Stable Isotopic Labeling by Amino Acids in Cultured Primary Neurons

Abstract

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics. Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we demonstrated that stable isotope labeling by amino acids in cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by brain-derived neurotrophic factor (BDNF), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitations comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase for BDNF, and others such as hepatocyte growth factor-regulated tyrosine kinase substrate and signal-transducing adaptor molecule, which are proteins known to regulate intracellular trafficking of receptor tyrosine kinases. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics. Primary culture usually refers to differentiated cells that are harvested from a living organism and maintained in culture (1Alberts B. Molecular Biology of the Cell.4th Ed. Garland Science, New York2002: 472Google Scholar). Dissociated embryonic neuronal cell culture is one of many primary culture systems researchers use to create model systems that more closely resemble in vivo biology, cellular makeup, and complexity than do immortalized cell lines (2Silva R.F. Falcao A.S. Fernandes A. Gordo A.C. Brito M.A. Brites D. Dissociated primary nerve cell cultures as models for assessment of neurotoxicity.Toxicol. Lett. 2006; 163: 1-9Crossref PubMed Scopus (65) Google Scholar). Compared with the intact organ or slice cultures, dissociated primary neuronal cell cultures represent a simplified model that allows evaluation, in a specific cell type, of features such as cell morphology, energy metabolism, signal transduction, and neurotransmitter release (2Silva R.F. Falcao A.S. Fernandes A. Gordo A.C. Brito M.A. Brites D. Dissociated primary nerve cell cultures as models for assessment of neurotoxicity.Toxicol. Lett. 2006; 163: 1-9Crossref PubMed Scopus (65) Google Scholar, 3Matteoli M. Verderio C. Krawzeski K. 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In combination with mass spectrometry, SILAC 1The abbreviations used are: SILAC, stable isotope labeling by amino acids in cell culture; BDNF, brain-derived neurotrophic factor; IP, immunoprecipitation; RTK, receptor tyrosine kinase; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; STAM, signal-transducing adaptor molecule; PLCγ, phospholipase C γ; HA, hemagglutinin; DIV, day in vitro; pTrkB, activated, tyrosine phosphorylated TrkB. 1The abbreviations used are: SILAC, stable isotope labeling by amino acids in cell culture; BDNF, brain-derived neurotrophic factor; IP, immunoprecipitation; RTK, receptor tyrosine kinase; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; STAM, signal-transducing adaptor molecule; PLCγ, phospholipase C γ; HA, hemagglutinin; DIV, day in vitro; pTrkB, activated, tyrosine phosphorylated TrkB. can be an effective means for characterization of cellular signaling and protein-protein interactions (5Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4475) Google Scholar, 6Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Crossref PubMed Scopus (600) Google Scholar, 7Mann M. Functional and quantitative proteomics using SILAC.Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958Crossref PubMed Scopus (744) Google Scholar). In a SILAC experiment, cells representing different biological conditions are grown in media supplemented with either “light” or “heavy” isotope-containing amino acids. Metabolic incorporation of labeled amino acids into all proteins from cells of one population and subsequent combination of differentially labeled samples in equal ratios enables quantification of proteins from each sample based on the intensities of the corresponding differentially labeled peptides. In the same mass spectrometric experiment, MS/MS can be carried out to obtain sequence information for protein identification (for a review, see Ref. 7Mann M. Functional and quantitative proteomics using SILAC.Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958Crossref PubMed Scopus (744) Google Scholar). The advantages of combining primary neuronal culture and SILAC (primary neuronal SILAC) are significant because this approach allows the expansion to a proteomics scale of established biochemical and cell biological experiments that are frequently used to address neuroscience problems. In a “standard” SILAC experiment, labeling of cells over five or more cell divisions ensures that even very stable proteins achieve nearly complete (97% or more) incorporation of labeled amino acids (8Ong S.E. Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).Nat. Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (660) Google Scholar, 9Schmidt F. Strozynski M. Salus S.S. Nilsen H. Thiede B. Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC).Rapid Commun. Mass Spectrom. 