BDNF Induces Widespread Changes in Synaptic Protein Content and

BDNF Induces Widespread Changes in Synaptic Protein Content and Up-Regulates Components of the Translation Machinery: An Analysis Using High-Throughput Proteomics Lujian Liao,† Julie Pilotte,‡ Tao Xu,† Catherine C. L. Wong,† Gerald M. Edelman,‡ Peter Vanderklish,*,‡ and John R. Yates, III*,† Departments of Cell Biology and Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 Received July 19, 2006

The brain-derived neurotrophic factor (BDNF) plays an important role in neuronal development, and in the formation and plasticity of synaptic connections. These effects of BDNF are at least partially due to the ability of the neurotrophin to increase protein synthesis both globally and locally. However, only a few proteins have been shown to be up-regulated at the synapse by BDNF. Using multidimensional protein identification technology (MudPIT) and relative quantification by spectra counting, we found that several hundred proteins are up-regulated in a synaptoneurosome preparation derived from cultured cortical neurons that were treated with BDNF. These proteins fall into diverse functional categories, including those involved in synaptic vesicle formation and movement, maintenance or remodeling of synaptic structure, mRNA processing, transcription, and translation. A number of translation factors, ribosomal proteins, and tRNA synthetases were rapidly up-regulated by BDNF. This up-regulation of translation components was sensitive to protein synthesis inhibitors and dependent on the activation of the mammalian target of rapamycin (mTOR), a regulator of cap-dependent mRNA translation. The presence of a subset of these proteins and their mRNAs in neuronal processes was corroborated by immunocytochemistry and in situ hybridization, and their up-regulation was confirmed by Western blotting. The data demonstrate that BDNF increases the synthesis of a wide variety of synaptic proteins and suggest that the neurotrophin may enhance the translational capacity of synapses. Keywords: BDNF • translation • synapse • MudPIT • spectra count • protein quantification • synaptoneurosomes • ribosomal proteins

Introduction Activity-dependent regulation of protein synthesis is crucial for the establishment of neural circuitry, the maturation of synaptic connections, and the consolidation of synaptic efficacy changes.1-3 The brain-derived neurotrophic factor (BDNF), a neurotrophin that is synthesized and released at glutamatergic synapses in an activity-dependent manner,4 plays a major role in regulating protein synthesis in these contexts.5-7 BDNF has been shown to enhance dendritic arbor formation,8 modulate iontropic transmission,5 and increase the levels of proteins associated with vesicular glutamate release9 via mechanisms that can involve local protein synthesis within dendrites and axons. BDNF also plays an essential role in the late, protein synthesis-dependent phase of long-term potentiation (LTP).10,11 The basis of these effects involves the ability of BDNF to potently activate the mammalian target of rapamycin (mTOR) and the extracellular signal regulated kinase (ERK) signaling pathways,12,13 both of which are critical regulators of translation * To whom correspondence should be addressed. E-mails: John R. Yates III, [email protected]; Peter Vanderklish, [email protected]. † Department of Cell Biology, The Scripps Research Institute. ‡ Department of Neurobiology, The Scripps Research Institute. 10.1021/pr060358f CCC: $37.00

 2007 American Chemical Society

during LTP formation.1 These pathways mediate phosphorylation of several translation initiation factors, including the eukaryotic initiation factor 4E (eIF4E), the eIF4E-binding protein (4EBP), and ribosomal protein S6, resulting in an increase in mRNA translation.12,14,15 BDNF can exert highly localized effects on translation within dendrites and axons.7,16 However, while a large set of mRNAs have been identified in dendrites and axons,17-20 and a large number of mRNAs exhibit increased polysome association after addition of BDNF in dissociated neurons,2 relatively few proteins have been directly demonstrated to be locally synthesized in response to BDNF. Locally synthesized proteins include the activity-regulated cytoskeletal protein (Arc),21,22 the Ca++/calmodulin-dependent kinase IIR (CaMKIIR),12 homer 2, and other proteins,2,18 all of which play important roles in synapse formation, maturation, and plasticity. Given the broad influence of BDNF on synaptic function, its ability to activate the translation machinery, and the abundance of dendritically localized mRNAs, we postulated that BDNF may up-regulate the synthesis of a much larger and more diverse set of proteins within the synaptic compartment than has been identified to date. Journal of Proteome Research 2007, 6, 1059-1071

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research articles Quantitative proteomics techniques are well-suited to study dynamic changes in protein expression on a large scale. Traditionally, local protein synthesis has been studied in isolated synaptic fractions using two-dimensional gel electrophoresis combined with either radioisotope labeling of newly synthesized proteins, protein staining, or Western blotting. However, these approaches are subject to limitations in sensitivity and throughput. We have developed an alternative approach for the relative quantification of large-scale protein expression level changes in synaptic fractions from BDNFtreated neurons using multidimensional protein identification technology (MudPIT).23 This approach utilizes spectra counting to obtain quantitative information of cellular proteomes.24,25 Spectra counts represent a measure of the repeated identification of the same peptide sequences in a given sample over the entire MudPIT run. Generally, abundant proteins will give higher spectra counts than less abundant proteins. A recent study has shown a strong correlation between the spectra counting method and peptide ion chromatograms derived from proteins labeled with stable isotopes.26 The advantage of spectra counting is that no isotopic labeling is needed, which greatly facilitates comparative proteomic studies where metabolic labeling is not convenient or not possible. Using MudPIT and relative quantification by spectra counting, we examined large-scale changes in protein levels in a synaptoneurosome preparation from cultured rat cortical neurons that were treated with BDNF for 30 min. The results show a rapid up-regulation of a large number of different proteins which fall into several categories. Cytoskeletal and synaptic proteins were prominent, as were proteins with binding functions (e.g., proteins that bind calmodulin, cytoskeletal components, or unfolded proteins). Interestingly, treatment with BDNF induced increases in the levels of many components of the translation machinery, including a subset of ribosomal proteins, initiation and elongation factors, and tRNA synthetases. Because of their potential relevance to the progression of protein synthesis-dependent events at synapses, these changes were investigated further using Western blots, in situ hybridization, and immunocytochemical methods. The results support the notion that BDNF enhances the local synthesis of a large number of proteins at synapses, and that it may also alter the translational capacity of synapses.

