Moll Cellproteomic PDF
Moll Cellproteomic PDF
Moll Cellproteomic PDF
001290
Synaptic panel Alzheimer’s disease CSF
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Synaptic panel Alzheimer’s disease CSF
Abbreviations
Alzheimer’s disease (AD), array tomography (AT), cerebrospinal fluid (CSF), false
discovery rate (FDR), gene ontology (GO), liquid chromatography (LC), mass
synaptic density (PSD-95), strong cation exchange (SCX), selected reaction monitoring
(SRM)
pathophysiology that precedes neuronal death and symptom onset, would be a much-
needed prognostic biomarker. With direct access to the brain interstitial fluid, the
study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD.
Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the
literature and public databases, we identified 251 synaptic proteins, from which we
selected 22 for further study. Verification: Twelve proteins were discarded due to poor
cohort of CSF from cognitively normal controls and subjects in the pre-clinical and
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(0.6 to 0.8-fold, p<0.04), a finding that was replicated (0.7 to 0.8-fold, p<0.05) for 6
reduced 0.8-fold (p<0.05) in preclinical AD, changes that precede clinical symptoms
and CSF markers of neurodegeneration. Therefore these proteins could have clinical
diseases including, but not limited to AD, Parkinson’s disease, Lewy body diseases,
synapse loss in living individuals has the potential to be a surrogate marker for disease
severity, which would make an excellent addition to the biomarker arsenal for a wide
selected AD as a disease model for synaptopathy. Synapse loss is an early event in AD,
which precedes neuronal death (1) and evidence from animal models indicates that the
synapse is the target of both AD pathological proteins, Aβ and tau (4). AD can be
this conceptualization, patients with mild cognitive impairment (5) or dementia (6) who
are positive for AD biomarkers are labelled as patients with MCI due to AD (prodromal
AD) or dementia due to AD (6). Likewise, the current guidelines from the National
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stages of AD (7) whereby cognitively normal subjects with signs of brain amyloidosis
markers as well as subtle cognitive decline (preclinical Stage 3). While markers of Aβ
and tau pathology are excellent diagnostic biomarkers for AD, a marker of synapse
In this regard, many researchers have turned to biochemical markers in CSF, a biofluid
with direct access to the central nervous system that can be extracted from living
findings support the idea that synaptic proteins in CSF may be informative in AD, the
previously reported correlation between CSF levels of synaptic proteins with CSF levels
of tau suggest that widespread neuronal loss could be a confounding factor when
studying synaptic proteins in the CSF, particularly at clinical disease stages. The CSF
neurodegeneration has taken hold has not been explored in detail. This is an important
system to evaluate the potential relationship between CSF levels of proposed synaptic
Here we report (i) a systematic proteomic study of the CSF with a thorough
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SRM assays for a set of CSF proteins whose expression was confirmed at the human
CSF profile of the synaptic panel in a clinical cohort that includes cognitively normal
subjects and AD preclinical and clinical stages (exploration) and (iv) confirmation in
Experimental Procedures
Experimental Design and Statistical Rationale. This study is divided into the following
CSF and pathological study of selected proteins in human post-mortem tissue from 6
donors. Exploration stage; SRM of selected peptides in CSF from cognitively normal
controls and pre-clinical and clinical stages of the AD continuum (n=80) prospectively
recruited from the Sant Pau Initiative in Neurodegeneration (SPIN) cohort at Hospital
Sant Pau, Barcelona and by the CITA Foundation, Donostia (exploratory cohort).
controls and preclinical stage 1 subjects (n=38) from the SPIN cohort. Where possible,
subjects included in each group were age and sex-matched. CSF samples were run on
technical replicates were included in the SRM study. Biological controls for SRM
n=18, validation cohort-2, n=20). Biological replicates for SRM included patients and
SRM, BSA controls were run between each sample (shotgun and targeted LC-MS/MS).
