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Vol. 19, Issue 6, 2444-2456, June 2008

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The Signaling Adaptor Protein CD3 Is a Negative Regulator of Dendrite Development in Young Neurons

Stéphane J. Baudouin^*,,, Julie Angibaud^*,,, Gildas Loussouarn^,,||, Virginie Bonnamain^*,,, Akihiro Matsuura^¶, Miyuki Kinebuchi^¶, Philippe Naveilhan^*,,, and Hélène Boudin^*,,

INSERM, *U643 and U915, Nantes, F44000 France; CHU Nantes, Institut de Transplantation et de Recherche en Transplantation, Nantes, F44000 France; Université de Nantes, Faculté de Médecine, Nantes, F44000 France; ^||Centre National de la Recherche Scientifique, ERL3147, F-44000, France; and ^¶Department of Pathology, Fujita Health University School of Medicine, Aichi, 470-1192, Japan

Submitted September 20, 2007; Revised March 17, 2008; Accepted March 19, 2008
Monitoring Editor: Erika Holzbaur

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A novel idea is emergxsing that a large molecular repertoire is common to the nervous and immune systems, which might reflect the existence of novel neuronal functions for immune molecules in the brain. Here, we show that the transmembrane adaptor signaling protein CD3, first described in the immune system, has a previously uncharacterized role in regulating neuronal development. Biochemical and immunohistochemical analyses of the rat brain and cultured neurons showed that CD3 is mainly expressed in neurons. Distribution of CD3 in developing cultured hippocampal neurons, as determined by immunofluorescence, indicates that CD3 is preferentially associated with the somatodendritic compartment as soon as the dendrites initiate their differentiation. At this stage, CD3 was selectively concentrated at dendritic filopodia and growth cones, actin-rich structures involved in neurite growth and patterning. siRNA-mediated knockdown of CD3 in cultured neurons or overexpression of a loss-of-function CD3 mutant lacking the tyrosine phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAMs) increased dendritic arborization. Conversely, activation of endogenous CD3 by a CD3 antibody reduced the size of the dendritic arbor. Altogether, our findings reveal a novel role for CD3 in the nervous system, suggesting its contribution to dendrite development through ITAM-based mechanisms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neuronal communication requires the formation of neuronal circuitry, achieved by a coordinated development of axons and dendrites. The ability of dendrites to receive and process neuronal signals is greatly determined by the dendritic architecture elaborated during development. It is now well established that dendrite formation is regulated by multiple factors that precisely control the extent of dendritic outgrowth and branching. Neuronal activity (Lohmann et al., 2002), diffusible cues such as semaphorins and Slits (Polleux et al., 2000; Whitford et al., 2002), and intracellular signaling molecules (Fink et al., 2003) have all been shown to regulate various steps of dendrite development. Interestingly, a large number of the aforementioned factors are also involved in the differentiation and function of other cell types in the immune system. For example, semaphorins and Slits are required for the maturation, migration, and activation of T lymphocytes and dendritic cells (Wu et al., 2001; Kumanogoh and Kikutani, 2003). This common molecular repertoire shared by different biological systems emphasizes the existence of similar mechanisms underlying fundamental functions in cell differentiation, maturation and activation. Conversely, the expression and the role of several proteins thought to be specific for the immune system have recently been extended to the CNS (Boulanger and Shatz, 2004). This is the case for the CD3 subunit CD3, a transmembrane adaptor signaling protein only characterized to date in T lymphocytes and natural killer (NK) cells, which associates to different cell surface receptors depending on the cell type (Lanier, 2001; Pitcher and van Oers, 2003). In T-cells, CD3 is a component of the CD3 complex, the signaling module of the T-cell receptor (TCR) that recognizes peptide fragments presented by major histocompatibility complex (MHC) molecules and is thus responsible for antigenic recognition (Samelson et al., 1985). CD3 is instrumental in these processes because it is required for receptor-mediated signal transduction through its three copies of immunoreceptor tyrosine-based activation motif (ITAM; Pitcher and van Oers, 2003). Moreover, CD3 also participates in intrathymic T-cell differentiation, which is arrested in mice lacking CD3 (Malissen et al., 1993). Brain analysis of these CD3-deficient mice showed a striking abnormal development of the retinogeniculate projections in the visual system (Huh et al., 2000). Defects in synaptic plasticity recorded in the hippocampus were also observed with an enhanced LTP and a lack of LTD (Huh et al., 2000; Barco et al., 2005). These findings suggest a novel role for CD3 in the development of the nervous system as well as in synaptic functions. However, the cerebral expression of CD3 proteins and the identity of CD3-expressing cells have yet to be elucidated. In this study, we show that CD3 is predominantly expressed in neurons where it selectively localized at dendritic growth cones and filopodia. CD3 loss-of-function experiments in cultured neurons increased dendritic arborization, whereas activating endogenous CD3 inhibited dendritic branching. Altogether, our findings reveal a novel role for CD3 in the nervous system, highlighting its contribution to neuronal development.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
All protocols were carried out in accordance with French standard ethical guidelines for laboratory animals (Agreement 75-669).

