The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity
2007; Springer Nature; Volume: 26; Issue: 9 Linguagem: Inglês
10.1038/sj.emboj.7601680
ISSN1460-2075
AutoresNicola Brandt, Kristin Franke, Mladen‐Roko Rašin, Jan Baumgart, Johannes Vogt, Sergey Khrulev, Burkhard Hassel, Elena E. Pohl, Nenad Šestan, Robert Nitsch, Stefan Schumacher,
Tópico(s)Nuclear Receptors and Signaling
ResumoArticle12 April 2007free access The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity Nicola Brandt Nicola Brandt Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Kristin Franke Kristin Franke Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Mladen-Roko Rašin Mladen-Roko Rašin Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jan Baumgart Jan Baumgart Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Johannes Vogt Johannes Vogt Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Sergey Khrulev Sergey Khrulev Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Burkhard Hassel Burkhard Hassel Institute of Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Elena E Pohl Elena E Pohl Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Nenad Šestan Nenad Šestan Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Robert Nitsch Robert Nitsch Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany These authors contributed equally to this work Search for more papers by this author Stefan Schumacher Corresponding Author Stefan Schumacher Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany These authors contributed equally to this work Search for more papers by this author Nicola Brandt Nicola Brandt Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Kristin Franke Kristin Franke Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Mladen-Roko Rašin Mladen-Roko Rašin Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jan Baumgart Jan Baumgart Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Johannes Vogt Johannes Vogt Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Sergey Khrulev Sergey Khrulev Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Burkhard Hassel Burkhard Hassel Institute of Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Elena E Pohl Elena E Pohl Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Nenad Šestan Nenad Šestan Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Robert Nitsch Robert Nitsch Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany These authors contributed equally to this work Search for more papers by this author Stefan Schumacher Corresponding Author Stefan Schumacher Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany These authors contributed equally to this work Search for more papers by this author Author Information Nicola Brandt1, Kristin Franke1, Mladen-Roko Rašin2, Jan Baumgart1, Johannes Vogt1, Sergey Khrulev1, Burkhard Hassel3, Elena E Pohl1, Nenad Šestan2, Robert Nitsch1,4 and Stefan Schumacher 1,4 1Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Berlin, Germany 2Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA 3Institute of Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany 4These authors contributed equally to this work *Corresponding author. Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité—Universitätsmedizin Berlin, Charitéplatz 1, Berlin 10117, Germany. Tel.: +49 30 450 528323; Fax: +49 30 450 528902; E-mail: [email protected] The EMBO Journal (2007)26:2371-2386https://doi.org/10.1038/sj.emboj.7601680 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF-like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC-induced stimulation of dendritic branching. In contrast, CALEB/NGC-induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events. Introduction The development of dendritic arbors is critical to neuronal circuit formation as dendrites are the primary sites of synaptic input (Scott and Luo, 2001; Whitford et al, 2002; Jan and Jan, 2003). Following dendritic tree elaboration, small protrusions called spines emerge from the dendritic shafts of many neurons. These spines are morphologically specialized, and represent the main postsynaptic compartment for excitatory input (Hering and Sheng, 2001; Yuste and Bonhoeffer, 2004). A great deal of data has been presented on the importance of transmembrane proteins for connecting extrinsic cues, which regulate spine morphogenesis, to intracellular mediators of cytoskeletal rearrangements (Ethell and Pasquale, 2005). Various