Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid
2019; Elsevier BV; Volume: 18; Issue: 3 Linguagem: Inglês
10.1074/mcp.ra118.001290
ISSN1535-9484
AutoresAlberto 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 Luís Molinuevo, Mikel Tainta, Jordi Clarimón, Tara L. Spires‐Jones, Rafael Blesa, Juan Fortea, Pablo Martínez‐Lage, Raquel Sánchez‐Valle, Eduard Sabidó, Àlex Bayés, Olivia Belbin,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoA biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this 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 because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (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 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were 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 value for assessing disease progression, especially in preclinical stages of AD. A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this 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 because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsyntenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (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 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were 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 value for assessing disease progression, especially in preclinical stages of AD. Synapse loss is a fundamental process underlying many neurological and psychiatric diseases including, but not limited to Alzheimers Disease (AD) 1The abbreviations used are:ADAlzheimer's diseaseATarray tomographyCSFcerebrospinal fluidFDRfalse discovery rateGOgene ontologyLCliquid chromatographyMS/MSmass spectrometryNIH-AANational Institute of Health-Institute of AgingPSD-95post-synaptic densitySCXstrong cation exchangeSRMselected reaction monitoring. 1The abbreviations used are:ADAlzheimer's diseaseATarray tomographyCSFcerebrospinal fluidFDRfalse discovery rateGOgene ontologyLCliquid chromatographyMS/MSmass spectrometryNIH-AANational Institute of Health-Institute of AgingPSD-95post-synaptic densitySCXstrong cation exchangeSRMselected reaction monitoring., Parkinson's disease, Lewy body diseases, schizophrenia and depression (1Selkoe D.J. Alzheimer's disease is a synaptic failure.Science. 2002; 298: 789-791Crossref PubMed Scopus (3380) Google Scholar, 2Calabrese F. Riva M.A. Molteni R. Synaptic alterations associated with depression and schizophrenia: potential as a therapeutic target.Expert. Opin. Ther. Targets. 2016; 20: 1195-1207Crossref PubMed Scopus (25) Google Scholar, 3Bellucci A. Mercuri N.B. Venneri A. Faustini G. Longhena F. Pizzi M. Missale C. Spano P. Review: Parkinson's disease: from synaptic loss to connectome dysfunction.Neuropathol. Appl. Neurobiol. 2016; 42: 77-94Crossref PubMed Scopus (125) Google Scholar). Therefore, a biomarker capable of detecting 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 range of neurological diseases. To search for novel synaptic biomarkers, we have selected AD as a disease model for synaptopathy. Synapse loss is an early event in AD, which precedes neuronal death (1Selkoe D.J. Alzheimer's disease is a synaptic failure.Science. 2002; 298: 789-791Crossref PubMed Scopus (3380) Google Scholar) and evidence from animal models indicates that the synapse is the target of both AD pathological proteins, Aβ and tau (4Spires-Jones T.L. Hyman B.T. The intersection of amyloid beta and tau at synapses in Alzheimer's disease.Neuron. 2014; 82: 756-771Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). AD can be conceptualized as a continuum of preclinical and clinical phases, based on the clinical syndrome and biomarkers of brain amyloidosis and tau-mediated neurodegeneration. In this conceptualization, patients with mild cognitive impairment (5Albert M.S. DeKosky S.T. Dickson D. Dubois B. Feldman H.H. Fox N.C. Gamst A. Holtzman D.M. Jagust W.J. Petersen R.C. Snyder P.J. Carrillo M.C. Thies B. Phelps C.H. The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 270-279Abstract Full Text Full Text PDF PubMed Scopus (6348) Google Scholar) or dementia (6McKhann G.M. Knopman D.S. Chertkow H. Hyman B.T. Jack Jr, C.R. Kawas C.H. Klunk W.E. Koroshetz W.J. Manly J.J. Mayeux R. Mohs R.C. Morris J.C. Rossor M.N. Scheltens P. Carrillo M.C. Thies B. Weintraub S. Phelps C.H. