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• W1999746231 abstract "Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development. Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development. Fragile X syndrome (FXS) 4The abbreviations used are: FXSfragile X syndromeFMRPfragile X mental retardation proteinAZactive zonePSDpostsynaptic densityEPSCexcitatory postsynaptic currentmEPSCminiature EPSCPPRpaired-pulse ratioWTwild typeKOknock-out. is the most common form of hereditary mental retardation disease, affecting one in 4000 males and one in 8000 females. Patients suffer from multiple behavioral problems including cognitive impairments, impaired visuo-spatial processing, hyperactivity, anxiety, and in a number of cases, autism and/or epilepsy (1Garber K.B. Visootsak J. Warren S.T. Eur. J. Hum. Genet. 2008; 16: 666-672Crossref PubMed Scopus (286) Google Scholar). FXS is caused by mutations in the Fmr1 gene resulting in the absence, significant down-regulation, or loss-of-function of the corresponding protein product, fragile X mental retardation protein (FMRP) (2Koukoui S.D. Chaudhuri A. Brain Res. Rev. 2007; 53: 27-38Crossref PubMed Scopus (51) Google Scholar). In particular, aberrant synapse function caused by reduced FMRP expression is considered to be the major factor underlying FXS. fragile X syndrome fragile X mental retardation protein active zone postsynaptic density excitatory postsynaptic current miniature EPSC paired-pulse ratio wild type knock-out. FMRP is expressed abundantly in neurons and travels into dendritic spines in an activity-dependent manner (3Dictenberg J.B. Swanger S.A. Antar L.N. Singer R.H. Bassell G.J. Dev. Cell. 2008; 14: 926-939Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar), where it affects local synaptic protein synthesis and plasticity. FMRP is an mRNA-binding protein, associated to many types of mRNAs, encoding both presynaptic and postsynaptic proteins (4Brown V. Jin P. Ceman S. Darnell J.C. O'Donnell W.T. Tenenbaum S.A. Jin X. Feng Y. Wilkinson K.D. Keene J.D. Darnell R.B. Warren S.T. Cell. 2001; 107: 477-487Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar). By controlling several aspects of mRNA biology, e.g. transport, translation, and metabolism, FMRP may regulate synaptic structure and function (5Bagni C. Greenough W.T. Nat. Rev. Neurosci. 2005; 6: 376-387Crossref PubMed Scopus (383) Google Scholar, 6Bassell G.J. Warren S.T. Neuron. 2008; 60: 201-214Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Indeed, both FXS patients and Fmr1 knock-out (KO) mice display an abnormal appearance of generally long and thin dendritic spines that resemble an immature postsynaptic phenotype (7Nimchinsky E.A. Oberlander A.M. Svoboda K. J. Neurosci. 2001; 21: 5139-5146Crossref PubMed Google Scholar, 8Irwin S.A. Galvez R. Greenough W.T. Cereb. Cortex. 2000; 10: 1038-1044Crossref PubMed Scopus (494) Google Scholar). Presynaptically, lack of FMRP significantly affects the motility and dynamics of axonal growth cone development in hippocampal neurons, and alterations in terminal branching of hippocampal mossy fibers are found in the Fmr1 KO mouse (9Antar L.N. Li C. Zhang H. Carroll R.C. Bassell G.J. Mol. Cell. Neurosci. 2006; 32: 37-48Crossref PubMed Scopus (200) Google Scholar, 10Ivanco T.L. Greenough W.T. Hippocampus. 2002; 12: 47-54Crossref PubMed Scopus (52) Google Scholar). Thus, the effects of FMRP upon both presynaptic and postsynaptic structure are likely to alter functional aspects of glutamatergic signaling during synaptic development in Fmr1 KO mice. Recent studies of molecular mechanisms underlying reduced cognitive function in FXS have focused on glutamatergic synapse function in several brain regions including hippocampus, cortex, and cerebellum. In these brain areas, long-term synaptic plasticity is strongly affected. The relationship between impaired FMRP expression and synaptic plasticity is complex (11Meredith R.M. de Jong R. Mansvelder H.D. Neurobiol. Dis. 2011; 41: 104-110Crossref PubMed Scopus (29) Google Scholar, 12Meredith R.M. Mansvelder H.D. Front. Synaptic Neurosci. 