylated Transactive Response DNA Binding Protein 43 (pTDP-43). This protein recently
gained an attention that can be served for monitoring the development of at least two
neurodegenerative diseases (i.e., AD and amyotrophic lateral sclerosis, ALS). We have
identified an assay platform and generated some preliminary data may suggest that the platelet
TDP-43 that were increased (<65%) in post-mortem AD brain regions and similar trends were
also observed in AD patient’s platelet. In this study, we propose that platelet phosphorylated
form of TDP-43 could be used as a potential surrogate biomarker that is easy to measure,
reproducible, sensitive, and cost effective for screening the patients with some early clinical
signs of AD and can be used to monitor disease prognosis.

1
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
INTRODUCTION
More than 5 million Americans are living in great demand for the early stages of
with Alzheimer’s disease (AD) and this neurodegenerative diagnosis and
number is projected to rise to 13.8 making dementia screening a viable
million by 2050. As the world population approach. We have turned to explore for
gets older, the incidence of AD is rising. identifying a biomarker molecule(s) that
Unfortunately, only 1 in 4 people with can be measurable in blood tissue.
AD have been diagnosed [1].The rising There are several fluid based biomarker
cost of health care for AD patients has a candidates for AD [4-7]; however, either
negative socioeconomic impact on the the milieu of the diagnostic biomolecules
world society as well as being burden on or the measurement platforms for them
caretakers. The early diagnosis of AD have made many of these biomarkers
could be critical for starting an effective unfavorable candidates. The popular AD
treatment with that of current options as biomarker candidates plasma Aβ
well as designing new competent oligomers and tau were recently found
disease-modifying approaches. There is to be not good contender due to
a great need to improve early detection measuring environment [8, 9].
in the course of neurodegenerative Serum/plasma contains substantial
diseases such as AD, Amyotrophic amount of albumin and
lateral sclerosis (ALS), Parkinson’s immunoglobulins, which interfere the
disease (PD), frontotemporal lobar measurement of the target protein.
disease (FTLD), and others so that the Removal of interfering biomolecules
timely application of disease-specific could easily reduce the measurable of
treatments would be effective. Current AD relevant signature proteins. These
diagnostic tests for AD rely on reported observations led us to explore
expensive brain imaging technology that new blood-based surrogate
is available only to a few patients [2], biomarker(s) that may be useful for AD
cognitive and psychiatric assessments, diagnosis.
the collection of cerebrospinal fluid
There is a new potential biomarker
(CSF) samples which requires invasive
candidate for AD, Trans-activation
and lumbar puncture that has a
response DNA/RNA binding protein
negative public perception in several
(TARDP). Due to its 43 kDa size, TDP-
countries [3]. Therefore, it becomes an
43 acronym will be used throughout this
urgent task to identify better biomarkers
paper. Substantial research has been
for AD as well as for other
conducted to decipher the role of TDP-
neurodegenerative diseases. More
43 in different cellular events as well as
specifically, the new biomarker should
its role(s) in neurodegenerative disease
be sensitive, selective, reliable,
states [10-14]. TDP-43 is ubiquitously
affordable, and involve a non-invasive
expressed in all nucleated cells [15, 16]
sampling method. Such biomarkers are
2
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
, it has the ability to shuttle in-and-out sampling blood platelets may serve a
between nucleus and cytoplasm due to feasible media where the aberrant TDP-43
having nuclear localization and nuclear is detectable. Platelets are anuclear blood
cell fragments that are derived from
export sequences [17-20] . Cytosolic TDP-
megakaryocytes [27] and share the
43 is not well described yet; however
biochemical properties of neurons [28-30].
cytosolic phosphorylated derivatives of
Accordingly, several investigators consider
TDP-43 in neurodegenerative diseases may
using platelets as a venue to study the
be responsible for hyperphosphorylated
pathogenesis of neurodegeneration. The
aggregate formation and could be
latest progress on platelets as a reliable
considered as a biomarker candidate. In
source for biomarkers was reviewed in the
neurodegenerative diseases such as AD,
literature [29, 31-35]. One of the notable
FTDL, and ALS, the tissue levels of TDP-43
research conducted on platelet-derived
are increased, mostly located in the cytosol,
secreted amyloid precursor protein-β (APP-
and consistently observed in inclusion
β) for AD [35]; however, their data does not
bodies in neurons [16] rather than the
tell much about which Aβ peptide was
nucleus. In Pick disease (PiD), the presence
measured.
