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Biochimica et Biophysica Acta
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Secretome profiling of differentiated neural mes-c-myc A1 cell line endowed withstem cell properties☆
Valeria Severino a ,1, Annarita Farina b,1, Luca Colucci-D'Amato a, Mafalda Giovanna Reccia a,Floriana Volpicelli c,d, Augusto Parente a, Angela Chambery a,⁎a Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, I-81100 Caserta, Italyb Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Geneva University, CH-1211 Geneva, Switzerlandc Department of Experimental Pharmacology, University of Naples “Federico II”, I-80131 Napoli, Italyd CNR, Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, I-80131 Napoli, Italy
☆ This article is part of a Special Issue entitled: An Up⁎ Corresponding author. Tel.: +39 0823 274535; fax:
E-mail address: [email protected] (A. Cham1 These Authors contributed equally to this work.
Neural stem cell proliferation and differentiation play a crucial role in the formation and wiring of neuronalconnections forming neuronal circuits. During neural tissues development, a large diversity of neuronalphenotypes is produced from neural precursor cells. In recent years, the cellular andmolecular mechanismsby which specific types of neurons are generated have been explored with the aim to elucidate the complexevents leading to the generation of different phenotypes via distinctive developmental programs that con-trol self-renewal, differentiation, and plasticity. The extracellular environment is thought to provide in-structive influences that actively induce the production of specific neuronal phenotypes.In this work, the secretome profiling of differentiated neural mes-c-myc A1 (A1) cell line endowed with stemcell properties was analyzed by applying a shotgun LC–MS/MS approach. The results provide a list of secretedmolecules with potential relevance for the functional and biological features characterizing the A1 neuronalphenotype. Proteins involved in biological processes closely related to nervous system development includ-ing neurites growth, differentiation of neurons and axonogenesis were identified. Among them, proteins be-longing to extracellular matrix and cell-adhesion complexes as well as soluble factors with well establishedneurotrophic properties were detected. The presented work provides the basis to clarify the complex extra-cellular protein networks implicated in neuronal differentiation and in the acquisition of the neuronal phe-notype. This article is part of a Special Issue entitled: An Updated Secretome.
Proliferation and differentiation of neural stem cells (NSCs) andprecursors are processes essential for the development of the nervoussystem and the maintenance of its adult functions.
It is now well-established that, besides embryonic brain, NSCs arepresent also in selective areas of the adult nervous systemwhere theyare contained within specialized structures called niches, namely thesubventricular zone of the lateral ventricle wall and the subgranularzone of the dentate gyrus the hippocampus [1]. Interactions betweenstem cells and other cells present within the niches and with extra-cellular matrix (ECM) play a pivotal role in regulating the fate ofstem cells including proliferation, migration and differentiation [1,2].
It is worth mentioning that in many pathological conditions such asstroke and inflammation, stem cells and/or precursors are recruitedfrom their niches, migrate toward the diseased areas and differentiate.
dated Secretome.+39 0823 274571.bery).
sevier B.V.
cretomeprofiling of differenti.org/10.1016/j.bbapap.2012.1
This process indicates that environmental changes are able to elicit pro-found effects on stem cells fate and biological functions [3].
Therefore, the study of molecular cues underlying NSCs and/or neu-ral precursor differentiation is important to unveil molecular mecha-nism(s) responsible for their physiological functions and may alsohelp to modify their biological properties for therapeutic goals.
Neural cells secrete proteins that play pivotal functions in the ner-vous system. Besides proteins secreted through classical secretorypathways via endoplasmic reticulum (ER)/Golgi-dependent routesand alternative vesicular transport systems, soluble factors are alsoreleased upon cleavage from cell surface of transmembrane proteinectodomains. In either case, secreted proteins modify the extracellu-lar environment thus affecting the interaction between neural cellsand their substrates and eventually functions such as differentiation,motility and neurite outgrowth [4,5].
In vitro studies of neural cells, including stem cells, precursors andneurons have provided important information about molecules in-volved in proliferation and differentiation.
In this study, we investigated the secretome profile of the differ-entiated neural cell line mes-c-myc A1 (A1) endowed with stemcell properties by applying a shotgun LC–MS/MS-based approach.
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A1 cell line, obtained frommesencephalic primary cultures generatedfrom 11-day-old mouse embryos, has been widely used as a model sys-tem of neurogenesis and has proven to be a powerful tool to study neuraldifferentiation [6–8]. A1 cells can be cultured under undifferentiated/proliferative or differentiated/non-proliferative conditions [6]. In thepresence of serum, these cells appear undifferentiated and proliferate,whereas cAMP stimulation and/or serum deprivation cause cell cyclearrest and neuronal differentiation, with the acquisition of neuronal elec-trophysiological properties and the expression of neuronal markers [6].Differentiated A1 cells present numerous neuronal features, such asperipherin, neuron-specific enolase, microtubule-associated protein 1and neural cell adhesion molecule [6,9]. In addition, morphologically dif-ferentiated A1 cells show functional voltage-gated channels, a neuronalhallmark. Finally, these cells synthesize and accumulate γ-aminobutyricacid (GABA), the principal inhibitory neurotransmitter in CNS [6].