2010; 10: 2-10Google Scholar). In the cortex, the absence of FMRP results in reduced long-term potentiation (13Wilson B.M. Cox C.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2454-2459Crossref PubMed Scopus (101) Google Scholar, 14Li J. Pelletier M.R. Perez Velazquez J.L. Carlen P.L. Mol. Cell. Neurosci. 2002; 19: 138-151Crossref PubMed Scopus (226) Google Scholar). In the hippocampus and cerebellum, enhanced metabotropic glutamate receptor-dependent long-term depression via an increased internalization of membrane-localized AMPA receptors is observed in Fmr1 KO mice (15Nosyreva E.D. Huber K.M. J. Neurophysiol. 2006; 95: 3291-3295Crossref PubMed Scopus (230) Google Scholar, 16Pilpel Y. Kolleker A. Berberich S. Ginger M. Frick A. Mientjes E. Oostra B.A. Seeburg P.H. J. Physiol. 2009; 587: 787-804Crossref PubMed Scopus (79) Google Scholar). Activity-dependent changes of synaptic efficacy are encoded by sequential molecular events at the synapse, including FMRP-mediated de novo protein synthesis during the late phase of long-term potentiation and long-term depression (17Costa-Mattioli M. Sossin W.S. Klann E. Sonenberg N. Neuron. 2009; 61: 10-26Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). Interestingly, in addition, Gq-coupled M1 muscarinic acetylcholine receptor-dependent long-term depression requires protein synthesis and is enhanced in Fmr1 KO mice (18Massey P.V. Bhabra G. Cho K. Brown M.W. Bashir Z.I. Eur. J. Neurosci. 2001; 14: 145-152Crossref PubMed Google Scholar, 19Volk L.J. Pfeiffer B.E. Gibson J.R. Huber K.M. J. Neurosci. 2007; 27: 11624-11634Crossref PubMed Scopus (133) Google Scholar). However, an overview of synaptic proteins that are affected by the lack of FMRP and to what extent their expression levels are altered is largely unknown. Many forms of short-term plasticity, such as synaptic facilitation, are affected by rapid changes occurring at the presynaptic terminal. FMRP is expressed at presynaptic sites (20Christie S.B. Akins M.R. Schwob J.E. Fallon J.R. J. Neurosci. 2009; 29: 1514-1524Crossref PubMed Scopus (154) Google Scholar), where it may regulate establishment and/or maintenance of synaptic connections (21Bureau I. Shepherd G.M. Svoboda K. J. Neurosci. 2008; 28: 5178-5188Crossref PubMed Scopus (150) Google Scholar, 22Gibson J.R. Bartley A.F. Hays S.A. Huber K.M. J. Neurophysiol. 2008; 100: 2615-2626Crossref PubMed Scopus (339) Google Scholar). A direct relation between the absence of FMRP and its regulated proteins upon synaptic ultrastructure and fast presynaptic-mediated forms of synapse function in Fmr1 KO mice has not been fully investigated in mammalian neurons. To address these issues, we have taken a quantitative proteomics approach to reveal changes in the hippocampal synaptic membrane proteome in Fmr1 KO mice as compared with the wild type (WT) mice during an early developmental period of synapse formation and refinement. We observed changes in a group consisting of known regulators of cell differentiation, filopodia protrusion, and neurite outgrowth by remodeling the actin cytoskeleton. Increase in their expression may retain synapses in an immature state, as we confirmed for Fmr1 KO synapses at the ultrastructural level. In addition, a second group of presynaptic vesicle-associated proteins was dysregulated, in line with our observed functional changes in presynaptic plasticity. Our findings show that changes in the expression levels of synaptic proteins in the Fmr1 KO mice