Antibodies
Two commercially available anti-CD3 affinity-purified rabbit polyclonal antibodies were used for CD3 immunodetection. The first antibody (Ab36) was raised against aa 36–54, corresponding to an intracellular sequence of the protein (Spring Bioscience, Fremont, CA). The second antibody (Ab22) was raised against aa 22–30, a sequence corresponding to the N-terminus extracellular region of the protein (Alexis Bioscience, San Diego, CA). The mouse monoclonal antibodies to tau (antibody tau-1, clone PC1C6), microtubule-associated protein 2 (MAP2, clone AP20), neuronal nuclei (NeuN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; clone 6C5) were purchased from Chemicon (Billerica, MA). The mouse monoclonal β-tubulin isotype III antibody (Tuj1, clone SDL.3D10) and the mouse monoclonal anti-glial fibrillary acidic protein (GFAP) were obtained from Sigma (St. Louis, MO) and the rabbit polyclonal tau from Dako (Glostrup, Denmark). The mouse monoclonal anti-oligodendrocyte (RIP) was purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) and anti-CD90 (clone OX7) was purchased from Bioatlantic (Nantes, France). The mouse anti-CD11b/c (clone OX42) and anti-CD161 (clone 3.2.3) were produced in our laboratory from hybridomas obtained from the European Collection of Animal Cell Culture (Salisbury, United Kingdom). The mouse mAb anti-CD45 receptor (CD45R, clone HIS24) was purchased from BD Biosciences (San Jose, CA). Peroxidase-conjugated goat anti-rabbit and biotinylated- and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit secondary antibodies were purchased from Jackson Laboratory (Bar Harbor, ME). The aminomethylcoumarin acetate (AMCA)-conjugated streptavidin was obtained from Beckman Coulter (Fullerton, CA) and the Alexa Fluor 568 goat anti-rabbit and goat anti-mouse were obtained from Invitrogen (Carlsbad, CA)

PCR Analysis
Total RNA was extracted from 50 to 100 mg of rat brain and spleen by homogenization in 1 ml of Trizol reagent (Invitrogen, Carlsbad, CA) for 5 min at room temperature followed by chloroform extraction and isopropanol precipitation. After centrifugation and washing in ethanol, RNA was resuspended in water and frozen at –20°C until use. After Turbo DNAse treatment (Invitrogen), 2 µg of RNA were reverse-transcribed with a MMLV-reverse transcriptase system (Invitrogen). The synthesized cDNA was denatured for 2 min at 92°C, and 100 ng was subjected to 35 cycles of PCR amplification (92°C for 30s, 60°C for 30s, 72°C for 1 min, and a final extension step at 72°C for 5 min) in a total volume of 25 µl containing 0.4 µM of sense and antisense primers, 0.5 U of Taq polymerase (Invitrogen), and 200 µM of dNTP. For the amplification of CD3 mRNA, the sense oligonucleotide primer was 5'TCGAGGAATTCCCACCATGAAGTGGACGGCATCAGTC-3' and the antisense oligonucleotide primer was 5'-TGACGACCGGTGCGCGAGGGGGCAGGGTCTG-3', giving rise to a 152-base pair fragment. Internal standards were generated by amplifying the hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA using the following set of primers: sense, 5'-TTCCTCCTCAGACCGCTTTT-3' and antisense, 3'-CTTATAGCCCCCCTTCAGCA-5', giving rise to a 262-base pair amplification product. PCR products were loaded on a 2% agarose gel.

Slice Preparation
Adult male Sprague-Dawley rats weighing 200 g were deeply anesthetized with Rompun-ketamine (1:4) at 1 ml/kg (i.p.). They were then perfused transcardially with 250 ml of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Brains were dissected and postfixed in the same solution for 1 h at room temperature and then were cryoprotected overnight at 4°C in a solution of 15% sucrose in PBS and then 48 h at 4°C in 30% sucrose in PBS. Coronal sections (30 µm thick) were cut on a cryostat and collected in 0.1 M phosphate buffer (PB), pH 7.4.

Cell Cultures
COS-7 cells were grown in DMEM containing 10% fetal calf serum (FCS), 100 µg/ml streptomycin, and 100 U/ml penicillin.

Neural stem cell cultures were prepared from whole brains of E15 rat embryos as previously described (Sergent-Tanguy et al., 2006). Briefly, tissues freed of meninges were incubated with 0.25% trypsin for 15 min at 37°C, and after addition of 10% FCS and 10 mg/ml DNAse I, were dissociated by mechanical trituration. Aggregates were removed by decantation and cells were further purified from small debris by centrifugation. The cell suspension was then incubated for 12 h in basal culture medium composed of DMEM containing Hams' F12 (1/1, vol/vol), 3 mM glucose, 5 mM HEPES (pH 7.2), 100 µg/ml streptomycin, and 100 U/ml penicillin (basal medium) and supplemented with 10% FCS (complete medium), and the uncoated cells were transferred to basal medium supplemented with N2 (Invitrogen) and 10 ng/ml FGF-2 to allow for the formation of neurospheres. After 5 d of culture, neurospheres were dissociated by trypsin and mechanical trituration to give a single-cell suspension. This was then plated on poly-L-ornithine–coated glass coverslips at a density of 3 x 10⁴ cells/cm² for 2 h in complete medium and subsequently fixed in 4% PFA and 4% sucrose in PBS.

Glia cultures were prepared from newborn rat forebrains. Briefly, cortexes were dissected and dissociated by trypsin and DNAse and the cells were plated at a density of 35 x 10³ cells/cm² in MEM containing 10% FCS, 0.6% D-glucose, 100 µg/ml streptomycin, and 100 U/ml penicillin. The medium was replaced with serum-free MEM containing N2 supplements 24 h before the neuron culture.

Rat hippocampal and cortical cultures were prepared from 18-d-old rat embryos by previously described methods (Goslin et al., 1998; Pujol et al., 2005). Briefly, hippocampi were dissected and dissociated by trypsin and trituration and plated on glass coverslips coated with poly-L-lysine. For calcium imaging assays and transfection, cells were plated at a density of 18 x 10³ cells/cm². For all other uses, cells were plated at 4 x 10³ cells/cm². The cells were allowed to attach on coverslips before being transferred to a dish containing a glial feeder layer, prepared as described above, and maintained for up to 21 d in serum-free MEM with N2 supplements. Neurons were used for immunocytochemical staining and morphology analysis 3 h after plating or after 1, 2, 4, 7, 10, and 21 d in vitro (DIV). For Western blot analysis, cells were collected at 7 DIV.

