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Proc Natl Acad Sci U S A. Apr 30, 2013; 110(18): 7464–7469.
Published online Apr 15, 2013. doi:  10.1073/pnas.1304422110
PMCID: PMC3645558
Neuroscience

A strategy to capture and characterize the synaptic transcriptome

Abstract

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.

Keywords: long-term memory storage, RNA transport, local protein synthesis, neurogenomics, cystoskeleton

Despite decades of research on the importance of local translation in learning-related synaptic plasticity and long-term memory storage, we know relatively little about the identity of the “synaptic transcriptome,” the various components of the total RNA population that is transported from the cell body, and how this transcriptome becomes localized to and translated at specific synapses (112). To develop a general strategy for isolating and characterizing all of the RNAs transported from the cell body to the synapse, we have focused on the RNA transport complexes that interact with the molecular motor kinesin that mediate transport of gene products from the cell body to synapses (13, 14).

Our approach offers four distinct advantages over previously described methods: (i) it allows the identification of RNAs based on their association with transport machinery that is destined for synapses; (ii) it reflects dynamically regulated RNAs; (iii) it allows for identification of the targeted RNAs; and (iv) it can be used in different regions of the central nervous system (CNS), thereby facilitating genomic characterization of synaptic transcriptome of the entire CNS or region of the CNS. These advantages should aid the study of stoichiometric changes in localized RNAs and their role of local translation, not only in memory storage, but also in a variety of other physiological conditions, such as development. Using this strategy, we have succeeded in obtaining a comprehensive collection of RNAs targeted to Aplysia CNS synapses. We further show that myosin heavy chain mRNA, a cargo of kinesin, is localized to sensory neuron processes and is required specifically for the induction of long-term facilitation (LTF) at sensory and motor neuron synapses.

Results

Strategy for Identifying Synaptically Targeted RNAs: Isolation and Characterization of RNA Transport Complexes.

We assumed that successful isolation of the RNA–protein complexes associated with the kinesin transport machinery would help identify the full repertoire of RNAs that are actively transported to synapses. Because the CNS contains both neuronal and nonneuronal cells, this approach will also yield RNAs found in the kinesin complex in nonneuronal cells, such as glia. We first optimized conditions for isolating RNA transport complexes from the CNS of Aplysia based on a previously described protocol for preparation of kinesin transport complexes Aplysia Kinesin Heavy Chain 1 (ApKHC1) that contain several synaptic proteins (15). To examine whether the transport complexes prepared from CNS contain RNA cargos (Fig. 1 A and B), we searched in the complex for the presence of three RNA-binding proteins known to be present at synapses—staufen, fragile X mental retardation protein (FMRP), and cytoplasmic polyadenylation element-binding protein (CPEB)—that have been associated with kinesin in the mouse (14, 16). Western blot analysis of the coimmunoprecipitated Kinesin Heavy Chain (KHC) complex revealed the presence of all three of these RNA-binding proteins (Fig. 1C) specifically in the kinesin coimmunoprecipitations (CoIPs), but not in the Aplysia Target of Rapamycin (ApTOR) Ab (used as a nonspecific Ab control) CoIPs. Thus, the kinesin complex immunoprecipitated from CNS contains several RNA-binding proteins and is likely to contain RNAs destined for synaptic sites.

Fig. 1.
Isolation and characterization of RNA transport complexes from CNS of Aplysia. (A) Cartoon representation of RNA transport complex. Components of the transport machinery, including the cytoskeleton, kinesin heavy chain and light chain, and different cargos ...

We next prepared RNAs from the ApKHC1 complexes isolated from Aplysia CNS and searched for the presence of Calcium/Calmodulin dependent protein kinase IIα (CaMK IIα) mRNA, a transcript reportedly transported by kinesin (14, 17). We used ApC/EBP mRNA, a transcription factor, as a negative control. Semiquantitative RT-PCR experiments showed the presence in the complex of CaMK IIα mRNA and absence of the Aplysia CCAAT/enhancer-binding protein (ApC/EBP) mRNA, suggesting that the complex contains some previously described RNA cargo of kinesin (Fig. 1D).