of TDP-43 inclusions suggests that TDP-43
accumulation and modification are an
We chose platelets for identifying and
important component of PiD [21]. Post-
measuring both total and phosphorylated
translationally modified TDP-43 aggregates
TDP-43 protein derivatives in AD for the
were also observed in post-mortem brain
following reasons: (i) platelets are easy to
tissue sections [15]. TDP-43 positive
repeatedly obtain from the patients with
inclusion bodies are becoming more
minimal distress (ii) their life span is short
detectable in about 75% of the brain tissues
(7-10 days) [36] which will reflect dynamic
from AD patients [11, 22]. The elevated
changes on phosphorylated TDP-43, (iii) it
level of TDP-43 in blood suggests that TDP-
was reported that platelets transiently open
43 may be considered as a potential
the blood brain barrier (BBB) [37],
surrogate biomarker in neurodegenerative
consequently, biomolecules may come in to
diseases [23, 24] . TDP-43 is prone to
contact with the blood stream and become
phosphorylation and undergoes truncation,
absorbed by platelets, (iv) serum/plasma
if it remains in the cytosol [25]. A recent
proteins and other biomolecules are
study has provided some evidence that
exposed to dilutions and results in large
TDP-43 mislocalization was an early or pre-
analytical challenges; hence, platelet
symptomatic event and was later associated
content are protected from such changes.
with neuron loss [26] . These studies
We have hypothesized that platelet TDP-43
suggest that post-translationally modified
and its phosphorylated derivatives may be
(i.e. phosphorylated) TDP-43 may be
considered as a viable surrogate dynamic
viewed as a disease specific protein.
biomarker. The elevated brain TDP-43
protein species in AD patients can be
To monitor disease relevant biomolecules in
determined in the platelet lysate so that
the central nervous system (CNS) is a
platelet TDP-43 readings could be used as
challenge due to unfeasibility and
a surrogate biomarker for AD that helps to
invasiveness of taking repeated samples
from brain and spinal cord tissues. Hence,
3
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
monitor the progress of disease during the treatment.
utilized as a negative disease control for
identifying a selective antibody for AD
MATERIAL METHODS
patient platelets. The baseline evaluation
included a thorough clinical examination by
Reagents: Anti hTARDBP polyclonal
a trained clinician was conducted. This
antibody (ProteinTech Group, Chicago, IL;
evaluation included a Clinical Dementia
Cat#1078-2-AP) and phosphorylated
Rating (CDR) to exclude the presence of
derivatives of the pTDP-43 antibodies
dementia was performed according to
(CosmoBio USA; Cat#TIP-TD-P09, TIP-TD-
clinical and cognitive variables and
P07, TIP-PTD-P05, TIP-PTD-P03, TIP-
descriptive data collected from Alzheimer
PTD-M01, TIP-PTD-P01, TIP-PTD-P02,
Disease Centers [39]. A trained
TIP-PTD-P04) (Abcam Cat# Ab184683 ,
psychometrician administered a
ProteinTech Cat# 10782-2-AP,66318-1-
comprehensive (National Alzheimer’s
Ig,22309-1-p(discontinued); Sigma Cat#
Coordinating Center Uniform Data Set,
T1705,SAB4200225; Biolegend Cat#
versions 2 and 3) cognitive testing battery
829901 ) were commercially purchased.
[39]. Clinical and psychometric test results
Citrate Wash Buffer (11mM glucose,
were reviewed and discussed at a weekly
128mM NaCl, 4.3 mM NAH PO , 7.5 mM
2 4 consensus conference that included
Na HPO , 4.5 mM sodium citrate, and
2 4 clinicians, a neuropsychologist, and raters.
2.4mM citric acid, pH 6.5)[38] and platelet
The ALS patients were clinically diagnosed
rupture Buffer (250 mM sucrose, 1 mM
by physicians and the subject identities for
EDTA, 10 mM Tris, pH 7.4) were prepared
the biosamples were de-identified. All
in our lab using reagent grade chemicals.
patients and otherwise healthy individuals
Phosphatase inhibitor cocktail (Calbiochem
were given a consent form before obtaining
# D00147804) (1:1,000) and protease
the blood samples. The sample collection
inhibitor cocktail (Calbiochem# 539134)
procedure was approved by the Institutional
(1:2,000) were added to the platelet rupture
Review Board of Kansas City University of
buffer just before use to preserve TDP-43
Medicine and Biosciences (KCU) and the
proteins from proteolytic degradation and
University of Kansas Medical Center
dephosphorylation processes.