2007; 21: 3919-3926Crossref PubMed Scopus (27) Google Scholar). To assure that SILAC could be applied to non-dividing neurons, which do not have the advantage of label dilution effects enjoyed by dividing cells, we tracked the levels of label incorporation for individual proteins and showed that the majority of proteins do indeed achieve significant levels of stable isotope labeling. Here we demonstrate the first application of this approach, and use it to assess BDNF-dependent phosphotyrosine-associated signaling events in dissociated neuronal cultures prepared from embryonic rat cortex and hippocampus. BDNF is a member of the neurotrophin family of protein growth factors. These proteins function as survival factors that ensure a match between surviving neurons and appropriate targets during development of the mammalian nervous system (10Huang E.J. Reichardt L.F. Neurotrophins: roles in neuronal development and function.Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3208) Google Scholar). They also regulate many other aspects of neural function such as cell fate decisions, axon growth, dendrite pruning, long term potentiation, synaptic plasticity, and the expression of proteins crucial for normal neuronal function, such as neurotransmitters and ion channels (10Huang E.J. Reichardt L.F. Neurotrophins: roles in neuronal development and function.Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3208) Google Scholar, 11Reichardt L.F. Neurotrophin-regulated signalling pathways.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1545-1564Crossref PubMed Scopus (1538) Google Scholar, 12McAllister A.K. Katz L.C. Lo D.C. Neurotrophins and synaptic plasticity.Annu. Rev. Neurosci. 1999; 22: 295-318Crossref PubMed Scopus (1141) Google Scholar, 13Poo M.M. Neurotrophins as synaptic modulators.Nat. Rev. Neurosci. 2001; 2: 24-32Crossref PubMed Scopus (1498) Google Scholar). BDNF-dependent phosphotyrosine-associated signaling events are primarily transmitted through the TrkB receptor tyrosine kinase (14Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors.Annu. Rev. Neurosci. 1995; 18: 223-253Crossref PubMed Scopus (758) Google Scholar). To assess BDNF-dependent phosphotyrosine-associated signaling events, we performed an LC-MS/MS analysis of Tyr(P) IPs from combined lysates of BDNF-treated and control neurons (Fig. 1). These experiments resulted in the identification of 13 proteins that were differentially immunoprecipitated (SILAC ratios above 1.5 or below 0.67) and 18 potential changers (SILAC ratios above 1.25 and below 0.80), suggesting BDNF-dependent changes in tyrosine phosphorylation state or association with a tyrosine phosphorylated protein. Proteins with changing abundances included a number of known and potentially novel pathway participants. We validated the SILAC results for six of these proteins with Western blot time course experiments. Two proteins with increasing abundance in Tyr(P) IPs after BDNF stimulation, Hrs and STAM, were further investigated through biochemical and cell biological approaches. The results suggested a role for the Hrs/STAM-mediated RTK degradative sorting pathway in neurons. Preparation of cortical and hippocampal cell cultures has been described previously (15Brewer G.J. Isolation and culture of adult rat hippocampal neurons.J. Neurosci. Methods. 1997; 71: 143-155Crossref PubMed Scopus (363) Google Scholar, 16Pereira D.B. Chao M.V. The tyrosine kinase Fyn determines the localization of TrkB receptors in lipid rafts.J. Neurosci. 2007; 27: 4859-4869Crossref PubMed Scopus (81) Google Scholar) (refer to supplemental experimental procedures section for details). Cells were cultured in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and 0.5 mm l-glutamine (Invitrogen) or an identical medium formulated without l-arginine and l-lysine (Specialty Media, Philipsburg, NJ) supplemented with either heavy 13C isotope-containing amino acids (Cambridge Isotope Laboratories, Andover, MA) or isotopically normal amino acids (Sigma-Aldrich). Culture media were refreshed every 3 days by removing half of the volume present on each plate and replacing it with fresh medium. After culturing cells for 10 days in vitro, BDNF (PeproTech, Rocky Hill, NJ) was added to neuronal cultures at a concentration of 25 ng/ml for 2, 5, 15 (for SILAC experiments), 30, and 45 min, and cells were washed with ice-cold PBS and immediately placed on ice in ice-cold lysis buffer containing 1.0% Nonidet P-40 (Sigma-Aldrich), 150 mm NaCl, 20 mm Tris-HCl, pH 8, 0.2 mm EDTA, 2 mm Na3VO4, 2 mm NaF, and protease for Tyr(P) experiments were either combined with with 20 of of for at or for SILAC experiments were first in a protein on after which were washed with lysis proteins were by in buffer for In a to assess incorporation of stable amino hippocampal neurons were grown in labeling medium for to and cell lysates conditions described were