Western Blot Analysis
For Western blot analysis, homogenates from CD3-transfected COS-7 cells, 7 DIV cultured neurons, T lymphocytes, and rat forebrain and spleen were prepared. Briefly, cultured neurons and COS-7 cells were scraped into PBS containing a cocktail of protease inhibitors and were then pelleted and resuspended in Laemmli buffer. T lymphocytes were isolated from total splenocytes after nylon wool adherence and depletion of CD161 (NK cells), CD11b/c (monocytes), and CD45R (B lymphocytes) positive cells using magnetic beads (Invitrogen/Dynal, Carlsbad, CA). For tissue preparation, rat forebrains and spleens were homogenized using a glass Teflon homogenizer in 10 mM Tris-HCl, pH 7.4, containing 320 mM sucrose and a cocktail of protease inhibitors. The resulting suspension was centrifuged at 700 x g for 10 min. The supernatant was collected and centrifuged at 150,000 x g for 30 min, and the pellet was resuspended in 10 mM Tris-HCl, pH 7.4. The membrane preparation was solubilized in Laemmli buffer.

Cell suspensions (500,000 cells) and tissue homogenates (30 µg of protein) were loaded on an SDS-PAGE (12% acrylamide) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% dried milk in 20 mM Tris-HCl, pH 7.4, containing 0.45 M NaCl and 0.1% Tween 20 (TBST). The membrane was then incubated overnight in Ab36 CD3 antibody (2.5 µg/ml) or with anti-GAPDH antibody (0,2 µg/ml), diluted in TBST containing 5% dehydrated milk, washed with TBST, incubated for 1 h in peroxidase-conjugated goat anti-rabbit antibody (1:3000) or in peroxidase-conjugated donkey anti-mouse antibody (1:2000), respectively, and visualized using a chemiluminescent substrate (Pierce, Rockford, IL) and exposure to x-ray films.

Immunostaining
For immunohistochemistry on rat brain sections, sections were first incubated in 0.3% H₂O₂ in 0.1 M PB for 30 min and, after two washes in PB, were additionally incubated for 30 min in 3% normal donkey serum (NDS) in PB. After two washes in PB, sections were incubated overnight at 4°C with Ab36 (0.2 µg/ml) or Ab22 (5 µg/ml) CD3 antibodies diluted in PB containing 3% NDS and 0.05% Triton X-100. The following day, sections were washed twice in PB and incubated for 45 min in biotinylated goat anti-rabbit IgG diluted at 1:200 in PB containing 0.5% NDS, followed by 45 min in streptavidin-biotine peroxidase solution (ABC Vectastain, Vector Laboratories, Burlingame, CA). After several rinses in PB, sections were further incubated in 0.05% 3,3'diaminobenzidine (Vector Laboratories) for 10 min at room temperature and were then mounted on gelatin-coated slides, dehydrated in graded ethanol, delipidated in xylene, cover-slipped with Eukit, and examined with a Zeiss microscope (Thornwood, NY). For control experiments, the primary antibodies were omitted during the procedure. For double immunofluorescence labeling, sections were incubated overnight at 4°C with Ab36 CD3 antibody and either anti-GFAP (1:500), anti-NeuN (1:200), anti-CD11b/c (1:500), or anti-RIP (1:500) antibodies diluted in PB containing 3% NDS and 0.05% Triton X-100. Slices were then incubated for 1 h with the appropriate FITC- or Alexa Fluor 568–conjugated secondary antibodies (1:200 and 1:1000, respectively) and mounted with Vectashield (Vector Laboratories) on glass slides for analysis on an Axioskop 2 plus microscope (Zeiss). Pictures were acquired with 10x and 20x objectives using a digital camera (AxioCam HRC, Zeiss) driven by AxioVision Release 4.2 software. Two animals were analyzed to determine the phenotype of CD3-expressing cells in the brain. For each animal, the number of CD3-positive cells was recorded in the neocortex from two slices (300 CD3-positive cells per slice), and the number of CD3-positive cells that were also immunoreactive for GFAP (astrocyte), NeuN (neuron), CD11b/c (microglia), or RIP (oligodendrocytes) was counted. Images for presentation were prepared for printing with Adobe Photoshop (San Jose, CA). For neural stem cells and cultured neurons, cells were fixed in 4% PFA for 15 min, permeabilized for 5 min in 0.25% Triton X-100 in PBS, and incubated for 30 min in 10% bovine serum albumin (BSA) in PBS at 37°C. Cells were then incubated overnight in Ab36 CD3 antibody (2.5 µg/ml) or CD90 antibody (3 µg/ml) diluted in PBS containing 3% BSA. For double labeling, Ab36 CD3 antibody was mixed with either MAP2 (1:200), tau-1 (1:500), or Tuj1 (1:1000) antibodies. F-actin was labeled with TRITC-phalloidin (1:10000, Sigma Aldrich) for 1 h at 37°C before immunostaining. Cells were then incubated with the appropriate secondary antibodies conjugated to FITC, biotin (1:200), or Alexa Fluor 568 (1:1000). When the biotin-conjugated antibody was used, cells were subsequently incubated in AMCA-conjugated streptavidin (1:200). For double labeling on brain sections or cultured cells, control experiments achieved by omitting one of the primary antibodies showed the lack of cross-reactivity between the secondary antibodies and the absence of signal spillover between the two channels. The coverslips were mounted with Vectashield on glass slides for analysis on an Axioskop 2 plus microscope (Zeiss). Pictures were acquired with 20x and 63x objectives using a digital camera as described above.

DNA Constructs and Small Interfering RNA
A plasmid encoding rat CD3 fused at the C terminus with enhanced green fluorescent protein (EGFP) was produced. The coding sequence of CD3 containing a Kozak sequence at the 5' end was obtained by PCR amplification and subcloned in frame into the EcoRI-AgeI sites of pEGFP-N1 to generate the CD3-GFP expression plasmid.