steps of dendrite development have been shown to be regulated by diffusible cues such as neurotrophins (Yacoubian and Lo, 2000; Horch and Katz, 2002; Ji et al, 2005), cell–cell interactions involving proteins such as Notch 1 (Šestan et al, 1999; Redmond et al, 2000) and β-catenin (Yu and Malenka, 2003) and neuronal activity (McAllister et al, 1996; Maletic-Savatic et al, 1999; Portera-Cailliau et al, 2003; Tolias et al, 2005). Among the proteins that transduce these signals into changes in dendritic shape are not only members of the Rho family of proteins (Govek et al, 2005), but also components of some key signaling pathways. One example is the Ras-Raf-MAP kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway, which has been shown to be involved in activity-dependent dendrite differentiation (Wu et al, 2001; Vaillant et al, 2002). Another likely candidate is the phosphatidylinositide 3-kinase (PI3K)-Akt signaling pathway. This pathway has gained attention in neuroscience as it was highlighted to be implicated in neuronal growth, survival, neurite outgrowth, and synaptic plasticity (Atwal et al, 2000; Kuruvilla et al, 2000; Markus et al, 2002; Sanna et al, 2002). The PI3K has also been shown to organize dendritic branching together with Rho GTPases (Leemhuis et al, 2004). One of the PI3K-Akt-regulated proteins is the protein kinase mammalian target of rapamycin (mTOR), which is thought to act primarily by regulating protein translation. PI3K-Akt-mTOR signaling not only controls synaptic plasticity (Tang et al, 2002; Hou and Klann, 2004), but also dendritic arborization (Jaworski et al, 2005; Kumar et al, 2005). However, the precise molecular mechanisms that transduce extracellular cues via transmembrane receptors to intracellular signaling pathways to shape dendritic arbors and spines during consecutive developmental events are not fully understood. In this study, we characterized CALEB/NGC (Chicken Acidic Leucine-rich EGF-like domain containing Brain protein/Neuroglycan C), a member of the neural transmembrane EGF family, in the processes of dendritic tree elaboration and spine formation. CALEB/NGC is highly expressed in brain, in particular in fiber-rich areas, and the expression of this protein is upregulated during times of dendrite differentiation (Schumacher et al, 1997; Aono et al, 2000). CALEB/NGC can bind to the extracellular matrix proteins tenascin-C and tenascin-R, and interacts with the intracellular PSD-95/Discs large/ZO-1 (PDZ) domain protein PIST (PDZ domain protein interacting specifically with TC10; Schumacher et al, 2001; Hassel et al, 2003; Schumacher and Stübe, 2003). Cell culture experiments suggested a function of CALEB/NGC in neurite formation (Schumacher et al, 1997; Nakanishi et al, 2006). Electrophysiological analysis of CALEB/NGC-deficient mice showed disturbances in maintaining normal release probability at early developmental stages (Jüttner et al, 2005). However, the physiological function of CALEB/NGC is still unclear. Using in utero electroporation, we show that CALEB/NGC stimulates dendritic tree and spine complexity in the mouse cortex in vivo. Further, studies in primary hippocampal neurons indicate that the enhancement of dendritic arborization by CALEB/NGC is mediated by the EGF-like domain. This effect is independent of electrical activity, but can be blocked by inhibitors of PI3K, Akt, and mTOR. It is also dependent on protein kinase C (PKC) but not on the MEK-MAPK pathway. In contrast to its effect on dendritic branching, CALEB/NGC increases the complexity of dendritic spines and filopodia independent of PI3K. Taken together, we present novel evidence for a physiological role of CALEB/NGC in mediating dendritic tree complexity in the rodent cortex and uncover mechanisms of how CALEB/NGC drives dendritic branching and spine formation. Results CALEB/NGC is expressed in hippocampal and neocortical neurons and increases dendritic arborizations In this study, we were interested in the expression of CALEB/NGC in rodent hippocampal and neocortical tissue. We found strong expression of CALEB/NGC in adult dentate gyrus (DG) and the CA1 and CA3 (Cornu Ammonis) regions, as demonstrated by indirect immunofluorescence staining with an affinity-purified polyclonal antibody to CALEB/NGC (Figure 1A1). CALEB/NGC was expressed in regions where basal or apical dendrites of pyramidal neurons elaborate (Figure 1A2). It was also expressed in