Experimental Section Primary Neuronal Culture, Drug Treatment, and Synaptoneurosomal Preparation. Cortical and hippocampal neurons were isolated from embryonic day 18 (E18) Sprague Dawley rats and cultured according to a previously described protocol with minor modifications.27 Dissociated neurons from either cortices or hippocampi were plated at a density of 50 000 cells/ cm2 (20 000 cells/cm2 for imaging) and maintained in Neurobasal Medium supplemented with B27, penicillin (50 µg/mL), streptomycin (50 U/mL), and glutamine (2 mM). After 15-17 days in vitro (DIV), neurons were treated with BDNF (50 ng/ mL) for 30 min, or in some cases, preincubated for 45 min with 10 ng/mL rapamycin, 10 µM anisomycin, or 34 µM cyclohexamide. Control neurons were treated with equal volumes of vehicle (dimethylsulfoxide; DMSO). For proteomic analyses, neurons were harvested, and synaptoneurosomes (SNS) were prepared based on a previously described method.28 Briefly, neurons were collected with HEPES-buffered sucrose (10 mM HEPES, pH 7.4, 0.32 M sucrose, protease inhibitor cocktails (Roche, Mannheim, Germany), 2 mM NaF, and 1 mM Na3VO4), 1060

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then homogenized and centrifuged at 500g for 5 min. The resulting supernatant fraction was passed through 30 µm and 5 µm nylon filters. The flow through was collected and spun down at 2000g for 15 min. The resuspended pellet was used as the SNS fraction. Comparative Analysis of SNS Proteins by Multidimensional Protein Identification Technology (MudPIT). Per treatment condition, 200 µg of SNS protein was solublized with 8 M urea/ Invitrosol (Invitrogen, Calsbad, CA), reduced with 10 mM dithiothreitol, alkylated with 10 mM iodoacetomide, diluted with 4× volumes of 100 mM Tris-HCl, and then digested with trypsin overnight. After digestion, the pH was adjusted to about 2.5 using 90% formic acid. Sixty micrograms of protein digest from each sample was analyzed by MudPIT. Briefly, digested proteins were pressure-loaded onto a fused silica capillary column packed with a 3 cm 5-µm Partisphere strong cation exchanger (SCX, Whatman, Clifton, NJ) and a 3 cm 5-µm Aqua C18 material (RP, Phenomenex, Ventura, CA), with a 2 µm filtered union (UpChurch Scientific, Oak Harbor, WA) attached to the SCX end. The column was washed with buffer containing 95% water, 5% acetonitrile, and 0.1% formic acid. After desalting, a 100-µm i.d. capillary with a 5-µm pulled tip packed with 10 cm 3-µm Aqua C18 material was attached to the filter union, and the entire split-column was placed inline with an Agilent 1100 quaternary HPLC (Agilent, Palo Alto, CA) and analyzed using a modified 12-step separation described previously.23 Three buffer solutions were used: 5% acetonitrile/0.1% formic acid (buffer A); 80% acetonitrile/0.1% formic acid (buffer B), and 500 mM ammonium acetate/5% acetonitrile/0.1% formic acid (buffer C). The first step consisted of a 100 min gradient from 0 to 100% buffer B. Steps 2-11 had the following profile: 3 min of 100% buffer A, 5 min of X% buffer C, a 10 min gradient from 0 to 15% buffer B, and a 97 min gradient from 15 to 45% buffer B. The 5 min buffer C percentages (X) were 5, 10, 15, 20, 25, 30, 35, 40, 55, and 75%, respectively, for the 12-step analysis. In the final step, the gradient contained: 3 min of 100% buffer A, 20 min of 100% buffer C, a 10 min gradient from 0 to 15% buffer B, and a 107 min gradient from 15 to 100% buffer B. As peptides were eluted from the microcapillary column, they were electrosprayed directly into an LTQ two-dimensional ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) with the application of a distal 2.4 kV spray voltage. A cycle of one fullscan mass spectrum (400-1400 m/z) followed by 3 datadependent MS/MS spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. MS/MS spectra were analyzed using the following software analysis protocol. Poor-quality spectra were removed from the data set using an automated spectra quality assessment algorithm.29 MS/MS spectra remaining after filtering were searched with the SEQUEST algorithm30 against the EBI rat IPI database (ftp://ftp.ebi.ac.uk/pub/databases/IPI/current/, version 3.05, released date July, 2005) concatenated to a decoy database in which the sequence for each entry in the original database was reversed.31 SEQUEST results were assembled and filtered using the DTASelect program32 with a peptide falsepositive score of 5%. Only proteins with two peptide hits or single hits with over 10 spectra counts were accepted. All peptides identified are at least half tryptic. Under such filtering conditions, the estimated false-positive rate at the protein level is about 0.2% based on decoy database hits. One of the output parameters from DTASelect for each protein was spectra count, which was applied to calculate the

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BDNF Induces Widespread Changes in Synaptic Protein Content

relative ratio of proteins identified from BDNF or BDNF plus inhibitor-treated neurons over control neurons. Although the total number of spectra was similar between any two samples, a normalization factor (F ) Total number of spectra in control sample/Total number of spectra in treated sample) was used to overcome the potential quantification error caused by the slight shift in total spectra count. The calculated spectra count ratio with control as the reference times the normalization factor gives the normalized ratio. Thereafter, proteins with expression level changes were filtered according to the following criteria: (1) if the same protein was identified in both control and BDNF-treated samples, spectra count ratios of 2 or above were considered as increased, while proteins with spectra ratios of 0.5 or less were considered as decreases; (2) otherwise, if a protein was not identified in one sample, a peptide count greater than 2 and spectra count greater than 4 were used to consider a significant change. Immunoprecipitation and Immunoblotting. DIV17 cortical neurons were lysed with RIPA buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% NP-40, protease inhibitor cocktails (Roche), 5 mM NaF, and 2 mM Na3VO4) and briefly sonicated, and the lysate was centrifuged at 13 000g for 10 min. For immunoprecipitation, supernatants containing 1.5 mg of protein were first precleared with 50 µL of protein G-sepharose beads, then incubated with 2 µg of monoclonal anti-phosphotyrosine antibody (Upstate, Charlottesville, VA), followed by incubation with 50 µL of protein G beads overnight on an orbital mixer at 4 °C. The beads were then collected by centrifugation and washed four times with 1% NP-40 in phosphate buffered saline (PBS). Immunoprecipitated complexes were eluted from the beads by boiling them in 30 µL of LDS sample buffer (Invitrogen), followed by centrifugation at 13 000g; 20 µL of the resulting supernatant was used for immunoblot analysis using a monoclonal antibody against total TrkB (Transduction Laboratories, San Diego, CA). For immunoblot analysis of whole cell lysates, 30 µg of protein was boiled in LDS buffer and analyzed by Western blot using the following antibodies: rabbit against eIF5 (1:1000, Upstate), L7A (1:500), S20 (1:500), tyr-tRNA synthase (try-TS, 1:1000; a generous gift from Francella Otero and Dr. Paul Schimmel), mouse antibodies against eEF1A (1:500), and eIF4E (1:1000) (Cell Signaling Technology, Danvers, MA). Western blot analyses were performed on samples from three separate experiments. The blots were scanned, and band intensities were analyzed using AlphaEaseFC (Alpha Innotech, San Leandro, CA), followed by Student’s t-test to assess the statistical significance. Immunocytochemistry and in Situ Hybridization. For immunocytochemistry, DIV17 cortical neurons grown on glass coverslips were fixed with 4% paraformaldehyde/4% sucrose, permeabilized with 0.5% Triton X-100 in PBS, and then blocked with PBS containing 3% normal goat serum. The coverslips were incubated overnight with antibodies against eIF5 and S20 at 1:250 overnight, then washed and incubated with FITCconjugated anti-rabbit secondary antibody. For in situ hybridization, DIV14 neurons were fixed with 4% paraformaldehyde in PBS and permeabilized with 70% ethanol overnight at 4 °C. DNA fragments encompassing 150 nucleotide regions of βIII tubulin, eIF5, TyrRS, ribosomal proteins L14, and S20 were amplified by PCR using cDNA from rat cortex, and in vitro transcribed using T7 RNA polymerase (Ambion). The probes were labeled with digoxigenin, and in situ hybridization was carried out as described.33 Digoxigenin-labeled RNA was de-

tected with the anti-digoxigenin antibody conjugated to rhodamine (Roche) and stained with the nuclear marker DAPI. Fluorescent images were collected on a Zeiss Axioplan II microscope fitted with a Cooke Sensicam, using the Slidebook software from Intelligent Imaging Innovations.