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 263-269Abstract Full Text Full Text PDF PubMed Scopus (9233) Google Scholar) who are positive for AD biomarkers are labeled as patients with MCI because of AD (prodromal AD) or dementia because of AD (6McKhann G.M. Knopman D.S. Chertkow H. Hyman B.T. Jack Jr, C.R. Kawas C.H. Klunk W.E. Koroshetz W.J. Manly J.J. Mayeux R. Mohs R.C. Morris J.C. Rossor M.N. Scheltens P. Carrillo M.C. Thies B. Weintraub S. Phelps C.H. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 263-269Abstract Full Text Full Text PDF PubMed Scopus (9233) Google Scholar). Likewise, the current guidelines from the National Institute on Aging-Alzheimer Association (NIA-AA) conceptualize three preclinical stages of AD (7Sperling R.A. Aisen P.S. Beckett L.A. Bennett D.A. Craft S. Fagan A.M. Iwatsubo T. Jack Jr, C.R. Kaye J. Montine T.J. Park D.C. Reiman E.M. Rowe C.C. Siemers E. Stern Y. Yaffe K. Carrillo M.C. Thies B. Morrison-Bogorad M. Wagster M.V. Phelps C.H. Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 280-292Abstract Full Text Full Text PDF PubMed Scopus (4647) Google Scholar) whereby cognitively normal subjects with signs of brain amyloidosis (preclinical Stage 1), amyloidosis and neurodegeneration (preclinical Stage 2) or both markers as well as subtle cognitive decline (preclinical Stage 3). Although markers of Aβ and tau pathology are excellent diagnostic biomarkers for AD, a marker of synapse degeneration would be invaluable for assessing disease progression in at-risk subjects. 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 individuals by lumbar puncture. Alzheimer's disease array tomography cerebrospinal fluid false discovery rate gene ontology liquid chromatography mass spectrometry National Institute of Health-Institute of Aging post-synaptic density strong cation exchange selected reaction monitoring. Alzheimer's disease array tomography cerebrospinal fluid false discovery rate gene ontology liquid chromatography mass spectrometry National Institute of Health-Institute of Aging post-synaptic density strong cation exchange selected reaction monitoring. Previous studies have reported elevated CSF levels of individual synaptic proteins such as neurogranin (8Thorsell A. Bjerke M. Gobom J. Brunhage E. Vanmechelen E. Andreasen N. Hansson O. Minthon L. Zetterberg H. Blennow K. Neurogranin in cerebrospinal fluid as a marker of synaptic degeneration in Alzheimer's disease.Brain Res. 2010; 1362: 13-22Crossref PubMed Scopus (156) Google Scholar), SNAP-25 (9Brinkmalm A. Brinkmalm G. Honer W.G. Frolich L. Hausner L. Minthon L. Hansson O. Wallin A. Zetterberg H. Blennow K. Ohrfelt A. SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer's disease.Mol. Neurodegener. 2014; 9: 53Crossref PubMed Scopus (178) Google Scholar), synaptotagmin-1 (10Ohrfelt A. Brinkmalm A. Dumurgier J. Brinkmalm G. Hansson O. Zetterberg H. Bouaziz-Amar E. Hugon J. Paquet C. Blennow K. The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer's disease.Alzheimers Res. Ther. 2016; 8: 41Crossref PubMed Scopus (100) Google Scholar) in AD dementia patients and neurexins 1, 2 and 3, and neurofascin (11Duits F.H. Brinkmalm G. Teunissen C.E. Brinkmalm A. Scheltens P. Van der Flier W.M. Zetterberg H. Blennow K. Synaptic proteins in CSF as potential novel biomarkers for prognosis in prodromal Alzheimer's disease.Alzheimers Res. Ther. 2018; 10: 5Crossref PubMed Scopus (61) Google Scholar) in prodromal AD patients. Although these 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 profile of synaptic proteins in preclinical stages of AD, before widespread neurodegeneration has taken hold has not been explored in detail. This is an important aspect because a good CSF marker of underlying synaptic degeneration should demonstrate changes that precede those of neurodegeneration markers. The progressive staging of neurodegeneration markers in AD, make this disease an excellent model system to evaluate the potential relationship between CSF levels of proposed synaptic biomarkers and existing markers of neurodegeneration. Here we report (1) a systematic proteomic study of the CSF with a thorough characterization of the synaptic composition (discovery), (2) development of Tier 2 SRM assays for a set of CSF proteins whose expression was confirmed at the human cortical synapse or in synapse-associated structures (verification), (3) assessment of the CSF profile of the synaptic panel in a clinical cohort that includes cognitively normal subjects and AD preclinical and clinical stages (exploration), and (4) confirmation in independent