Therefore, deciphering the extracellular components released bydifferentiated A1 cell line may deepen our understanding on solublefactors relevant for neural phenotype.
2. Materials and methods
2.1. A1 cell cultures and differentiation
Mouse immortalized A1 cell line was obtained from mouse embry-onic mesencephalon primary culture as previously described [6]. A1cells were cultured and differentiated as previously reported [6]. Briefly,A1 cells were cultured in MEM/F12 (Gibco-BRL, Milan, Italy)supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad,CA, USA) andwere differentiated by serumwithdrawal and stimulationwith 1 mM dibutyryl cyclic adenosine 3′,5′-monophosphate (cAMP,Sigma, Milan, Italy) and N2 supplement (Gibco-BRL). All tissue culturereagents were from Invitrogen. The time interval chosen for secretomeanalysis was between days 3–6, when morphological and physiologicalevents underlying neuronal differentiation (i.e. neurite outgrowth andneuronal electrophysiological properties) are evident (day 3) andmarkedly displayed (day 6) [6]. Thus, collected secretome samplescontained proteins released between days 3 and 6. Beforemedia collec-tion, cells were starved for 16 h by removing cAMP and N2 and A1 cellswere extensively washedwith PBS 1× as previously described [10]. TheA1 differentiated cells were generated from three independent culturepreparations and then pooled to address biological variation. Further-more, data validation by quantitative real-time PCR (QRT-PCR) wasperformed on additional, independent, biological replicates.
2.2. Cell viability assay
The number of living and dead cellswas determined by cell countingafter trypan blue staining, as previously described [11]. Briefly, A1 cellswere dissociated with trypsin solution 10× (Sigma) and the resultingcell suspension was diluted 1:5 in 0.4% trypan blue solution, incubatedfor 2 min and counted using a Burker chamber. Cell viability was deter-mined on the basis of the total cell count, the dilution factor and thetrypan blue dye exclusion.
2.3. A1 neurosphere cell culture
To generate neurospheres, A1 cells were counted and cultured insuspension (2.5×105/mL) in 25 cm2
flasks in MEM/F12 and with N2supplement in the presence of bFGF (20 ng/mL, R&D System, Milan,Italy) and EGF (25 ng/mL, Sigma) and formed after 4–5 days.
2.4. RNA isolation and quantitative real-time PCR (QRT-PCR)
Total RNA was isolated from A1 cells using Tri-Reagent (Sigma)according to the manufacturer's instructions. The yield and integrityof RNA were determined by spectrophotometric measurements of
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
absorbance at 260 nm and agarose gel electrophoresis, respectively.Then, RNA (2 μg) was reverse transcribed, using random hexa-nucleotides (6 mM, New England Biolabs, Milan, Italy) and 200 U ofMoloney-murine leukemia virus reverse transcriptase (Ambion, LifeTechnologies, Milan, Italy). Gene specific primer sets used forQRT-PCR (Applied Biosystem, Milan, Italy) were designed using OLIGO6 software (Molecular Biology Insights Inc., Cascade, CO, USA), inorder to obtain amplified fragments with comparable length (around80–120 bps). SYBR Green QRT-PCR reactions were performed in96-well plates using 7900HT Fast Real-Time PCR System (AppliedBiosystem).
Thermal cycling conditions comprised initial steps at 50 °C for2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for15 sec and 60 °C for 1 min. All samples were run in triplicate. Ampli-fication efficiency of each primer pair was verified by performingQRT-PCR using different template dilutions. Gene expression levelswere quantified from QRT-PCR data by the comparative thresholdcycle (CT) method using Hypoxanthine phosphoribosyl transferase(Hprt) as internal control gene.
The fractional number of PCR cycles CT required to obtain a givenamount of QRT-PCR product in the exponential phase of amplificationwas determined for the gene of interest and for Hprt in each RNAsample.
The relative expression level of the gene of interest was thenexpressed as 2−ΔCT where ΔCT=CT gene of interest - CT Hprt. Theprimers used for QRT-PCR are reported in Supplementary Table 1 ofSupporting information. All the statistical analyses were performedusing GraphPad Prism 3.0 (GraphPad Software Inc., La Jolla, CA, USA).Control cultures were compared to treated cultures by un-pairedStudent's t-test; data were expressed as mean±SE.