Results Characterization of Synaptoneurosomes Prepared from Cultured Cortical Neurons. BDNF induces protein synthesis in axon terminals and in postsynaptic elements. In consideration of this, we chose to conduct our high-throughput proteomic analyses on a synaptic fraction that contains resealed pre- and postsynaptic elements. This fraction, termed synaptoneurosomes (SNS28), was prepared from cultured cortical neurons after they were treated with BDNF, and from controls. Before conducting proteomic analyses, we characterized the SNS fraction by electron microscopy and Western blot analyses of different subcellular markers. Electron micrographs of the SNS preparation (Figure 1A) revealed the presence of intact synaptic contacts as marked by opposing elements with presynaptic vesicles (arrowhead) and postsynaptic densities (arrow). Figure 1B shows Western blot analyses of different subcellular markers in the starting material and SNS fractions. In fractions from untreated neurons, the endoplasmic reticulum (ER) marker calnexin exhibited little or no change in SNS compared with starting homogenates, indicating that a similar amount of ER was present per unit protein. Prior treatment with BDNF did not change this pattern. The mitochondrial marker complex VI-I was reduced in SNS fractions compared with homogenates, but it was increased in SNS fractions prepared from BDNF-treated neurons. This indicates that mitochondria levels are reduced during preparation of SNS under baseline conditions, and that their levels in the synaptic fraction are elevated by BDNF. Importantly, there were large increases in the presynaptic marker synapsin I and the postsynaptic markers PSD95 and CaMKII in SNS fractions, compared with homogenates, confirming an enrichment of pre- and postsynaptic endings in the SNS preparation. An even greater enrichment in synapsin I, PSD95, and CaMKII in the SNS fraction was observed after cortical neurons were treated with BDNF, suggesting that the neurotrophin increases the synthesis or recruitment of these proteins to synapses. Synthesis of CaMKII in response to BDNF has been demonstrated previously.16 Several previous studies on BDNF-induced synaptic plasticity utilized hippocampal preparations.22,34,35 Because large amounts of sample are needed in proteomic assays, we used cortical neurons instead. To determine whether the response of cortical neurons to BDNF treatment is similar to that of hippocampal neurons, we analyzed the levels and phosphorylation of its receptor, TrkB, and of the 90K ribosomal protein S6 kinase (p90RSK), a kinase that regulates the translation machinery in response to synaptic activity.15,36,37 In both cortical and hippocampal neurons, BDNF greatly increased the level of phosphorylated p90RSK and the amount of TrkB that could be immunoprecipitated with an anti-phosphotyrosine antibody. BDNF also slightly increased total levels of full-length TrkB (Figure 1C). MudPIT Analysis Reveals Large-Scale Protein Expression Changes in SNS from BDNF-Treated Neurons. Multidimensional protein identification technology (MudPIT) is uniquely suited to analyze changes in protein expression within SNS because it can resolve and identify several thousand proteins at the peptide level. As an initial test of the MudPIT method Journal of Proteome Research • Vol. 6, No. 3, 2007 1061

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Figure 1. Characterization of synaptoneurosomes (SNS) prepared from cortical neurons. (A) Electron micrograph showing the presence of intact pre- and postsynaptic elements in the SNS preparation. Presynaptic vesicles (arrowhead) and postsynaptic densities (arrow) are shown. (B) Western blot analyses of three fractions from the SNS preparation procedure: whole cell homogenate (Hom), lowspeed pellet (Pell), and the final SNS fraction. Levels of markers for ER (calnexin) and mitochondria (complex VI-1) were similar in SNS and homogenate fractions, while synaptic markers were increased in SNS relative to homogenates indicating a relative enrichment of synapses. BDNF treatment increased synaptic proteins, especially synapsin, in the low-speed pellet and SNS fraction. Calnexin was unaltered by BDNF, while complex VI-I was slightly increased in SNS after BDNF treatment. BDNF also caused phosphorylation of P90RSK. (C) Western blots of TrkB in whole cell lysates of cortical and hippocampal neurons treated with or without BDNF, and of TrkB immunoprecipitated from these lysates using an anti-phosphotyrosine antibody. In both hippocampal and cortical neurons, BDNF increased levels of full-length (FL) and truncated (Trunc) isoforms of TrkB, and levels of phosphorylated full-length TrkB. (D) Normalized spectra count ratios (BDNF/control) of subcellular markers derived by MudPIT analyses of the same SNS fractions tested in panels B and C. All proteins selected for Western analysis were identified; when a protein was not identified in a sample, the spectra count number was listed in the format (BDNF/control).

on SNS, samples from the same SNS fractions that were characterized by Western blot and electron microscopy techniques were used for MudPIT analyses. These analyses were repeated three times, with control and BDNF-treated samples analyzed alternately. All proteins selected for Western blot analyses of SNS fractions were identified and, by and large, the spectra count ratio was consistent with Western blot results (Figure 1D). With the exception of synapsin, the standard deviations of the spectra count ratios for a majority of the proteins are reasonably small, regardless of the semiquantita1062


tive nature of the spectra count approach. While a statistical method for defining thresholds at which ratios (e.g., ratio > 1.4) can be considered significantly different from 1 has recently been demonstrated for this approach,38 we chose a stringent, albeit arbitrary, cutoff value of changes (ratio > 2); these data were supported by replicates and studies with inhibitors, as well as bioinformatics analysis (GoMiner) of pathway changes to evaluate their biological significance. To obtain a comprehensive analysis of BDNF-induced changes in synaptic protein expression, we conducted MudPIT


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Figure 2. Proteomic analysis of SNS reveals widespread changes in synaptic protein content after BDNF treatment. (A-C) Comparison of the number and degree of overlap of proteins identified by MudPIT in SNS from four neuronal treatment groups: control, BDNF, BDNF + anisomycin (BDAN), and BDNF + rapamycin (BDRA). A majority of proteins identified in BDNF-treated neurons were also identified in the other treatment groups, greatly facilitating relative quantification by spectra counting. (D) Pie chart showing the relative distribution of SNS proteins that were increased by BDNF among 9 categories of cellular components. (E) A plot of the fold change in the spectra count ratios of ribosomal subunit proteins as a function of treatment condition. Ribosomal proteins for which a spectra count ratio could be calculated were included in the plot; among them, 13 proteins with >1.6-fold up-regulation were listed in the figure legend.