clinical AD cohorts (clinical validation). This study is divided into the following stages: Discovery stage; peptide and protein identification in 7 pools of 60 CSF samples. Verification stage; Tier 2 SRM assay development for selected peptides in 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). Validation stage; SRM of selected peptides in an independent collection of CSF (n = 60) prospectively recruited from the collection at Hospital Clinic, Barcelona (validation cohort-1) and an independent selection (validation cohort-2) of cognitively normal 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 the mass spectrometer in a randomized order with respect to diagnostic group. No technical replicates were included in the SRM study. Biological controls for SRM included cognitively normal individuals (exploratory Cohort, n = 20, validation cohort-1, n = 18, validation cohort-2, n = 20). Biological replicates for SRM included patients and volunteers grouped according to established diagnostic criteria. As technical controls for SRM, BSA controls were run between each sample (shotgun and targeted LC-MS/MS). All participants gave their written consent. The study (IIBSP-BIO-2015–76) was approved by the local ethics committee following the ethical standards recommended by the Helsinki Declaration. All subjects were evaluated by neurologists with expertise in neurodegenerative diseases, by neuropsychologists using a previously published neuropsychological battery (12Sala I. Illan-Gala I. Alcolea D. Sanchez-Saudinos M.B. Salgado S.A. Morenas-Rodriguez E. Subirana A. Videla L. Clarimon J. Carmona-Iragui M. Ribosa-Nogue R. Blesa R. Fortea J. Lleo A. Diagnostic and Prognostic Value of the Combination of Two Measures of Verbal Memory in Mild Cognitive Impairment due to Alzheimer's Disease.J. Alzheimers Dis. 2017; 58: 909-918Crossref PubMed Scopus (21) Google Scholar) and assessed for established AD 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 distinguish AD dementia patients from controls (13Alcolea D. Martinez-Lage P. Sanchez-Juan P. Olazaran J. Antunez C. Izagirre A. Ecay-Torres M. Estanga A. Clerigue M. Guisasola M.C. Sanchez Ruiz D. Marin Munoz J. Calero M. Blesa R. Clarimon J. Carmona-Iragui M. Morenas-Rodriguez E. Rodriguez-Rodriguez E. Vazquez Higuera J.L. Fortea J. Lleo A. Amyloid precursor protein metabolism and inflammation markers in preclinical Alzheimer disease.Neurology. 2015; 85: 626-633Crossref PubMed Scopus (112) Google Scholar). Diagnoses of prodromal AD and AD dementia were made according to NIA-AA guidelines (5Albert M.S. DeKosky S.T. Dickson D. Dubois B. Feldman H.H. Fox N.C. Gamst A. Holtzman D.M. Jagust W.J. Petersen R.C. Snyder P.J. Carrillo M.C. Thies B. Phelps C.H. The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 270-279Abstract Full Text Full Text PDF PubMed Scopus (6348) Google Scholar, 6McKhann G.M. Knopman D.S. Chertkow H. Hyman B.T. Jack Jr, C.R. Kawas C.H. Klunk W.E. Koroshetz W.J. Manly J.J. Mayeux R. Mohs R.C. Morris J.C. Rossor M.N. Scheltens P. Carrillo M.C. Thies B. Weintraub S. Phelps C.H. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 263-269Abstract Full Text Full Text PDF PubMed Scopus (9233) Google Scholar). Subjects within the normal range following formal neuropsychological evaluation, when accounting for age and education (mostly recruited among patients' caregivers), were classified into preclinical AD stages in accordance with NIA-AA guidelines (7Sperling R.A. Aisen P.S. Beckett L.A. Bennett D.A. Craft S. Fagan A.M. Iwatsubo T. Jack Jr, C.R. Kaye J. Montine T.J. Park D.C. Reiman E.M. Rowe C.C. Siemers E. Stern Y. Yaffe K. Carrillo M.C. Thies B. Morrison-Bogorad M. Wagster M.V. Phelps C.H. Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 280-292Abstract Full Text Full Text PDF PubMed Scopus (4647) Google Scholar). CSF samples were collected following international consensus recommendations (14Teunissen C.E. Tumani H. Bennett J.L. Berven F.S. Brundin L. Comabella M. Franciotta D. Federiksen J.L. Fleming J.O. Furlan R. Hintzen R.Q. Hughes S.G. Jimenez C.R. Johnson M.H. Killestein J. Krasulova E. Kuhle J. Magnone M.C. Petzold A. Rajda C. Rejdak K. Schmidt H.K. van Pesch V. Waubant E. Wolf C. Deisenhammer F. Giovannoni G. Hemmer B. Consensus Guidelines for CSF and Blood Biobanking for CNS Biomarker Studies.Mult. Scler. Int. 2011; 2011: 246412PubMed Google Scholar) as previously described (13Alcolea D. Martinez-Lage P. Sanchez-Juan P. Olazaran J. Antunez C. Izagirre A. Ecay-Torres M. Estanga A. Clerigue M. Guisasola M.C. Sanchez Ruiz D. Marin Munoz J. Calero M. Blesa R. Clarimon J. Carmona-Iragui M. Morenas-Rodriguez E. Rodriguez-Rodriguez E. Vazquez Higuera J.L. Fortea J. Lleo A. Amyloid precursor protein metabolism and inflammation markers in preclinical Alzheimer disease.Neurology. 