Research Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer’s Disease Cerebrospinal Fluid Authors Alberto Lleó, Raúl Núñez-Llaves, Daniel Alcolea, Cristina Chiva, Daniel Balateu-Paños, Martí Colom-Cadena, Gemma Gomez-Giro, Laia Muñoz, Marta Querol-Vilaseca, Jordi Pegueroles, Lorena Rami, Albert Lladó, José L. Molinuevo, Mikel Tainta, Jordi Clarimón, Tara Spires-Jones, Rafael Blesa, Juan Fortea, Pablo Martínez-Lage, Raquel Sánchez-Valle, Eduard Sabidó, Àlex Bayés, and Olivia Belbin Correspondence Graphical Abstract alleo@santpau.cat; obelbin@santpau.cat In Brief In the search for biomarkers of synapse loss associated with Alz- heimer’s disease (AD), we used shotgun proteomics to identify a panel of 9 synaptic proteins de- tectable in human cerebrospinal fluid (CSF). Expression at the hu- man synapse was verified using super resolution microscopy. Using SRM, we monitored the panel in CSF from 3 independent clinical cohorts. We report 6 synaptic bio- markers that demonstrate changes in preclinical AD prior to markers of neurodegeneration, which could have clinical value for assessing disease progression. Highlights • Proteomic analysis of cerebrospinal fluid and identification of synaptic component. • Use of super resolution microscopy to verify synapse-specificity in human tissue. • Selective reaction monitoring MS (SRM) of synaptic panel in 3 cohorts of Alzheimer’s disease cerebro- spinal fluid. • Synaptic protein changes precede tau in preclinical Alzheimer’s disease. Lleó et al., 2019, Molecular & Cellular Proteomics 18, 546 –560 March 2019 © 2019 Lleó et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. https://doi.org/10.1074/mcp.RA118.001290 This is an Open Access article under the CC BY license. los Research Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer’s Disease Cerebrospinal Fluid* Alberto Lleó‡§ §§§¶¶¶, Raúl Núñez-Llaves§¶ ¶¶¶, Daniel Alcolea‡§, Cristina Chiva储**, Daniel Balateu-Paños¶, Martí Colom-Cadena§¶, Gemma Gomez-Giro¶, Laia Muñoz§¶, Marta Querol-Vilaseca§¶, Jordi Pegueroles§¶, Lorena Rami‡‡, Albert Lladó‡‡, José L. Molinuevo‡‡, Mikel Tainta§§¶¶, Jordi Clarimón§¶, Tara Spires-Jones储储, Rafael Blesa‡§, Juan Fortea‡§, Pablo Martínez-Lage§§, Raquel Sánchez-Valle‡‡, Eduard Sabidó储**, Àlex Bayés***‡‡‡, and Olivia Belbin§¶储储储 A biomarker of synapse loss, an early event in Alzheimer’s cross-cohort meta-analysis, 6 synaptic proteins (Calsyn- disease (AD) pathophysiology that precedes neuronal tenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B death and symptom onset, would be a much-needed and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical prognostic biomarker. With direct access to the brain AD, changes that precede clinical symptoms and CSF interstitial fluid, the cerebrospinal fluid (CSF) is a potential markers of neurodegeneration. Therefore, these proteins source of synapse-derived proteins. In this study, we could have clinical value for assessing disease progres- aimed to identify and validate novel CSF biomarkers of sion, especially in preclinical stages of AD. Molecular & synapse loss in AD. Discovery: Combining shotgun pro- Cellular Proteomics 18: 546–560, 2019. DOI: 10.1074/mcp. teomics of the CSF with an exhaustive search of the RA118.001290. literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). Synapse loss is a fundamental process underlying many We confirmed the specific expression of 9 of the remain- neurological and psychiatric diseases including, but not lim- ing proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, ited to Alzheimers Disease (AD)1, Parkinson’s disease, Lewy Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at body diseases, schizophrenia and depression (1–3). There- the human synapse using Array Tomography microscopy fore, a biomarker capable of detecting synapse loss in living and biochemical fractionation methods. Exploration: Us- individuals has the potential to be a surrogate marker for ing SRM, we monitored these 9 synaptic proteins (20 disease severity, which would make an excellent addition to peptides) in a cohort of CSF from cognitively normal con- the biomarker arsenal for a wide range of neurological dis- trols and subjects in the pre-clinical and clinical AD eases. To search for novel synaptic biomarkers, we have stages (n ⴝ 80). Compared with controls, peptides from 8 selected AD as a disease model for synaptopathy. Synapse proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodro- loss is an early event in AD, which precedes neuronal death (1) mal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, and evidence from animal models indicates that the synapse p ⴝ 0.04) in an independent cohort (n ⴝ 60). Moreover, 7 is the target of both AD pathological proteins, A␤ and tau (4). proteins were reduced at preclinical stage 1 (0.6 to 0.8- AD can be conceptualized as a continuum of preclinical and fold, p < 0.04), a finding that was replicated (0.7 to 0.8- clinical phases, based on the clinical syndrome and biomark- fold, p < 0.05) for 6 proteins in a third cohort (n ⴝ 38). In a ers of brain amyloidosis and tau-mediated neurodegenera- From the ‡Memory Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; §Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain; ¶Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025Barcelona, Spain; 储Proteomics Unit, Center for Genomics Regulation, Barcelona Institute of Science and Technology, 08003 Barcelona; **University Pompeu Fabra, 08003 Barcelona; ‡‡Alzheimer’s Disease and Other Cognitive Disorders Unit, Neurology Department, Hospital Clínic-Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 08015 Barcelona, Spain; §§Department of Neurology, Center for Research and Advanced Therapies, CITA-Alzheimer Foundation, 20009 San Sebastian, Spain; ¶¶Servicio de Neurologia, Orga- nización Sanitaria Integrada Goierri-Alto Urola, Osakidetza, Zumárraga, España; 储 储Centre for Discovery Brain Sciences and UK Dementia Research Institute, University of Edinburgh EH8 9JZ, UK; ***Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025, Barcelona, Spain; ‡‡‡Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain Received December 20, 2018 Published, MCP Papers in Press, January 3, 2019, DOI 10.1074/mcp.RA118.001290 546 Molecular & Cellular Proteomics 18.3 © 2019 Lleó et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Synaptic Panel Alzheimer’s Disease CSF tion. In this conceptualization, patients with mild cognitive EXPERIMENTAL PROCEDURES impairment (5) or dementia (6) who are positive for AD bio- Experimental Design and Statistical Rationale—This study is di- markers are labeled as patients with MCI because of AD vided into the following stages: Discovery stage; peptide and protein (prodromal AD) or dementia because of AD (6). Likewise, the identification in 7 pools of 60 CSF samples. Verification stage; Tier 2 current guidelines from the National Institute on Aging-Al- SRM assay development for selected peptides in CSF and patholog- ical study of selected proteins in human post-mortem