2.5. Sample preparation and tryptic digestion
Conditioned media collected from A1D were lyophilized andresuspended in 1 mL of 50 mM NH4HCO3. Proteins were then precipi-tated with 20% trichloroacetic acid (TCA) for 30 min on ice andcentrifuged for 15 min at 17530 g [10,11]. Pellets washed with diethylether and acetone, were air dried at room temperature, resuspendedin 50 mMNH4HCO3, pooled and thoroughly sonicated into an ultrason-ic bath for 10–15 min. Proteins were reduced with 2.5 mM DTT (finalconcentration) at 60 °C for 30 min and carbamidomethylated with7.5 mM iodoacetamide (final concentration) at room temperature inthe dark for 30 min. Enzymatic hydrolysis was performed by the addi-tion of 10 μL of Tosyl Phenylalanyl Chloromethyl Ketone (TPCK)-treatedtrypsin solution (5 ng/μL) to the reduced and alkylated mixture. Diges-tion was performed by incubation at 37 °C for 16 h, followed by a sec-ond addition of 10 μL of trypsin incubated at 37 °C for 3 h. Afterdigestion, sample was dried under vacuum in a SpeedVac Vacuum (Sa-vant Instruments, Holbrook, NY, USA), resuspended in 20 μL of 0.1%formic acid (FA) and centrifuged at 17530 g for 15 min. Aliquots ofthe supernatant (6 μL) were analyzed by ESI LTQ-OT MS [10].
2.6. LC–MS configurations
ESI LTQ-OTMSwas performed on a LTQOrbitrap velos from ThermoElectron (San Jose, CA, USA) equippedwith a NanoAcquity system fromWaters (Waters Corporation, Manchester, UK). Peptides were trappedon a home-made 5 μm 200 Å Magic C18 AQ (Michrom, Auburn, CA,USA) 0.1×20 mm pre-column and separated on a home-made 5 μm100 Å Magic C18 AQ (Michrom) 0.75×150 mm column with agravity-pulled emitter. The analytical separation was run for 65 min,using a gradient of H2O/FA 99.9%/0.1% (solvent A) and CH3CN/FA99.9%/0.1% (solvent B), at a flow rate of 220 nL/min as follows: 5% Bfor 1 min, from 5 to 35% B in 54 min, from 35 to 80% B in 10 min. ForMS survey scans, the Orbitrap resolution was set to 60000 and the ionpopulation was set to 5×105 with an m/z window from 400 to 2000.
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Five precursor ions were selected for collision-induced dissociation(CID) in the LTQ. For this, the ion populationwas set to 7×103 (isolationwidth of 2 m/z). The normalized collision energies were set to 35% forCID.
2.7. Data processing and protein identification
Peak lists were generated from raw data using ReAdW4.2.1 (http://sourceforge.net). The monoisotopic masses of the selected precursorionswere corrected using an in-house scriptmodified froma previouslypublished report [12]. Briefly, the precursor ion m/z ratio was deter-mined using SuperHirn 1.0 [13]. Then, miss-assigned precursor ionsfrom the instrument control software were corrected using this value.In cases of ambiguity, all precursor m/z values were used for databasesearching using the Mascot software (Matrix Science, London, UK; ver-sion 2.2.0). The corrected peaklist files were searched against theuniprot_sprot database release 2011_09 (21-Sep-2011, selected forMus musculus, 16394 entries) assuming trypsin as digestion enzyme.Mascot was searched with a fragment ion mass tolerance of 0.60 Daand a parent ion tolerance of 25 ppm. Iodoacetamide derivative of cys-teine was specified in Mascot as a fixed modification. Oxidation of me-thionine was specified in Mascot as a variable modification.
Scaffold software (version 3.5.1, Proteome Software Inc., Portland,OR) was used to validate MS/MS based peptide and protein identifica-tions. Peptide identifications were accepted if they could be establishedat greater than 90 %probability as specifiedby the Peptide Prophet algo-rithm [14]. Protein identifications were accepted if they could beestablished at greater than or equal to 90 % probability in at least one in-jection and contained at least 2 identified peptides in at least two injec-tions. For proteins identified in a single injection, a further constrainwas applied by considering as reliable only proteins identified with anumber of peptides greater than or equal to 3. Protein probabilitieswere assigned by the Protein Prophet algorithm [15]. Proteins thatcontained similar peptides and could not be differentiated based onMS/MS analysis alone were grouped to satisfy the principles of
Fig. 1. Phase-contrast photomicrographs (20×) showing A1-derived neurospheres (A) and Arefractive cell bodies can be observed. Morphological changes are paralleled by the appearanlevels of typical stemness markers (Nestin, Nanog and Sox2) were analyzed in A1 neurospheboth NS and differentiated A1 (A1D), **p≤0.01.
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentiBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
parsimony. For the MS/MS validation, the false discovery rate (FDR)was evaluated by Scaffold software, yielding a calculated FDR value of0.1%.
2.8. Bioinformatics analyses of identified proteins
The pathway analysis was performed using the Ingenuity PathwaysAnalysis (IPA) software (Ingenuity Systems Inc., Redwood City, CA,USA; www.ingenuity.com). The list of A1D secreted proteins wasimported into the IPA platform for batch analysis and identification ofthe canonical pathways as previously reported [10,16].