correlate to changes in both excitatory synapse structure and excitatory synapse function, demonstrating a presynaptic locus for mechanisms underlying FXS. Fmr1 KO1 mice on a background of C57Bl/6J from Harlan Netherlands (23Dutch-Belgian Fragile X Consortium Cell. 1994; 78: 23-33PubMed Google Scholar), a kind gift of Dr. B. Oostra and Dr. R. Willemsen, were bred in our facility. Pregnant WT mice C57Bl/6JOlaHsd were obtained from Harlan Netherlands, and pups were born in our facility. For the proteomics experiment, 16 Fmr1 KO mice and 16 WT mice were used. Synaptic membranes from predominately glutamatergic synapses were isolated from PN14 Fmr1 KO and WT mice as described previously (24Li K.W. Miller S. Klychnikov O. Loos M. Stahl-Zeng J. Spijker S. Mayford M. Smit A.B. J. Proteome Res. 2007; 6: 3127-3133Crossref PubMed Scopus (46) Google Scholar, 25Klychnikov O.I. Li K.W. Sidorov I.A. Loos M. Spijker S. Broos L.A. Frants R.R. Ferrari M.D. Mayboroda O.A. Deelder A.M. Smit A.B. van den Maagdenberg A.M. Proteomics. 2010; 10: 2531-2535Crossref PubMed Scopus (18) Google Scholar). A short overview of the experimental setup is represented in Fig. 1. In brief, for each sample, hippocampi from two mice were pooled and homogenized in a glass Potter-Elvehjem homogenizer containing 5 ml of ice-cold homogenization buffer (320 mm sucrose in 5 mm Hepes, pH 7.4) at 900 rpm with 12 up and down strokes of the piston. The lysate was centrifuged at 1000 × g, 4 °C, for 10 min. Supernatant was loaded on top of a sucrose step gradient consisting of 0.85 and 1.2 m sucrose. After ultracentrifugation at 100,000 × g, 4 °C for 2 h, the synaptosome fraction at the interface of 0.85/1.2 m sucrose was collected, diluted six times with 5 mm HEPES buffer (pH 7.4), and centrifuged at 80,000 × g, 4 °C for 40 min. The osmotic shock of synaptosomes was done by resuspending the pellet in HEPES buffer and stirring slowly over ice for 15 min. The resulting synaptic membrane fraction was recovered by ultracentrifugation using the sucrose step gradient as outlined above. Protein concentration was determined by Bradford measurement. The obtained synaptic membranes were subjected to trypsin digestion and iTRAQ reagent tagging or quantitative immunoblotting. In two independent 8-plex iTRAQ experiments, we have compared eight WT samples with eight Fmr1 KO samples. The relatively high number of biological replicas (n = 8) facilitates subsequent statistical analysis. The digestion and iTRAQ labeling of the synaptic membranes have been described in previous studies (24Li K.W. Miller S. Klychnikov O. Loos M. Stahl-Zeng J. Spijker S. Mayford M. Smit A.B. J. Proteome Res. 2007; 6: 3127-3133Crossref PubMed Scopus (46) Google Scholar, 25Klychnikov O.I. Li K.W. Sidorov I.A. Loos M. Spijker S. Broos L.A. Frants R.R. Ferrari M.D. Mayboroda O.A. Deelder A.M. Smit A.B. van den Maagdenberg A.M. Proteomics. 2010; 10: 2531-2535Crossref PubMed Scopus (18) Google Scholar). In short, for each sample, 75 μg of membrane proteins was solubilized in 28 μl of 0.85% RapiGest (Waters Corp.) reconstituted with dissolution buffer (iTRAQ reagent kit, AB Sciex). A 2-μl cleavage reagent (iTRAQ reagent kit, AB Sciex) was added and incubated at 55 °C for 1 h, after which 1 μl of Cys blocking reagent (iTRAQ reagent kit, AB Sciex) was added and samples were vortexed for 10 min. Subsequently, 5 μg of trypsin (sequencing grade, Promega) was added and incubated overnight at 37 °C. The trypsinized peptides were then tagged with iTRAQ reagents dissolved in 85 μl of ethanol. After incubation for 3 h, the samples were pooled and acidified with 10% trifluoroacetic acid to pH 2.5–3. After 1 h, the sample was centrifuged, and the supernatant was dried in a SpeedVac. We performed two 8-plex iTRAQ experiments to cover the 16 