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Clinical CSF cohorts. All participants gave their written consent. The study (IIBSP-
BIO-2015-76) was approved by the local ethics committee following the ethical
biomarkers, namely brain amyloidosis (low CSF levels of Aβ1-42 or positive amyloid
PET imaging) and neurodegeneration (high CSF levels of total tau or phosphorylated
tau) based on local cut-offs. These cut-offs have high specificity and sensitivity to
normal range following formal neuropsychological evaluation, when accounting for age
and education (mostly recruited among patients’ caregivers), were classified into
CSF collection, biomarker assessment and APOE genotyping. CSF samples were
described (13). Samples had been previously stored at -80°C and had not been thawed
prior to analysis. Commercially available ELISA kits were used to determine levels of
Post-mortem human brain samples. All post-mortem brain tissue used in this study
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Committee of both the Tissue Bank and Sant Pau Research Institute. Fresh brain tissue
used for array tomography was collected from the superior frontal cortex of a female
donor who died at the age of 83 and showed low AD pathology (Braak stage II). Pre-
collected frozen tissue blocks from 6 donors (4 male, 2 female, mean age-at-death 66
years) without AD pathology were used for synaptosome and PSD enrichment.
individuals were precipitated with acetone and protein content was quantified by
Bradford assay. Samples (50ug) were reduced in 10mM DTT, alkylated with 55 mM
IAA, and, digested in-solution with trypsin and LysC overnight and desalted using a
Desalted MicroSpin Column (GE healthcare, UK). Where indicated, samples were
Sigma-Aldrich). An equivalent of 5ul of each CSF Sample was analysed using a LTQ-
Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, San Jose, CA)
μm), and a reversed-phase chromatography 25cm C18 column (Nikkyo Technos, Japan;
μm i.d., 1.9 μm) using a data-dependent acquisition mode. Acquired data were analyzed
using the Proteome Discoverer software suite (v1.4.1.14, Thermo Fisher Scientific), and
the Mascot search engine (v2.5.1, Matrix Science) was used for peptide identification.
Data were searched against the Swiss Prot Human Protein database plus the most
common contaminants (version 2014, 20884 entries). A precursor ion mass tolerance of
7 ppm at the MS1 level was used, and up to three missed cleavages for trypsin were
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allowed. The fragment ion mass tolerance was set to 0.5 Da. Oxidation of Methionine
peptides were filtered at 5% FDR calculated using a target-decoy database strategy. For
each identified peptide, peptide peak areas were obtained as extracted ion
chromatograms and protein abundances were estimated with the average peak area of
the three most intense peptides per protein. For characterization of the CSF proteome,
proteins without a reviewed Uniprot identifier were excluded. The mass spectrometry
proteomics data have been deposited to the ProteomeXchange Consortium via the
identifier PXD010356.
for proteomic studies was performed using the search terms “Cerebrospinal
fluid”/“CSF”, & “proteome”/“proteomics” & “human”, for the CSF proteome (April
“proteomics” & and “human” / ”mouse” / ”rat”, for the synapse proteome (April 2014).
Only publications in English were reviewed. All identified proteins were extracted
either directly from the published material or where available, from the PRIDE
repository. All proteins with a known function related to the synapse were retrieved
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signalling” according to the Kyoto Encyclopedia of Genes and Genomes were retrieved
(www.uniprot.org). Since each data source used distinct protein identifiers that, in some
cases, had been retired or updated, a unique, reviewed Uniprot identifier was assigned to
each protein using the bioinformatic gene identification conversion tools, PIR
duplication across studies. Proteins without a reviewed Uniprot identifier were removed
Synaptosome enrichment. All steps were performed at 4°C. 200mg chunks were cut
from frozen frontal cortex tissue blocks and homogenized in cold Buffer A (0.32M
10 minutes) and the supernatant transferred to a new tube. The pellet was resuspended
in cold Buffer A and the previous step repeated with centrifugation at 710 x g. The two
supernatants were combined and centrifuged (710 x g). The supernatant was subjected
B (0.32M sucrose, 1mM NaHCO3), layered over a sucrose gradient (0.85M, 1M, 1.2M)
and centrifuged (82,500 x g, 2 hours). The synaptosomal fraction (a thick white band at
the 1-1.2M interface) was collected, diluted in 4x volume of Buffer B. An aliquot was
C (50mM Tris pH 7.4, 1% SDS) and stored at -80ºC. The remaining aliquot was diluted
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Synaptic panel Alzheimer’s disease CSF
incubated for 10 minutes and centrifuged (maximum velocity, 30 minutes). The pellet
Array Tomography. The array tomography protocol was applied using previously
described methods (17, 18). Briefly, a 1cm3 section was taken from the superior frontal
cortex was fixed, dehydrated and polymerised in 100% LR-white resin. The embedded
samples were sectioned using a diamond knife (Diatome, UK) creating 20 serial 70nm