Proteins with a predicted N-terminal signal sequence were identi-fied by using SignalP 4.0 [17], available at http://www.cbs.dtu.dk/services/SignalP/ (set for eukaryotes using both neural networks andhidden Markov models), and were considered to be secreted via a clas-sical pathway (endoplasmic reticulum/Golgi-dependent pathway).Identified proteinswere also analyzed for secretion pathways accordingto SecretomeP 2.0 Server [18] (http://www.cbs.dtu.dk/services/SecretomeP/). If the neural network exceeded or was equal to a valueof 0.5 (NN-score≥0.50), but no signal peptide was predicted, proteinswere considered potentially secreted via a non-classical pathway.
2.9. Effect of A1 conditioned media on neural differentiation
For the preparation of the A1 conditioned media (CM), cells werecultured and differentiated as described in Section 2.1. In this instance,cAMP, endowed with potent pro-differentiative properties, was notused for differentiation in order to avoid to mask the effect of A1 CMon neural differentiation. Indeed, previous work has shown that A1cell morphological differentiation and cell growth arrest can beachieved also by removing cAMP from the differentiation medium[6,7]. At first, conditioned media at day 3 (CM3) or day 6 (CM6) aswell as control MEM/F12 were collected during a standard differentia-tion process performed by serum withdrawal and stimulation with N2supplement (Gibco-BRL). Then, CM were lyophilized with LIO 5P
1 differentiated cells (B). Under differentiated conditions, neurites sprouting from highce of functional and molecular features of differentiated neurons [6]. mRNA expressionres (NS); mRNA expression of neuronal markers (TuJ1 and L1cam) was also analyzed in
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Table 1Proteins identified in the A1D secretome by high resolution LC–MS/MS. The Scaffold Software was used to improve protein identification by filtering data following the acceptancecriteria of probability greater than 90.0%, as specified by the Protein-Prophet algorithm [1]. The higher number of unique peptides for each protein identification is reported. Mr,Relative molecular mass; Pr, Protein identification probability.
Accession Protein name Pr (%) Mr (kDa) # Peptide Secretome Pa Signal Pb
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Please cite this article as: V. Severino, et al., Secretomeprofiling of differentiated neuralmes-c-mycA1 cell line endowedwith stem cell properties,Biochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.12.005
a Secretion prediction according to Secretome P 2.0 server. Proteins with NN-score≥0.5 are predicted as secreted by non-classical secretory pathways.b Secretion prediction according to Signal P 4.0 server. Numbers in parenthesis indicate the signal peptide probability.
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lyophilizer (VWR International, PBI, Milan, Italy), resuspended in freshMEM/F12 supplemented with N2. To investigate the effect of A1 CMon neural differentiation, CM3 and CM6 were added to theundifferentiated/proliferating A1 at day 0 of differentiation (DIV0). A1differentiation was also carried out under standard conditions byadding, at DIV0, MEM/F12 supplemented with N2 previously subjectedto the same preparation procedure described for CM samples. After day3 (DIV3) and day 6 (DIV6) of differentiation, RNAwas isolated from cellcultures for QRT-PCR analyses as described in Section 2.3. QRT-PCRanalyses were also performed on RNA isolated at day 3 (CTRL3) andday 6 (CTRL6) from A1 cell cultures differentiated under standardconditions.
3. Results
A1 cells represent a pluripotent neural cell line useful to study themolecular mechanisms underlying CNS differentiation, plasticity, aswell as the neural cell-fate determination and neurotransmission. In-deed, A1 cells have properties of neural progenitors since morphologi-cally differentiated and proliferating A1 cells express markers ofneural precursors (e.g. Vimentin and Nestin) [6].
To ascertain whether A1 cells were endowed with neural stem cellpotential, we investigate their ability to form neurospheres. To thisaim, cells where cultured in suspension in a serum free medium withthe addition of EGF and bFGF. Under these growing culture conditions,A1 cells were able to form neurospheres (Fig. 1A). Hence, the expres-sion of typical markers of stemness in A1-derived neurospheres (NS)was examined by QRT–PCR. The expression of known neural stem cellmarkers (i.e. Nestin, Nanog and Sox2) was revealed in NS (Fig. 1A).Moreover, an increase of the expression of the neuronal markersNeuron-specific class III beta-tubulin (TuJ1) and Neural cell adhesionmolecule L1 (L1cam) was revealed in differentiated A1 cells (A1D,Fig. 1B) with respect to NS accordingly with their restricted differentia-tion capacity for neuronal lineages.
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentiBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
The differentiation and morphogenesis of neural tissues involve aplethora of interactions between neural cells and their extracellularenvironment. Signals arising from ECM or cell surfaces and solublefactors released from neighbour cells play instructive and/or permis-sive roles for cell-fate determination [19]. However, the signaling cas-cades triggered by extracellular factors are not fully elucidated. The invitro culturing of CNS stem cells and stem cell lines offers a useful toolfor the study of neural differentiation.