tissue from 6 zheimer Association (NIA-AA) conceptualize three preclinical donors. Exploration stage; SRM of selected peptides in CSF from stages of AD (7) whereby cognitively normal subjects with cognitively normal controls and pre-clinical and clinical stages of the signs of brain amyloidosis (preclinical Stage 1), amyloidosis AD continuum (n ⫽ 80) prospectively recruited from the Sant Pau and neurodegeneration (preclinical Stage 2) or both markers Initiative in Neurodegeneration (SPIN) cohort at Hospital Sant Pau, as well as subtle cognitive decline (preclinical Stage 3). Al- Barcelona and by the CITA Foundation, Donostia (exploratory cohort). Validation stage; SRM of selected peptides in an independent collec- though markers of A␤ and tau pathology are excellent diag- tion of CSF (n ⫽ 60) prospectively recruited from the collection at nostic biomarkers for AD, a marker of synapse degeneration Hospital Clinic, Barcelona (validation cohort-1) and an independent would be invaluable for assessing disease progression in selection (validation cohort-2) of cognitively normal controls and pre- at-risk subjects. In this regard, many researchers have turned clinical stage 1 subjects (n ⫽ 38) from the SPIN cohort. Where possible, to biochemical markers in CSF, a biofluid with direct access to subjects included in each group were age and sex-matched. CSF the central nervous system that can be extracted from living samples were run on the mass spectrometer in a randomized order with respect to diagnostic group. No technical replicates were included in individuals by lumbar puncture. the SRM study. Biological controls for SRM included cognitively normal Previous studies have reported elevated CSF levels of in- individuals (exploratory Cohort, n ⫽ 20, validation cohort-1, n ⫽ 18, dividual synaptic proteins such as neurogranin (8), SNAP-25 validation cohort-2, n ⫽ 20). Biological replicates for SRM included (9), synaptotagmin-1 (10) in AD dementia patients and neur- patients and volunteers grouped according to established diagnostic exins 1, 2 and 3, and neurofascin (11) in prodromal AD pa- criteria. As technical controls for SRM, BSA controls were run between tients. Although these findings support the idea that synaptic each sample (shotgun and targeted LC-MS/MS). Clinical CSF Cohorts—All participants gave their written consent. proteins in CSF may be informative in AD, the previously The study (IIBSP-BIO-2015–76) was approved by the local ethics reported correlation between CSF levels of synaptic proteins committee following the ethical standards recommended by the Hel- with CSF levels of tau suggest that widespread neuronal loss sinki Declaration. All subjects were evaluated by neurologists with could be a confounding factor when studying synaptic pro- expertise in neurodegenerative diseases, by neuropsychologists us- teins in the CSF, particularly at clinical disease stages. The ing a previously published neuropsychological battery (12) and as- CSF profile of synaptic proteins in preclinical stages of AD, sessed for established AD biomarkers, namely brain amyloidosis (low CSF levels of A␤1– 42 or positive amyloid PET imaging) and neuro- before widespread neurodegeneration has taken hold has not degeneration (high CSF levels of total tau or phosphorylated tau) been explored in detail. This is an important aspect because based on local cut-offs. These cut-offs have high specificity and a good CSF marker of underlying synaptic degeneration sensitivity to distinguish AD dementia patients from controls (13). should demonstrate changes that precede those of neurode- Diagnoses of prodromal AD and AD dementia were made according generation markers. The progressive staging of neurodegen- to NIA-AA guidelines (5, 6). Subjects within the normal range following formal neuropsychological evaluation, when accounting for age and eration markers in AD, make this disease an excellent model education (mostly recruited among patients’ caregivers), were classi- system to evaluate the potential relationship between CSF fied into preclinical AD stages in accordance with NIA-AA guidelines levels of proposed synaptic biomarkers and existing markers (7). of neurodegeneration. CSF Collection, Biomarker Assessment, and APOE Genotyping— Here we report (1) a systematic proteomic study of the CSF CSF samples were collected following international consensus rec- with a thorough characterization of the synaptic composition ommendations (14) as previously described (13). Samples had been previously stored at ⫺80 °C and had not been thawed prior to anal- (discovery), (2) development of Tier 2 SRM assays for a set of ysis. Commercially available ELISA kits were used to determine levels CSF proteins whose expression was confirmed at the human of CSF A␤1– 42 (InnotestTM A␤1– 42, Fujirebio-Europe, Belgium), total cortical synapse or in synapse-associated structures (verifi- Tau (InnotestTM hTAU Ag), Tau phosphorylated at threonine residue cation), (3) assessment of the CSF profile of the synaptic 181 (InnotestTM Phospho-Tau 181P). Our laboratory has extensive panel in a clinical cohort that includes cognitively normal experience in CSF biomarker determination and participates in the subjects and AD preclinical and clinical stages (exploration), Alzheimer’s Association external quality control program for CSF biomarkers (15). and (4) confirmation in independent clinical AD cohorts (clin- Post-mortem Human Brain Samples—All post-mortem brain tissue ical validation). used in this study was collected by the Neurological Tissue Bank at Hospital Clínic (IDIBAPS, Barcelona). The study (IIBSP-ATM-2012– 46) was approved by the local Ethics Committee of both the Tissue 1 Bank and Sant Pau Research Institute. Fresh brain tissue used for The abbreviations used are: AD, Alzheimer’s disease; AT, array tomography; CSF, cerebrospinal fluid; FDR, false discovery rate; GO, array tomography was collected from the superior frontal cortex of a gene ontology; LC, liquid chromatography; MS/MS, mass spectrom- female donor who died at the age of 83 and showed low AD pathology etry; NIH-AA, National Institute of Health-Institute of Aging; PSD-95, (Braak stage II). Pre-collected frozen tissue blocks from 6 donors (4 post-synaptic density; SCX, strong cation exchange; SRM, selected male, 2 female, mean age-at-death 66 years) without AD pathology reaction monitoring. were used for synaptosome and PSD enrichment. Molecular & Cellular Proteomics 18.3 547 Synaptic Panel Alzheimer’s Disease CSF Shotgun Liquid Chromatography Mass Spectrometry (LC-MS/ avoid duplication across studies. Proteins without a reviewed Uniprot MS)—Where indicated, immunodepletion was performed using the identifier were removed from the study. ProteoPrep® Immunoaffinity Albumin & IgG Depletion Kit (Sigma- Synaptosome Enrichment—All steps were performed at 4 °C. 200 Aldrich, Missouri). Six pools each containing CSF from 10 individuals mg chunks were cut from frozen frontal cortex tissue blocks and were precipitated with