(KUMC).
Sample preparations:
Platelets donors were consisting of five
female AD patients with average age of
Human Platelets: Human blood-platelet
75.2±10.5, five male AD patients with
samples were obtained from the following
average age of 75.8 ±8.6. Non-demented
sources; (1) The Bio-specimen Bank of
control subjects were consisting of two
University of Kansas Medical Center
males with average age of 77.5 ±2.13 and
(KUMC); platelets were previously collected
eight non-demented females with average
from AD patients and age-matched with
age of 73.6 ± 3.5.
otherwise healthy subjects (non-demented)
and stored at -800C and (2) ALS clinic at the
Platelets were isolated from freshly drawn
University of Kansas Medical Center,
blood into ACD containing vacutainer tube
Kansas City. ALS patient platelets were
from clinically diagnosed patients and
4
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
otherwise healthy subjects according to a respectively under the reducing conditions.
standard two-step low speed centrifugation The proteins were transferred onto a PVDF
method adopted by Bio-Specimen bank membrane and subsequently the membrane
facilities of the University of Kansas Medical was probed with both pan anti-TDP-43 and
Center. The platelet pellets were gently several anti-phosphorylated TDP-43
washed with 1 ml of citrate was buffer [38] antibodies listed in reagents section. The
and re-centrifuged at 200xg for 20 min. The protein bands were visualized by enhanced
washed platelet pellet was ruptured, chemiluminescence and infrared dye based
sonicated in 0.6 ml of rupturing buffer with fluorescence methods, and they were
protease and phosphatase inhibitors, and analyzed by NIH’s ImageJ (V.1.46r) and
subjected to high speed centrifugation Image StudioTM Lite software ( V. 4.0)
(16,000 x g; 30 min; 40C) to obtain platelet
cytosol. Protein concentrations were Capillary Electrophoresis: The platelet
determined by the bicinchoninic acid (BCA) lysates from AD, ALS patients and
spectrophotometric method [40]. The otherwise healthy subject cohort were
samples were aliquot and stored at -800C analyzed by a simple western system, a
until use. new technology developed by
ProteinSimple, Inc., USA. This technology
Human Brain Sample preparation: The does not require classical SDS/PAGE and
human brain tissue samples from post- Western blotting components. It uses very
mortem AD patients and age-matched little sample-mix volume (~3-5 µl) and
control subjects were obtained from the Bio- provides high-throughput style sample run.
Specimen bank of the University of Kansas The platelet lysate proteins (0.2 mg/ml)
Medical Center. The 100-200 mg samples were analyzed in duplicate and both
of excised tissue from three brain regions capillary electropherogram and pseudo
(frontal cortex, cerebellum, and protein bands were generated and analyzed
hippocampus) were received and by the system software (Compass for
homogenized in a Teflon-pestle glass Simple Western, v.3.0.9).
homogenizer containing. ice-cold buffer
(0.32 M sucrose, 0.5 mM MgSO , 10 mM Statistical analysis: Mann-Whitney U rank
4
epsilon-caproic acid, 0.1 mM EGTA, sum test was employed for statistical
protease inhibitor cocktail 0.1% v/v, 10 mM analysis.
HEPES, pH 7.4). Tissue : Homogenate
buffer ratio was kept at 1:20 for achieving RESULTS:
efficient solubilization The homogenization
TDP-43 protein level was differentially
was carried out in an ice bucket with 8-10
increased in AD-patient brain tissue and
strokes. The homogenate was aliquoted
this trend was reflected in platelets. In
and stored in -800C until use. Protein
early stages of this work, we have shown
concentrations were analyzed by the BCA
that total TDP-43 protein levels were
method [40].