To generate a mutant form of CD3 with the six tyrosine residues substituted by phenylalanine in the three ITAMs, 100 ng of the following three primers bearing the mutations—5'-AACCAGCTCTTTAACGAGCTCAATCTAGGGCGAAGAGAGGAATTTGATGTTTTG-3', (5'-AAGGCGTGTTCAATGCACTGCAGAAAGACAAGATGGCAGAGGCCTTCAGTGAGATT-3', and (5'-ACGGCCTTTTCCAGGGTCTCAGCACTGCCACCAAGGACACCTTTGACGCCCTG-3'—were mixed with 100 ng of CD3-GFP DNA template. The reaction was performed using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. All the CD3 cDNA constructs were confirmed by sequencing.

For small interfering RNA (siRNA) experiments, four sense and antisense oligonucleotides corresponding to the following cDNA sequences were purchased from Ambion (Austin, TX): 5'-GCAAAAUUCAGCAGGAGUGtt-3' and 5'-CACUCCUGCUGAAUUUUGCtc-3', siRNA1, nucleotides 276–294; 5'-GCUCUAUAACGAGCUCAAUtt-3' and 5'-AUUGAGCUCGUUAUAGAGCtg-3'; siRNA2, nucleotides 329–347; 5'-GGUAACAUGUAAUUAGGUCtt-3' and 5'-GACCUAAUUACAUGUUACCtt-3', siRNA3, nucleotides 1066–1084; and 5'-GGCGUGUACAAUGCACUGCtt and 5'-GCAGUGCAUUGUACACGCCtt; siRNA4, nucleotides 444–462). A control siRNA (Ambion), which showed no homology to any known gene sequences from rat, mouse, or human, was used as a negative control.

Transfections and Pharmacological Treatments
Transfection of COS-7 cells with the CD3-expressing plasmid pRZ-3 (Itoh et al., 1993) was performed using the Lipofectamine 2000 reagent (Invitrogen) as previously described (Baudouin et al., 2006). To determine which siRNA was the most effective in reducing CD3 protein expression, COS-7 cells were cotransfected with 3 µg of pRZ-3 and 50 nM of either siRNA1, 2, 3, or 4 or negative control using the Lipofectamine 2000 reagent. One day after transfection, cells were collected to analyze CD3 protein expression by Western blot.

Neuron transfection was performed at 3 DIV with 3 µg of either CD3-GFP cDNA or 3 µg of CD3-6Y6F-GFP or 3 µg of membrane-targeted GFP (mGFP) cDNA [GAP-GFP(S65T), a gift from Dr. K. Moriyoshi, Kyoto University, Japan; Moriyoshi et al., 1996], using 3 µl of the Lipofectamine 2000 reagent. The cells were fixed in 4% PFA and 4% sucrose in PBS at 5 DIV for MAP2 and tau double labeling.

For siRNA assays, neurons were cotransfected at 3 DIV with 3 µg of mGFP cDNA and 50 nM of siRNA1 or siRNA3 using 3 µl of the Lipofectamine 2000 reagent. The cells were fixed in 4% PFA and 4% sucrose in PBS at 5 DIV for MAP2 and CD3 double immunolabeling.

For antibody treatments, either 5 µg/ml Ab22 CD3 antibody or 5 µg/ml control antibody to human CD16 (3g8) were added to neuron cultures at 1 and 3 DIV. In control cultures the same volume of vehicle was added. Cells were fixed in 4% PFA and 4% sucrose in PBS at 5 DIV and were immunolabeled for MAP2 morphological analysis.

To assess the effects of inhibiting protein kinases on CD3 distribution, neurons were treated at 7 DIV for 5, 15, 30, 60, and 120 min with 10 µM piceatannol (Sigma) an inhibitor of the Syk/ZAP70 family of the protein tyrosine kinases, 100 nM damnacanthal (Calbiochem, San Diego, CA) a specific inhibitor of lck, a protein tyrosine kinase of the Src family, 1 µM PP2 (Sigma), a broad inhibitor of the Src-family protein tyrosine kinases (Sigma), or 50 nM wortmannin (Sigma), an inhibitor of phosphoinositide 3-kinase. All the kinases inhibitors were added directly to the culture medium from a concentrated DMSO stock. To test the role of the actin cytoskeleton on CD3 localization, neurons were treated for 30 min and 3 and 24 h with 1 µM cytochalasin D (Sigma) added to the culture medium from a concentrated DMSO stock.

Quantification of CD3 Expression and Neuronal Morphology Analysis
To quantify CD3 or GAPDH expression in CD3-expressing COS-7 cells after siRNA transfection, the signal intensity obtained on Western blot membranes was measured from four experiments using Image J software (http://rsb.info.nih.gov/ij/) and expressed as a percentage of the intensity obtained from control cells without siRNA. To quantify CD3 immunoreactivity in neurons after siRNA transfections, coverslips were scanned on the microscope with a 10x lens and pictures of transfected cell were acquired with a 40x objective. The intensity of CD3 immunofluorescence was measured with the Image J software in well-isolated GFP-positive neurons cotransfected with either control or CD3 siRNAs and in nontransfected neurons located nearby the transfected cells. The intensity of CD3 immunoreactivity was measured in the dendritic field, but excluding the cell body, because CD3 was mainly targeted to dendrites and because the thickness of the cell body is a frequent source of nonspecific labeling compared with the nerve processes. A total of 16–34 cells per experimental condition were analyzed from two independent experiments. For each individual experiment, 3–7 cells were scored from at least two coverslips. Results are presented as average ± SEM of the ratio of anti-CD3 fluorescence intensity in GFP-positive neurons relative to nearby nontransfected neurons.