acetone and protein content was quantified by homogenized in cold Buffer A (0.32 M sucrose, 1 mM NaHCO3, 1 mM Bradford assay. Samples (50 ug) were reduced in 10 mM DTT, alky- MgCl2, 0.5 mM CaCl2, 1:2500 phenylmethylsulfonyl fluoride, 1 ␮g/ml lated with 55 mM IAA, and, digested in-solution with trypsin and LysC aprotinin, 1 ␮g/ml leupeptin). Homogenates were centrifuged (1400 ⫻ overnight and desalted using a Desalted MicroSpin Column (GE g, 10 min) and the supernatant transferred to a new tube. The pellet healthcare, UK). Where indicated, samples were fractionated by was resuspended in cold Buffer A and the previous step repeated strong cation exchange (SCX; Empore Disks Anion Exchange-SR, with centrifugation at 710 ⫻ g. The two supernatants were combined Sigma-Aldrich). An equivalent of 5 ␮l of each CSF Sample was and centrifuged (710 ⫻ g). The supernatant was subjected to a final analyzed using a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo centrifugation (30,000 ⫻ g, 15 min). The pellet was resuspended in Fisher Scientific, San Jose, CA) coupled to a nano-LC (Proxeon, Buffer B (0.32 M sucrose, 1 mM NaHCO3), layered over a sucrose Odense, Denmark) equipped with a reversed-phase chromatography gradient (0.85 M, 1 M, 1.2 M) and centrifuged (82,500 ⫻ g, 2 h). The 2-cm C18 pre-column (Acclaim PepMap-100, Thermo; 100 ␮m i.d., 5 synaptosomal fraction (a thick white band at the 1–1.2 M interface) ␮m), and a reversed-phase chromatography 25 cm C18 column was collected, diluted in 4x volume of Buffer B. An aliquot was (Nikkyo Technos, Japan; ␮m i.d., 1.9 ␮m) using a data-dependent centrifuged (48,200 ⫻ g, 20 min) and the pellet (synaptosome) resus- acquisition mode. Acquired data were analyzed using the Proteome pended in Buffer C (50 mM Tris pH 7.4, 1% SDS) and stored at Discoverer software suite (v1.4.1.14, Thermo Fisher Scientific), and ⫺80 °C. The remaining aliquot was diluted in equal volume Buffer D the Mascot search engine (v2.5.1, Matrix Science) was used for (50 mM Tris pH 7.4) and 1⫻ volume of 2% Triton-X, incubated for 10 peptide identification. Data were searched against the Swiss Prot min and centrifuged (maximum velocity, 30 min). The pellet (post- Human Protein database plus the most common contaminants (ver- synaptic density; PSD) was re-suspended in Buffer C and stored at sion 2014, 20884 entries). A precursor ion mass tolerance of 7 ppm at ⫺80 °C. the MS1 level was used, and up to three missed cleavages for trypsin Array Tomography—The array tomography protocol was applied were allowed. The fragment ion mass tolerance was set to 0.5 Da. using previously described methods (17, 18). Briefly, a 1 cm3 section Oxidation of Methionine and N-terminal protein acetylation was de- was taken from the superior frontal cortex was fixed, dehydrated and fined as variable modification and carbamidomethylation of Cysteines polymerized in 100% LR-white resin. The embedded samples were was set as fixed modification. The identified peptides were filtered at sectioned using a diamond knife (Diatome, UK) creating 20 serial 70 5% FDR calculated using a target-decoy database strategy. For each nm thick sections, which were mounted onto coverslips. The ultrathin identified peptide, peptide peak areas were obtained as extracted ion ribbons were washed with Tris buffer and blocked for 5 min. Primary chromatograms and protein abundances were estimated with the antibodies used were as mentioned above with the following excep- average peak area of the three most intense peptides per protein. For tions; anti-Tenascin-R (Abcam, UK; ab121916) anti-GluR2, anti-Neuroli- characterization of the CSF proteome, proteins without a reviewed gin-2, anti-Neurexin2 (Merck Millipore, Massachusetts; MAB397, Uniprot identifier were excluded. The mass spectrometry proteomics AB15510, ABN97), anti-synaptophysin (Osenses, Australia; oss00029w) data have been deposited to the ProteomeXchange Consortium via and anti-PSD95 (Synaptic Systems, Germany; 124014), alexa-tagged the PRIDE (16) partner repository (https://www.ebi.ac.uk/pride/ secondary antibodies (Thermo Fisher Scientific). Coverslips were archive) with the data set identifier PXD010356. mounted onto the slides using Slowfade Gold with DAPI (Thermo Fisher Database and Literature Curation—A systematic search of PubMed Scientific). Images were captured using a fully automated epifluores- (www.pubmed.com) for proteomic studies was performed using the cence upright microscope (custom adapted BX51, Olympus, Pennsyl- search terms “Cerebrospinal fluid”/“CSF,” & “proteome”/“proteom- vania) with a 64 ⫻ 1.2 NA Plan Apochromat objective. Image analysis ics” & “human,” for the CSF proteome (April 2013) and “synapto- was performed using Matlab (Mathworks). The script has been depos- some”/”synapse”/“post-synaptic density” & “proteome”/“proteom- ited at https://github.com/MemoryUnitSantPau with the name SynSeg. ics” & and “human”/”mouse”/”rat,” for the synapse proteome (April Images from serial sections were stacked, aligned, thresholded and the 2014). Only publications in English were reviewed. All identified pro- nonspecific staining (not present in at least 2 consecutive sections) teins were extracted either directly from the published material or removed using a local threshold-based algorithm. 3-D reconstructions where available, from the PRIDE repository. All proteins with a known of representative synapses were generated using the ImageJ Volume function related to the synapse were retrieved from 4 publicly avail- Viewer plug-in with tricubic smooth interpolation. able databases. Specifically, all proteins annotated with a GO related SDS-Page and Western Blotting—The total protein content of ho- to the synapse were retrieved from AmiGO2 version 2.3.2 (http:// mogenate, synaptosome and PSD enriched fractions was quantified amigo2.geneontology.org/amigo) using the search terms “dendritic by bicinchoninic acid assay. Aliquots containing 20 ␮g total protein spine,” “synapse,” “synaptic,” transmission of nerve impulse” and were boiled, diluted in loading buffer (100 mM Tris-HCL, 4% SDS, “neurotransmitter”. All proteins annotated to “Glutamatergic syn- 20% glycerol, 200 mM DTT and 200 mM ␤-mercaptoethanol) and apse,” “Cholinergic Synapse,” “Serotonergic synapse,” “GABergic loaded onto a 12% Tris-Tricine gel and electrophoresed. Proteins synapse,” “Dopaminergic synapse,” “Synaptic vesicle cycle,” ”Long- were transferred to a nitrocellulose membrane, which was immuno- term potentiation,” ”Long-term depression,” “Retrograde endocan- stained using the following antibodies; anti-CLSTN1, anti-Synapto- nabinoid signaling” according to the Kyoto Encyclopedia of Genes physin, anti-Thy1, (Abcam; ab134130, ab8049, ab133350), anti- and Genomes were retrieved from http://www.kegg.jp. All proteins GluR2, anti-GluR4, anti-Vamp2, anti-PSD95 (Cell Signaling; 13607, with “neurotransmitter release,” “synapse,” “synaptic,” or “synapto- 8070, 13508, 3450), Anti-neuroligin-2, anti-Syntaxin-1B (Synaptic genesis” as an annotated