In this context, the secretome analysis of the A1D cell line wasperformed with the aim to identify a hallmark of neuronal phenotype.To this aim, A1 cells were induced to differentiate under establishedconditions promoting neural differentiation as described in theMaterials and methods section. In particular, the conditioned mediasamples were collected between days 3 and 6 of differentiation whenthe appearance of morphological and physiological events underlyingneuronal differentiation (i.e. neurite outgrowth and neuronal electro-physiological properties) are evident (day 3) and markedly displayed(day 6)within A1 cell cultures [6]. To rule out the possibility that differ-entiation factors might affect the secretome analysis, A1D cells werestarved overnight (16 h) by removing cAMP and N2 before media col-lection. Cell viability assays performed on unstarved and starved cellsrevealed that starvation did not affect cell viability, thus minimizingthe possibility of potential contamination of the conditioned media byintracellular proteins occurring following cell lysis (data not shown).
The secretome protein profiling of A1D cells was performed by ap-plying a shotgun proteomics approach. A1D conditioned media wereprocessed for protein precipitation by TCA and proteins were subjectedto tryptic digestion and analyzed by LC–MS/MS. Using high resolutionmass spectrometry and Mascot database search, 222 proteins wereidentified in the A1D secretome. Datawere filtered by using the Scaffoldsoftware on the basis of probability of protein identification and techni-cal reproducibility, allowing the selection of 104 non-redundant pro-teins. The list of proteins identified in the A1D cell line secretome isreported in Table 1 and Supplementary Table 2 of Supporting informa-tion. In eukaryotic cells, proteins are actively secreted in the
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Fig. 2. A)Bar chart of the significantly enrichedbiological functions of proteins secreted byA1D cell line; the black line indicates thep-values reported inB. B) Biological functions, p-valuesand molecules mapped on the enriched categories. Molecules are named according to IPA software classification as follows: App, Amyloid beta A4 protein; CSRP1, Cysteine andglycine-rich protein 1; DPYSL2, Dihydropyrimidinase-related protein 2; EZR, Ezrin; FN1, Fibronectin; HMGB1, High mobility group protein B1; MYH9, Myosin-9; NCAM1, Neural cell ad-hesion molecule 1; PDIA3, Protein disulfide-isomerase A3; S100A4, Protein S100-A4; SEMA7A, Semaphorin-7; SERPINE2, Glia-derived nexin; TNC, Tenascin; VIM, vimentin; YWHAZ,14-3-3 protein zeta/delta; ACTB, Actin, cytoplasmic 1; ACTR3, Actin-related protein 3; CLU, Clusterin; MSN, Moesin; PFN1, Profilin-1; RDX, Radixin; TNC, Tenascin; UCHL1, Ubiquitincarboxyl-terminal hydrolase isozyme L1; CFL1, Cofilin-1; PPIA, Peptidyl-prolyl cis-trans isomerase A; SERPINF1, Pigment epithelium-derived factor; YWHAG, 14-3-3 protein gamma;SOD1, superoxide dismutase [Cu–Zn]; ANXA2, Annexin A2; MIF, Macrophage migration inhibitory factor; CST3, Cystatin-C; YWHAE, 14-3-3 protein epsilon.
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extracellular space through a well characterized secretory pathway viaan ER/Golgi-dependent route [20]. In addition, several non-classical se-cretion pathways have been described, extending the definition of cellsecretome to proteins deriving from the ECM or shed from the cell sur-face, as well as to proteins released through vesicles [21]. Therefore, theidentified proteinswere analyzed for classical (i.e. signal peptide-drivensecretion) and non-classical secretion pathways by using the SignalPand SecretomeP prediction servers (Table 1). These analyses revealedthat approximately 49% (51/104) of the identified proteins were pre-dicted to be secreted. Among them, twenty three proteins (23/51,~45%) were predicted to have a signal peptide for secretion throughthe ER/Golgi pathway. On the other hand, 28 proteins (28/51, ~55%)were released from cells by non-classical secretion mechanisms(NN-score≥0.5). In addition, significantly enriched categories for iden-tified proteins were related to biological functions using the IPA soft-ware. As expected, a significant number of proteins was found to beinvolved in nervous system development and function processes(Fig. 2), being the growth and outgrowth of neurites, the neuritogenesisand neurons differentiation and morphogenesis the most representa-tive categories.
Based on these findings, we decided to investigate the temporal ex-pression profiles of a set of proteins known to be involved in neuron dif-ferentiation processes and/or neurite outgrowth by QRT-PCR (see
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
Discussion). To this aim, specific primers were designed for Amyloidbeta A4 protein (App), Calsyntenin-1 (Clstn1), Glia-derived nexin(Serpine2), Clusterin (Clu), Neural cell adhesion molecule 1 (Ncam1)and Pigment epithelium-derived factor (Serpinf1). The presence of allselected proteins was revealed at both days 3 and 6 of A1 cell differen-tiation with no significant differential expression levels at the two timepoints of neural differentiation (Fig. 3).