samples. In each iTRAQ experiment, four WT samples were labeled respectively with iTRAQ reagents 113–116 and four Fmr1 KO mice samples were labeled respectively with iTRAQ reagents 117–119 and 121 (Fig. 1). The dried iTRAQ labeled sample was dissolved in 250 μl of loading buffer (20% acetonitrile, 10 mm KH2PO4, pH 2.9), whereas 200 μl was injected into a strong cation exchange column (2.1 × 150-mm PolySULFOETHYL A column, PolyLC Inc.). Peptides were eluted with a linear gradient of 0–500 mm KCl in 20% acetonitrile, 10 mm KH2PO4, pH 2.9, over 25 min at a flow rate of 200 μl/min. Fractions were collected at 1-min intervals and dried in a SpeedVac. The peptides were redissolved in 20 μl of 0.1% TFA, delivered with a FAMOS autosampler at 30 μl/min to a reverse phase C18 trap column (1 mm × 300-μm inner diameter column), and separated on an analytical capillary reverse phase C18 column (150 mm × 100 μm-inner diameter column) at 400 nl/min using the LC-Packing Ultimate system. The peptides were separated using a linear increase in concentration of acetonitrile from 6 to 45% in 50 min and to 90% in 1 min. The eluent was mixed with matrix (7 mg of recrystallized α-cyano-hydroxycinnamic acid in 1 ml of 50% acetonitrile, 0.1% TFA, 10 mm ammonium dicitrate) delivered at a flow rate of 1.5 μl/min and deposited off-line to an Applied Biosystems metal target every 15 s using an automatic robot (Probot, Dionex). The sample was analyzed on an ABI 4800 proteomics analyzer (AB Sciex). Peptide collision induced dissociation was performed at 2 kV; the collision gas was air. MS/MS spectra were each collected from 2500 laser shots. Peptides with signal-to-noise ratio above 50 at the MS mode were selected for an MS/MS experiment; a maximum of 25 MS/MS was allowed per spot. The precursor mass window was 200 relative to resolution (full width at half maximum). To annotate spectra, Mascot (MatrixScience) searches were performed against the SwissProt database (release 12.6) and the larger but more redundant National Center for Biotechnology Information (NCBI) database (release 081003) using the GPS Explorer (AB Sciex version 3.6). MS/MS spectra were searched against mouse databases with trypsin specificity and fixed iTRAQ modifications at lysine residues and N termini of the peptides. Mass tolerance was 150 ppm for precursor ions and 0.5 Da for fragment ions; one missed cleavage was allowed. The false discovery rate (percentage) for peptide identification was calculated using a randomized database. Protein redundancy in the result files was removed by clustering the precursor protein sequences at a threshold of 90% sequence similarity over 85% of the sequence length (blastclust, version 20041205). Subsequently, all peptides were matched against the protein clusters; those that were matched to more than one protein cluster were not considered for protein identification and quantification, leaving only “unique” peptides in the analysis. Only proteins identified with at least two peptides with a confidence interval ≥95% (AB Sciex CI, percentage) were considered identified, and of these proteins, only those with three or more quantifiable peptides in both iTRAQ experiments were included in subsequent quantitative analyses. Peak areas for each iTRAQ signature peak (m/z 113.1, 114.1, 115.1, 116.1, 117.1, 118.1, 119.1, 121.1) were obtained and corrected according to the manufacturers' instructions to account for the differences in isotopic overlap. To compensate for the possible variations in the starting amounts of the samples, the individual peak areas of each iTRAQ signature peak were log 2-transformed and normalized to the total peak area of the signature peak. Peptides with iTRAQ signature peaks of less than 2000 were not considered for quantification. Within each