combined with samples were by using were with and were into for Tyr(P) and for of label incorporation in were into and in 25 mm with and The were with 10 in 25 mm and at were with by a with A. M. O. Mann M. Mass spectrometric of proteins PubMed Scopus Google Scholar). were by and in of for sample The from were using of samples from label experiments was performed in the same as described previously using MA) systems to and mass G. T.A. phosphotyrosine proteomics of signaling by stable isotope labeling with amino acids in cell culture Res. 2006; PubMed Scopus Google Scholar). analysis of phosphotyrosine proteome experiments were performed using a system to a mass mass were using Proteins were using Science, and using (refer to supplemental experimental procedures for of labeled and were tracked in the LC-MS/MS and label incorporation for individual proteins was for labeling experiments 1). Tyr(P) SILAC quantification was carried out using the by and Mann an additional to achieve ratios were for proteins in more than one and in experiments a control to the label incorporation of proteins in Tyr(P) experiments were performed in cells grown in labeling medium and with BDNF in the same as described were using for proteins that had with of than of for were for labeling Cell culture, cell and were the same as described proteins were by and to were in pH 20 containing or with the corresponding primary kinase from Hrs C. A. H. Hrs to 2001; PubMed Scopus Google C. A. H. of Hrs of the growth factor 2002; PubMed Scopus Google Scholar) from and and and with Cells were in in PBS for 15 at with in PBS for 2 washed with in normal in for and with the for at in Cells were for min, and with activated, tyrosine phosphorylated TrkB labeling Molecular at Cells were washed with in and using an with a were using and of neurons at were with 0.5 of from and 0.5 of (Invitrogen) in Neurobasal medium for 45 and to media Cells were in medium for with 25 ng/ml BDNF for the and as described proteins from hippocampal neuronal cultures were tracked for of incorporation of stable amino acids for a proteins from cell lysates demonstrated significant that levels of label incorporation by days in (Fig. analysis was to proteins containing at one that could be tracked through of the 10 time 1). These results that proteins from a differentiated, non-dividing cell population such as cultured primary neurons can be labeled by amino acids in time days and proteins a number of label incorporation of protein and and demonstrated that primary neuronal SILAC can be used to such We used SILAC to study the intracellular signaling events by BDNF stimulation in primary cultures of neurons. We performed one neurons and scale experiments neurons in which Tyr(P) IPs of combined lysates from and control neurons were by and by conditions were between to out the of from SILAC labeling. Proteins were using the using and for labeling We proteins in with a of as by the number of the these were Tyr(P) proteins by one sequence in label of the proteins were such as and and were not Proteins by one were also We for proteins with label for of this After for incorporation of label into individual proteins in these non-dividing neurons, 13 proteins demonstrated protein abundance ratios above 1.50 or below and an additional 18 proteins by more than 3 from the of all proteins The of ratios from experiments was between experiments. Proteins abundance in Tyr(P) IPs after BDNF included the known receptor for BDNF, TrkB a of to and other proteins such as Hrs and of and These results demonstrated that primary neuronal SILAC can be a powerful tool for the proteomics study of intracellular signaling in of abundance in Tyr(P) IPs from primary cultures after 15 of BDNF in a To further the SILAC ratios we by proteins in were for Western blot analysis based on of Western and IPs using the as well as Western for previously known and potentially novel BDNF signaling were performed on lysates of neuronal cultures from the same as used for SILAC experiments after with BDNF for 2, 5, or The proteins included the known BDNF signaling TrkB PLCγ, and and novel Hrs, STAM, and in Western analysis performed on Tyr(P) IPs and neuronal cell using Tyr(P) and receptor and tyrosine phosphorylation after stimulation by These results are with that the TrkB receptor and are after BDNF L.F. Neurotrophin-regulated signalling pathways.Philos. 