In order to investigate the potential involvement of the identifiedsecreted proteins in the activation of known molecular pathways andalso to point out the mutual interactions of identified proteins, a net-work map was constructed in silico using the IPA software (Fig. 4). Aspecific subset of secreted proteins was mapped on a network con-verging, at intracellular level, on core molecules previously involvedin neuronal differentiation including extracellular-signal-regulatedkinases ERK1/2 and Rho Kinase (ROCK) [22,23]. Additional networknodes were found to include cytoplasmic partners of adhesion mole-cules and cell–matrix adhesion complexes (i.e. alpha catenin andfocal adhesion kinase, FAK) that mediate the ECM regulatory effectson cell behavior through the control of the actin cytoskeleton [24].
It has been reported that soluble factors secreted by terminally orpartially differentiated CNS cells are able to promote and/or enhanceneural stem cell differentiation [25,26]. We therefore investigated theeffect of A1 conditioned media on neural differentiation process by
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Fig. 3. mRNA expression levels of Amyloid beta A4 protein (A, App), Calsyntenin-1 (B, Clstn1), Glia-derived nexin (C, Serpine2), Clusterin (D, Clu), Neural cell adhesion molecule 1(E, Ncam1) and Pigment epithelium-derived factor (F, Serpinf1) were analyzed in A1 cells differentiated at days 3 and 6 by QRT-PCR.
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adding the conditioned media collected at day 3 (CM3) or day 6 (CM6)of differentiation to the undifferentiated/proliferating A1 cells (Fig. 5).After day 3 and day 6 of differentiation, cell cultures were subjected toQRT-PCR for the detection of Nestin, Neurofilament L (NFL-L) andTuJ1 (Fig. 6). Following the addition of A1 cells CM6, a significant in-crease of mRNA expression levels of Tuj1 and NFL-L was revealed atday 3 of differentiation (DIV3/CM6) with respect to controls attestingan enhancement of neural differentiation. This effect was not observedat day 6 likely because the cells, at this timepoint, were already fully dif-ferentiated. Furthermore, a reduction of Nestin expression, a marker ofneural precursors, was observed at day 6 of differentiation by treatingcultures with CM6. This finding is in agreement with an enhanced pro-gression from the A1 undifferentiated phenotype toward the acquisi-tion of a neuronal fate.
4. Discussion
The differentiation andmorphogenesis of neural tissues is finely reg-ulated by serial events mediated by highly coordinated signaling path-ways whose deregulation may lead to the onset of neurologicaldiseases. Most of these intracellular signaling cascades are activated
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by the binding of extracellular signals to their cognate receptors. There-fore, it is increasingly clear that a key role in nervous system develop-ment and neurite outgrowth is played by secreted molecules either byneurons and supporting glial cells [5]. Indeed, both stimulatory and in-hibitory extracellular stimuli affect the initiation and guidance ofneurites exerting positive (permissive or attractive), negative (inhibito-ry or repulsive), or guiding (affecting the advance of the growth cone)actions on developing neural tissues [19].
Intensive research activities have been focused on the under-standing of cell signaling pathways regulating embryonic stem cellsand neural stem cell differentiation.
Although new insights atmolecular level enhanced our understand-ing of neural differentiation andmaturation, the role played by extracel-lular proteins on neuron survival, differentiation and proliferationremains elusive.
In this work, the secretome profiling of differentiated neuralmes-c-myc A1 cell line endowed with stem cell properties was ana-lyzed by applying a shotgun LC–MS/MS approach with the aim toidentify secreted proteins potentially characterizing the neuronalphenotype. The ability of the A1 cells to form neurospheres as wellas the presence of typical stemness markers was at first ascertained.
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Fig. 4. Protein interaction network including proteins secreted by A1D cells. The core molecules of the network were found to converge on the ERK1/2 signaling pathway. Proteinsidentified in the A1D secretome with intracellular or extracellular localization (according to IPA categorization) are coloured in blue and green, respectively. MIF and GPI proteinswere categorized as secreted according to Secretome P (NN-score≥0.5). In violet are reported identified proteins with transmembrane domains. The pathway nodes identified bythe algorithm and not detected in the analysis are reported in yellow. Molecules are named according to IPA software as follows: CLSTN1, Calsyntenin-1; FBLN1, Fibulin-1; CNBP,Cellular nucleic acid-binding protein; BGN, Biglycan; FSTL1, Follistatin-related protein 1; GPI, Glucose-6-phosphate isomerase (Neuroleukin); HNRNPAB, Heterogeneous nuclearribonucleoprotein A/B; GDNF, Glial cell line-derived neurotrophic factor; HSPG2, Basement membrane-specific HSPG; TPM3, Tropomyosin alpha-3 chain; PARK, Protein DJ-1;LDH, L-lactate dehydrogenase A chain. Other protein names are reported in the legend of Fig. 2.