increased in the brain regions of post-
mortem AD patients (n=3). The most
Western Blot: The brain homogenate and
noticeable TDP-43 increase was observed
platelet lysate proteins were resolved in 12
in the hippocampus while the frontal cortex
% SDS-PAGE and 4-20% SDS-PAGE,
and cerebellum reflected a slight TDP-43
5
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
increase as compare to non-symptomatic Therefore, we have tested several anti-
control subjects (Fig. 2A). Total TDP-43 phosphorylated TDP-43 antibodies, raised
aggregates were observed in three different for several different sequence of the C-
brain regions and the most notable terminal of TDP-43 protein, purchased from
aggregates were observed in the various vendors (ProteinTech, Abcam,
hippocampus (Fig.2B). We have also Cosmobio-USA, Sigma, and Biolegend) to
noticed that the platelet lysate TDP-43 identify an AD-selective antibody that can
levels were increased by <65 % in AD be used for screening assays. An anti-
patients (n=3) (Fig. 2C) in the early phase phospho (S409/410-2) TDP-43 antibody
of this study. Readers should be advised (ProteinTech Cat# 22309-1-AP) was
that platelet lysates were obtained from a identified as a potential antibody that
separate AD patient cohort, because the discriminates AD platelet lysate phospho-
University of Kansas Medical Center Bio- TDP-43 profile from that of amyotrophic
specimen repository did not have the lateral sclerosis (ALS) (negative disease
matching post-mortem tissue and platelet control) (Fig. 3A) and from that of non-
lysates from the same AD patients and non- symptomatic, otherwise healthy age-
symptomatic control individuals. matched subjects (Fig.3B). A prominent
protein peak at about 62 kDa position was
A sequence specific anti-phosphorylated
consistently observed in AD patient platelet
TDP-43 Ab distinguished AD from other
lysates (Fig.3A).
neurodegenerative disease. In the next
phase of this study, we have focused on DISCUSSION:
identifying an AD selective anti-
phosphorylated TDP-43 Ab as a screening Misfolded aberrant protein aggregation is
tool in a small number of subject platelets frequently observed in neurodegenerative
(n= 10 in each group). First, we have diseases [43-45]. Pathologically misfolded
employed a computer based Predictor of protein aggregate formation occurs long
Natural Disordered Region (PONDR®) before any measurable cognitive decline
algorithm using TDP-43 sequence (NCBI [46-48]. Current brain imaging methods are
accession code: Q5R5W2.1). Disordered expensive and not easily accessible in
Enhanced Phosphorylation Predictor many medical facilities. Therefore, it is
(DEPP) analysis predicted 28 potential essential to develop a blood-based
phosphorylation sites and a majority of them biomarker assay platform that is feasible,
were Ser amino acid enriched on the C- cost-effective, and selective. This assay
terminus (aa 369-410) (Fig.1A). Another may aid medical evaluations to predict AD
algorithm (PONDR® VL3-BA) was employed before the clinical manifestations are
to predict 152 aa long regions of disorder revealed as well as to monitor the potential
that were characterized by other methods AD treatments.
(Fig.1B). Nuclear magnetic resonance
(NMR) studies also revealed that a ~ 80 aa In this preliminary study, we have tested our
sequence from the C-terminus region of hypothesis and provided some new findings
TDP-43 was identified as the most that human blood-derived platelet TDP-43
disorderly region [41, 42] where the majority and its derivatives may reflect the changes
of phosphorylation sites were located. in the TDP-43 profile in human AD brain.
There are several reports in the literature
6
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
supporting the link between TDP-43 that have reported TDP-43 levels in serum
pathology and diagnosed AD cases [11, 49- and brain samples obtained from AD
51]. In early stage of this study, we have patients. All of these studies provide
demonstrated that TDP-43 protein considerable supporting evidence that a
aggregation in select brain regions (Fig.2A, notable percent of AD cases are linked to
2B). Hippocampus had relatively high level altered TDP-43. However, the peripheral
of TDP-43 aggregation as compare to the measurement of TDP-43 and its
other brain region, and the difference was phosphorylated derivatives are relatively
found statistically significant (P≤0.015; new. In this preliminary study, we have
Mann-Whitney U rank sum test) (Fig. 2A). tested our overarching hypothesis stating
We are aware of the insufficient sample that peripheral TDP-43 profile may mirror in
numbers (n=3) analyzed in this study. It was that of brain tissue so that the early
a challenge for us to obtain more AD patient diagnosis of AD would be possible before
brain samples. We continue to the clinical manifestations are revealed.