analyses on SNS derived from neurons divided into four treatment conditions: vehicle controls (CONT), BDNF-treated neurons (BDNF), and BDNF-treated neurons preincubated with the translation inhibitors anisomycin (BDAN) or rapamycin (BDRA). Anisomycin and rapamycin were included to determine whether increased expression of proteins was dependent on de novo protein synthesis, as opposed to protein translocation into the synaptic compartment. All samples were processed and analyzed in parallel by MudPIT. The analyses resulted in identification of 2166, 2372, 2433, and 2137 proteins within CONT, BDNF, BDAN, and BDRA samples, respectively. There was an approximate 67% overlap of proteins identified in SNS from BDNF-treated neurons with proteins identified in samples from each of the other conditions (Figure 2A-C). This result greatly facilitates quantification using spectra counting. To obtain a measure of the relative expression of identified proteins after BDNF treatment, an expression ratio was calculated for each protein using spectra counts from controls as the denominator and spectra counts from each of the BDNF treatments as numerators to estimate its relative changes after different treatments. A comparison between control and BDNFtreated samples reveals that, among proteins identified in both samples, there are 200 proteins with a spectra count ratio above 2, and 174 proteins with a ratio less than 0.5. In addition, 740 proteins were identified in the BDNF-treated sample only, while 432 proteins were identified solely in the control. The overall results suggest that, after neurons are treated with BDNF, more proteins are up-regulated than down-regulated.

Most of the long-lasting physiological effects of BDNF on synapses require protein synthesis. In light of this, we focused our subsequent analyses of proteomic data on proteins that are up-regulated by BDNF. After filtering the proteins with differential expression levels based on the criteria described in the Experimental Section, which includes proteins that are identified in both samples and those identified only in one or the other, 230 proteins were found to be up-regulated in SNS by BDNF. These proteins together with the down-regulated proteins were sorted by decreasing spectra count ratio and listed in Supporting Information (Table S1). The proteins upregulated after BDNF treatment were categorized using GoMiner software39 based on a combination of cellular location and molecular function (Figure 2D). A comparison of the distribution of up-regulated proteins across nine cellular component categories revealed enrichment in the proportion of synaptic proteins, ribonucleoproteins, and ribosomal proteins. Proteins known to influence synaptic efficacy and dendritic spine morphology are well-represented, such as neurabin,40 F-actin capping protein,41 cadherin-2,42 and neural cell adhesion molecules.43 Nuclear, mitochondrial, and ER proteins occupied a relatively smaller percentage of all the up-regulated proteins. Using GoMiner, we compared the number of up-regulated proteins in a particular group versus all the proteins identified in that group to assess whether particular pathways were upregulated by BDNF. GoMiner assigns a Fisher’s Exact p-Value to changed pathways39 for a statistical evaluation of the likelihood that they have been altered. For example, 6 out of Journal of Proteome Research • Vol. 6, No. 3, 2007 1063

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Table 1. Spectra Count and Ratio of Spectra Count for Ubiquitin Related Proteins after Treatmenta locusID

protein name

SpCCTb

SpCBDc

SpCANd

SpCRAe

BD/CTf

AN/CTg

RA/CTh

IPI00361888 IPI00370134 IPI00369881 IPI00390118 IPI00360081 IPI00204375 IPI00363473 IPI00358610

Polyubiquitin similar to potential ubiquitin ligase similar to ubiquitin protein ligase E3C similar to ubiquitin-conjugating enzyme E2 variant 1 similar to ubiquitin-conjugating enzyme Ubiquitin carboxyl-terminal hydrolase isozyme L1 Ubiquitin specific protease 14 similar to Ubiquitin-conjugating enzyme E2 L3 (Ubiquitin-protein ligase L3) similar to ubiquitin specific protease 9, X-linked (fat facets-like, Drosophila) similar to Ubiquitin carboxyl-terminal hydrolase 5

0 0 0 2 2 14 2 3

20 10 4 5 4 22 3 4

10 5 0 0 0 10 5 5

10 4 2 2 2 13 0 3

n/a n/a n/a 2.4 1.9 1.5 1.4 1.3

n/a n/a n/a 0.0 0.0 0.7 2.3 1.5

n/a n/a n/a 1.0 1.0 0.9 0.0 1.0

6

8

8

6

1.3

1.2

1.0

12

8

8

16

0.6

0.6

1.3

IPI00204923 IPI00207657

Table lists spectra counts (SpC) for: control (CT), BDNF (BD), BDNF + Anisomycin (AN), BDNF + rapamycin (RA). Spectra count ratios of the three BDNF treatment groups (numerators) relative to control (denominator) were calculated, normalized and are listed as fBD/CT, gAN/CT, and hRA/CT. a

b

c

d

16 proteins in the RNA splicing pathway were up-regulated, resulting in a probability of being wrong of 0.0014, a statistically significant assignment. The pathways or groups of proteins upregulated with a confidence level of over 95% are listed in Supporting Information Table S2. Some other significantly upregulated pathways include protein synthesis, cell adhesion, regulation of Wnt receptor signaling pathway, and cell communication. In addition, polyubiquitin was greatly increased, and this increase was largely blocked by either rapamycin or anisomycin. Several variants of ubiquitin conjugating enzymes and ubiquitin ligases were also increased (Table 1). Overall, about 25% of changed pathways are known to be influenced by BDNF. BDNF Up-Regulates Components of the Translation Machinery. Increases in the levels of a large number of proteins that function in translation were observed in SNS fractions from BDNF-treated neurons (p-Value ) 0.0058, Table S2 of Supporting Information). This result was particularly interesting because of the known role of local translation in BDNF-induced synaptic plasticity. Table 2 lists the spectra count results of all the proteins identified that are components of the translation machinery. These include 20 translation factors and 69 ribosomal subunit components, representing almost all aspects of the translation machinery. The ribosomal proteins identified by our proteomic analysis overlap with 80% of the ribosomal protein mRNAs that have been previously identified in dendrites.17 While translation factors show similar numbers of proteins up- or down-regulated by BDNF, as well as 4 unchanged factors, ribosomal subunits show an overall trend of up-regulation. Among them, 13 proteins were increased by >1.6-fold, whereas only 3 proteins showed a < 0.6-fold decrease (Table 2). Figure 2E shows the fold change of 53 ribosomal proteins for which a spectra count ratio could be calculated, plotted as a function of treatment. Those that increased by >1.6-fold are listed in the legend. Interestingly, all of the BDNFinduced increases in translation components were blocked by anisomycin or rapamycin, and there was a similar but reciprocal effect for the down-regulated proteins. For example, eukaryotic initiation factor 5 (eIF5) was identified only in the BDNF-treated sample, and both isoforms of eukaryotic elongation factor 1R (eEF1R) increased about 1.6-fold. However, eEF2 appears decreased by 2-fold. Each of these differences was reversed by the translation inhibitors. In addition to primary translation components, several mRNA-binding proteins, including multiple isoforms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were significantly up-regulated by BDNF. These results indicate that BDNF induces significant up1064