function were retrieved from Uniprot Systems; 129203, 110403), anti-Neurexin3, anti-Tenascin R (Thermo (www.uniprot.org). Because each data source used distinct protein Fisher; PA5– 47714, PA5– 47546), fluorescent dye-conjugated sec- identifiers that, in some cases, had been retired or updated, a unique, ondary antibodies (Li-COR Biosciences, Nebraska). reviewed Uniprot identifier was assigned to each protein using the Targeted Liquid Chromatography Mass Spectrometry (LC-SRM)— bioinformatic gene identification conversion tools, PIR (http://pir. Fifty-four proteotypic peptides (7 to 20 amino acids long) with tryptic georgetown.edu) and Bio-Mart (http://www.ensembl.org/biomart) to terminals corresponding to 22 proteins brought forward from the 548 Molecular & Cellular Proteomics 18.3 Synaptic Panel Alzheimer’s Disease CSF shotgun data were selected based on previous LC-MS/MS data and RESULTS database searches (Peptide Atlas). For each targeted peptide, corre- sponding crude heavy peptides were synthesized with 13C6 15N4 Discovery Stage - Characterizing the Synaptic Component (Arg), or 13C6 15N2 (Lys) isotopes (Peprotech SRM custom peptides, of the CSF—To characterize the CSF proteome, we per- grade 2, Thermo Fisher Scientific) for use as reference internal stand- formed a discovery proteomics screen of 7 CSF pools each ards in the CSF samples (Thermo Fisher), and to generate a library of containing samples from 10 individuals extracted from either MS/MS spectra for the selection of interference-free transitions for cognitively normal controls (5 pools), AD patients (1 pool) or a the peptides or interest. Individual CSF samples were precipitated mixture of both (1 pool), varying the LC-MS/MS conditions to with acetone and redissolved in 6 M urea before reduction (10 mM, DTT), alkylation (55 mM, IAA) and in-solution digestion with LysC and optimize protein yield. Further details of the sample pools, Trypsin (1:10, 37 °C, overnight). Isotopically-labeled peptides were LC-MS/MS conditions and identified contaminants can be spiked in each sample, and an equivalent of 5 ␮l of each sample was found in supplemental Table S1. We found that depletion of analyzed in a 120-min gradient (0 –35% ACN⫹0.1%FA) in SRM mode abundant IgGs and albumins increased the protein yield by using a triple quadrupole-Qtrap mass spectrometer (5500 QTrap, 115% and that SCX fractionation had a greater effect on Sciex, Massachussetts) coupled to a nano-LC chromatography col- umn (300 ␮l/min, 25-cm C18 column, 75 ␮m I.d., 2 ␮m particle size). protein yield (186% increase compared with runs without BSA control samples were analyzed between runs for instrument SCX, 6 h LC) than extended LC times (141% increase follow- quality control. Transitions were visualized and analyzed using Sky- ing SCX ⫹ 6 h LC compared with SCX ⫹ 2 h LC). Overall, IgG line 3.5 and peak picking was manually reviewed based on the and albumin depletion, SCX fractionation with 6 h LC resulted co-elution of the reference and endogenous peptides, peak shape in the greatest protein yield (mean yield ⫽ 1,259 proteins, and correlation of transition rank intensities between the reference range ⫽ 702–1,615). Across all LC-MS/MS runs, we identified and endogenous transitions. Of the initial 54 targeted peptides, 32 were eliminated from the study because of poor detection in a pilot 18,785 peptides (supplemental Table S2) corresponding to detection experiment with 5 CSF samples. The remaining 22 peptides 2742 unique proteins (supplemental Table S3). were taken forward for further monitoring in the exploratory and To gain a more complete picture of the CSF proteome, we validation cohorts. Peptide stability was assessed by injecting a pool performed a complementary search of published proteomic of all the samples over the duration of the mass spectrometric meas- studies of CSF extracted from cognitively or neurologically nor- urements and monitoring the peak area of the standard peptides. SRM transitions were processed using the dataProcess function of mal individuals. The search identified 10 independent studies MSstats v3.5 package in R (19). Specifically, the EqualizeMedians (20 –29) listed in Table IA. We extracted 3662 unique proteins function was used to normalize the data using the transition intensi- supplemental Table S3) from the 9 studies for which data were ties of the isotope-labeled standard peptides. This method was cho- publicly available. By combining our dataset with the datasets sen as it assumes that the samples of a data set are separated by a from the literature, we report 4315 proteins that have been constant and scales the samples so that they have the same median. identified in cognitively/neurologically normal CSF and a further The results were not significantly altered when compared with normal- ization using the alternative Quantile method in MSstats. The normal- 296 proteins that we detected only in pools from AD patients ized data were summarized (TMP: Tukey’s Median Polish) according to and/or mixed pools. either peptide or protein. Transitions with between run interference were To identify the synaptic component of the CSF, we next identified using the “betweenRunInterferenceScore” (cut-off ⫽ ⬍0.8). sought to characterize the human synaptic proteome also Samples with log base-2 endogenous intensities under the cut-off des- using publicly available data, but in this case related to the ignated by the MSstats package for each cohort (9.5211, 8.4337, and 8.5219, respectively) were considered as censored missing values. The characterization of the synapse. Here we have defined the mass spectrometry-based targeted proteomics data have been depos- synaptic proteome as proteins that (1) have an annotated ited to the Panorama repository (https://panoramaweb.org/5Jugvs.url) synaptic function according to 4 selected public databases and the PRIDE repository with the identifier PXD012138. (see Experimental Procedures), and (2) have been detected in Statistical Analysis—To determine enrichment of synaptic prteins in at least 1 of 23 published proteomic studies (30 –50) of syn- synaptosome and PSD-enriched fractions isolated from post-mortem brain tissue, the intensity of bands corresponding to the synaptic apse-enriched fractions from rodent or human brain tissue proteins on SDS-PAGE membranes were quantified using Odyssey (Table IB–ID). supplemental Table S4 shows the 537 proteins software (Li-COR Biosciences). The ratio of each fraction relative to that satisfy these criteria. Cross-referencing the CSF with this that of the homogenate (enrichment) was calculated. Because enrich- shortlist, we identified 251 synaptic proteins that are detect- ment ratios for the 10 synaptic proteins did not clearly deviate from a able in the CSF by shotgun mass spectrometry (supplemental Gaussian distribution (Kolmogorov-Smirnov p ⬎ 0.1), mean enrich- Table S5). Using these criteria, we report that ⬃6% of CSF ment ratios were compared in synaptosome and PSD fractions versus homogenates