2015; 85: 626-633Crossref PubMed Scopus (112) Google Scholar). 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 CSF Aβ1–42 (InnotestTM Aβ1–42, Fujirebio-Europe, Belgium), total Tau (InnotestTM hTAU Ag), Tau phosphorylated at threonine residue 181 (InnotestTM Phospho-Tau 181P). Our laboratory has extensive experience in CSF biomarker determination and participates in the Alzheimer's Association external quality control program for CSF biomarkers (15Mattsson N. Andreasson U. Persson S. Carrillo M.C. Collins S. Chalbot S. Cutler N. Dufour-Rainfray D. Fagan A.M. Heegaard N.H. Robin Hsiung G.Y. Hyman B. Iqbal K. Kaeser S.A. Lachno D.R. Lleo A. Lewczuk P. Molinuevo J.L. Parchi P. Regeniter A. Rissman R.A. Rosenmann H. Sancesario G. Schroder J. Shaw L.M. Teunissen C.E. Trojanowski J.Q. Vanderstichele H. Vandijck M. Verbeek M.M. Zetterberg H. Blennow K. CSF biomarker variability in the Alzheimer's Association quality control program.Alzheimers Dement. 2013; 9: 251-261Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). All post-mortem brain tissue 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 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. Where indicated, immunodepletion was performed using the ProteoPrep® Immunoaffinity Albumin & IgG Depletion Kit (Sigma-Aldrich, Missouri). Six pools each containing CSF from 10 individuals were precipitated with acetone and protein content was quantified by Bradford assay. Samples (50 ug) were reduced in 10 mm 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 fractionated by strong cation exchange (SCX; Empore Disks Anion Exchange-SR, Sigma-Aldrich). An equivalent of 5 μl of each CSF Sample was analyzed using a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a nano-LC (Proxeon, Odense, Denmark) equipped with a reversed-phase chromatography 2-cm C18 pre-column (Acclaim PepMap-100, Thermo; 100 μm i.d., 5 μm), and a reversed-phase chromatography 25 cm 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 allowed. The fragment ion mass tolerance was set to 0.5 Da. Oxidation of Methionine and N-terminal protein acetylation was defined as variable modification and carbamidomethylation of Cysteines was set as fixed modification. The identified 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 PRIDE (16Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar) partner repository (https://www.ebi.ac.uk/pride/archive) with the data set identifier PXD010356. A systematic search of PubMed (www.pubmed.com) for proteomic studies was performed using the search terms "Cerebrospinal fluid"/"CSF," & "proteome"/"proteomics" & "human," for the CSF proteome (April 2013) and "synaptosome"/"synapse"/"post-synaptic density" & "proteome"/"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 from 4 publicly available databases. Specifically, all proteins annotated with a GO related to the synapse were retrieved from AmiGO2 version 2.3.2 (http://amigo2.geneontology.org/amigo) using the search terms "dendritic spine," "synapse," "synaptic," transmission of nerve impulse" and "neurotransmitter". All proteins annotated to "Glutamatergic synapse," "Cholinergic Synapse," "Serotonergic synapse," "GABergic synapse," "Dopaminergic synapse," "Synaptic vesicle cycle," "Long-term potentiation," "Long-term depression," "Retrograde endocannabinoid signaling" according to the Kyoto Encyclopedia of Genes and Genomes were retrieved from http://www.kegg.jp. All proteins with "neurotransmitter release," "synapse," "synaptic," or "synaptogenesis" as an annotated function were retrieved from Uniprot (www.uniprot.org). Because 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 (http://pir.georgetown.edu) and Bio-Mart (http://www.ensembl.org/biomart) to avoid duplication across studies. Proteins without a reviewed Uniprot identifier were removed from the study. All steps were performed at 4 °C. 200 mg chunks were cut from frozen frontal cortex tissue blocks and homogenized in cold Buffer A (0.32 m sucrose, 1 mm NaHCO3, 1 mm MgCl2, 0.5 mm CaCl2, 1:2500 phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). Homogenates