New Approach to Capture and Characterize Synaptic Proteome
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New approach to capture and characterize synaptic proteome Xin-An Liua, Beena Kadakkuzhaa,1, Bruce Pascalb,1, Caitlin Stecklerc, Komolitdin Akhmedova, Long Yand, Michael Chalmersc, and Sathyanarayanan V. Puthanveettila,2 aDepartment of Neuroscience, bInformatics Core, and cProteomics Core Facility, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458; and dOptical Workshop and Light Microscopy Facility, Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458 Edited* by Eric R. Kandel, Columbia University, New York, NY, and approved October 8, 2014 (received for review January 23, 2014) Little is known regarding the identity of the population of proteins abundant in mouse hippocampus. We studied the expression of 31 that are transported and localized to synapses. Here we describe different kinesins in mouse hippocampus using specific primers (SI a new approach that involves the isolation and systematic proteomic Appendix, Dataset S1). Quantitative real-time PCR (qRT-PCR) characterization of molecular motor kinesins to identify the popula- analysis of RNAs isolated from mouse hippocampus suggests that tions of proteins transported to synapses. We used this approach to Kif5C is highly abundant in hippocampus (normalized expression identify and compare proteins transported to synapses by kinesin level, 5.3 ± 0.14; SI Appendix,Fig.S1A). We then confirmed the (Kif) complexes Kif5C and Kif3A in the mouse hippocampus and pre- expression of Kif5C by RNA in situ hybridization using digoxigenin- frontal cortex. Approximately 40–50% of the protein cargos identified labeled sense and antisense riboprobes (SI Appendix,Fig.S1B)and in our proteomics analysis of kinesin complexes are known synaptic by immunohistochemical and Western blot analyses using an anti- proteins. We also found that the identity of kinesins and where they Kif5C antibody (SI Appendix,Fig.S1C and D and Dataset S1). are expressed determine what proteins they transport. Our results Kif5C transport complexes were then immunoprecipitated reveal a previously unappreciated role of kinesins in regulating the from the hippocampus (Fig. 1A) following a previously published composition of synaptic proteome. protocol (13) and analyzed by silver stain and Western blot analysis (Fig. 1B). Immunoprecipitated beads alone and immu- kinesin | protein transport | synapse | proteome | hippocampus noprecipitated anti-CREB antibody (SI Appendix, Dataset S1) served as specificity controls for the immunoprecipitation (IP). NEUROSCIENCE number of researchers have reported specific roles of pro- The specific protein band corresponding to Kif5C was present Ateins that are localized to synapses, termed the “synaptic only in IP of Kif5C, and not in the beads alone or in antibody proteome,” in determining synaptic function and plasticity (1–6). controls (Fig. 1B). We analyzed the protein bands corresponding Identifying the composition and dynamics of the synaptic pro- to Kif5C by mass spectrometry, which identified ∼80% of the teome is key to understanding the remodeling of existing syn- predicted tryptic peptides of Kif5C (SI Appendix, Fig. S1E). apses and growth of new synapses, as well as identifying novel To identify the population of proteins transported by Kif5C, therapeutic targets for synaptopathies (7, 8). The composition of we performed a systematic proteomic analysis of Kif5C com- the synaptic proteome is determined by proteins transported plexes from hippocampus. We performed four biological repli- from the cell body and local synthesis of proteins at the synapses. cations of Kif5C IPs and four beads-alone control IPs. Scaffold Several studies have shown the identification and localization of software identified 72 proteins present only in the Kif5C IPs and RNAs at the synapse (9–12). Comprehensive knowledge of the not in the control IPs. proteins that are actively targeted to synapses will help elucidate We considered two factors affecting the abundance of proteins the dynamics of signal transduction at the synapse, as well as the associated with kinesin in individual IP biological replications: (i) molecules that are critical for the remodeling of synapses and the IP efficiency of samples and the stability of specific proteins formation of new synapses that accompany long-term memory interacting with kinesin and (ii) the physiological state of the storage. Despite this perceived significance, however, there are neurons. Thus, to identify the maximum number of possible no currently available methodologies for studying populations of cargos associated with Kif5C in the hippocampus, we analyzed proteins targeted to synapses. the proteomics dataset (SI