experiment, for each peptide, the peak area in each sample was mean-centered. Protein averages were calculated from these mean-centered peak areas of multiple peptides. Finally, the eight mutant and eight wild type protein means of both experiments were used to calculate the average difference between WT and Fmr1 KO mice. To assess whether this difference had occurred by chance due to the multiple testing problem or could be deemed significant, we calculated the permutation-derived false discovery rate (q-value) using the Excel plug-in of the Significance Analysis of Microarrays (SAM) program (26Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar, 27Roxas B.A. Li Q. BMC Bioinformatics. 2008; 9: 187Crossref PubMed Scopus (50) Google Scholar). The settings for the SAM program were: two class unpaired, log 2-scaled, t-statistic, 1000 permutations, automatic estimation of s0 factor, 10 k-nearest neighbors. Samples were mixed with SDS sample buffer and heated to 90 °C for 5 min. Proteins were separated on an SDS-polyacrylamide gel in a Mini-PROTEAN electrophoresis system (Bio-Rad) and electroblotted onto the PVDF membrane overnight at 40 V. Membranes were incubated with primary antibodies followed by an alkaline phosphatase-conjugated secondary antibody (Dako; 1:1000). The signal was developed with ECF substrate (GE Healthcare); the image was scanned with an FLA 5000 instrument (Fujifilm) and analyzed with Quantity One software (Bio-Rad). The following antibodies were used: mouse anti-calcium/calmodulin-dependent protein kinase type II subunit β (Zymed Laboratories Inc.; 1:1000), mouse anti-PSD-95 (Abcam; 1:1000), mouse anti-glutamate receptor (NMDAR1) (BD Biosciences; 1:1000), rabbit anti-syntaxin 1B (gift from M. Verhage; 1:1000), rabbit anti-synaptophysin (gift from M. Verhage; 1:2000), rabbit anti-synapsin I and II (gift from M. Verhage; 1:1000), rabbit anti-NAP-22 (Basp1) (Chemicon; 1:1000), rabbit anti-BM88 (Cend1; gift from R. Matsas; 1:2000), and mouse anti-β-catenin (BD Biosciences; 1:1000). Mice (n = 3/genotype) were subjected to transcardiac perfusion with a mixture of glutaraldehyde (2.5%) and paraformaldehyde (4%) in 0.1 m cacodylate buffer, pH 7.4. From the fixed brain, 50-μm-thick coronal slices containing the stratum radiatum were post-fixed (1% OsO4) and stained with 1% uranyl acetate. After embedding in Epon, ultrathin sections (∼90 nm) were collected on 400-mesh copper grids and stained with uranyl acetate and lead citrate. For each condition, docked vesicles, total vesicle number, postsynaptic density (PSD), active zone (AZ) length, and vesicle cluster surface (nm2) were measured on digital images taken at 100,000× magnification using software in individual electron micrographs, in which a single synapse was counted from each of the three perfusion-fixed mice per genotype using a Jeol (Peabody, MA) 1010 electron microscope. The observer was blind to genotype during the procedure. Horizontal hippocampal brain slices (300 μm thick) were prepared from 13–15-day-old WT and Fmr1 KO mice. Brains were rapidly removed and dissected in ice-cold artificial cerebrospinal fluid containing (in mm): 125 NaCl; 3 KCl; 1.25 NaH2PO4; 3 MgSO4; 1 CaCl2; 26 NaHCO3; 10 glucose (∼300 mosm). Slices were cut on a vibrating microtome and placed in artificial cerebrospinal fluid in a submerged-style holding chamber, bubbled with carbogen (95% O2, 5% CO2) containing the following (in mm): 125 NaCl; 3 KCl; 1.25 NaH2PO4; 1 MgSO4; 2 CaCl2; 26 NaHCO3; 10 glucose. Slices were left for 1 h to recover before recording began. Tetrodotoxin (1 μm) was added to the recording artificial cerebrospinal fluid (same as holding chamber), and slices were perfused in a submerged recording chamber at 28–32 °C. SR-95531 (gabazine, 10 μm, Tocris) was added to the bath