by One-Way Analysis of Variance (with Dunnett’s post- proteins are of synaptic origin. hoc test). For SRM data, log2 fold-changes for each preclinical and Verification Stage - Selecting a Panel of Synapse-specific clinical AD stage relative to controls were calculated using a mixed Proteins Suitable for SRM—The 251 synaptic proteins that effect linear regression model (GroupComparison function in were detectable in the CSF were manually curated and an MSstats). For meta-analyses of the SRM data from the three cohorts, initial list of 22 proteins that participate in one or more core p values were calculated according to Fisher’s method using the “metap” package in R. Where stated, adj.p values refers to p values synaptic process (Calsyntenin-1 (51), GABAB receptor 1 (52), adjusted using the Benjamini-Hochberg method to account for mul- GABAA receptor A2 (53), GABAA receptor A3, GluR2 (54), tiple testing. GluR4 (55), mGluR1 (56), Munc18 (57), Neurexin-2A (58) Neur- Molecular & Cellular Proteomics 18.3 549 Synaptic Panel Alzheimer’s Disease CSF TABLE I Details of published proteomic studies of the CSF and synapse. The number of proteins identified by at least 1 proteotypic peptide in each study of (A) the CSF, (B) whole synapse, (C) active zone and synaptic vesicles and (D) PSD/MAGUK-associated signalling complexes (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 Clinical status (A)/ #Proteins Study Species (n) Tissue Protein identification method Fraction analyzed (B–D) published (A) Proteomic studies of the CSF (20) Begcevic Human (6) CSF Non-pathological SCX⫹LC-MS/MS 2615* (21) Zhang 2015 Human (14) CSF Non-neurological Surgery HPLC⫹LC-MS/MS 2513 (22) Guldbrandsen Human (21) CSF Neurologically healthy RP-AX LC-MS/MS 3081 (23) Holtta Human (3) CSF Neurologically healthy LC-MALDI TOF/TOF 104 (24) Schutzer Human (11) CSF Non-neurologic SCX ⫹ LC-MS/MS 2630 (25) Stoop Human (40) CSF Non-neurological Surgery LC-MS & MALDI-FT-ICR-MS 178 (26) Pan 2006 Human (19) CSF Cognitively normal LC-MS/MS 216 (27) Xu Human (22) CSF Cognitively normal SCX ⫹ LC-MS/MS 784 (28) Zhang 2005 Human (22) CSF Cognitively normal SCX ⫹ LC-MS/MS 312 (29) Wenner Human (10) Postmortem CSF Cognitively normal SCX ⫹ LC-MS/MS 249 (B) Proteomic studies of the whole synapse (30) DiGiorgis Human (3) cortex Synaptosome MS/MS 24 (phosphoproteins) (31) Wilhelm Rat (4) cortex &cerebellum Synaptosome iBAQ 1113 (32) Abdul-Husn Mouse (10) hippocampus Presynapse In-gel(26)/In-solution: LC-MS/MS 131 Rat (10) striatum Presynapse In-gel(26)/In-solution: LC-MS/MS 113 (C) Proteomic studies of the active zone/synaptic vesicles (SV) (33) Weingarten Mouse (12) whole brain Presynaptic active microOTOF-Q II/MALDI-TOF/TOF 482 zone, SV (34) Blondeau Rat whole brain SV (Ficoll) In-gel(61): LCMS/MS 209 (35) Boyken Rat whole brain SV (fraction 4–7, 19–21, iTRAQ AP⫹LC-MS/MS 491 vGLUT1/VGAT⫹) (36) Coughenour Rat forebrain SV (fraction 13–21) In-gel NanosprayMS/MS QTOF 35 (37) Morciano 2005 Rat (3) whole brain SV (fraction 5–11, In-gel(36) MALDI-TOF 93 28–34/SV2-complex) (38) Morciano 2009 Rat whole brain SV (fraction 5–11, In-gel MALDI-TOF-MS/WB 218 28–34/SV2-complex) (39) Takamori Rat whole brain SV 2D-GE Q-TOF MS 400 (D) Proteomic studies of the post-synaptic density (PSD)/ MASC (40) Bayés 2011 Human (9) neocortex PSD LC-MS 1451 (41) Fernandez Mouse forebrain PSD (PSD-95⫹complexes) AP ⫹ LC-MS/MS LTQ-FT 117 (42) Cheng Rat Forebrain/cerebellum PSD ICAT-LC-MS/MS 238 (44) Li 2004 Rat forebrain PSD 2D-GE⫹MALDI-TOF/TOF 92 (43) Li 2005 Rat forebrain PSD ICAT LC-MS TOF/TOF - ICAT 113 (45) Peng Rat forebrain PSD LC-MS/MS LCQ-DECA XP Ion trap 365 (46) Walikonis Rat forebrain PSD 2D-GE, MALDI-TOF MS 26 (47) Bayés 2012 Mouse (3) cortex PSD LC-MS 1546 (48) Collins 2006 Mouse whole brain PSD SDS-PAGE-LC-MS/MS Q-TOF 617 (49) Trinidad Mouse whole brain PSD SCX Nano-LC-ESI-QTOF MS/MS 242 (50) Bayés 2014 Human neocortex MASC AP-Orbitrap LCMS/MS 289 (48) Collins 2006 Mouse whole brain MASC SDS-PAGE-LC-MS/MS Q-TOF 186 exin-3A (59), Neuroligin-1 (60), Neuroligin-2 (61), Neuroplastin To investigate the specificity of the expression of the re- (62), Synapsin-1 (63), Synaptic Ras-GAP 1 (64), Synaptic ves- maining 10 proteins in human synapses, we applied array icle glycoprotein 2B (65), Synaptotagmin-1 (66), Syntaxin-1A tomography (AT) microscopy to our panel of proteins in hu- (67), Syntaxin-1B, Tenascin-R (68), Thy-1 and Vamp-2 (69)) man cortical tissue. AT is particularly suited to the study of were selected with the goal of defining a subset of proteins synapses as it provides improved spatial resolution in the that is amenable for multiplex quantification. To confirm de- axial plane compared with other light microscopy techniques tection of the 22 proteins in CSF by targeted mass spectrom- (i.e. confocal microscopy). By obtaining ultrathin (70 nm) tis- etry, we developed SRM assays using 54 isotopically-labeled sue sections, single synapse terminals may be identified. peptides corresponding to the 22 selected proteins to be Representative 3D reconstructions of single synapse termi- used as internal references. We monitored the peptides in a nals clearly show the expression of 9 of the panel proteins preliminary set of 5 CSF samples and eliminated 32 peptides directly at the synapse, marked by pre (synaptophysin) and from the study because of poor detection (supplemental Ta- post (PSD-95) synaptic markers (Fig 1A). In contrast, Tenas- ble S6). cin-R was found surrounding the synapse without making 550 Molecular & Cellular Proteomics 18.3 Synaptic Panel Alzheimer’s Disease CSF FIG. 1. Expression of the panel proteins at the human cortical synapse. A, Using AT microscopy, ultrathin tissue slices (70 nm) 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 representative 3-D reconstruction of a representative synapse is shown for each panel protein. The segmented immunofluorescence of the 3 proteins in each individual stack (at 70 nm increments) is shown to the right of the reconstruction. Scale bars representing 1 ␮m are shown at the bottom of each z stack. B, Mean fold-enrichment plotted for each panel protein in homogenate (H), synaptosome-enriched (S) and PSD-enriched (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 p values are shown for significant pair-wise comparisons (␣ ⫽ 0.05). Molecular & Cellular Proteomics 18.3 551 Synaptic Panel Alzheimer’s Disease CSF direct contact. This is consistent with the literature where 7 ⫻ 10⫺4), Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Tenascin-R has been reported to reside in extracellular Neuroligin-2 and Thy-1 in particular (␳ ⫽ 0.66 to 0.90). perineuronal nets that surround the synapse (70). Validation Phase - Validation of the Synaptic CSF Profile in Specificity of synaptic expression was further evaluated by an Independent AD Cohort—All 20 peptides were brought quantifying the enrichment of the panel proteins in synapto- forward for validation in an independent clinical cohort com- some and PSD fractions extracted from 6 human cortical