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Then, for the secretome analysis, A1 cell differentiation toward aneuronal fate was induced. Under these conditions, morphologicallydifferentiated A1 cells acquire typical physiological properties (i.e.neurite outgrowth and neuronal electrophysiological properties) ofdifferentiated neurons. Accordingly, the A1D secretome analysisprovides a list of candidates potentially associated to the acquisitionof the neuronal phenotype. Proteins involved in biological processesclosely related to nervous system development including growth,outgrowth and morphogenesis of neurites, differentiation of neu-rons and axonogenesis were identified. Among them, proteins in-volved in cell adhesion and ECM-interaction were detected. Anemerging role of ECM components and cell adhesion molecules(CAMs) as regulators of neurite outgrowth has been proposed[19,27–29]. Indeed, besides their involvement in themechanical reg-ulation of cell–cell and cell–ECM adhesion, CAMs also trigger intra-cellular signaling cascades inducing neurite outgrowth and neuralcell migration [19,27]. Among proteins identified in the A1Dsecretome, the Neural cell adhesion molecule 1 (N-CAM1) plays acrucial role in neuronal development and synaptic plasticity[19,27,30]. N-CAM1 is a member of the immunoglobulin superfamilyexpressed on the neural cells surface containing five Ig-like domainsand two fibronectin type III repeats. Besides the regulation ofcell adhesion by homophilic binding, additional functional roles
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
have been attributed to N-CAM1 including the activation of intracel-lular signaling pathways, the regulation of cytoskeletal dynamicsand of the growth factor signaling by heterophilic binding to Glialcell line-derived neurotrophic factor (GDNF) [31], Brain-derivedneurotrophic factor (BDNF) [32], and Platelet-derived growth factor(PDGF) [33]. Other permissive cues promoting neurite extension andbelonging to ECMwere identified in the A1D secretome including Fi-bronectin and Tenascin [19,34]. In addition, adhesion-modulatingproteins belonging to the matricellular protein family, including Se-creted protein acidic and rich in cysteine (SPARC) and Galectin-1,were secreted by A1D cells. In particular, Galectins are a taxonomi-cally widespread family of glycan-binding proteins regulating celladhesion, spreading, and migration [35]. It has been reported thatthe interaction between β1 Integrin and Galectin-1 plays an impor-tant role in regulating the number of neural progenitor cells in theadult mouse subependymal zone through mechanisms includingcell adhesion [36].
Furthermore, all the components of the Ezrin–Radixin–Moesin(ERM) complex, a crucial nexus between the extracellular environmentand the cytoskeleton, were identified. ERM proteins are involved in thetransmission of signals from the cellmembrane to intracellular cascadesthrough their ability to bind transmembrane receptors thus assuring anefficient signal transduction on the cytoplasmic face of the plasma
iated neuralmes-c-mycA1 cell line endowedwith stem cell properties,2.005
Fig. 5. Schematic diagram of the experimental workflow applied to evaluate the effect of A1 conditioned media on neural differentiation. A) For the preparation of the A1 condi-tioned media, cells were cultured and differentiated as described in the Materials and methods section. Conditioned media at day 3 (CM3) or day 6 (CM6) as well as controlMEM/F12 were collected during a standard differentiation process, lyophilized and resuspended in fresh MEM/F12 supplemented with N2. B) CM3 and CM6 were added to theundifferentiated A1 at day 0 of differentiation (DIV0). RNA was isolated from cell cultures for QRT-PCR analyses at day 3 (DIV3) and day 6 (DIV6) of differentiation, as well as atday 3 (CTRL3) and day 6 (CTRL6) from A1 cell cultures differentiated under standard conditions.
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membrane [37]. It has been recently reported that the activation of ERMproteins by phosphorylation promotes attractive growth cone guidanceacting asmediators of neurotrophin-induced F-actin remodeling in sen-sory neurons [38].
Several neurotrophic factors were also detected in the A1Dsecretome. Among them, Pigment epithelium-derived factor (PEDF,gene name Serpinf1), a potent neurotrophic and neuroprotectiveprotein, was identified. PEDF regulates processes associated with an-giogenesis, neuronal cell survival and differentiation and also in-duces increased expression of Nerve growth factor (NGF), BDNFand GDNF [39]. Furthermore, both culture and animal studies sup-port the neuroprotective role of PEDF in acute and chronic forms ofneurodegenerative disorders, including Parkinson's disease [39,40].Neuroleukin (NLK), a multifunctional protein involved in neuronalgrowth, glucose metabolism, cell motility and differentiation, wasalso identified as a soluble factor secreted by A1D cells. NLK is 100%identical to Glucose-6-Phosphate Isomerase (GPI) that catalyze the
Fig. 6.mRNA expression levels of Nestin, NFL-L and TuJ1 performed on A1 cell cultures differaccording to the experimental procedure described in Fig. 5. Data are means±SE, (n=3);
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentiBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
interconversion of glucose-6-phosphate to fructose-6-phosphate inthe glycolytic pathway [41]. However, while GPI dimers are respon-sible for the enzymatic activity, the monomer is endowed with theneurotrophic activity by promoting growth of embryonic spinaland sensory neurons [41].