To adopt a genomics approach to identifying all RNAs present in the ApKHC1 complex, we first used Aplysia microarrays containing probes corresponding to 56,000 unique neuronal transcripts described previously (18). Fig. 1E shows the results of the hybridization analysis carried out on this array showing ~200 RNAs (blue) with at least twofold enrichment in the kinesin complex over control (P ≤ 0.05) immunoprecipitates from CNS. The RNAs specifically represented in the complex included several neuropeptide precursors, kinases, phosphatases, ion channels, and regulatory factors involved in protein synthesis (Dataset S1, Tables S1, S2, and S3). In this collection, we identified several RNAs that are localized to neuronal processes of sensory neurons, such as elongation factor 1α (19) and sensorin (2023).

RNA deep sequencing (RNA-seq) Analysis of ApKHC1 Transport Complex Reveals the Complex Composition of Transported RNAs.

Our Aplysia microarray contained 50–60% of all of the genes predicted to be expressed in the CNS of Aplysia (18). An inherent limitation of microarrays is the inability to identify novel and low-abundance transcripts. In addition, the Aplysia genome has not yet been fully annotated, and a complete list of Aplysia protein-coding genes is not available. Thus, we used the Roche 454 pyrosequencing platform to directly identify all RNAs transported by kinesin. Using 454 sequencing followed by transcript assembly, we identified 5,657 nonredundant sequences (i.e., contigs) of RNAs associated with the kinesin complex prepared from CNS (Dataset S1, Table S4). Approximately 90% of the ~200 RNAs that we identified from the microarray experiments (described above) were found in this collection of RNAs (cf. Dataset S1, Tables S1, S2, S3, and S5), providing independent validation of the microarray results. Therefore, we are reasonably confident that the majority of transcripts identified by RNA-seq indeed comprise a significant portion of the kinesin transport complex. Our RNA-seq analysis has identified ~2,000 transcripts not previously represented in the Aplysia EST database.

Using BLAST searches (e-value <10−4) in GenBank and SwissProt databases, among the 5,657 identified RNA sequences associated with the CoIP kinesin complex, we were able to identify 1,184 transcripts encoding predicted proteins with known homologs from other organisms (Fig. 2A and Dataset S1, Table S5). Based on their predicted physiological functions, these RNAs code for signaling molecules (15.8%), components of the protein synthesis machinery (13.5%), channels and receptors (5.9%), neuropeptides (2.5%), elements of the cytoskeleton (3.9%), components of the protein degradation machinery (1.9%), metabolic and other processes (32.2%), and unknown proteins (24.3%). Importantly, the majority of known Aplysia secretory signaling molecules, including neuropeptide precursors and other secretory peptides (e.g., tolloid/bone morphogenetic proteins) (Dataset S1, Table S6) were found in the complex. In comparison, neuropeptide RNAs represent <0.5% of the total transcripts that we recently characterized from the CNS (18) These data suggest that there is substantial enrichment for this class of RNAs in the kinesin complex, and that in general, most neuropeptide RNAs may be transported to the synapses.

Fig. 2.
Genomic characterization of the kinesin transport complex. (A) Pie chart showing different functional groups of gene products identified from RNA-seq analysis of RNAs prepared from the kinesin complex coimmunoprecipitated from CNS. (B and C) Characterization ...