to block GABAA receptor-mediated synaptic currents. Whole-cell patch clamp recordings were made from pyramidal cells in the CA1 region under visual guidance by differential interference contrast microscopy. Patch pipettes (3–5 megaohms) were pulled from standard-wall borosilicate tubing and were filled with intracellular solution containing the following (in mm): 140 potassium gluconate; 9 KCl; 10 HEPES; 4 K2-phosphocreatine; 4 ATP (magnesium salt); 0.4 GTP (pH 7.2–7.3, pH adjusted with KOH; 290–300 mosm). For recording mEPSCs, cells were held in a voltage clamp and recorded for on average 8 min at −70 mV. Events were analyzed using Mini Analysis (Synaptosoft, NJ) with event detection levels for synaptic currents set at 8 pA. Rise and decay times were calculated from average event fits. Statistical comparisons were made using analyses of variance with post hoc t tests and Levene's analysis of variance as appropriate in SPSS software with p values < 0.05 being considered statistically significant. Synaptic current parameters are reported as mean ± S.E. Horizontal hippocampal brain slices (300 μm thick) were prepared from 14–18-day-old WT and Fmr1 KO mice, and CA1 pyramidal cells were selected and recorded as described previously for mEPSCs but without the addition of tetrodotoxin to artificial cerebrospinal fluid. Patch pipettes were filled with intracellular solution containing the following (in mm): 129 cesium gluconate; 1 CsCl; 10 HEPES; 4 K2-phosphocreatine; 10 tetraethylammonium; 4 ATP (magnesium salt); and 0.4 GTP (pH 7.2–7.3, pH adjusted to 7.3 with CsOH; 290–300 mosm). After whole-cell configuration, the membrane potential was held at −70 mV, and the internal solution was allowed to diffuse for 5 min into the cell prior to the onset of recording. The low intracellular chloride concentration in the pipette allowed identification and exclusion of experiments in which inhibitory events occurred close to the EPSCs measured. Schaffer collateral fibers were stimulated using an extracellular electrode positioned in the stratum radiatum on the CA3 side. Moderate stimulation was used to measure synaptic facilitation or depression in response to presynaptic trains (of up to 10 pulses) over a range of frequencies (5–50 Hz). For each frequency, the stimulus train was repeated 20 times, with a 15-s delay between each sweep. Sweeps at each frequency were equally divided into two groups, one at the start and one at the end of the experiment, allowing time-dependent changes in the responses to be identified. Synaptic currents sampled at intervals of 100 μs were digitized and analyzed off-line as described previously (28Meredith R.M. Holmgren C.D. Weidum M. Burnashev N. Mansvelder H.D. Neuron. 2007; 54: 627-638Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). To assess significance, analysis of variance with repeated measures was used (for within subjects effect test, Huynh-Feldt was used after Mauchly's test for sphericity) as well as post hoc unpaired Student's t test. Data are given as mean ± S.E., with p < 0.05 indicating significance. To reveal the molecular basis of synapse dysfunction that underlies the FXS phenotype, we compared the proteomes of the synaptic membrane fractions of hippocampus from Fmr1 KO and WT mice. In previous studies, we showed the robustness of the synaptic proteome to resist perturbation, which argues for the use of a large sample size to detect subtle changes (24Li K.W. Miller S. Klychnikov O. Loos M. Stahl-Zeng J. Spijker S. Mayford M. Smit A.B. J. Proteome Res. 2007; 6: 3127-3133Crossref PubMed Scopus (46) Google Scholar, 25Klychnikov O.I. Li K.W. Sidorov I.A. Loos M. Spijker S. Broos L.A. Frants R.R. Ferrari M.D. Mayboroda O.A. Deelder A.M. Smit A.B. van den Maagdenberg A.M. Proteomics. 