e

regulation of components of the translational machinery, and proteins involved in mRNA processing and transport, in synaptic fractions within 30 min. Validation of Changes in the Protein Synthesis Machinery Detected by MudPIT. To validate the proteomics findings and to gain a more extensive temporal view of the changes induced by BDNF, Western blot analyses of a subset of translation components representing each aspect of the translation machinery were conducted on whole cell lysates from cortical neurons treated with BDNF for 15, 30, 60, and 120 min (Figure 3A). The average fold increase as a function of time for these components in three separate experiments is plotted in Figure 3B. One of the initiation factors, eIF4E, increased gradually up to 2-fold after 1 h of BDNF treatment and maintained this high level at 2 h. Another initiation factor, eIF5, showed a sharp increase starting as early as 15 min, and the uptrend continued for 2 h. The elongation factor eEF1R-1 showed no change over the course of the treatment, in contrast to our spectra count data which showed an increase. This may be due to the fact that eEF1R-1 is more abundant than the other factors (see spectra count data), and synaptic increases may therefore be masked in blots of whole cell lysates. Two ribosomal subunits S20 and L7A showed steady increases over the entire course of BDNF treatment, as did tyrosyl tRNA synthetase (TyrRS). Overall, the Western blot results are consistent with our proteomics findings and indicate that BDNF treatment of cortical neurons results in a steady increase in components of the translation machinery. To confirm that proteins involved in translation were themselves up-regulated at the translational level, as suggested by our proteomic data (Figure 2E and Table 2), rapamycin and two protein synthesis inhibitors that have distinct mechanisms of action were applied 30 min before BDNF treatment. For all the proteins tested by Western blot analysis, there was a 1.2to 1.6-fold increase after BDNF treatment compared with control neurons. Pretreatment with either rapamycin, anisomycin or cyclohexamide blocked the BDNF-induced increases (Figures 3C,D). These data indicate that up-regulation of the translation machinery by BDNF occurs at the translational level and involves the mTOR pathway of translational control. In Situ Detection of Translation Components and Their mRNAs in Neuronal Processes. To further investigate whether BDNF locally increases the translation machinery in dendritic processes, we conducted immunocytochemical staining. Previous studies have shown that eIF4E has a dendritic localization,44 and that eEF1R is translationally up-regulated in dendritic compartments upon high-frequency stimulation of hippocam-

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Table 2. Spectra Count and Ratio of Spectra Count for Translation Machinery Components after Treatmenta locusID

protein name

SpCCTb

SpCBDc

SpCANd

SpCRAe

BD/CTf

AN/CTg

RA/CTh

IPI00370315 IPI00194880 IPI00372810

Eukaryotic translation initiation factor 4H Eukaryotic translation initiation factor 5 PREDICTED: similar to eukaryotic translation initiation factor 3, subunit 10 theta, 150/170 kDa PREDICTED: similar to eukaryotic translation initiation factor 3, subunit 5 epsilon, 47 kDa 40S ribosomal protein S12 40S ribosomal protein S23 40S ribosomal protein S4, X isoform 60S acidic ribosomal protein P1 60S ribosomal protein L10 60S ribosomal protein L10a Mitochondrial 39S ribosomal protein L23 Mitochondrial ribosomal protein L9 PREDICTED: similar to 40S ribosomal protein S19 PREDICTED: similar to 60S ribosomal protein L27a PREDICTED: similar to mitochondrial ribosomal protein L1 PREDICTED: similar to Mitochondrial ribosomal protein L12 PREDICTED: similar to mitochondrial ribosomal protein L15 Ribosomal protein S27a Ribosomal protein, mitochondrial, L2 60S ribosomal protein L7a 40S ribosomal protein S20 60S ribosomal protein L6 60S ribosomal protein L14 PREDICTED: similar to 60S ribosomal protein L12 40S ribosomal protein S17 60S ribosomal protein L23a 60S ribosomal protein L24 PREDICTED: similar to 40S ribosomal protein S16 40S ribosomal protein S2 60S ribosomal protein L13 Eukaryotic translation Elongation factor 1 alpha 2 60S ribosomal protein L7 PREDICTED: eukaryotic translation elongation factor 1 gamma 40S ribosomal protein S10 40S ribosomal protein S25 Elongation factor 1-alpha 1 Eukaryotic translation initiation factor 4A2 40S ribosomal protein S11 PREDICTED: similar to 60S ribosomal protein L11 60S acidic ribosomal protein P2 60S ribosomal protein L18 60S ribosomal protein L8 PREDICTED: similar to Tu translation elongation factor, mitochondrial PREDICTED: similar to 60S ribosomal protein L37a PREDICTED: similar to eukaryotic translation initiation factor 2, subunit 3, structural gene X-linked 60S acidic ribosomal protein P0 60S ribosomal protein L18a PREDICTED: similar to 40S ribosomal protein S9 Similar to mitochondrial ribosomal protein L13 60S ribosomal protein L23 60S ribosomal protein L28 Similar to ribosome-binding protein p34-rat 40S ribosomal protein S3a Eukaryotic translation initiation factor 4A1 40S ribosomal protein S3 40S ribosomal protein S9 Ribosomal pRotein S7 40S ribosomal protein S15 60S ribosomal protein L17 40S ribosomal protein S13 40S ribosomal protein S14 60S ribosomal protein L3 60S ribosomal protein L35 PREDICTED: ribosomal protein S19 Eukaryotic translation elongation factor 1 delta

0 0 0

2 3 4

0 0 2

4 0 0

n/a n/a n/a

n/a n/a n/a

n/a n/a n/a

0

2

3

3

n/a

n/a

n/a

0 0 0 0 0 0 0 0 0 0 0

0 2 2 3 2 2 0 2 0 2 0

0 0 0 0 3 2 2 0 3 0 2

2 0 0 0 0 2 2 0 0 0 0

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a



0

0

0

3

n/a

n/a

n/a

0

2

0

2

n/a

n/a

n/a

0 0 2 2 3 2 2 3 2 2 2 3 3 29 5 4

2 2 8 7 8 5 5 7 4 4 4 6 6 54 9 7

0 0 4 2 3 2 3 4 0 3 4 5 3 24 6 5

0 2 4 2 5 0 0 2 0 0 3 2 4 30 5 5

n/a n/a 3.8 3.3 2.5 2.4 2.4 2.2 1.9 1.9 1.9 1.9 1.9 1.8 1.7 1.6

n/a n/a 1.8 0.9 0.9 0.9 1.4 1.2 0.0 1.4 1.8 1.5 0.9 0.8 1.1 1.2

n/a n/a 2.0 1.0 1.6 0.0 0.0 0.7 0.0 0.0 1.5 0.7 1.3 1.0 1.0 1.2

3 3 34 5 2 2 4 3 4 10

5 5 55 8 3 3 6 4 5 12

3 2 28 7 0 0 5 2 4 10

0 3 36 4 2 0 5 0 6 17

1.6 1.6 1.5 1.5 1.4 1.4 1.4 1.3 1.2 1.1

0.9 0.6 0.8 1.3 0.0 0.0 1.2 0.6 0.9 0.9

0.0 1.0 1.0 0.8 1.0 0.0 1.2 0.0 1.5 1.7

6 3

7 3

5 0

0 2

1.1 0.9

0.8 0.0

0.0 0.7

2 2 2 2 4 3 3 4 11 5 4 4 10 3 3 3 3 3 3 6

2 2 2 2 4 3 3 4 9 4 3 3 7 2 2 2 2 2 2 4

2 0 3 0 3 0 4 4 8 5 4 3 9 2 2 3 4 0 5 3

2 0 0 0 2 2 6 3 6 0 0 4 9 0 4 2 2 3 2 4

0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.8 0.8 0.7 0.7 0.7 0.6 0.6 0.6 0.6 0.6 0.6 0.6