thick sections, which were mounted onto coverslips. The ultrathin ribbons were washed
with Tris buffer and blocked for 5 minutes. Primary antibodies used were as mentioned
(Thermo Fisher Scientific). Coverslips were mounted onto the slides using Slowfade
Gold with DAPI (Thermo Fisher Scientific). Images were captured using a fully
Pennsylvania) with a 64x 1.2 NA Plan Apochromat objective. Image analysis was
sections were stacked, aligned, thresholded and the non-specific staining (not present in
at least 2 consecutive sections) removed using a local threshold based algorithm. 3-D
SDS-Page and Western blot. The total protein content of homogenate, synaptosome
and PSD enriched fractions was quantified by bicinchoninic acid assay. Aliquots
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Synaptic panel Alzheimer’s disease CSF
containing 20ug total protein were boiled, diluted in loading buffer (100mM Tris-HCL,
4% SDS, 20% glycerol, 200mM DTT and 200mM β-mercaptoethanol) and loaded onto
anti-GluR4, anti-Vamp2, anti-PSD95 (Cell Signaling; 13607, 8070, 13508, 3450), Anti-
brought forward from the shotgun data were selected based on previous LC-MS/MS
data and database searches (Peptide Atlas). For each targeted peptide, corresponding
crude heavy peptides were synthesised with 13C6 15N4 (Arg) or 13C6 15N2 (Lys) isotopes
(Peprotech SRM custom peptides, grade 2, Thermo Fisher Scientific) for use as
reference internal standards in the CSF samples (Thermo Fisher), and to generate a
library of MS/MS spectra for the selection of interference-free transitions for the
peptides or interest. Individual CSF samples were precipitated with acetone and re-
dissolved in 6M urea prior redution (10 mM, DTT), alkylation (55 mM, IAA) and in-
solution digestion with LysC and Trypsin (1:10, 37ºC, overnight). Isotopically-labeled
peptides were spiked in each sample, and an equivalent of 5ul of each sample was
particle size). BSA control samples were analyzed between runs for instrument quality
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Synaptic panel Alzheimer’s disease CSF
control. Transitions were visualised and analysed using Skyline 3.5 and peak picking
was manually reviewed based on the co-elution of the reference and endogenous
peptides, peak shape and correlation of transition rank intensities between the reference
and endogenous transitions. Of the initial 54 targeted peptides, 32 were eliminated from
the study due to poor detection in a pilot detection experiment with 5 CSF samples. The
remaining 22 peptides were taken forward for further monitoring in the exploratory and
validation cohorts. Peptide stability was assessed by injecting a pool of all the samples
over the duration of the mass spectrometric measurements and monitoring the peak area
of the standard peptides. SRM transitions were processed using the dataProcess function
to normalise the data using the transition intensities of the isotope-labeled standard
peptides. This method was chosen as it assumes that the samples of a data set are
separated by a constant and scales the samples so that they have the same median. The
results were not significantly altered when compared to normalisation using the
alternative Quantile method in MSstats. The normalised data were summarised (TMP:
Tukey’s Median Polish) according to either peptide or protein. Transitions with between
off=<0.8). Samples with log base-2 endogenous intensities under the cut-off designated
by the MSstats package for each cohort (9.5211, 8.4337 and 8.5219, respectively) were
proteomics data have been deposited to the Panorama repository with the dataset
identifier PXD012138.
PSD-enriched fractions isolated from post-mortem brain tissue, the intensity of bands
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Odyssey software (Li-COR Biosciences). The ratio of each fraction relative to that of
the homogenate (enrichment) was calculated. Since enrichment ratios for the 10
synaptic proteins did not clearly deviate from a Gaussian distribution (Kolmogorov-
Smirnov p>0.1), mean enrichment ratios were compared in synaptosome and PSD
hoc test). For SRM data, log2 fold-changes for each preclinical and clinical AD stage
relative to controls were calculated using a mixed effect linear regression model
(GroupComparison function in MSstats). For meta-analyses of the SRM data from the
Results
CSF pools each containing samples from 10 individuals extracted from either
pool), varying the LC-MS/MS conditions to optimise protein yield. Further details of
the sample pools, LC-MS/MS conditions and identified contaminants can be found in
Table S1. We found that depletion of abundant IgGs and albumins increased the protein
yield by 115% and that SCX fractionation had a greater effect on protein yield (186%
increase compared to runs without SCX, 6 hour LC) than extended LC times (141%
increase following SCX + 6 hour LC compared to SCX + 2 hour LC). Overall, IgG and
albumin depletion, SCX fractionation with 6 hour LC resulted in the greatest protein
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Synaptic panel Alzheimer’s disease CSF
yield (mean yield = 1,259 proteins, range =702-1,615). Across all LC-MS/MS runs, we
identified 18,785 peptides (Table S2) corresponding to 2,742 unique proteins (Table
S3).