prising 60 CSF samples controls and preclinical and clinical tissue samples without pathology (supplemental Table S7). stages of the AD continuum (Table 3B). Supplemental Table The 2A isoform of Neurexin-2 could not be analyzed because S8B lists the transitions included and excluded from the anal- of the lack of a commercially-available specific antibody suit- ysis. Fig 2B shows the relative fold-change in CSF levels of able for Western blotting. Fig 1B shows that all 9 proteins the individual peptides and summarized proteins at each AD tested were enriched in synaptic fractions compared with the stage compared with the cognitively normal, AD biomarker homogenate (p ⬍ 0.03). Specifically, Calsyntenin-1, Neurexin- negative control group (n ⫽ 18). Supplemental Table S9B 3A, Syntaxin-1B and Vamp-2 were enriched 2.0 to 5.0-fold in shows the raw values. Like the exploratory cohort, levels of synaptosomes (p ⬍ 0.02) with Vamp-2 in particular, showing peptides from the same protein were highly correlated across greater enrichment (5-fold, p ⬍ 0.0001) than the widely-used all samples (␳ ⫽ 0.52 to 0.98) and all 20 peptides showed pre-synaptic marker, synaptophysin (3-fold, p ⬍ 0.0001). Cal- comparable CSF profiles at all disease stages. All proteins syntenin-1, GluR2, GluR4, Neuroligin-2, Tenascin-R and were positively correlated (n ⫽ 60, all ␳⬎0.36 all p ⬍ 0.005), Thy-1 were enriched 1.8 to 5.0-fold in the PSD fraction (p ⬍ GluR4, Neurexin-2A, Neuroligin-2, and Thy-1 in particular (␳ ⫽ 0.03). Therefore all 10 proteins are expressed specifically at 0.61 to 0.80). The greatest fold-change was observed in indi- viduals at preclinical stage 1 (n ⫽ 9) in whom Calsyntenin-1, the human synapse or in the surrounding area (Tenascin R). GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1, Exploration Phase - Characterizing the CSF Profile of the and Thy-1 peptides were reduced 0.6 to 0.8-fold (p ⬍ 0.05, Synaptic Panel in AD—Because Tenascin-R, was not directly adj.p ⫽ 0.03 to 0.09). In individuals at preclinical stage 2 (n ⫽ expressed at the synapse, this protein was not brought for- 8), Calsyntenin-1 and Vamp-2 peptides were elevated 1.3-fold ward for evaluation. Table II provides further information re- (p ⬍ 0.05, adj.p ⬎ 0.1). Although all peptides showed positive garding the 9 synaptic proteins and their corresponding pep- fold-changes (1.1 to 1.3-fold) in prodromal AD patients (n ⫽ tides that were monitored by SRM in the exploratory cohort 10), the only peptide to validate the elevated levels observed comprising 80 CSF samples from controls and all preclinical in the exploratory analyses was for GluR4, which was ele- and clinical stages of the AD continuum (Table IIIA). Supple- vated 1.3-fold (p ⫽ 0.04, adj.p ⫽ 0.5). None of the peptides mental Table S8A lists the transitions included and excluded were significantly elevated in patients at the dementia stage from the analysis. Fig 2A shows the relative fold-change in (1.0 to 1.2-fold, p ⬎ 0.09). CSF levels of the 20 individual peptides and summarized Although, all synaptic proteins moderately correlated with protein levels at each AD stage compared with the cognitively total tau levels, in both cohorts (exploratory; n ⫽ 80, ␳ ⫽ 0.45 normal, AD biomarker negative, control group (n ⫽ 20). sup- to ␳ ⫽ 0.67, validation; n ⫽ 60, ␳ ⫽ 0.36 to 0.56), by definition, plemental Table S9A shows the raw values. Peptides were not total tau levels are unaltered in individuals at preclinical AD significantly altered in individuals at preclinical AD stage 1 stage 1. Therefore, a reduction of synaptic proteins at preclin- (n ⫽ 10) compared with controls, albeit that 17 peptides from ical stage 1 would be of increased clinical value because 8 proteins were subtly reduced 0.8 to 0.9-fold (p ⬎ 0.1). At individuals at this early stage are asymptomatic and have yet preclinical AD stage 2 (n ⫽ 10), no differences were observed to demonstrate widespread neuronal degeneration. We there- (0.9 to 1.1-fold, p ⬎ 0.2). In prodromal AD patients (n ⫽ 20), 19 fore sought to replicate this finding in a larger set of controls peptides from 8 proteins were elevated 1.3 to 1.6-fold (p ⬍ and preclinical AD stage 1 (n ⫽ 38) from the SPIN cohort. 0.04, adj.p ⬍ 0.04). In patients at the dementia stage (n ⫽ 20), Supplemental Table S8C lists the transitions included and Calsyntenin-1 (1.2 to 1.3-fold, p ⬍ 0.02, adj.p ⫽ 0.1), GluR4 excluded from the analysis. Fig 2C shows that compared with (1.3-fold, p ⬍ 0.04, adj.p ⫽ 0.1), Neurexins-2A and 3A and control subjects, Calsyntenin-1, GluR4, Neurexin-2A, Neur- Thy-1 (1.2 to 1.3-fold, p ⬍ 0.04, adj.p ⫽ 0.1) peptides re- exin-3A, Syntaxin-1 and Thy-1 were reduced 0.7 to 0.8-fold mained elevated. Overall, levels of peptides from the same (p ⬍ 0.05). Supplemental Table S9C shows the raw values. protein were highly correlated across all samples (␳ ⫽ 0.77 to Finally, we performed a cross-cohort meta-analysis by 0.98). Excepting the GluR2 peptide, which showed relatively combining the p values (Fisher’s p value) at each disease elevated levels (p ⬎ 0.05) at preclinical stage 1, the fold- stage for peptides that demonstrated fold-changes in the changes of all peptides and proteins were comparable to same direction across all cohorts. Supplemental Table S9D each other at all disease stages, differing only in significance shows the raw values. The mean of fold-change for each level, indicating very little peptide or protein-specific differ- peptide at each disease stage is plotted in Fig 2D. In this ences. In fact, the levels of all proteins across all disease meta-analysis, Calsyntenin-1, GluR4, Neurexin-2A, Neurexin- stages were positively correlated (n ⫽ 80, all ␳⬎0.37, all p ⬍ 3A, Syntaxin-1B, and Thy-1 peptides were reduced (0.8-fold, 552 Molecular & Cellular Proteomics 18.3 TABLE II 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) (A) Protein (B) Peptides Dendritic AMPA Synaptic Position; Domain; Protein spine Postsynaptic receptor transmission Synaptic Presynaptic Length; Isoform; Protein products Name; UniprotID assembly, Ca2⫹ endocytosis and Synaptogenesis vesicle differentiation Coverage Sequence where the maintenance signalling and plasticity exocytosis peptide resides and maturation trafficking Calsyntenin-1; O94985 (51) (87) 981aa; 3% LTVTAYDCGK 235–244; extracellular;1,2; Mature, sAlc-␣ GNLAGLTLR 537–545; extracellular;1,2; Mature, sAlc-␣ Molecular & Cellular Proteomics 18.3 IISTITR 684–690; extracellular;1,2; Mature, sAlc-␣ GluR2; P42262 (88) (89) (90) 883aa; 2% YTSALTYDAVQVMTEAFR 295–312; extracellular; Flip,Flop,3 4; Mature GluR4; P48058 (90) 902aa; 5% LQNILEQIVSVGK 218–230; extracellular;1,2; Mature GYHYIIANLGFK 234–245; extracellular;1,2; Mature IQGLTGNVQFDHYGR 354–368; extracellular;1,2; Mature Neurexin-2A; Q9P2S2 (58) (91) 1712aa; 2% LSALTLSTVK 161–170; extracellular;1A,2A; Mature, sNRX2A LGERPPALLGSQGLR 184–198; extracellular;1A,2A; Mature, sNRX2A LQGDLSFR 478–485; extracellular;1A,2A; Mature, sNRX2A Neurexin-3A; Q9Y4C0 (58) (59) (91) 1643aa; 3% SDLSFQFK 