Consistent with previous findings on mRNA localization in the sensory neuron processes (18, 21), we identified transcripts encoding nearly all of the ribosomal proteins and the majority of components of the protein synthesis machinery and RNA-binding proteins, such as staufen and zinc finger dsRNA-binding proteins, as well as components of the cytoskeleton, and cytoskeleton-associated regulatory proteins, including myosin, tubulin, actin, and kinesin-like protein (Table S5). Also present in the complex were components of signal transduction pathways, including kinases (e.g., protein kinase A, C, G), phosphatases, lipases, calmodulin, ion channels (e.g., hyperpolarization-activated ion channel, Phe-Met-Arg-Phe-NH2 (FMRFamide)-gated Na+ channel), neurotransmitter transporters (e.g., vesicular acetylcholine transporter), synaptic proteins (e.g., synaptotagmin-1, synaptobrevin, NMDA-type glutamate, other receptors, including seven transmembrane helix receptors), and cell adhesion molecules (e.g., neural cell adhesion molecule [NCAM]-related adhesion molecule). We also discovered several natural antisense/noncoding RNAs (e.g., natural antisense RNAs for beta tubulin, S6 kinase, protein kinase A type II) in the complex (Fig. S1), suggesting that they also might be transported and contribute to synaptic physiology and memory storage. We next compared recently published data on RNA localization in rat hippocampal neurons with that of our current dataset on kinesin-associated RNAs identified from Aplysia CNS. Bioinformatics analysis suggest that they share ~40% Gene Ontology terms (Fig. S2 and Dataset S1, Table S10), suggesting a major overlap of signaling pathways present in hippocampal and Aplysia CNS neuronal processes.

RNA Cargo-Enriched Microarray Analysis Suggests Enrichment of Specific RNAs in the ApKHC1 Complex from Aplysia CNS.

To determine whether there is a substantial enrichment of certain RNAs in the transport complex, we compared gene expression profiles of total RNAs expressed in the CNS and RNAs present in the ApKHC1 complex using Aplysia microarrays (18) supplemented with the ~2,000 additional gene probes identified by 454 sequencing. We then used this kinesin cargo-enriched array to compare RNAs present in the immunoprecipitated complex from CNS and total RNA from CNS.

Our microarray results suggest a significant enrichment of many RNAs in the kinesin immunoprecipitates, supporting our initial characterization (Fig. 2 B and C and Fig. S3). The enrichment of 317 transcripts in the kinesin immunoprecipitation (IP) complex (shown in red in Fig. 2 B and C) was statistically significant, with a median false discovery rate (FDR) <0.62%, which would correspond to approximately two false-positive results in the set. The functional assignment was based on homology searches and information obtained from EST sequencing. These transcripts in the kinesin IP complex included homologs for ash1 (asymmetric synthesis of HO endonuclease1), SFRS8 (splicing factor, arginine/serine-rich 8), cyclin-dependent kinase activator, cactin, and heat shock protein 60 (HSP60) (Dataset S1, Table S7). Of note, 40% of the transcripts that we identified as enriched in the kinesin complex compared with the total RNA were among the 2,000 additional features that we added to make the cargo-enriched array, thereby further validating our initial microarray and RNA-seq results.

We next studied the 317 transcripts identified as enriched in the ApKHC1 complex to identify biological pathways possibly mediated by these RNA cargos. Because Aplysia currently does not have a complete well-annotated genome and transcriptome, we identified human homologs of the Aplysia ESTs using blastx searches against the human transcriptome. Out of 317 cargo-enriched transcripts (significant at 1% FDR), 147 transcripts produced hits in the human transcriptome (Dataset S1, Table S8). We further analyzed these 147 transcripts using the Expression Analysis Systematic Explorer (EASE) (30) to search for predominant biological themes in gene annotation databases and the Kyoto Encyclopedia of Genes and Genomes (KEGG) using Aplysia array projection into the human transcriptome, as a background for statistical estimations (Dataset S1, Table S9). From this analysis, we identified 16 homologous pathways, including pathways involved in basic cellular processes, such as protein export, calcium signaling, axon guidance, endocytosis, cytoskeletal regulation, and RNA splicing, and diseases, such as Huntington’s disease. Not surprisingly, our findings suggest that RNAs transported by kinesin could regulate several different physiological processes at the CNS synapses.

Myosin Heavy Chain, an RNA Cargo of Kinesin, Is Required for the Establishment of LTF.

We next localized three candidate RNA cargos of ApKHC1 by RNA in situ hybridization, using a hybridization probe for sensorin as a positive control. We found that synaptobrevin, α-tubulin, and myosin heavy chain (MHC) RNAs localized to sensory neuron processes (Fig. 3). α-tubulin has previously been found to be localized to sensory neurons (18, 21), which is of interest in understanding molecular motor-dependent transport and localization of proteins and RNAs to distal neuronal processes. MHCs are actin-dependent molecular motors critical for hippocampal long-term potentiation (LTP) (24, 25).