listed in Table 1A. We extracted 3,662 unique proteins (Table S3) from the 9 studies
for which data were publicly available. By combining our dataset with the datasets from
cognitively/neurologically normal CSF and a further 296 proteins that we detected only
To identify the synaptic component of the CSF, we next sought to characterize the
human synaptic proteome also using publicly available data, but in this case related to
the characterization of the synapse. Here we have defined the synaptic proteome as
databases (see Experimental Procedures), and ii) have been detected in at least 1 of 23
human brain tissue (Table 1B-D). Table S4 shows the 537 proteins that satisfy these
criteria. Cross-referencing the CSF with this shortlist, we identified 251 synaptic
proteins that are detectable in the CSF by shotgun mass spectrometry (Table S5). Using
these criteria, we report that approximately 6% of CSF proteins are of synaptic origin.
The 251 synaptic proteins that were detectable in the CSF were manually curated and an
initial list of 22 proteins that participate in one or more core synaptic process
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Synaptic panel Alzheimer’s disease CSF
receptor A3, GluR2 (54), GluR4 (55), mGluR1 (56), Munc18 (57), Neurexin-2A
Vamp-2 (69)) was selected with the goal of defining a subset of proteins that is
eliminated 32 peptides from the study due to poor detection (Table S6).
improved spatial resolution in the axial plane compared to other light microscopy
synapse terminals clearly show the expression of 9 of the panel proteins directly at the
synapse, marked by pre (synaptophysin) and post (PSD-95) synaptic markers (Fig 1A).
In contrast, Tenascin-R was found surrounding the synapse without making direct
contact. This is consistent with the literature where Tenascin-R has been reported to
of the panel proteins in synaptosome and PSD fractions extracted from 6 human cortical
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Synaptic panel Alzheimer’s disease CSF
tissue samples without pathology (Table S7). The 2A isoform of Neurexin-2 could not
Western blotting. Fig 1B shows that all 9 proteins tested were enriched in synaptic
1.8 to 5.0-fold in the PSD fraction (p<0.03). Therefore all 10 proteins are expressed
Since Tenascin-R, was not directly expressed at the synapse, this protein was not
brought forward for evaluation. Table 2 provides further information regarding the 9
synaptic proteins and their corresponding peptides that were monitored by SRM in the
exploratory cohort comprising 80 CSF samples from controls and all preclinical and
clinical stages of the AD continuum (Table 3A). Table S8A lists the transitions
included and excluded from the analysis. Fig 2A shows the relative fold-change in CSF
levels of the 20 individual peptides and summarized protein levels at each AD stage
Table S9A shows the raw values. Peptides were not significantly altered in individuals
proteins were subtly reduced 0.8 to 0.9-fold (p>0.1). At preclinical AD stage 2 (n=10),
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adj.p=0.1), GluR4 (1.3-fold, p<0.04, adj.p=0.1), Neurexins-2A and 3A and Thy-1 (1.2
from the same protein were highly correlated across all samples (ρ=0.77 to 0.98).