0.9 0.0 1.4 0.0 0.7 0.0 1.2 0.9 0.7 0.9 0.9 0.7 0.8 0.6 0.6 0.9 1.2 0.0 1.5 0.5

1.0 0.0 0.0 0.0 0.5 0.7 2.0 0.7 0.5 0.0 0.0 1.0 0.9 0.0 1.3 0.7 0.7 1.0 0.7 0.7

IPI00371204 IPI00421719 IPI00210238 IPI00475474 IPI00200145 IPI00230915 IPI00339012 IPI00211861 IPI00370636 IPI00363921 IPI00368848 IPI00361793 IPI00203773 IPI00364774 IPI00190240 IPI00365066 IPI00363949 IPI00475776 IPI00390343 IPI00475722 IPI00475561 IPI00324983 IPI00203523 IPI00230939 IPI00421451 IPI00392390 IPI00230916 IPI00476815 IPI00199543 IPI00470317 IPI00191142 IPI00215184 IPI00195372 IPI00193595 IPI00197713 IPI00359103 IPI00188804 IPI00230917 IPI00215208 IPI00371236 IPI00559050 IPI00188016 IPI00200147 IPI00192257 IPI00368950 IPI00366411 IPI00207980 IPI00555189 IPI00206020 IPI00231693 IPI00369618 IPI00212776 IPI00421626 IPI00476418 IPI00231692 IPI00210946 IPI00366014 IPI00201500 IPI00395285 IPI00212783 IPI00559098 IPI00471525

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Table 2 (Continued) locusID

protein name

SpCCTb

SpCBDc

SpCANd

SpCRAe

BD/CTf

AN/CTg

RA/CTh

IPI00202512 IPI00188058 IPI00214654 IPI00231202 IPI00197900 IPI00203214 IPI00214582 IPI00192486 IPI00231360 IPI00390823 IPI00200552 IPI00370003

60S ribosomal protein L4 40S ribosomal protein S18 Splice Isoforms of 40S ribosomal protein S24 40S ribosomal protein S8 Translation elongation factor 1-delta subunit Elongation factor 2 40S ribosomal protein S7 40S ribosomal protein S6 60S ribosomal protein L27 60S ribosomal protein L38 PREDICTED: similar to 60S ribosomal protein L26 PREDICTED: similar to mitochondrial ribosomal protein L45 Eukaryotic translation initiation factor 2 subunit 1 PREDICTED: similar to Eukaryotic translation elongation factor 1 beta 2 PREDICTED: similar to Eukaryotic translation initiation factor 3, subunit 8 Translation initiation factor eIF-2B beta subunit Eukaryotic translation initiation factor 4B 40S ribosomal protein SA 60S ribosomal protein L15 60S ribosomal protein L5 PREDICTED: similar to ribosomal protein S24 60S ribosomal protein L19 60S ribosomal protein L21

6 5 5 5 4 17 10 2 3 2 2 2

4 3 3 3 2 7 4 0 0 0 0 0

5 6 0 5 2 16 7 3 0 0 0 0

4 4 2 4 2 12 8 2 3 0 2 0

0.6 0.6 0.6 0.6 0.5 0.4 0.4 0.0 0.0 0.0 0.0 0.0

0.8 1.1 0.0 0.9 0.5 0.9 0.6 1.4 0.0 0.0 0.0 0.0

0.7 0.8 0.4 0.8 0.5 0.7 0.8 1.0 1.0 0.0 1.0 0.0

2 2

0 0

2 0

0 0

0.0 0.0

0.9 0.0

0.0 0.0

2

0

2

0

0.0

0.9

0.0

2 4 3 3 3 3 4 8

0 0 0 0 0 0 0 0

0 0 3 0 5 0 0 4

0 4 0 0 3 0 0 2

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.9 0.0 1.5 0.0 0.0 0.5

0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.2

IPI00230830 IPI00476899 IPI00197490 IPI00209122 IPI00373045 IPI00215107 IPI00231445 IPI00230914 IPI00564947 IPI00202214 IPI00421720

a Table lists spectra counts (SpC) for: bcontrol (CT), cBDNF (BD), dBDNF + Anisomycin (AN), eBDNF + rapamycin (RA). Spectra count ratios of the three BDNF treatment groups (numerators) relative to control (denominator) were calculated, normalized and are listed as fBD/CT, gAN/CT, and hRA/CT.

pal slices.45 We focused on eIF5 and one of the small ribosomal subunit proteins, S20; these proteins have not been characterized in dendrites or shown to be up-regulated by synaptic activity. Antibodies against eIF5 or S20 labeled both somata and their processes in control and BDNF-treated neurons (Figure 4); cell body labeling was more prominent than labeling in dendrites. Treatment with BDNF resulted in enhanced antieIF5 and anti-S20 labeling of dendrites in a subset of neurons; this labeling was found in immunoreactive puncta distributed along processes (Figure 4D,H). This pattern of labeling was not found in control neuronal processes (Figure 4C,G). To help ascertain whether local mRNA translation may be involved in producing the BDNF-induced increases in translation machinery, we conducted in situ hybridization on cultured cortical neurons. In contrast to the somatic localization of the mRNA encoding βIII tubulin (our negative control46), mRNAs encoding eIF5, L14, S20, and TyrRS were observed in dendritic processes (Figure 5A-E). This is best seen by comparing the distribution of these mRNAs with immunocytochemical labeling of the microtubule associated protein 2 (MAP2), which we used to identify dendritic processes (Figure 5F-J). Thus, we identified mRNAs for members of each component of the translation machinery up-regulated by BDNFslarge and small ribosomal subunits, initiation and elongation factors, and tRNA synthetasessas candidates for local translation. These data add to a growing list of mRNAs that are known to be localized in dendrites including some translation components.17-20,47