were centrifuged (1400 × g, 10 min) 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 × g. The two supernatants were combined and centrifuged (710 × g). The supernatant was subjected to a final centrifugation (30,000 × g, 15 min). The pellet was resuspended in Buffer B (0.32 m sucrose, 1 mm NaHCO3), layered over a sucrose gradient (0.85 m, 1 m, 1.2 m) and centrifuged (82,500 × g, 2 h). The synaptosomal fraction (a thick white band at the 1–1.2 M interface) was collected, diluted in 4x volume of Buffer B. An aliquot was centrifuged (48,200 × g, 20 min) and the pellet (synaptosome) resuspended in Buffer C (50 mm Tris pH 7.4, 1% SDS) and stored at −80 °C. The remaining aliquot was diluted in equal volume Buffer D (50 mm Tris pH 7.4) and 1× volume of 2% Triton-X, incubated for 10 min and centrifuged (maximum velocity, 30 min). The pellet (post-synaptic density; PSD) was re-suspended in Buffer C and stored at −80 °C. The array tomography protocol was applied using previously described methods (17Colom-Cadena M. Pegueroles J. Herrmann A.G. Henstridge C.M. Munoz L. Querol-Vilaseca M. Martin-Paniello C.S. Luque-Cabecerans J. Clarimon J. Belbin O. Nunez-Llaves R. Blesa R. Smith C. McKenzie C.A. Frosch M.P. Roe A. Fortea J. Andilla J. Loza-Alvarez P. Gelpi E. Hyman B.T. Spires-Jones T.L. Lleo A. Synaptic phosphorylated alpha-synuclein in dementia with Lewy bodies.Brain. 2017; 140: 3204-3214Crossref PubMed Scopus (61) Google Scholar, 18Pickett E.K. Henstridge C.M. Allison E. Pitstick R. Pooler A. Wegmann S. Carlson G. Hyman B.T. Spires-Jones T.L. Spread of tau down neural circuits precedes synapse and neuronal loss in the rTgTauEC mouse model of early Alzheimer's disease.Synapse. 2017; 71: e21965Crossref Scopus (33) Google Scholar). Briefly, a 1 cm3 section was taken from the superior frontal cortex was fixed, dehydrated and polymerized in 100% LR-white resin. The embedded samples were sectioned using a diamond knife (Diatome, UK) creating 20 serial 70 nm thick sections, which were mounted onto coverslips. The ultrathin ribbons were washed with Tris buffer and blocked for 5 min. Primary antibodies used were as mentioned above with the following exceptions; anti-Tenascin-R (Abcam, UK; ab121916) anti-GluR2, anti-Neuroligin-2, anti-Neurexin2 (Merck Millipore, Massachusetts; MAB397, AB15510, ABN97), anti-synaptophysin (Osenses, Australia; oss00029w) and anti-PSD95 (Synaptic Systems, Germany; 124014), alexa-tagged secondary antibodies (Thermo Fisher Scientific). Coverslips were mounted onto the slides using Slowfade Gold with DAPI (Thermo Fisher Scientific). Images were captured using a fully automated epifluorescence upright microscope (custom adapted BX51, Olympus, Pennsylvania) with a 64 × 1.2 NA Plan Apochromat objective. Image analysis was performed using Matlab (Mathworks). The script has been deposited at https://github.com/MemoryUnitSantPau with the name SynSeg. Images from serial sections were stacked, aligned, thresholded and the nonspecific staining (not present in at least 2 consecutive sections) removed using a local threshold-based algorithm. 3-D reconstructions of representative synapses were generated using the ImageJ Volume Viewer plug-in with tricubic smooth interpolation. The total protein content of homogenate, synaptosome and PSD enriched fractions was quantified by bicinchoninic acid assay. Aliquots containing 20 μg total protein were boiled, diluted in loading buffer (100 mm Tris-HCL, 4% SDS, 20% glycerol, 200 mm DTT and 200 mm β-mercaptoethanol) and loaded onto a 12% Tris-Tricine gel and electrophoresed. Proteins were transferred to a nitrocellulose membrane, which was immunostained using the following antibodies; anti-CLSTN1, anti-Synaptophysin, anti-Thy1, (Abcam; ab134130, ab8049, ab133350), anti-GluR2, anti-GluR4, anti-Vamp2, anti-PSD95 (Cell Signaling; 13607, 8070, 13508, 3450), Anti-neuroligin-2, anti-Syntaxin-1B (Synaptic Systems; 129203, 110403), anti-Neurexin3, anti-Tenascin R (Thermo Fisher; PA5–47714, PA5–47546), fluorescent dye-conjugated secondary antibodies (Li-COR Biosciences, Nebraska). Fifty-four proteotypic peptides (7 to 20 amino acids long) with tryptic terminals corresponding to 22 proteins 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 synthesized with 13C615N4 (Arg), or 13C615N2 (Lys) isotopes (Peprotech SRM custom peptides, grade 2, Thermo Fisher Scientific) for
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