Excepting the GluR2 peptide, which showed relatively elevated levels (p>0.05) at
preclinical stage 1, the fold-changes of all peptides and proteins were comparable to
each other at all disease stages, differing only in significance level, indicating very little
peptide or protein-specific differences. In fact, the levels of all proteins across all
disease stages were positively correlated (n=80, all ρ>0.37, all p<7x10-4), Calsyntenin-
All 20 peptides were brought forward for validation in an independent clinical cohort
comprising 60 CSF samples controls and preclinical and clinical stages of the AD
continuum (Table 3B). Table S8B lists the transitions included and excluded from the
analysis. Fig 2B shows the relative fold-change in CSF levels of the individual peptides
biomarker negative control group (n=18). Table S9B shows the raw values. Similar to
the exploratory cohort, levels of peptides from the same protein were highly correlated
across all samples (ρ=0.52 to 0.98) and all 20 peptides showed comparable CSF profiles
at all disease stages. All proteins were positively correlated (n=60, all ρ>0.36 all
0.80). The greatest fold-change was observed in individuals at preclinical stage 1 (n=9)
Syntaxin-1 and Thy-1 peptides were reduced 0.6 to 0.8-fold (p<0.05, adj.p=0.03 to
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were elevated 1.3-fold (p<0.05, adj.p>0.1). While all peptides showed positive fold-
changes (1.1 to 1.3-fold) in prodromal AD patients (n=10), the only peptide to validate
the elevated levels observed in the exploratory analyses was for GluR4, which was
elevated 1.3-fold (p=0.04, adj.p=0.5). None of the peptides were significantly elevated
While, all synaptic proteins moderately correlated with total tau levels, in both cohorts
since individuals at this early stage are asymptomatic and have yet to demonstrate
larger set of controls and preclinical AD stage 1 (n=38) from the SPIN cohort. Table
S8C lists the transitions included and excluded from the analysis. Fig 2C shows that
Syntaxin-1 and Thy-1 were reduced 0.7 to 0.8-fold (p<0.05). Table S9C shows the raw
values.
p-value) at each disease stage for peptides that demonstrated fold-changes in the same
direction across all cohorts. Table S9D shows the raw values. The mean of fold-change
for each peptide at each disease stage is plotted in Fig 2D. In this meta-analysis,
and Vamp-2 peptides were elevated (1.2 to 1.4-fold, Fisher’s p<0.04) in prodromal AD
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Synaptic panel Alzheimer’s disease CSF
(1.2 to 1.3-fold, p=0.04) at the dementia stage. Therefore, these data support a biphasic
whereby compared to controls, CSF peptide levels are reduced at the earliest preclinical
stage 1 of AD when neurodegeneration has yet to take hold, as indicated by total tau
Discussion
This is the first systematic study of the CSF proteome and thorough characterisation of
the synaptic proteins at the human cortical neuronal synapse therefore increasing the
probability that their CSF levels are directly related to synapse integrity. A thorough
evaluation of the CSF profile of the 9 synaptic proteins in three independent clinical
cohorts comprising controls and pre-clinical and clinical stages of AD revealed a set of
1B and Thy-1) that were reduced 0.8-fold (p<0.05) at the earliest preclinical stage of
Five of these proteins were also elevated in patients at the clinical stages (cognitive
levels in preclinical stage 1 may reflect reduced synaptic density in these individuals
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Synaptic panel Alzheimer’s disease CSF
In the discovery stage of this study, a thorough proteomic screen of 60 CSF samples
combined with a literature search of proteomic studies of the CSF identified 4,315
proteins in cognitively normal individuals. To put this into context, the latest release of
the Human Protein Atlas (v17) comprises RNA expression for 10,226 unique genes that
report that 26% are detectable in the CSF. According to the Human Protein Atlas data,
we estimate that 79% of the brain-expressed proteins detected in the CSF are known to
be expressed in the cortex, 65% in the hippocampus and 67% in the cerebellum.
characterised synaptic proteins. The 537 proteins retrieved represent 4% of those with
positive RNA brain expression data included in the Human Protein Atlas database,
suggesting that this is a relatively stringent classification. Regional expression data from
the Human Protein Atlas provide evidence for 95% of these synaptic transcripts in the
cerebral cortex, 75% in the hippocampus and 87% in the cerebellum. We conclude that
the majority of the proteins we have classified as synaptic are expressed across multiple
brain regions. This lack of regional specificity offers the possibility that these proteins
could be informative across the range of neurological and psychiatric diseases, which
demonstrate distinct regional susceptibility. Notably, our CSF database included 47% of
these synaptic proteins. When compared with the 26% yield of brain-expressed proteins
in CSF, we conclude that the coverage of synaptic proteins in our CSF database was
relatively high.
study single synapses (17, 18) to verify the synaptic expression of a set of proteins
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Synaptic panel Alzheimer’s disease CSF
detectable in the CSF by SRM and with a known synaptic function at the human cortical
synapse. With improved antibody penetration compared with other super resolution
report with high confidence that our panel of 9 proteins are expressed directly at or
surrounding human cortical neuronal synapses. The synaptic panel includes proteins
with a range of synaptic functions, including but not limited to, spine assembly and
function. The protein expression of 7 of these proteins has been assessed by the Human
Neuroligin-2 were the only proteins detected, at low levels, in glial cells. Consistent
with the extracellular expression in perineuronal nets, Tenascin-R was detected in the
neuropil but not in neurons or glia, further supporting its exclusion from the remainder
of the study. In AD, reduced brain expression at the transcriptomic or proteomic level
has been reported for GluR2, GluR4, Neurexin-2A, Syntaxin-1B, Tenascin-R, Thy-1
and VAMP-2, while increased expression has been reported for Neuroligin-2 (71-74).