To analyze the morphology of neurons transfected with control siRNA, siRNA1, siRNA3, mGFP, CD3-GFP, or CD3-6Y6F-GFP, each coverslip was systematically scanned with a 10x lens and acquired with a 40x or 63x objective. Images from mGFP-labeled neurons were projected on the computer screen, all primary dendritic branches were traced with the mouse using the ImageJ software program, and the number and length of dendritic branches were scored. As defined in previous studies using a model of hippocampal neuron culture, protrusions with a length <10 µm likely corresponded to filopodia and dendritic spines, whereas protrusions with a length >10 µm were defined as dendritic branches (Jaworski et al., 2005; Terry-Lorenzo et al., 2005). Consequently, only the protrusions with a length >10 µm were taken into account to quantify the number and length of dendritic branches. Between 10 and 15 cells were scored from at least two coverslips for each experimental condition. A total of 29–63 cells were analyzed for each experimental condition from two to four independent experiments, and the data were presented as average ± SEM. To analyze experiments involving neuron cultures treated with either 3g8 or CD3 antibody, images from MAP2-labeled neurons were randomly acquired with a 20x and 40x lens, and the number and length of dendritic branches was measured as above. Data are presented as average ± SEM of a total of 40–52 cells per experimental condition recorded from two to four coverslips (5–10 cells per coverslips) obtained from two to three independent neuron cultures. Data analysis was performed using Excel (Microsoft, Redmond, WA) and GraphPad Prism 4 (San Diego, CA), and statistical analyses were done by Student's t test. Images were processed and prepared for printing using Adobe Photoshop.

Fluorescence Measurements of Intracellular Calcium
The effects of Ab22 CD3 antibody on intracellular calcium concentration ([Ca²⁺]_i) in 7 DIV cultured neurons were assessed using the calcium-sensitive fluorescent probe Fluo-3 AM (Molecular Probes). Neurons were loaded with 4 µM Fluo-3 AM in Tyrode (145 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM HEPES, and 5 mM glucose) and 0.02% Pluronic at 37°C for 30 min. The coverslips were mounted on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan) equipped for fluorescence and illuminated at 450–490 nm. The emitted light (>515 nm) was collected by a high-resolution image intensifier coupled to a video camera (Extended ISIS camera system; Photonic Science, Roberts-bridge, United Kingdom) and connected to a digital image processing board controlled by FLUO software (Imstar, Paris, France). Cells were maintained at 37°C and continuously superfused with Ca²⁺-free Tyrode. A microperfusion system allowed for local application and rapid change of the different experimental media. Microperfusion solutions were identical to the Ca²⁺-free Tyrode except that they contained 20 mM mannitol for efficient local perfusion of the neurons under analysis. Single-cell fluorescence intensity was measured from digital image processing and displayed against time. Fluorescence intensity was normalized to the intensity measured before antibody application. Calcium variations were quantified by a linear regression of the first 10 points of the fluorescence curve after antibody application (F/F/t in s^–1).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of CD3 in the Rat Brain and in Cultured Neurons
To examine whether CD3 mRNA was expressed in the brain, RT-PCR amplifications of CD3 was performed on total RNA extracted from rat forebrain and compared with the spleen. Amplification of HPRT transcripts served as an internal control to confirm RNA integrity for each preparation. In brain samples, a band of the expected size of 152 base pairs was detected, although exhibiting a lower intensity than in spleen preparations (Figure 1A). The expression of CD3 at the protein level was investigated by Western blot experiments under nonreducing conditions to preserve the disulfide bridge of the homodimer forms of CD3 known to be prevalent in immune cells (Baniyash, 2004). Western blots of COS-7 cells transfected with rat CD3 cDNA showed that the Ab36 CD3 antibody yielded a single band of 32 kDa, whereas no signal was apparent in nontransfected cells (Figure 1B). In the spleen, T-cells and cultured neurons, the 32-kDa form was also detected, whereas a 36-kDa band was present in rat brain homogenates (Figure 1B), a molecular weight in agreement with the 37-kDa form of CD3 reported in hippocampus preparations (Sourial-Bassillious et al., 2006). The characterization of the molecular forms of CD3 expressed in heterologous COS-7 cell systems, splenocytes, and peripheral T-cells have been well established, which showed that the nonphosphorylated CD3 homodimer exhibited an apparent molecular weight of 32 kDa and that activation-induced tyrosine phosphorylation of the ITAMs resulted in the appearance of multiple forms with molecular weights ranging from 36 to 45 kDa depending on the number of phosphorylated tyrosine residues (Sancho et al., 1993; Furukawa et al., 1994; Garcia and Miller, 1997; van Oers et al., 2000). Although the 32-kDa form of CD3 detected in CD3-transfected COS-7 cells, spleen, T-cells, and cultured neurons likely corresponds to a nonphosphorylated CD3 homodimer, the 36-kDa band detected in the brain might reflect the existence of a constitutively phosphorylated form of CD3 in cerebral tissue.

View larger version (28K): [in this window] [in a new window]   Figure 1. Expression of CD3 in the rat brain and in cultured neurons. (A) Expression of CD3 mRNA in the rat brain. PCR amplification of CD3 mRNA extracted from adult rat brain and spleen (positive control). HPRT amplification products indicated that PCR was performed on comparable levels of RNA. (B) Identification of heterologously expressed and endogenous CD3 by Western blot. Cell homogenates of nontransfected (COS-WT) and CD3-transfected (COS-CD3) COS-7 cells, as well as membrane homogenates prepared from rat spleen, brain, 7 DIV cultured neurons, and T-cells were resolved on a 12% SDS-PAGE and immunoblotted with Ab36 CD3 antibody. An immunoreactive band can be observed at 32 kDa in CD3-transfected COS-7 cells, spleen, cultured neurons, and T-cells and at 36 kDa in brain homogenates. Molecular weight markers are indicated on the right.