Fig. 3.
In situ hybridization analysis of cargo candidates. Three different cargos (ApMHC, α-tubulin, and synaptobrevin) were analyzed by in situ hybridization of sensory neurons using digoxigenin-labeled riboprobes. After hybridization and washing, these ...

We first examined whether knockdown of ApKHC1 would affect the localization of Aplysia MHC (ApMHC). We injected ApKHC1 antisense oligonucleotides (15) into sensory neurons to disrupt kinesin-mediated transport and examined localization of MHC RNAs at the processes by in situ hybridization analysis. Analysis of the confocal projection images showed a ~30% decrease in ApMHC staining [unpaired t test, two-tailed P value = 0.014; t(7) = 3.28; mean fluorescence intensity ± SEM of ApMHC staining: control, 21.7 ± 2.03, (n = 4); ApKHC knockdown, 15.1 ± 0.78 (n = 5)] in the processes of sensory neurons that received ApKHC1 antisense oligonucleotide injection (Fig. 4 A and B).

Fig. 4.
Characterization of ApMHC localization at sensory neuron processes. (A) Confocal projection image showing ApMHC RNA staining using fluorescent antisense riboprobes in sensory neuron processes. Antisense oligonucleotides against ApKHC1 (ApKHC1 AS) were ...

We next examined whether repeated exposure to serotonin (5HT), a modulatory neurotransmitter that produces LTF at sensory and motor neuron synapses, regulates the expression of ApMHC RNA in sensory neurons. Quantitative RT-PCR (qRT-PCR) showed that exposure to five pulses of 5HT (5× 5HT) increased ApMHC RNA levels in sensory neuron clusters (normalized fold increase after 1 h of 5HT exposure, 1.8 ± 0.13; after 6 h of exposure, 0.93 ± 0.37; n = 6; Student t test) (Fig. 5A). Expression of ApC/EBP was used as a positive control for 5× 5HT treatment.

Fig. 5.
MHC, an RNA cargo of kinesin, is required for establishment of LTF. (A) Transcriptional regulation of ApMHC by 5 pulses of 5HT (5× 5HT). RNAs were extracted from sensory neuron clusters 1 h (red bars) or 6 h (black bars) after 5× 5HT treatment. ...

To explore the significance of ApMHC up-regulation during memory storage, we studied the electrophysiological consequences of specific knockdown of ApMHC in sensory neurons during short-term facilitation (STF) and initiation and persistence of LTF of sensory and motor neuron synapses. We injected antisense oligonucleotides that specifically degrade ApMHC [unpaired t test, two-tailed P values: control vs antisense-injected, P = 0.0031, t (10) = 3.87; sense oligonucleotide vs antisense-injected, P = 0.0005, t (10) = 5.11; mean fluorescence intensity ± SEM of ApMHC staining: control, 19.5 ± 1.73 (n = 6); sense oligonucleotide, 20.8 ± 1.48 (n = 6); antisense oligonucleotide, 12.1 ± 0.84 (n = 6)] into sensory neurons and measured excitatory post synaptic potentials (EPSPs) (Fig. 4 C and D).

We began by examining STF. For this, ApMHC oligonucleotides (sense and antisense) were injected into sensory neurons cocultured with L7 motor neurons (31). At 4 h after oligonucleotide injection, the cultures were treated with 10 μM 5HT for 5 min. EPSPs were recorded (Fig. 5B) after 10 min of 5HT treatment. An uninjected sensory neuron synapsing with the same motor neuron was used as an internal control for injection. We found the following percent changes in mean EPSP amplitudes: control (untreated), −10.3 ± 7.9 (n = 7); antisense MHC oligonucleotide alone: 7.5 ± 6.4 (n = 10); sense MHC oligonucleotide alone, 1.9 ± 6.1 (n = 6); one pulse of 5HT (1× 5HT), 139.14 ± 51.5 (n = 7); antisense MHC + 1× 5HT, 137.2 ± 27.4 (n = 10); sense MHC + 1× 5HT, 143 ± 25.1 (n = 12). Our data shows that neurons that were exposed to single pulses of 5HT, 5HT + antisense ApMHC, and 5HT + sense ApMHC showed no statistically significant differences in EPSPs (F = 0.1924; P = 0.8264, repeated-measures ANOVA; P > 0.05, Tukey’s multiple-comparison test), suggesting that ApMHC RNA levels are not critical for STF.