Discussion Over the past few decades, mounting evidence has shown that protein synthesis within axons and dendrites is an essential mechanism underlying synapse formation, maturation, and the consolidationofvariousformsoflong-termsynapticplasticity.17-20,47 BDNF is thought to influence synaptic function at each of these levels through its effects on local mRNA translation.5-7 Using 1066


a mass spectrometry-based relative quantification methodology, we show that treatment of cultured neurons with BDNF induces a large-scale up-regulation of 230 proteins in a synaptic fraction. Many of the identified proteins have functions compatible with their having a direct role in modulating synaptic structure and function. In addition, several classes of proteins were identified that may have indirect roles in synaptic remodeling. We also identified many proteins involved in protein turnover, a process which has been implicated in the remodeling of the postsynaptic density.48 Of particular note was that components of the translation machinery were among the proteins up-regulated by BDNF. Taken together, these data indicate that BDNF induces widespread changes in the synaptic proteome and that an increase in synaptic translation capacity may be part of this response. While it is true that synapses in primary neurons have some physiological differences compared with synapses in the mature brain (notably in transmitter release, ability to express LTP, the percentage of silent synapses, etc.), the effects of BDNF on synaptic protein synthesis are likely to be very comparable due to the fact that BDNF activates mTOR and MAP kinase cascades in both contexts. This study used spectra counting in consecutive MudPIT runs to obtain a relative quantification of a large set of proteins in SNS from control and BDNF-treated neurons. The majority of proteins identified in BDNF-treated neurons were also identified in the other three treatment groups, which provides a strong basis for relative quantification by spectra counting. However, while a recent study has established that spectra counting provides a larger dynamic range than quantification using ion chromatograms via stable isotope labeling,26 we found that spectra count ratios could not be derived for a subset of protein spectra. This was because in one sample the protein was not identified, while in the other sample multiple peptides from the same protein were found (see Table 2 for examples); thus, a statistical analysis for the corresponding proteins was


research articles

Figure 3. Western blot analyses confirm that BDNF induces a time- and translation-dependent increase in translation machinery components. (A) DIV17 cortical neurons were treated with BDNF and harvested after 15, 30, 60, and 120 min, with vehicle treatment as control. Antibodies against selected translation machinery components were used for Western blot analysis on total cell lysate. Shown are representative blot results for each individual protein tested, with ponseau red (PR) staining as loading control. (B) Time plot of the mean density value at each time point from Western blot analyses of three independently cultured and BDNF-treated cortical neuron preperations. (C) Western blots of the same translation components in lysates from cortical neurons that were treated with BDNF with or without rapamycin (RAP), anisomycin (ANI), or cyclohexamide (CHX). (D) Bar chart of the mean density value from Western blot analyses of three independently cultured and treated cortical neurons preperations. (* significantly different from control, Student’s t-test p < 0.02).

hindered. This problem is generally believed to be the result of peptide mixture complexity versus sampling efficiency in data-dependent data acquisition (e.g., scan speed of the mass spectrometer).26 Therefore, in this study, for those proteins, an arbitrary cutoff value was used to identify changes in protein levels. After normalization and stringent filtering, spectra count results are generally consistent with immunoblot analyses, which can be seen by comparing synaptic proteins in Figure 1B,D, and translation components in Table 2 and Figure 3. BDNF can affect global and local translation in neurons, and it strongly activates the translation machinery. Taken with the fact that MudPIT is much more sensitive than two-dimensional gel approaches used previously to assay BDNF-induced changes in protein expression, it is reasonable to expect that we would detect changes in a large number of proteins after BDNF treatment. Nevertheless, it is surprising that hundreds of proteins were identified by MudPIT and spectra counting as up-regulated. Currently, the number of mRNAs with an axonal or dendritic distribution, including both validated candidates and those that await confirmation, is around 20016,19,21,22 (and many others), and it has been estimated that the dendritic mRNA population could be ≈400 messages.19 However, only a

handful of mRNAs have been shown to be translated locally in response to BDNF.16,19,21,22 Although our studies did not discriminate between true local synthesis of all up-regulated proteins and the possibility that existing proteins were trafficked to synapses during the BDNF treatment, our data suggest that there could be many more proteins synthesized at synapses in an activity-dependent manner than are currently known. We identified up-regulation of multiple proteins with synaptic functions, including synaptotagmin, glutamate receptors, neurabin, piccolo, drebrin-like protein, and others. This is consistent with previous findings that BDNF induces a variety of translation-dependent synaptic changes, including synaptogenesis which requires synthesis of new proteins involved in presynaptic vesicle formation and trafficking, as well as postsynaptic proteins involved in PSD remodeling.2,49 The most intriguing finding and the one which we chose for initial validation was that BDNF up-regulates components of the translation machinery. Ribosomal proteins from the 40S and 60S subunits, tRNA synthetases, and initiation and elongation factors were detected in the SNS fraction, and a majority of these were up-regulated by the brief BDNF treatment. In accord with our MudPIT data, Western blot assays demonstrated that selected translation components were up-regulated Journal of Proteome Research • Vol. 6, No. 3, 2007 1067

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Figure 4. Immunocytochemistry of BDNF-treated and untreated neurons. Neurons were labeled with antibodies against initiation factor eIF5 or one of the ribosomal small subunit proteins, S20 (green), and double-labeled with antibodies to MAP2 (red). (A, B, E, F) Neuronal soma and processes; (C, D, G, H) neuronal processes. Scale bar: 50 µm.

by BDNF in a time-dependent manner. It is likely that many of these components are locally synthesized at or near synapses, because mRNAs encoding members of each type of component were detected in dendrites by in situ hybridization, and the increases were sensitive to protein synthesis inhibitors and the mTOR inhibitor rapamycin. Our findings corroborate results from several studies that observed specific translation components,44,50-52 or mRNAs for translation machinery in neurites,18,53 and we provide additional data showing that many more components exhibit this distribution. We also observed decreases in the levels of some ribosomal proteins. These decreases were similarly blocked by translation inhibitors and rapamycin, suggesting that the BDNF-induced synthesis of other proteins regulates their degradation. One possibility that we are currently evaluating is that BDNF-induced increases in ubiquitin system components (see Table 1) mediate decreases in ribosomal and other proteins. Our proteomic data revealed a 1.6-fold increase in the level of eEF1R-1 (Table S1 in Supporting Information), a highly expressed elongation factor that also functions as an actin binding protein.54 This is consistent with a recent report demonstrating that high-frequency stimulation of hippocampal slices caused an approximate 2-fold increase in eEF1R-1;45 mRNA for eEF1R-1 is located in dendrites and translated in response to stimuli that induce LTP and long-term depression (LTD).55 However, our Western blot data on total cell lysate from BDNF-treated neurons failed to show significant upregulation, while blots of SNS from BDNF-treated neurons revealed only an approximate 20% increase (data not shown). The discrepancy between MudPIT and Western blot data for this translation factor may be due to the fact that, as indicated 1068


Figure 5. In situ hybridization of cultured cortical neurons shows localization of mRNAs for translation machinery components in neuronal processes. (A) mRNA for β-tubulin shows a somatic localization. (B-E) mRNAs for eIF5, L14, S20, and Tyr-RS show localization in both somata and neuronal processes. (F-J) Immunocytochemistry of the same neurons using an antibody against MAP2 highlights dendritic morphology. Scale bar: 50 µm.