Pertinently, there is evidence in the literature that some of the panel proteins may be
GluR4 (76) and Neurexin-2A (77) have been postulated as direct targets of Aβ
oligomers. Additionally, there is evidence that Calsyntenin-1 (78, 79) and Vamp-2
(80) play a role in Aβ production, trafficking or secretion and a role for the Neurexin-
Neuroligin complex in regulating Aβ metabolism and function has also been postulated
proteins particularly attractive candidate biomarkers for AD. That being said, this does
not exclude their potential as biomarkers of synapse loss in other neurological diseases.
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Synaptic panel Alzheimer’s disease CSF
Defective GluR2 RNA editing may be relevant to amyotrophic lateral sclerosis (82),
been implicated in schizophrenia (83), autism spectrum disorder (84) and epilepsy(85),
respectively.
In the exploration and validation stages of this study, we quantified the 9 synapse-
verified proteins in three independent clinical cohorts of CSF samples from cognitively
normal controls and individuals from the AD continuum. As has been reported for other
synaptic proteins, such as neurogranin (86), synaptotagmin-1 (10) and SNAP-25 (9), we
compared with controls. The moderate correlation of the synaptic panel proteins with a
marker of tau-mediated neurodegeneration suggests that synapse and neuronal loss are
interrelated in AD and that the panel proteins partially reflect both processes. That being
correlation with CSF total tau than that reported for neurogranin, synaptotagmin and
proteins from CSF tau levels compared with other synaptic proteins provides additional
clinical value when used as biomarkers. We also report that 12 peptides from
preclinical AD stage 1. Our data support that reduced levels of synaptic proteins in the
CSF reflect a reduced synaptic density in these individuals, which is only apparent
valuable markers for monitoring disease progression in at-risk individuals before the
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In conclusion, we have identified and provided clinical validation for a set of synaptic
provide added value in the clinical setting to assess disease progression in individuals
at-risk for AD and AD patients and could improve enrichment and monitoring of drug
efficacy in pharmaceutical drug trials. To our knowledge, this is the first study to
the CSF. Since the cross-sectional design of the current study does not allow assessment
of the prognostic capacity of the synaptic panel proteins, future longitudinal studies in
large cohorts are necessary to further explore the potential of the synaptic panel proteins
neurological and psychiatric disease opens up many avenues for further investigation.
Acknowledgements
The authors are indebted to all the patients and their families for their participation in
this study. This work was supported by the following research grants: Programa 1
Funds for Regional Development (FEDER) and the Instituto de Salud Carlos III,
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Synaptic panel Alzheimer’s disease CSF
(4560/6393) and “La Marató” organized by the television channel, TV3 (201426 10).
OB is supported by the Miguel Servet program (CP13/00091) from the ISCIII and
Integration Grant from the European Union (ref. 304111, Marie Curie Actions), by the
Generalitat de Catalunya. RN-L is supported by the research grant from the Generalitat
in the CRG/UPF Proteomics Unit which is part of the Proteored, PRB3 and is supported
Data Availability
The shotgun proteomics data have been deposited to the ProteomeXchange Consortium
dataset identifier PXD010356. The targeted proteomics data have been deposited to the
PXD012138. The script used for Matlab image analysis has been deposited at
Disclosures
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Synaptic panel Alzheimer’s disease CSF
The authors declare that the Biomedical Research Institute Sant Pau (IIB Sant Pau) has
protect the intellectual property included in this manuscript. O Belbin, A Lleó, Á Bayés,
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Synaptic panel Alzheimer’s disease CSF
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Synaptic panel Alzheimer’s disease CSF
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40
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Figures
AT microscopy, ultrathin tissue slices (70nm) from human post-mortem cortical tissue
from one brain donor were immunostained for pre- (synaptophysin, red) and post-
(PSD95, green) synaptic markers and the synaptic panel proteins (blue). A
41
Synaptic panel Alzheimer’s disease CSF
(at 70nm increments) is shown to the right of the reconstruction. Scale bars representing
1μm are shown at the bottom of each z stack. (B) Mean fold-enrichment plotted for
(PSD) fractions taken from post-mortem human cortex (n=6). S/H and PSD/H; intensity
in S or PSD fractions relative to H for the same sample. H/H; intensity in the H fraction
for each sample relative to the mean intensity in the H fraction across all samples.