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These also demonstrated increasing levels of tyrosine phosphorylated proteins with equal protein demonstrated by a of receptor (Fig. To of with Tyr(P) Tyr(P) IPs were performed for each time were and the same was used for all an additional the signal for from the was for that demonstrated for this that used an Western blot intensities well with SILAC ratios for all proteins additional for SILAC quantification (Fig. cell lysates with BDNF for and 2, 5, and as described above were used for Hrs IPs by Western to Hrs and These demonstrated a BDNF-dependent in the between Hrs and (Fig. was as an in for in Hrs IPs after BDNF and as a of the signal from Hrs Western of an Hrs also demonstrated that Hrs is tyrosine phosphorylated BDNF (Fig. experiments using and Hrs Western also showed the of Hrs into a BDNF 1). and showed and Hrs to many of which after 15 of BDNF to in the cell and in neuronal by demonstrated of these proteins with suggesting that and Hrs can in after 15 of BDNF (Fig. of Hrs and and of neuronal cultures demonstrated of Hrs and in after 15 of BDNF can be in the cell and in of of additional demonstrated of Hrs, pTrkB, and for after 15 of BDNF in the of neurons were with a day after cells were for by BDNF for were and for and (Fig. and cells were present on the same with for control and of BDNF after to of receptors in control cells (Fig. with Cells had levels of with that receptor BDNF was by of Here we to the of the first application of SILAC to the characterization of cell signaling in non-dividing primary cell cultures such as the primary neurons we used in The primary neuronal cellular model system has been used for in neuroscience and is with a number of established experimental The use of these in combination with can a of cellular function than has advantages over previously described cell SILAC experiments in neuronal cellular it a system that more closely the in vivo intracellular which is a factor in signal has been that many through biological be to of or to localization of signaling proteins in specific intracellular or to a specific combination of by receptor and Full Text Full Text PDF PubMed Scopus Google Scholar, the kinase pathway in 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, K. 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H. in using stable to individual protein PubMed Scopus Google Scholar). an approach could be for to the of neuronal function, such as protein at We used primary neuronal SILAC to study neuronal signal from BDNF stimulation of dissociated cultured neurons. After stimulation with BDNF, 13 proteins had SILAC ratios of than 1.50 or than and an additional 18 proteins had ratios that 1.25 or than in Tyr(P) IPs a number of novel in BDNF this of proteins were known of the BDNF pathway such as TrkB (14Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors.Annu. Rev. Neurosci. 1995; 18: 223-253Crossref PubMed Scopus (758) Google PLCγ, (10Huang E.J. Reichardt L.F. Neurotrophins: roles in neuronal development and function.Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3208) Google Scholar, 11Reichardt L.F. Neurotrophin-regulated signalling pathways.Philos. Trans. R. Soc. Lond. B Biol. 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Biol. PubMed Scopus Google Scholar, S. S. G. of signaling Biol. Cell. 2003; PubMed Scopus Google Scholar). To role for this degradative pathway has been in neurons, and results that TrkB be in a The SILAC ratios for Hrs, STAM, and heavy in Tyr(P) IPs after BDNF stimulation and are with tyrosine phosphorylation of one or more of these proteins in response to BDNF Hrs and to for each other in a BDNF-dependent as demonstrated by and Hrs is tyrosine phosphorylated in response to BDNF (Fig. was with Hrs after BDNF (Fig. this can be in suggesting that of and Hrs is in (Fig. Hrs function in a to Hrs A.C. M. role of Hrs in a functional of cell 24: PubMed Scopus Google Scholar, B. growth factor-regulated tyrosine kinase substrate trafficking of receptor 2 and Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. of mammalian proteins in trafficking of a receptor to Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Cells had levels of and receptor BDNF suggesting that of Hrs has a on (Fig. TrkB receptors have been to to a pathway in to which is to the cell after stimulation, and these trafficking have been to biological A. M. A novel signal biological of neurotrophin Biol. Cell. PubMed Scopus Google Scholar). be to role the in this of family We a for a more assessment of intracellular trafficking of TrkB in neuronal We have demonstrated that it is to label with a differentiated cell population such as primary neurons with stable isotope-containing amino acids. The of primary neurons such as development and long term survival in to the labeling and other cells with have potential for use in SILAC experiments. We used primary neuronal SILAC to study signal of BDNF stimulation of dissociated cultured neurons. Proteins changing SILAC ratios in Tyr(P) after BDNF stimulation include a number of known and novel of the TrkB The role of of these proteins in the of the receptor to localization after is of We primary neuronal SILAC a powerful tool for the assessment of neuronal function and cellular on a proteomics scale and in neuroscience