To determine the role of ApMHC in LTF, we next injected antisense and sense oligonucleotides into sensory neurons in the coculture and then applied five pulses of 10 μM 5HT at 4 h after oligonucleotide injection. Measurements of EPSPs at 24 h and 48 h after the 5HT exposure revealed the following percent changes in mean EPSP amplitudes: 5HT alone: at 24 h, +57 ± 7.5 (n = 8); at 48 h, +51.4 ± 6.7 (n = 8); 5HT + ApMHC antisense oligonucleotide: at 24 h, +3.14 ± 8.5 (n = 12); at 48 h, +3.9 ± 11 (n = 12); 5HT + ApMHC sense oligonucleotide: at 24 h, +52.9 ± 14.7 (n = 8); at 48 h, +46 ± 10.7 (n = 8) (Fig. 5C). Neither the antisense nor the sense MHC oligonucleotides affected basal synaptic transmission. However, the antisense oligonucleotides blocked the 5HT-dependent increase in EPSPs [F(8, 88) = 4.3825; P = 0.00017, repeated-measures ANOVA; P < 0.001, Newman–Keuls post hoc test at both 24 h and 48 h], suggesting that ApMHC is required for the establishment of LTF (Fig. 5C).

We then asked whether inhibition of MHC would affect the persistence of LTF. We injected MHC antisense oligonucleotides at 24 h after induction of LTF with exposure to 5HT and measured EPSPs at 48 h. Interestingly, we found that MHC inhibition did not affect the persistence of LTF. Percent changes in EPSP amplitudes measured at 48 h after exposure to 5HT alone and to 5HT + antisense oligonucleotide injected at 24 h were not significantly different [5HT + ApMHC antisense oligonucleotide injected at 24 h, +73 ± 17.3 (n = 9); at 48 h, +58 ± 11 (n = 9); t (11) = 0.04; two-tailed P value = 0.9722, unpaired t test] (Fig. 5C). These results suggest that ApMHC levels are important for the initiation of LTF, but not for persistence of LTF at Aplysia sensory and motor neuron synapses.

Discussion

Characterization of ApKHC1 Transport Complex Has Led to Identification of RNAs Actively Targeted to Synapses of Aplysia CNS.

Several previous studies that characterized RNAs prepared from dissected processes of cultured neurons have identified distally localized RNAs in neurons (2, 18, 21, 26). The methodologies applied in those studies are not useful for directly identifying RNAs targeted to synapses of intact brain or brain regions, however. Our NextGen sequence analysis of RNAs isolated from kinesin immunoprecipitates allowed us to identify approximately 5,657 transcripts from Aplysia CNS, 1,184 of which were annotated using Gene Ontology terminology. This analysis will improve as the availability of Aplysia genome sequence and gene annotation increases. The RNAs identified in the kinesin complex constitute ~2–5% of the predicted transcriptome of the Aplysia genome. We also identified several naturally occurring antisense RNA transcripts with functions at synapses that remain to be determined. Our analyses suggest that the transported RNAs are surprisingly diverse and pose the question of why so many different RNAs are targeted to synapses.

One possible explanation is that these different mRNAs are stored at the synapses for later use. Translation of these RNAs might be regulated at specific synapses during the process of storing, maintaining, and reconsolidating long-term memories not only for modifying preexisting synaptic connections, but also for the formation of new ones. Consistent with this idea, Si et al. (27, 28) have demonstrated learning-dependent activation of synaptically localized RNAs by polyadenylation, which is mediated by CPEB.

Role of Active Molecular Transport in Initiation and Persistence of Long-Term Memories.