Enrichment of the pre- (synaptophysin) and post (PSD-95) markers are also shown.
Degrees of freedom (df), F statistic and p-values for the ANOVA are shown at the top
of each plot and Dunnett’s pvalues are shown for significant pair-wise comparisons
42
Synaptic panel Alzheimer’s disease CSF
synaptic panel peptides and summarized protein levels are plotted for each preclinical and clinical AD stage versus cognitively normal controls
43
Synaptic panel Alzheimer’s disease CSF
for (A) the exploratory cohort (Controls n=20, Stage1 n=10, Stage2 n=10, Prodromal n=20, AD dementia n=20), (B) validation cohort-1
(Controls n=18, Stage1 n=9, Stage2 n=8, Prodromal n=10, AD dementia n=15), (C) validation cohort-2 (Controls n=20, Stage1 n=18) and (D)
The linestyle of the error bars were determined by p-value cut-offs for pair-wise group comparisons using a mixed effect linear regression model
(see legend). Stage1; preclinical AD stage 1, Stage2; preclinical stage 2,Prodromal; prodromal AD, Dementia; AD dementia.
44
Synaptic panel Alzheimer’s disease CSF
Study Species (n) Tissue Clinical status (A) /Fraction analyzed (B-D) Protein identification method #Proteins published
(A) Proteomic studies of the CSF
(20) Begcevic Human (6) CSF Non-pathological SCX+LC-MS/MS 2,615*
(21) Zhang 2015 Human (14) CSF Non-neurological Surgery HPLC+LC-MS/MS 2,513
(22) Guldbrandsen Human (21) CSF Neurologically healthy RP-AX LC-MS/MS 3,081
45
Synaptic panel Alzheimer’s disease CSF
(MASC) is shown. *Data were not publicly available for retrieval. The species, tissue, clinical status (of CSF donors), synaptic fraction
(synapse), protein identification and number of proteins are shown for each study.
46
(A) Protein (B) Peptides
AMPA receptor endocytosis
maintenance and maturation
Presynaptic differentiation
Synaptic transmission and
Dendritic spine assembly,
Position;
Postsynaptic Ca2+
Length; Domain;
Name; UniprotID Sequence
Synaptogenesis
and trafficking
plasticity
Vamp-2; P63027 (69) 116aa; 14% LQQTQAQVDEVVDIMR 32-47; cytoplasmic, v-SNARE motif;1; Mature
Table 2. Synaptic proteins and corresponding peptides monitored by SRM in CSF samples. The proteins (A) are listed with a
summary of the reported synaptic function of the protein and coverage achieved by quantifying the peptides listed in (B).
47
Amyloid- Neuro- Cognitive N (% female, Age at Mean +/- SD (range)
Clinical stage collection
osis degeneration decline %E4+) MMSE score Aβ42 (ng/ml) total tau (ng/ml) p-tau (ng/ml)
Table 3. Demographic and biomarker levels for CSF cohorts used for Targeted SRM. The number of samples, percentage female
and APOE ε4 (E4+) carriers as well as the mean, standard deviation (SD) and range of the age-at-lumbar puncture, mini-mental stage
examination (MMSE) score, and CSF levels of Aβ42, total tau and phosphorylated tau (p-tau) are shown for each diagnostic group of
the exploratory (A) and validation cohorts 1 (B) and 2 (C). Diagnostic groups were classified according to AD biomarkers for
amyloidosis (+/-) and neurodegeneration (+/-) and degree of cognitive impairment (-; cognitively normal, +; amnestic mild cognitive
impairment, ++; Dementia). Local biomarker cut-offs for positivity were as follows; Aβ42. <550ng/ml (Exploratory), <500ng/ml
48
Synaptic panel Alzheimer’s disease CSF
(Validation). PET positive, total tau >350ng/ml (Exploratory), >450ng/ml (age50-70) />500ng/ml (age>70) (Validation),