Other proteins promoting neurite growth and axon outgrowthwereidentified, including Glia-derived nexin (GDN, gene name Serpine2)and Semaphorin 7A. Glia-derived nexin is a secreted serine protease in-hibitor with activity toward thrombin, trypsin, and urokinase that isable to promote neurite extension by inhibiting thrombin [42].Semaphorin 7A belongs to the semaphorin family endowed with im-munomodulatory effects. Several semaphorins molecules have beenalso found to have a critical role in neuronal functions, suggesting com-mon cellular signaling mechanisms between immune and nervous sys-tem [43]. While most semaphorins act as repulsive guidance cues,Semaphorin 7A enhances axon outgrowth through integrin receptorsand the activation of MAPK signaling pathways [43].
entiated under standard conditions and following the addition of A1 conditioned media*p≤0.05; **p≤0.01.
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Interestingly, the Amyloid beta A4 Protein (APP) was identified inthe A1D secretome with all peptides identified in the soluble region.APP is a type 1 transmembrane glycoprotein with a large extracellu-lar domain and a small cytoplasmic domain [44]. Although the phys-iological functions of APP are not fully understood, accumulatingevidences suggest that the products of the non-amyloidogenic path-way, deriving from the sequential cleavage of α- and γ-secretases,have a neuroprotective effect and also are able to increase neuriteoutgrowth, neuronal adhesion and axonogenesis [45–47]. Besidesits physiological role, deposition of amyloid-β peptides (Aβ) withinamyloid plaques is implicated in the pathogenesis of Alzheimer's dis-ease (AD) [45]. Thus, the understanding of the APP physiologicalfunction and of its binding partners is of pivotal importance for pro-viding insights leading to improved therapeutic strategies for AD.Calsyntenin-1 and Clusterin, two proteins reported to be involvedin APP/Aβ processing, transport and binding were also detected inthe A1D secretome. Calsyntenin-1 is a type-1 transmembrane pro-tein acting as a cargo-docking protein for Kinesin-1-mediated axonaltransport of endosomal vesicles, thus contributing to axonal growthand pathfinding in the adult nervous system [48,49]. Furthermore, ithas been recently postulated that Calsyntenin-1 mediates axonaltransport of APP and regulates Aβ production opening new perspec-tives for the treatment of AD [48]. Clusterin, also known as apolipo-protein J, is a secreted and highly glycosylated chaperone moleculefrom long time known to be involved in preventing Aβ fibrillization[50]. Recent evidences demonstrated that Clusterin also enhancesneuronal differentiation and neurite outgrowth of neural precursorcells, thus suggesting the potential application of this protein forCNS repair [26].
Overall, the identified proteins from A1D secretome hold greatpromise to further investigate themechanisms of neural differentiation,as well as to provide useful suggestions for clarifying the role of extra-cellular signals in the determination of neuronal phenotype.
5. Conclusions
The characterization of the “secretome” (i.e. the sub-populations of aproteome secreted from the cell) of neural models is one of the mainchallenges of the proteomic approach applied to neuroscience. Thephysiological cellular heterogeneity of neural tissues and primary cul-tures may render difficult the interpretation of proteomic results, ham-pering the correlation of the altered proteins with a given neuronalphenotype. Neuronal cell lines formed by homogeneous populationrepresent a valid complementary alternative and a useful tool to over-come these problems.
In this context, the secretome profiling of differentiated A1,performed by shotgun LC–MS/MS approach, provides a list of candi-dates with potential relevance for the functional and biological featuresof differentiated A1 neural cells.
Several proteins belonging to ECM and cell-adhesion complexeswere identified, supporting their role as potential regulators of neuriteoutgrowth, axogenesis and neural differentiation. In addition, solublefactors with well established neurotrophic properties were alsodetected in the A1D secretome.
The perspectives of the present work will focus on the setting upof functional studies on the identified proteins, with the aim to com-pare their expression levels in proliferating/undifferentiated vs.non-proliferating/differentiated mes-c-myc A1 cell line to better un-derstand their implications in the A1 differentiation process.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbapap.2012.12.005.
Acknowledgements
We thank theGeneva Proteomics Core Facility for technical assistance.
Please cite this article as: V. Severino, et al., Secretomeprofiling of differentBiochim. Biophys. Acta (2012), http://dx.doi.org/10.1016/j.bbapap.2012.1
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