49–56; extracellular;1A,4A; Mature, sNRXN3 NGLILHTGK 293–301; extracellular;1A,4A; Mature, sNRXN3 ANDGEWYHVDIQR 537–549; extracellular;1A,3A,4A; Mature, sNRXN3 Neuroligin-2; Q8NFZ4 (92) (61) (93) 835aa; 4% ELVDQDVQPAR 336–346; extracellular;1; Mature, sNLGN2 TLLALFTDHQWVAPAVATAK 450–469; extracellular;1; Mature, sNLGN2 Syntaxin-1B; P61266 (94) (95) 288aa; 5% AIEQSIEQEEGLNR 94–107; cytoplasmic;1,2; Mature Thy-1; P04216 (96) (97) (98) 161aa; 24% VTSLTACLVDQSLR 22–35; polypeptide chain;1; Mature HVLFGTVGVPEHTYR 61–75; polypeptide chain;1; Mature VLYLSAFTSK 88–97; polypeptide chain;1; Mature Vamp-2; P63027 (69) 116aa; 14% LQQTQAQVDEVVDIMR 32–47; cytoplasmic, v-SNARE motif;1; Mature 553 Synaptic Panel Alzheimer’s Disease CSF 554 TABLE III Demographic and biomarker levels for CSF cohorts used for Targeted SRM. The number of samples, percentage female and APOE ⑀4 (E4⫹) carriers as well as the mean, standard deviation (S.D.) and range of the age-at-lumbar puncture, mini-mental stage examination (MMSE) score, and CSF levels of A␤42, 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; A␤42. ⬍550 ng/ml (Exploratory), ⬍500 ng/ml (Validation). PET positive, total tau ⬎350 ng/ml (Exploratory), ⬎450 ng/ml (age50 –70) /⬎500 ng/ml (age⬎70) (Validation), Synaptic Panel Alzheimer’s Disease CSF phosphorylated tau (p-tau) ⬎61 ng/ml (Exploratory), ⬎75 ng/ml (Validation) Amyloid- Neuro- Cognitive N (% female, Age at Mean ⫹/⫺ S.D. (range) Clinical stage osis degeneration decline %E4⫹) collection MMSE score A␤42 (ng/ml) total tau (ng/ml) p-tau (ng/ml) (A) Controls ⫺ ⫺ ⫺ 20 (75,0) 67⫹/⫺5 (60–77) 29⫹/⫺1 (28–30) 899⫹/⫺175 (618–1147) 205⫹/⫺35 (144–274) 38⫹/⫺6 (30–49) Preclinical 1 ⫹ ⫺ ⫺ 10 (60,86) 69⫹/⫺4 (64–75) 28⫹/⫺2 (26–30) 406⫹/⫺92 (245–509) 232⫹/⫺67 (155–318) 43⫹/⫺7 (30–52) Preclinical 2/3 ⫹ ⫹ ⫺ 10 (50,75) 68⫹/⫺5 (63–76) 29⫹/⫺2 (26–30) 412⫹/⫺43 (361–485) 601⫹/⫺591 (323–2270) 85⫹/⫺42 (56–201) Prodromal AD ⫹ ⫹ ⫹ 20 (65,83) 70⫹/⫺4 (60–77) 28⫹/⫺2 (24–30) 449⫹/⫺62 (300–548) 716⫹/⫺355 (352–1771) 108⫹/⫺45 (66–230) AD dementia ⫹ ⫹ ⫹⫹ 20 (65,70) 72 (62–81)⫹/⫺6 23⫹/⫺2 (19–28) 400⫹/⫺87 (178–530) 708⫹/⫺398 (353–1784) 91⫹/⫺36 (45–206) (B) Controls ⫺ ⫺ ⫺ 18 (61,17) 60⫹/⫺5 (52–68) 29⫹/⫺1 (26–30) 788⫹/⫺227 (528–1351) 235⫹/⫺79 (111–429) 54⫹/⫺13 (37–71) Preclinical 1 ⫹ ⫺ ⫺ 9 (78,44) 64⫹/⫺7 (55–73) 28⫹/⫺1 (26–30) 375⫹/⫺84 (263–466) 182⫹/⫺60 (103–258) 43⫹/⫺14 (26–61) Preclinical 2/3 ⫹ ⫹ ⫺ 8 (38,38) 76⫹/⫺6 (68–85) 28⫹/⫺1 (26–29) 362⫹/⫺94 (229–486) 677⫹/⫺539 (389–2093) 100⫹/⫺45 (64–213) Prodromal AD ⫹ ⫹ ⫹ 10 (50,100) 67⫹/⫺9 (53–77) 25⫹/⫺3 (18–29) 357⫹/⫺73 (243–456) 773⫹/⫺217 (451–1253) 111⫹/⫺20 (75–144) AD dementia ⫹ ⫹ ⫹⫹ 15 (60,60) 66⫹/⫺9 (51–83) 23 (18–26) 322⫹/⫺85 (206–441) 1039⫹/⫺484 (430–2000) 142⫹/⫺49 (76–222) (C) Controls ⫺ ⫺ ⫺ 20 (55,25) 57⫹/⫺8 (45–76) 29⫹/⫺1 (28–30) 835⫹/⫺137 (614–1151) 151⫹/⫺58 (79–290) 31⫹/⫺11 (4–59) Preclinical 1 ⫹ ⫺ ⫺ 18 (56,39) 57⫹/⫺8 (40–74) 29⫹/⫺1 (27–30) 516⫹/⫺81 (384–734) 136⫹/⫺ (61–292) 29⫹/⫺10 (18–57) Molecular & Cellular Proteomics 18.3 Synaptic Panel Alzheimer’s Disease CSF FIG. 2. Synaptic panel peptide levels in the CSF across the AD continuum. The log2 fold-change (⫾ standard error; S.E.) in CSF levels of the synaptic panel peptides and summarized protein levels are plotted for each preclinical and clinical AD stage versus cognitively normal controls 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 combined mean log2 fold-change across the 3 cohorts. For ease of interpretation, the natural values are labeled on the y axis on a log2 scale. 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. Fisher’s p ⬍ 0.05) in individuals at preclinical AD stage 1, cohorts comprising controls and pre-clinical and clinical Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, stages of AD revealed a set of six synaptic proteins (Calsyn- Neuroligin-2, Thy-1, and Vamp-2 peptides were elevated (1.2 tenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and to 1.4-fold, Fisher’s p ⬍ 0.04) in prodromal AD patients and Thy-1) that were reduced 0.8-fold (p ⬍ 0.05) at the earliest Calsyntenin-1, GluR4, Neurexin-2A, and Neurexin-3A were preclinical stage of AD (stage 1, cognitively normal with pos- elevated (1.2 to 1.3-fold, p ⫽ 0.04) at the dementia stage. itive amyloid markers) compared with controls. Five of these Therefore, these data support a biphasic profile for 5 proteins proteins were also elevated in patients at the clinical stages (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, and Thy-1) (cognitive impairment and positive neurodegeneration mark- whereby compared with controls, CSF peptide levels are re- ers). We propose that the reduced levels in preclinical stage 1 duced at the earliest preclinical stage 1 of AD when neurode- may reflect reduced synaptic density in these individuals who generation has yet to take hold, as indicated by total tau already show signs of brain amyloidosis, an effect that is levels, but elevated at the prodromal AD stage when neuro- confounded by widespread neurodegeneration at later dis- degeneration is widespread. ease stages. In the discovery stage of this study, a thorough proteomic DISCUSSION screen of 60 CSF samples combined with a literature search This is the first systematic study of the CSF proteome and of proteomic studies of the CSF identified 4,315 proteins in thorough characterization of its synaptic component. Using cognitively normal individuals. To put this into context, the this resource, we selected a panel of 10 synaptic proteins for latest release of the Human Protein Atlas (v17) comprises evaluation as potentially novel biomarkers of synapse degen- RNA expression for 10,226 unique genes that were positively eration in neurological diseases characterized by synaptopa- detected in the brain (cortex, cerebellum or hippocampus), of thy. We confirmed the specific expression of 9 of the synaptic which we report that 26% are detectable in the CSF. Accord- proteins at the human cortical neuronal synapse therefore ing to the Human Protein Atlas data, we estimate that 79% of increasing the probability that their CSF levels are directly the brain-expressed proteins detected in the CSF are known related to synapse integrity. A thorough evaluation of the CSF to be expressed in the cortex, 65% in the hippocampus and profile of the 9 synaptic proteins in three independent clinical 67% in the cerebellum. Molecular & Cellular Proteomics 18.3 555 Synaptic Panel Alzheimer’s Disease CSF We then searched the literature and public databases for genesis makes these proteins particularly attractive candidate proteins that are both physically and functionally related to the biomarkers for AD. This does not exclude their potential as synapse, thereby prioritizing well-characterized synaptic pro- biomarkers of synapse loss in other neurological diseases. teins. The 537 proteins retrieved represent 4% of those with Defective GluR2 RNA editing may be relevant to amyotrophic positive RNA brain expression data included in the Human lateral sclerosis (82), whereas variations in genes encoding