2010; 10: 2531-2535Crossref PubMed Scopus (18) Google Scholar). Therefore, we analyzed eight independent pools of both Fmr1 KO and WT mice. Altogether ∼50,000 MS/MS analyses were performed, and 347 proteins were identified with at least two distinct peptides with a confidence of ≥95% present in both iTRAQ experiments. The false discovery rate (percentage) for peptide identification using randomized database from NCBI and SwissProt was about 2% (the false discovery rate from the first iTRAQ experiment is 1.6% for NCBI and 2.1% for SwissProt; for the second iTRAQ experiment, it is 1.9 and 2.1%, respectively). As iTRAQ-based quantitation relies on the iTRAQ signature peak intensities generated from MS/MS analysis of a single selected parent ion, the presence of a co-eluting but distinct peptide of similar mass would contribute to the noise depending on the intensity and mass difference of the interfering peptide. For the sake of increased reliability of quantification of protein abundance, we focused on proteins with ≥3 distinct peptides in both iTRAQ experiments (n = 205, supplemental Table 1). The observed -fold changes were low with the largest difference of 1.24-fold (Table 1; log 2 = 0.31), but these were similar as observed previously for relevant biological changes (29Van den Oever M.C. Goriounova N.A. Li K.W. Van der Schors R.C. Binnekade R. Schoffelmeer A.N. Mansvelder H.D. Smit A.B. Spijker S. De Vries T.J. Nat. Neurosci. 2008; 11: 1053-1058Crossref PubMed Scopus (152) Google Scholar). Using the Significance Analysis of Microarray program (SAM, q-value) (27Roxas B.A. Li Q. BMC Bioinformatics. 2008; 9: 187Crossref PubMed Scopus (50) Google Scholar) with false discovery rate <5%, 23 proteins were considered to be significantly different between KO and WT mice. Among them, several presynaptic proteins were present, including syntaxin, synapsin, synaptophysin, piccolo, Rab3A, and vacuolar proton pump subunit E, as well as proteins involved in cell differentiation/neurite outgrowth including Cend1, Basp1, and Gap43.TABLE 1Proteins with significant changes in the hippocampal synaptic membrane fraction of Fmr1 KO miceUniProt accession numberProtein name% of coverage of the proteinNo. of peptides CI ≥ 95%Difference KO-WT (log 2 scale)Standard deviation KO-WT (log 2 scale)q-value (%) using SAMCell differentiation/neurite outgrowthQ91XV3Brain acid-soluble protein 158.1110.310.200.00Q9JKC6Cell cycle exit and neuronal differentiation protein 161.370.270.180.00Q02248Catenin β-131.119−0.160.120.00P18760Cofilin-155.770.130.110.00P06837Gap4386.8140.230.230.00Presynaptic transmissionP61264Syntaxin-1B252.6110.160.140.00P63011Ras-related protein Rab-3A30.350.150.130.00Q9QYX7Protein piccolo8.1170.130.121.80O88935Synapsin-146.1220.110.123.50Q62277Synaptophysin21.050.120.143.50Q9DBG3AP-2 complex subunit β-18.650.150.193.50P50518Vacuolar ATP synthase subunit E47.690.180.140.00Mitochondrial/metabolic proteinsQ9CR68Ubiquinol-cytochrome c reductase iron-sulfur subunit30.270.150.120.00O35143ATPase inhibitor, mitochondrial precursor28.030.190.150.00Q9WUA36-Phosphofructokinase type C16.980.130.100.00Q9CQ69Ubiquinone-binding protein QP-C54.250.170.170.00P17182α-Enolase29.2100.140.151.80P05064Fructose-bisphosphate aldolase A74.2190.130.141.80P16858Glyceraldehyde-3-phosphate dehydrogenase43.4110.120.111.80Q9CPQ8ATP synthase subunit g, mitochondrial59.640.140.131.80OtherP17742Peptidyl-prolyl cis-trans isomerase A81.2110.170.140.00P05132cAMP-dependent protein kinase, α-catalytic subunit15.330.180.180.00P58252Elongation factor 215.050.180.150.00 Open table in a new tab Quantitati" @default.
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• W1999746231 title "Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype" @default.
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