Appendix, Dataset S1) to identify To identify actively transported proteins, we focused on the protein cargos that were 1.5-fold enriched in kinesin IPs com- protein complexes associated with kinesins, molecular motor pared with the control IPs. We believed that this analysis would proteins that move from the cell body to distal neuronal pro- cesses and mediate the transport of various gene products (9, Significance 13). We considered that proteins associated with kinesins include both synaptic proteins and proteins localized to dendrites and Here we report a strategy for isolating and characterizing axons. Our methodology involved isolating specific kinesin populations of proteins targeted to synapses. Using this ap- complexes by coimmunoprecipitation (co-IP) from different proach, we isolated and characterized multiple protein trans- regions of the mouse brain and systematically identifying the port complexes from mouse brain, providing novel insights A protein cargos by mass spectrometry (Fig. 1 ). Using this strat- into the synaptic targeting of proteins and composition of egy, we have successfully isolated and characterized the Kif5C synaptic proteome. kinesin complex from hippocampus and prefrontal cortex (PFC) and the Kif3A kinesin complex from hippocampus. The cargos Author contributions: S.V.P. designed research; X.-A.L., B.K., C.S., K.A., and S.V.P. per- include organelles and proteins involved in translation, signaling, formed research; L.Y. contributed new reagents/analytic tools; X.-A.L., B.P., M.C., and and ion channels. Several of these proteins are implicated in S.V.P. analyzed data; and X.-A.L., M.C., and S.V.P. wrote the paper. neuropsychiatric disorders (14). The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. Results 1B.K. and B.P. contributed equally to this work. Isolation and Characterization of Protein Complexes of Molecular Motor 2To whom correspondence should be addressed. Email: [email protected]. Kif5C in the Mouse Hippocampus. To identify the population of syn- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. aptically targeted proteins, we first searched for kinesins that are 1073/pnas.1401483111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1401483111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Fig. 1C, we found that these proteins are indeed specifically asso- ciated with the Kif5C complex and are not present in the beads- alone control IPs. Examination of the protein expression and dis- tribution of Kif5C (SI Appendix,Fig.S1C) and the selected cargos in hippocampus by immunohistochemical staining (SI Appendix,Fig. S2) revealed that they are expressed in hippocampal neurons as well. We studied the expression levels of Kif5C in primary neuron cultures by qRT-PCR and found that, consistent with our data on Kif5C levels in dissected hippocampus, Kif5C is abundantly expressed in primary hippocampal neurons (SI Appendix,Fig.S1F). Using specific siRNAs, we next knocked down the expression of Kif5C in primary hippocampal neurons, and evaluated the knockdown by Western blot and immunohistochemistry analyses. We found that Kif5C siRNA, but not the scrambled siRNA control, was effective in knocking down Kif5C protein levels in hippocampal neurons. Analysis of Western blot data by two-way ANOVA followed by the Tukey post hoc test indicated statisti- cally significant Kif5C knockdown (Kif5C SiRNA, scrambled and no siRNA control, F(2, 63) = 16.866, P < 0.0001) at 72 h (0, 48, and 72 h, F(2, 63) = 12.622, P < 0.0001) after the siRNA transfection (SI Appendix, Fig. S3 A and B; 37.22 ± 4.69% knockdown, Kif5C siRNA vs. scrambled siRNA and control without siRNA transfection; n = 8), which was further supported by Kif5C immunohistochemistry data (SI Appendix, Fig. S3 C and D; F(2, 30) = 81.035, ***P < 0.001, one-way ANOVA). We then examined the distributions of PURα, DIC1, and GluR1 in primary neurons (Fig. 2). As shown in SI Appendix, Fig. S3 A–D, we observed an ∼40% decrease in Kif5C expression Fig. 1. Identification of the Kif5C complex in mouse hippocampus. (A) after siRNA transfection at 72 h. We measured the intensity Strategy used to analyze Kif5C-specific protein cargos in the mouse hippo- level of these cargos on random spots in the neurites that were at campus. The schematic diagram shows the workflow for profiling Kif5C- least 10 μm away from the soma, and found that the mean transported proteins by proteomics. The processes include brain dissection, fluorescence intensities of the three cargos of Kif5C in neurites hippocampal tissue extraction and homogenization, Kif5C IP, complex sep- were significantly decreased after Kif5C knockdown compared aration by SDS/PAGE, isolation of specific bands followed by in-gel tryptic with those in the neurons without siRNA transfection