by our proteomics data, levels of eEF1R-1 in SNS fractions are many times higher than any other translation component,24 which may hinder the detection of subtle changes by Western blot. This observation is consistent with previous reports that eEF1R-1 is expressed at very high levels within many cell types, which may be related to its function as an actin binding protein or functions other than enlongation. Our proteomic data also indicate that BDNF induced a decrease in the eEF1R-2, a splice variant of eEF1R that is developmentally regulated and, for the most part, restricted to cell types in which translational responses are regulated by tension in the actin network, such as skeletal and cardiac myocytes.56,57

research articles


Although our results may be relevant to a variety of protein synthesis-dependent processes triggered by BDNF, the observation that BDNF up-regulates components of the translation machinery may be of particular significance to activity-dependent synaptic plasticity. Local translation is a requirement for the consolidation of several forms of long-term efficacy change,58 and local synthesis of the translation machinery could affect this consolidation process in several ways. For example, increasing the abundance of ribosomal proteins, translation factors, and tRNA synthetases could increase net translation capacity at synapses and, therefore, contribute to the consolidation phase of LTP, which requires BDNF signaling and a finite period of increased local translation.1 Inasmuch as BDNF release and local synthesis of translation components can be site-specific in vivo, it is also possible that the new translation components endow some synapses with the ability to preferentially utilize dendritically localized mRNAs. Less obvious, but potentially significant, functions of the translation machinery may also be altered by BDNF. Several noncanonical functions of translation components have been described which may be relevant to the actions of BDNF at synapses. For example, TyrRS, which was up-regulated in a translation-dependent manner by BDNF, has been implicated in extracellular signaling.59,60 It is also noteworthy that some, but not all, ribosomal proteins were up-regulated by BDNF. Given the data that initiation of translation can occur through mechanisms that involve complementation between ribosomal RNA and mRNA,61 it is possible that the new ribosomal proteins may exert a bias in the profile of mRNAs that are translated by changing the accessibility of regions of the ribosomal RNA. The notion that ribosomes act as a “filter” on gene expression in such a way has been presented in general form.62 The data presented here suggest that such mechanisms may operate at synapses, a possibility that would help to explain how translation responses are tailored locally for the consolidation of functionally distinct forms of plasticity. We are investigating at present whether the ribosomal proteins up-regulated by BDNF are integrated into 40S and 60S subunits in synaptic fractions, and whether this alters ribosomal function. As a broad screen of proteins up-regulated by BDNF in the synaptic compartment, our proteomics approach provides insights into the mechanisms by which BDNF exerts its varied effects on synaptic development and remodeling. Pending validation on a protein-by-protein basis, our data may also provide significant insights into the functions of proteins that are of interest in other contexts. For example, spectra counts indicate that BDNF enhances the synthesis of multiple proteins that have been linked to neurological conditions. Notable among these is the protein DJ-1, a highly conserved 27 kDa protein initially characterized as an oncogene product capable of transforming NIH3T3 cells.63 DJ-1 is thought to have neuroprotective functions, and mutations in the DJ-1 gene have been identified as the second most frequent monogenetic cause of familial Parkinson’s disease.64 Future studies on the roles of DJ-1 and other proteins in mediating the cellular effects of BDNF will help refine our understanding of their functions in disease-related and other contexts.

Acknowledgment. We appreciate Dr. Kathryn Crossin for providing antibodies against phosphotyrosine and TrkB. This work was supported by NIH grants P41RR11823-10, 5R01MH067880, U01DE016267-03, and by a grant from the

FRAXA research foundation to P.W.V., and the Skaggs Institute for Chemical Biology to J.P.

Supporting Information Available: Proteins found to be up- or down-regulated in SNS by BDNF were listed in Table S1. The pathways or groups of proteins up-regulated with a confidence level of over 95% are listed in Table S2. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Kelleher, R. J., III; Govindarajan, A.; Tonegawa, S. Translational regulatory mechanisms in persistent forms of synaptic plasticity. Neuron 2004, 44 (1), 59-73. (2) Schratt, G. M.; Nigh, E. A.; Chen, W. G.; Hu, L.; Greenberg, M. E. BDNF regulates the translation of a select group of mRNAs by a mammalian target of rapamycin-phosphatidylinositol 3-kinasedependent pathway during neuronal development. J. Neurosci. 2004, 24 (33), 7366-77. (3) Campbell, D. S.; Holt, C. E. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 2001, 32 (6), 1013-26. (4) Malcangio, M.; Lessmann, V. A common thread for pain and memory synapses? Brain-derived neurotrophic factor and trkB receptors. Trends Pharmacol. Sci. 2003, 24 (3), 116-21. (5) Kang, H.; Schuman, E. M. A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 1996, 273 (5280), 1402-6. (6) Alsina, B.; Vu, T.; Cohen-Cory, S. Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. Nat. Neurosci. 2001, 4 (11), 1093-101. (7) Bramham, C. R.; Messaoudi, E. BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog. Neurobiol. 2005, 76 (2), 99-125. (8) Sanchez, A. L.; Matthews, B. J.; Meynard, M. M.; Hu, B.; Javed, S.; Cory, S. C. BDNF increases synapse density in dendrites of developing tectal neurons in vivo. Development 2006, 133 (13), 2477-86. (9) Tartaglia, N.; Du, J.; Tyler, W. J.; Neale, E.; Pozzo-Miller, L.; Lu, B. Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor. J. Biol. Chem. 2001, 276 (40), 37585-93. (10) Korte, M.; Carroll, P.; Wolf, E.; Brem, G.; Thoenen, H.; Bonhoeffer, T. Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (19), 8856-60. (11) Patterson, S. L.; Abel, T.; Deuel, T. A.; Martin, K. C.; Rose, J. C.; Kandel, E. R. Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 1996, 16 (6), 1137-45. (12) Takei, N.; Inamura, N.; Kawamura, M.; Namba, H.; Hara, K.; Yonezawa, K.; Nawa, H. Brain-derived neurotrophic factor induces mammalian target of rapamycin-dependent local activation of translation machinery and protein synthesis in neuronal dendrites. J. Neurosci. 2004, 24 (44), 9760-9. (13) Patterson, S. L.; Pittenger, C.; Morozov, A.; Martin, K. C.; Scanlin, H.; Drake, C.; Kandel, E. R. Some forms of cAMP-mediated longlasting potentiation are associated with release of BDNF and nuclear translocation of phospho-MAP kinase. Neuron 2001, 32 (1), 123-40. (14) Raught, B.; Gingras, A. C.; Sonenberg, N. The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (13), 703744. (15) Shahbazian, D.; Roux, P. P.; Mieulet, V.; Cohen, M. S.; Raught, B.; Taunton, J.; Hershey, J. W.; Blenis, J.; Pende, M.; Sonenberg, N. The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J. 2006, 25 (12), 2781-91. (16) Aakalu, G.; Smith, W. B.; Nguyen, N.; Jiang, C.; Schuman, E. M. Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 2001, 30 (2), 489-502. (17) Zhong, J.; Zhang, T.; Bloch, L. M. Dendritic mRNAs encode diversified functionalities in hippocampal pyramidal neurons. BMC Neurosci. 2006, 7, 17. (18) Willis, D.; Li, K. W.; Zheng, J. Q.; Chang, J. H.; Smit, A.; Kelly, T.; Merianda, T. T.; Sylvester, J.; van Minnen, J.; Twiss, J. L. Differential transport and local translation of cytoskeletal, injuryresponse, and neurodegeneration protein mRNAs in axons. J. Neurosci. 2005, 25 (4), 778-91.


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BDNF Induces Widespread Changes in Synaptic Protein Content and

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