communicate with bio-repositories in other Peripheral TDP-43 measurement in AD was
health centers to obtain more brain tissues. reported [23]; however, the ELISA method
We have shown the hippocampus region was employed for measuring total TDP-43
was very vulnerable to oxidative stress in in AD patient’s serum. Serum contains
the aging process [52]. This also partially some very abundant biomolecules such as
explains why increased level of TDP-43 albumin and immunoglobulins. These
aggregation was observed in the biomolecules may mask the levels of TDP-
hippocampus region, which is the most 43 in serum based assays so that positive
important region for memory storage. recognition of TDP-43 by its selective
antibody may be greatly reduced. That is
In the next stage of this study, we have why we have justified for utilizing anuclear
tested whether AD patient brain TDP-43 platelet as a biological milieu, which will
protein profile could be observed in reflect a more concentrated and
peripheral tissues such as blood-derived encapsulated population of TDP-43 with
platelets. If we could demonstrate the same minimum or no interference of serum
or similar profile in platelets, then we will albumin, immunoglobulins, and nuclear
have an assay platform by which TDP-43 TDP-43 contribution. We thought that a cell-
and its derivatives can be easily measured based TDP-43 chemical modification and
and analyzed in platelet-lysate during the aggregation model may be a good strategy
disease prognoses. Intracellular TDP-43 [55] to investigate whether peripheral cells
species such as aggregates, truncated would be considered as a platform where
TDP-43 fragments, and post-translationally the surrogate biomarker such as TDP-43
modified TDP-43 have been found in can be analyzed. Therefore, we have
neurodegenerative diseases [53]. The hypothesized that platelet TDP-43 and its
characteristics of post-translationally phosphorylated derivatives may be
modified TDP-43 may affect the considered as a viable surrogate dynamic
pathological course of the diseases much biomarker.
earlier than previously thought [54] . There
are several supporting studies in the To identify an AD-selective antibody was a
literature cited in the body of this manuscript major undertaking in this study. Eight
7
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
antibodies raised against to different regions relevant disorders [60]. In our view, there
of TDP-43 as well as phosphorylated are several enzymatic cleavages of TDP-43
derivatives of TDP-43 were tested. They that produces cleaved toxic TDP-43
were purchased from different vendors fragments that may be easily
(ProteinTech, Cosmobio-USA, Abcam). We phosphorylated. Subsequently, these
have identified an anti-phospho (S409/410- fragments will first form an aggregation
2) TDP-43 antibody purchased from nucleus through protein-protein interactions
ProteinTech (Cat#22309-1AP) as an AD- yielding TDP-43 enriched plaques in CNS
discriminating antibody for AD-platelet tissue. All of these cited studies as well as
lysate phosphorylated TDP-43 content. This many others strengthened the conception
antibody did not well recognize platelet that TDP-43 protein profile in Alzheimer’s
lysate phosphorylated TDP-43 in another disease may be a good dynamic biomarker
neurodegenerative disease such as ALS. that ought to be comprehensively studied. In
We have used ALS patient platelet lysate as tissue, cytosolic TDP-43 protein, especially
a negative disease control for testing the toxic monomers [61], begin to form
selectivity of the antibody that recognized hyperphosphorylated species. They are
the high levels of pTDP-43 in AD platelet sequestered into inclusion bodies as part of
lysate. This observation was unexpected the defense mechanism of the organism,
and requires more in depth analysis in suggesting that cytosolic pTDP-43 or
larger patient cohort. At this preliminary detergent-soluble TDP-43 protein is toxic
stage, we may offer an explanation that this [10]. We neither verified inclusion body
may be due to either the antibody producing presence in platelets nor existing literature
clone would be different or TDP-43 reported. What we observed that cytosolic
modification differs in AD than ALS. TDP-43 was present in platelets and
phosphorylated species of TDP-43 was
TDP-43 proteinopathy is characterized by elevated in Alzheimer’s disease. We
decreased solubility, hyper-phosphorylation speculate that anuclear platelet cytosol
and the generation of 25 kDa C-terminal represents the toxic form of TDP-43
fragment [16, 56-58]. This section of TDP- species.