View larger version (66K): [in this window] [in a new window]   Figure 2. Distribution of CD3 immunoreactivity in the rat brain. (A) In the parietal cortex, CD3-immunoreactive cells are detected throughout the six layers, but immunolabeled neuronal processes predominate in layers V and VI. (B–E') The comparison between the distribution of CD3 immunoreactivity in rat brain slices as revealed by Ab36 (B–E) and Ab22 (B'–E') antibodies. Both antibodies give rise to a virtually identical labeling pattern, as shown here in the cerebral cortex (B, B', C, and C') and hippocampus (D, D', E, and E'). At high magnification in the cerebral cortex (C and C') and in the dentate gyrus of the hippocampus immunolabeled with Ab36 (F), most CD3-expressing cells exhibit a neuronal morphology and are immunolabeled within both perikarya (arrows in C, C', and F) and dendritic processes (arrowheads in C, C', and F). (G–J) Double immunolabeling of hippocampal sections with Ab36 CD3 antibody (green) and the markers (red) NeuN for neurons (G), RIP for oligodendrocytes (H), GFAP for astrocytes (I), and CD11b/c for microglia (J). Scale bars, (A) 300 µm; (B and E) 75 µm; (C, F, and G–J) 15 µm; (D) 1 mm.

CD3 Is Selectively Enriched at Growth Cones and Filopodia during Neuronal Development

View larger version (63K): [in this window] [in a new window]   Figure 3. Developmental distribution of CD3 in neurons derived from neural stem cells and in hippocampal neurons in culture from 1 to 21 DIV. (A) Neural stem cells cultured as neurospheres were dissociated, plated on glass coverslips, and fixed 2 h later in PFA for the immunodetection of CD3 and Tuj1 (Tubulin β III), used as a neuronal marker. The immunopositive CD3 cells are all immunoreactive for Tuj1 (arrows), corresponding to newly differentiated neurons. (B–E) Hippocampal neuron cultures were fixed at 1 (B), 2 (C), 4 (D), 7 (E), and 21 (F) DIV and were immunolabeled for CD3. From 1–7 DIV, the paired phase-contrast image is shown to visualize cell morphology. At 1 and 2 DIV (B and C), CD3 immunoreactivity is selectively associated with minor neurite endings, axonal growth cones (arrowhead in C), and protrusions emerging along the growing axon (arrows in C). At 4 DIV (D), CD3 immunoreactivity is highly enriched at the tips of dendrites and short branches. At 7 DIV (E) CD3 immunoreactivity is still observed at the tip of dendrites and branches and is also abundantly associated with filopodia and spine-like protrusions distributed along dendritic processes, as shown at higher magnification of the boxed region (E'). (F) Neurons at 21 DIV were fixed and immunolabeled for CD3 (green) and the postsynaptic marker PSD-95 (red). Numerous CD3 clusters are distributed along dendritic processes that partially colocalized with PSD-95 at synaptic sites as shown at higher magnification of the boxed region (F'). Scale bars, (A and B) 5 µm; (C) 10 µm; (D–F) 15 µm.

View larger version (67K): [in this window] [in a new window]   Figure 4. CD3 is concentrated at dendritic tips and filopodia and is selectively associated with the peripheral region of the growth cone. (A) Neurons at 7 DIV were fixed and immunolabeled for CD3 (green) and the dendritic marker MAP2 (red). CD3-immunoreactivity is mostly confined to the somatodendritic compartment and is not detected in the axon, defined as a MAP2-negative process (arrows). (B) Double labeling of CD3 (green) and the axonal marker tau-1 (red) of 7 DIV neurons show that the axonal trunk and growth cone (arrow) are devoid of CD3 immunoreactivity. (C) At growth cones of 2–3 DIV neurons, CD3 immunoreactivity (green) is localized to the peripheral actin-rich region labeled with TRITC-phalloidin (red), but is mostly excluded from the extending filopodia. (D) The central region of the growth cone, defined as a microtubule-rich region immunolabeled with Tuj1 antibody (red), is devoid of CD3 immunoreactivity. Scale bars, (A and B) 20 µm; (C and D) 2.5 µm.

CD3 Clustering at the Tips of Neurites Is Dependent on Filamentous Actin and Protein Tyrosine Kinases of the Syk/ZAP-70 Family

View larger version (69K): [in this window] [in a new window]   Figure 5. The selective concentration of CD3 at dendritic tips depends on filamentous actin and on protein tyrosine kinases of the Syk/ZAP-70 family. (A) Neurons at 7 DIV were either left untreated (control) or were exposed for 24 h to 1 µM of cytochalasin D to depolymerize actin (cyto D), and fixed and immunolabeled for CD3 (green) and MAP2 (red). Cytochalasin D treatment induced a redistribution of the large CD3 clusters localized at dendritic tips to small puncta dispersed along dendrites, whereas MAP2 immunostaining was unaffected. A 3-h washout after cytochalasin D treatment resulted in significant reversal that shows reaccumulation of CD3 at dendritic tips (3-h washout). (B) Control neurons and cytochalasin D–treated neurons are double-labeled for either CD3 or CD90 (green) and for F-actin with TRITC-phalloidin (red). In control neurons CD3 strictly colocalizes to F-actin–rich subdomains identified as filopodia and dendritic tips. In cytochalasin-treated neurons, CD3 remains associated to the dispersed F-actin puncta, which become evenly distributed within dendrites. The distribution of CD90 shows no relationship with F-actin labeling in control neurons and is unaffected by the cytochalasin D treatment. (C) Neurons at 7 DIV were either left untreated (control) or treated for 5 min with 10 µM piceatannol, an inhibitor of the protein tyrosine kinases of the Syk/ZAP-70 family, and were then immunolabeled for CD3 (green) and MAP2 (red). Blockade of the Syk/ZAP-70 kinases induces a rapid loss of CD3 aggregates, an effect reversed after 4 h of washout. By contrast, CD3 distribution was unaffected by 1 µM PP2, an inhibitor of the Src-family of protein tyrosine kinases. Scale bars, (A and C) 20 µm; (B) 2 µm.