Protein Atlas database, suggesting that this is a relatively Neuroligin-2, Neurexin-3 and Syntaxin-1B have been impli- stringent classification. Regional expression data from the cated in schizophrenia (83), autism spectrum disorder (84) Human Protein Atlas provide evidence for 95% of these syn- and epilepsy (85), respectively. aptic transcripts in the cerebral cortex, 75% in the hippocam- In the exploration and validation stages of this study, we pus and 87% in the cerebellum. We conclude that most of the quantified the 9 synapse-verified proteins in three independ- proteins we have classified as synaptic are expressed across ent clinical cohorts of CSF samples from cognitively normal multiple brain regions. This lack of regional specificity offers controls and individuals from the AD continuum. As has been the possibility that these proteins could be informative across reported for other synaptic proteins, such as neurogranin (86), the range of neurological and psychiatric diseases, which synaptotagmin-1 (10) and SNAP-25 (9), we observed an in- demonstrate distinct regional susceptibility. Notably, our CSF crease in CSF levels of 8 of the panel proteins in prodromal database included 47% of these synaptic proteins. When AD patients compared with controls. The moderate correla- compared with the 26% yield of brain-expressed proteins in tion of the synaptic panel proteins with a marker of tau- CSF, we conclude that the coverage of synaptic proteins in mediated neurodegeneration suggests that synapse and neu- our CSF database was relatively high. ronal loss are interrelated in AD and that the panel proteins In the verification stage, we employed AT microscopy, a partially reflect both processes. GluR2, Neurexin-2A, Neuroli- technique especially suited to study single synapses (17, 18) gin-2, Syntaxin-1B, and Vamp-2 showed a weaker correlation to verify the synaptic expression of a set of proteins detect- with CSF total tau than that reported for neurogranin, synap- able in the CSF by SRM and with a known synaptic function totagmin and SNAP-25. It remains to be determined whether at the human cortical synapse. With improved antibody pen- the greater independence of these proteins from CSF tau etration compared with other super resolution techniques and levels compared with other synaptic proteins provides addi- improved resolution compared with traditional confocal mi- tional clinical value when used as biomarkers. We also report croscopy, we report with high confidence that our panel of 9 that 12 peptides from Calsyntenin-1, GluR4, Neurexin-2A, proteins are expressed directly at or surrounding human cor- Neurexin-3A, and Thy-1 were reduced at preclinical AD stage tical neuronal synapses. The synaptic panel includes proteins 1. Our data support that reduced levels of synaptic proteins in with a range of synaptic functions, including but not limited to, the CSF reflect a reduced synaptic density in these individu- spine assembly and maturation, synaptic transmission and als, which is only apparent when not being masked by wide- plasticity, synaptogenesis and synaptic vesicle exocytosis. spread neurodegeneration. These proteins could be valuable No clear difference in CSF profile could be attributed to any markers for monitoring disease progression in at-risk individ- function. The protein expression of 7 of these proteins has uals before the appearance of cognitive symptoms. been assessed by the Human Protein Atlas, all of which were In conclusion, we have identified and provided clinical val- detected in the cortical neuropil. Calsyntenin-1 and Neuroli- idation for a set of synaptic proteins (Calsyntenin-1, GluR4, gin-2 were the only proteins detected, at low levels, in glial Neurexin-2A, Neurexin-3A, Syntaxin-1B, and Thy-1) that can cells. Consistent with the extracellular expression in perineu- be detected in CSF by targeted liquid chromatography mass ronal nets, Tenascin-R was detected in the neuropil but not in spectrometry without depletion, immunoprecipitation or frac- neurons or glia, further supporting its exclusion from the re- tionation of the sample that could provide added value in the mainder of the study. In AD, reduced brain expression at the clinical setting to assess disease progression in individuals transcriptomic or proteomic level has been reported for at-risk for AD and AD patients and could improve enrichment GluR2, GluR4, Neurexin-2A, Syntaxin-1B, Tenascin-R, Thy-1, and monitoring of drug efficacy in pharmaceutical drug trials. and VAMP-2, whereas increased expression has been re- To our knowledge, this is the first study to demonstrate a ported for Neuroligin-2 (71–74). Pertinently, there is evidence non-linear profile of a subset of synaptic proteins in AD with in the literature that some of the panel proteins may be in- changes observed at the earliest preclinical AD stage before volved in mechanisms directly related to AD pathogenesis. neurodegeneration is widespread. This study serves as a For example, GluR2, (75) GluR4 (76), and Neurexin-2A (77) basis for future research on the dynamics of synaptic proteins have been postulated as direct targets of A␤ oligomers. Ad- in the CSF. Because the cross-sectional design of the current ditionally, there is evidence that Calsyntenin-1 (78, 79) and study does not allow assessment of the prognostic capacity Vamp-2 (80) play a role in A␤ production, trafficking or secre- of the synaptic panel proteins, future longitudinal studies in tion and a role for the Neurexin-Neuroligin complex in regu- large cohorts are necessary to further explore the potential of lating A␤ metabolism and function has also been postulated the synaptic panel proteins as prognostic biomarkers that (81). The potential involvement of these proteins in AD patho- precede markers of neurodegeneration. The potential of the 556 Molecular & Cellular Proteomics 18.3 Synaptic Panel Alzheimer’s Disease CSF proteins described herein to be useful prognostic biomarkers L.M., M.Q.-V., J.P., L.R., A. 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Pau (IIB Sant Pau) has filed a patent application (pending) to the (2016) The pre-synaptic vesicle protein synaptotagmin is a novel bio- European Patent Office (EP18382175.0) to protect the intellectual marker for Alzheimer’s disease. Alzheimers Res. Ther. 8, 41 11. Duits, F. H., Brinkmalm, G., Teunissen, C. E., Brinkmalm, A., Scheltens, P., property included in this manuscript. O Belbin, A Lleó, Á Bayés, J Van der Flier, W. M., Zetterberg, H., and Blennow, K. (2018) Synaptic Fortea and D Alcolea are the named inventors. proteins in CSF as potential novel biomarkers for prognosis in prodromal §§§ To whom correspondence may be addressed: Laboratorio Al- Alzheimer’s disease. Alzheimers Res. Ther. 10, 5 zheimer, Memory Unit, Hospital de la Santa Creu I Sant Pau, c/Sant 12. Sala, I., Illan-Gala, I., Alcolea, D., Sanchez-Saudinos, M. B., Salgado, S. A., Antoni Ma Claret 167, 08025, Barcelona, Spain. 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