43 protein was identified as the disordered
How does aberrant brain TDP-43 appear in
region (Fig.1B) which is also a target for a
peripheral blood cells? One explanation
caspase enzyme cleavage [59]. We also
might be that the TDP-43 protein has a C-
have observed ~35 and ~25 kDa TDP-43
terminus Q/N rich region [62]; therefore, this
fragments in platelet lysates obtained from
protein may have the characteristics of
AD patients in early stage of this work (data
prion-like proteins that propagates itself [63,
not shown); we thought that they may
64] and transfects other cells. Kanouchi et
represent the degradation products of TDP-
43 due to either storage of samples at -800C al., have reviewed the recent findings about
the prion-like characteristics of TDP-43
for extended period of time or the
propagation and offered the concepts of
degradation could be due to the old age of
contiguous and non-contiguous propagation
the subjects (Fig. 2C). These fragmented
of misfolded proteins including TDP-43 [65].
TDP-43 species are more likely
Considering the leaky BBB in
encapsulated in immunoreactive inclusion
neurodegenerative diseases as well as the
bodies that may be associated TDP-43
8
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
ability of platelets to transiently open the us to plan longitudinal studies in future.
BBB via releasing platelet activating factors Perhaps the platelet TDP-43 approach will
[37], it is conceivable that aberrant TDP-43 make these kinds of studies feasible. As
in astrocytes may transfect the blood cells discussed by Budini et al., cell-based TDP-
by means of cell-to-cell infection through 43 aggregation and modifications model is a
having access to the blood stream. This powerful tool [55] to test novel therapeutic
concept was recently reviewed on how the strategies aimed at preventing and/or
aberrant proteins infect the cells [66]. We reducing TDP-43 aggregation in AD.
are well aware that we were unable to
obtain the platelets and post-mortem brain In the near future, research teams may
tissues from the same subject, which could consider some therapeutic approaches by
be the ideal representation of the TDP-43 which cell permeable chemical chaperons
profile. However, we are searching other AD that bind to misfolded protein and stabilize
centers whether such paired samples would the folded state reduce protein misfolding
be available for us to verify our preliminary [70] . In normal circumstances, the
results. molecular chaperons and other
housekeeping mechanisms ensure that
In this study, we have provided a trend of potentially toxic aberrant proteins or pre-
TDP-43 profiles in AD and age-matched fibrillary aggregates are neutralized before
healthy subjects (Fig. 2A, 2C).To our they can do cellular damage [71, 72].
knowledge, we are the first research group Therefore, the researchers need to know
to identify the TDP-43 profile in platelets the folding features of protein of interest. If
which could be considered as a surrogate we know the folding features of TDP-43 and
dynamic biomarker to monitor the disease can measure the occurrence of misfolded,
progress as well as the pharmacological disease prone TDP-43 early enough, we
treatment response. Our findings about the may be able to stabilize the misfolded
presence of phosphorylated TDP-43 in protein by potential chemical chaperons,
platelets from AD patients are intriguing and which may open up new therapeutical
led us to question whether AD is an venues for neurodegenerative disease
exclusively CNS or peripheral system treatment.
disease? This issue is currently questioned
and would require some very FUTURE PERSPECTIVE
comprehensive studies [67, 68].
Our results suggest that peripheral blood-
It is well known fact that protein derived platelets could be used a venue to
aggregations occurs long before the clinical identify AD-relevant biomarker candidate
manifestations are revealed [43] . In vitro proteins. We have identified an AD-selective
biophysical studies in cell culture and anti-phospho (S409/410-2) TDP-43 Ab in
mouse brain have suggested that TDP-43 platelet lysates of AD patients. In future
naturally tends to form a dimeric protein as studies, we would like to utilize this antibody
cited in a recent review [69]. Could we as a screening tool in large AD patient
monitor TDP-43 modifications and population. The challenge is to identify a
aggregations during disease progression? reliable and validated blood-based
This issue was always a challenge and led biomarker(s) during next 4-6 years.
9
bioRxiv preprint first posted online May. 1, 2017; doi: http://dx.doi.org/10.1101/132837. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license.
EXECUTIVE SUMMARY from discovery to clinic. Alzheimers
Dement, 2017. 13(1): p. 45-58.
 TDP-43 and its phosphorylated (**) This comprehensive article reviews
the recent literature on blood-based
derivative can be measured in
biomarkers in AD to provide a current
platelet lysate
state of art.
 A-Phospho (S409/410-2) TDP-43 is
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 This AD-selective antibody may be fluid biomarkers and to examine how
fluid biomarkers have been
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incorporated into clinical trials.
strengthen AD diagnosis along with
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