CD3 siRNA and a CD3 Mutant Lacking the Sites of Tyr Phosphorylation Impairs Dendrite Formation
On the basis of our finding that CD3 proteins are specifically enriched at dendritic tips and protrusions during the stage of dendrite elaboration and maturation, we examined whether CD3 might play a role in dendrite development. For this purpose, we used an siRNA approach to knockdown CD3 protein expression in cortical neurons culture. Four CD3 siRNA sequences against different regions of CD3 were tested for their efficacy and specificity in suppressing CD3 expression in CD3-transfected COS-7 cells (Figure 6A). We found by immunoblot analysis that two of them, siRNA1 and siRNA3, caused a marked reduction of the CD3 protein expression reaching 30.7 ± 6.1 and 7.5 ± 5.2%, respectively, of the signal intensity of CD3-expressing cells transfected with a control siRNA (Figure 6A). To test whether these siRNAs can decrease the expression level of endogenous CD3 in neurons, cultured neurons at 3 DIV were cotransfected with either siRNA1 or siRNA3 and mGFP as a marker of transfection, and CD3 immunoreactivity was quantified in the dendritic arbor at 5 DIV. The addition of siRNA1 or siRNA3 induced a severe reduction of endogenous CD3 level that dropped to 21.7 ± 6.0% (n = 23) and 14.5 ± 5.1% (n = 16), respectively, of the averaged CD3 signal intensity measured in nearby untransfected neurons (Figure 6, B and C). The control siRNA did not affect CD3 immunostaining as compared with neighboring untransfected cells (98.7 ± 14.8%, n = 34; Figure 6, B and C). The siRNA transfections were performed at 3 DIV, and the cells were analyzed at 5 DIV, a period corresponding to a stage of dendrite differentiation in this neuronal culture model (Dotti et al., 1988). Therefore, these experiments allowed us to test the contribution of CD3 to dendrite formation. We observed that CD3 siRNA1 and siRNA3 significantly increased the size and the complexity of the dendritic arbor analyzed after immunostaining for CD3 and the dendritic marker MAP2. The total length of dendritic branches recorded per cell was increased by 90 and 123% upon transfection with siRNA1 or siRNA3, respectively, compared with control cells transfected with a control siRNA (control siRNA, 86.5 ± 11.1 µm, n = 63 cells; CD3 siRNA1, 163.3 ± 16.3 µm, n = 52 cells, p < 0.001; CD3 siRNA3, 192.6 ± 31.6 µm, n = 29 cells, p < 0.01, Student's t test; Figure 6, C–E). Moreover, the amount of dendritic branching recorded per cell was increased by 80 and 99% after transfection with CD3 siRNA1 or siRNA3, respectively, compared with siRNA control cells (control siRNA, 2.94 ± 0.34, n = 63 cells; CD3 siRNA1, 5.29 ± 0.45, n = 52 cells, p < 0.001; CD3 siRNA3, 5.86 ± 0.89, n = 29 cells, p < 0.01; Figure 6, C–E). There was no detectable difference in the dendritic arbor between either siRNA1 and siRNA3 (p = 0.43 for the branching length and p = 0.57 for the dendritic branchpoint number) or between mGFP and control siRNA (p = 0.63 for the branching length and p = 0.60 for the dendritic branchpoint number; values for mGFP are 79.1 ± 10.5 µm for the branching length, n = 56 cells, and 2.70 ± 0.31 for the number of dendritic branches, n = 56 cells), supporting that the changes in dendritic morphology induced by CD3-targeted siRNAs were specifically linked to the reduced CD3 expression.

View larger version (57K): [in this window] [in a new window]   Figure 6. CD3 siRNA1 and siRNA3 promoted dendritic branching. (A) The efficacy and specificity of CD3 siRNA was tested in CD3-transfected COS-7 cells by immunoblot analysis. An equal amount of protein was loaded for each lane, and the same samples were blotted against CD3 and GAPDH antibodies. CD3 siRNA1 and siRNA3 induced a strong suppression of CD3 expression. The signal intensity for each CD3 siRNA is plotted below as a percent of the signal intensity obtained from control CD3-expressing COS-7 cells without siRNA. The data are expressed as mean ± SEM from four experiments (* p < 0.05 compared with cells transfected with CD3 plasmid alone). (B) Quantification of CD3 expression in cultured cortical neurons transfected at 3 DIV and immunostained at 5 DIV. Data are presented as a ratio of anti-CD3 fluorescence intensity in mGFP-positive cells relative to nearby nontransfected cells ± SEM (for each condition n > 15 cells from at least two cultures; *** p < 0.001). (C) Knockdown of neuronal CD3 expression by siRNA1 and siRNA3. Neurons were cotransfected at 3 DIV with mGFP (green) and either control siRNA or CD3 siRNA1 or siRNA3, and were immunolabeled at 5 DIV for CD3 (red) and the dendritic marker MAP2 (blue). Neurons transfected with a control siRNA show a robust CD3 immunoreactivity on dendrites (arrowheads). Neurons transfected with either CD3 siRNA1 or siRNA3 show a decreased CD3 immunoreactivity in dendrites (arrows) and a more complex dendritic arbor compared with control cells. (D) mGFP-labeled cultured cortical neurons transfected at 3 DIV and fixed at 5 DIV sampled from cultures transfected with mGFP alone, or mGFP + control siRNA, or mGFP + CD3 siRNA1, or mGFP + CD3 siRNA3. (E) Quantification of the total length and number of dendritic branches per cell. Data are presented as mean ± SEM of 29–63 cells from two to four independent cultures. ** p < 0.01, *** p < 0.001 (Student's t test) compared with corresponding measurements in cells cotransfected with mGFP and the control siRNA. Scale bar, 20 µm.