We previously found that microtubule-dependent kinesin motors are essential for the initiation of LTF, but not for its persistence (15). This finding led us to consider the possibility that during the persistence phase, actin/MHC-dependent delivery of cargos might be important. Contrary to our expectation, however, we found that MHC antisense oligonucleotides blocked initiation of LTF, but did not affect persistence of LTF when injected into sensory neurons at 24 h after initiation. These results suggest that MHC levels are no longer critical during the persistence phase. Consistent with the requirement of ApMHC for the initiation of LTF, MHC has been found to be critical for LTP of the CA1 region of mouse hippocampus (24, 25). Taken together, our results lead us to conclude that during the early initiation phase of memory storage, the neuron uses active transport mechanisms, such as microtubule and actin-dependent motors, for delivery of cargos to synapses, but that once gene products arrive at their destination, enhanced microtubule- and actin-dependent delivery of molecules is no longer necessary for the persistence of memory storage. At this point, other mechanisms, including basal levels of transport, local regulation of translation, and local protein synthesis, become key determinants of persistence. Thus, these motors control the late phases of plasticity indirectly by supplying the population of mRNAs and proteins that are required for maintenance.

Experimental Procedures

Details of 5HT stimulation, isolation of proteins and RNAs, cloning, antibodies, oligonucleotides, qRT-PCR, Western blot analysis, in situ hybridization, microinjection, and electrophysiology are provided in SI Experimental Procedures.

IP of Kinesin Complex and RNA Isolation.

We modified previously described buffer conditions to isolate kinesin complexes from the Aplysia CNS (15). The modified buffer contained 50 mM Tris[center dot]HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM KCl, 0.5% Nonidet P-40, 1 mM DTT, 1% ultrapure BSA (Ambion), 1% yeast tRNA, and inhibitors of proteases (Roche), phosphatases (Sigma-Aldrich), and RNAses (Ambion). All of the experiments were conducted at 4 °C in an RNAse-free environment. Precleared extracts prepared from two adult (50–90 g) Aplysia CNS (for one IP experiment, n = 1) were incubated with 20 µg of affinity-purified anti-kinesin Ab (15). After 6 h of incubation on a rotator, protein A/G beads (Pierce) were added to the immunoprecipitates, followed by another 1 h of rotation. The immunoprecipitates were washed three times using the same buffer, followed by brief centrifugation (100 × g for 2 min). These washes were carried out in separate Eppendorf tubes (siliconized Rnase-free) to minimize nonspecific binding of RNA to the plastic. After the third wash, the beads were incubated with Trizol to prepare RNA for Agilent Bioanalyzer, 454 sequencing, microarrays, and qRT-PCR analyses. Equivalent amounts of a rabbit polyclonal Ab against ApTOR protein was used in control IPs.

cDNA Library Construction for 454 Sequencing.

We used commercial kits (Marathon cDNA Amplification Kit, catalog no. 634913; Clontech) to ensure consistency and reproducibility of library preparations. Methods used for 454 library construction and sequencing have been described previously (29). Three independent biological replicates were sequenced. All original sequence reads were submitted to the National Center for Biotechnology Information’s Sequence Read Archive (project no. SRA009823.3). A total of 40,204 reads were generated, assembled, and used for analysis. The Velvet assembly (32) generated 5,657 unique transcripts.

Microarray Analysis.

Microarray and sample preparation and hybridization were performed as described previously (18, 29). Custom microarrays supplemented with 2,000 additional features containing probes (Agilent) for kinesin-immunoprecipitated transcripts identified by 454 sequencing were used in later experiments.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Kaben Schwartz for the initial characterization of cargo RNAs by qRT-PCR and preparation of probes for in situ hybridization, and Vivian Zhu and Eddy Konstantinov for preparation of Aplysia neuronal cultures. This work was supported by the Howard Hughes Medical Institute, National Institutes of Health (Grants P50 HG002806, MH075026, 1R01 NS060762, 1R01 GM097502, R21RR025699, and 5R21DA030118), McKnight Brain Research Foundation, Whitehall Foundation, and National Science Foundation (Grant 0744649).

Footnotes

The authors declare no conflict of interest.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. GSE30440 and GSE30389).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1304422110/-/DCSupplemental.

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