View larger version (75K): [in this window] [in a new window]   Figure 7. Overexpression of a CD3 mutant lacking the Tyr phosphorylation sites in the ITAMs increased dendritic branching. Neurons transfected at 3 DIV with mGFP or CD3-GFP or CD3-6Y6F-GFP were fixed at 5 DIV and were visualized by the GFP signal. The dendritic arborization was increased in neurons expressing CD3-6Y6F-GFP but reduced in neurons expressing CD3-GFP compared with control neurons transfected with mGFP. The length and number of dendritic branches were measured and data are expressed as the mean ± SEM of 32–58 cells from three independent experiments (bottom right). * p < 0.05, ** p < 0.01 (Student's t test) compared with corresponding measurements in cells transfected with mGFP. Scale bar, 15 µm.

Application of CD3 Antibody to Cultured Neurons Elicits Intracellular Calcium Increase and Inhibits Dendrite Development

View larger version (20K): [in this window] [in a new window]   Figure 8. Application of CD3 antibody to hippocampal cultured neurons induces an intracellular calcium increase and reduces the complexity of the dendritic arbor. (A) Cultured neurons at 7 DIV were loaded with Fluo-3 AM and exposed sequentially to the control antibody 3g8 (5 µg/ml) and to the CD3 Ab22 antibody (5 µg/ml). Left, representative relative fluo-3 fluorescence in response to 3g8 and CD3 antibodies shows a rise in [Ca2+]i after CD3 antibody application. [Ca2+]i increase was observed in 46 of 53 neurons from three different cultures. Right, bar graph comparing the slope of fluorescence increase triggered by 3g8 and CD3 antibodies. (B) MAP2-immunolabeled 5 DIV hippocampal neurons sampled from cultures treated with a control antibody (control) or with CD3 Ab22 antibody (CD3 antibody). (C) The length and number of dendritic branches were measured on neurons immunostained for MAP2. Results are shown as the mean ± SEM of 40–52 cells from two experiments. *** p < 0.001 (Student's t test) compared with corresponding measurements in 3g8 antibody-treated cells. Scale bar, 80 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Neuronal Expression of CD3 and Its Selective Association with Growth Cones and Filopodia in Young Neurons
We provide biochemical and immunohistochemical evidence for the expression of CD3 in the CNS. Previous studies reported the detection of CD3 mRNA in feline and mouse brain sections (Corriveau et al., 1998; Huh et al., 2000), but our study is the first to demonstrate expression of CD3 protein in brain slices and to identify the CD3-expressing cells, which were predominantly neurons and to a lesser extent oligodendrocytes. The cellular distribution of CD3 immunoreactivity analyzed in developing hippocampal neurons showed a prominent enrichment at dendritic growth cones and filopodia in young neurons and at dendritic spines in mature neurons. These aforementioned neuronal subdomains are all actin-rich structures, and we indeed found in young neurons a striking colocalization of CD3 with F-actin. Treatment with the actin depolymerization agent cytochalasin D caused a dispersal of CD3 clusters, indicating that the accumulation of CD3 at growth cones and filopodia was dependent on F-actin. Interestingly, CD3 remained colocalized with F-actin puncta spread throughout the neuropile upon actin disruption, suggesting a close molecular interaction between CD3 and F-actin. Association of CD3 with the actin cytoskeleton has been reported in lymphocytes and proposed to play a role in the dynamic rearrangements of the actin cytoskeleton required for the formation and stabilization of the immunological synapse at the interface between antigen-presenting cells and T lymphocytes (Caplan et al., 1995; Rozdzial et al., 1995; Krummel and Davis, 2002). In neurons, the localization of CD3 at cytoskeletal F-actin–rich structures positions it ideally for a role in the regulation of dendritic shaping driven by actin-based mechanisms. CD3 could directly bind actin filaments to regulate actin network assembly or act as an adaptor molecule that would connect membrane receptors and signaling proteins linked to the assembly/stability of actin cytoskeleton. In the latter case, one possibility is that the effects of CD3 recruitment on neuronal morphogenesis involves the actin-related RhoA GTPase, which has been documented to inhibit dendritic growth and branch extension (Li et al., 2000; Wong et al., 2000).

Novel Function of CD3 in Dendrite Patterning
Our finding that CD3 was enriched at dendritic growth cones and filopodia in young neurons suggests a potential role for this molecule in dendrite morphogenesis. Accordingly, the inhibition of CD3 expression in cultured neurons by siRNA increased dendrite outgrowth, a phenotype similarly obtained by expression of a CD3 mutant lacking the Tyr phosphorylation sites within the ITAMs. The effects of the CD3-6Y6F-GFP mutant were likely due to the competition with the endogenous protein and suggest that the role of CD3 on dendritic shaping required tyrosine-based signaling motifs. Conversely, CD3 overexpression, which presumably mimicked an increased recruitment of the protein, and CD3 antibody application, which acts through the activation of endogenously expressed CD3, both reduced the length and number of dendritic branches. Together, these data suggest that CD3 acts as a negative regulator of dendritic arbor complexity through an ITAM-based mechanisms.

A critical role for CD3 in the establishment of the neuronal network has been described in the retinogeniculate projections of the visual system (Huh et al., 2000). In mice lacking CD3, the area of retinal projections in the lateral geniculate nucleus is abnormally larger and ectopic clusters of inputs were observed, indicating that CD3 is required to precisely restrict the field of retinal projections (Huh et al., 2000). The fact that CD3 mRNA was detected in the lateral geniculate nucleus suggests a postsynaptic expression of CD3 proteins in this area (Huh et al., 2000). In light of our results, one could hypothesize that CD3 deletion might result in an excessive dendritic arborization of lateral geniculate nucleus neurons, which in turn might induce a broader extension of axonal inputs, as observed in mutant mice.

Potent