postnatal day 10 (P10) mouse hippocampal tissue (Figure 1A3) and in the neocortex, in particular in the upper layers (Figure 1A4). Furthermore, CALEB/NGC was expressed in primary hippocampal neurons at 9 days in vitro (DIV9) and, in addition to being present in axons and cell bodies, strongly localized to dendrites (Figure 1A5–A8). Figure 1.CALEB/NGC is expressed in hippocampal and neocortical neurons and increases dendritic arborizations. (A) A section of adult rat hippocampus was stained by indirect immunofluorescence with an antibody to CALEB/NGC (green, A1). In a high-magnification view of the CA1 region (A2) CALEB/NGC staining was found predominantly in fiber-rich areas. CALEB/NGC was also present in P10 mouse hippocampus (A3) and cortex (A4). When hippocampal cells in culture at DIV9 were probed with two different anti-CALEB/NGC antibodies (A5, A8), cell bodies and dendrites were clearly decorated. Anti-microtubule-associated protein 2 (MAP2) antibody stainings (red, A6) and overlay of anti-CALEB/NGC and MAP2 stainings (A7) confirmed dendritic localization of CALEB/NGC. (B) Examples of hippocampal neurons in culture transfected at DIV7 with either EGFP-encoding (left panel) or mCALEBb-encoding plasmid (right panel) and analyzed at DIV7+2. (C) Quantification of TNDET of hippocampal neurons transfected as described above; n=150, ***P<0.0001. (D) Cumulative frequency plot of TNDET in neurons examined as described. (E) Neurons transfected as described above were analyzed by Sholl analysis; n=15, **P<0.001, *P<0.01. (F, G) Effect of CALEB/NGC on total number of apical and basal dendritic branches (F) and on higher order dendritic branches (G); n=32, **P<0.005, *P<0.05. Scale bars, 200 μm (A1), 80 μm (A2), 150 μm (A3, A4), 25 μm (A5–A8), 15 μm (B). CA, cornu ammonis; DG, dentate gyrus; Cx, cortex. Download figure Download PowerPoint To find out whether CALEB/NGC is involved in the development of dendritic arborizations, we ectopically expressed the CALEB/NGC isoform mCALEBb or EGFP in DIV7 hippocampal neurons. After two more days in culture (DIV7+2), neurons were fixed and stained for mCALEBb or GFP. The dendritic trees of neurons expressing mCALEBb were much more elaborated than those of EGFP-expressing cells (Figure 1B). Compared to neurons expressing EGFP, mCALEBb-expressing cells had more complex dendritic branches, as measured by total number of dendritic end tips (TNDET) of branches longer than 8 μm (Figure 1C and D; mean values and s.e.m. of all statistical calculations can be found in Supplementary Figure S6). We also coexpressed mCALEBb together with EGFP, and compared these neurons to those that only expressed EGFP (Figure 3B). A similar increase in TNDET was found (Figure 3C and D). The effects of CALEB/NGC on dendritic arbor elaboration were further analyzed using Sholl analysis, which quantifies the number of times dendrites from a neuron cross concentric circles of increasing diameter (Sholl, 1953). With this analysis, we confirmed that expression of mCALEBb enhanced dendritic tree complexity (Figure 1E). Figure 2.Knockdown of CALEB/NGC reduces dendritic tree complexity. (A, B) Hippocampal neurons in culture were transfected at DIV9 with the shRNA constructs CAL1sh (A) and CAL3sh (B) and analyzed 3 days later. A GFP staining was performed to visualize neuron morphology (A1 and A2 green, B1–B5 green). Endogenous CALEB/NGC expression was shown by staining of the culture with the anti-CSPG5 monoclonal antibody (A1 and A2 red, B1–B5 red, arrows label transfected neurons), which recognizes an epitope in the cytoplasmic domain of CALEB/NGC (see Materials and methods). (C) Quantification of TNDET of hippocampal neurons transfected as described above; n=40, ***P<0.0001. (D) Western blot of mCALEBb levels in HEK293 cells co-transfected with control shRNA construct CAL1sh or CALEB/NGC-specific shRNA construct CAL3sh and mCALEBb-encoding plasmid. The immunoblot performed 24 h after transfection was probed with either anti-FLAG antibody or anti-β-tubulin antibody (loading control). Both the mCALEBb band (doublet, arrows) and the proteoglycan variant of CALEB/NGC (*) were stained. (E) Western blot of endogenous CALEB/NGC levels in primary hippocampal neurons transfected at DIV10 with control siRNA CAL1 or CALEB/NGC-specific siRNA CAL3 and analyzed 2 days later. The immunoblot was probed with either anti-CALEB/NGC monoclonal antibody (BD Biosciences) or anti-β-tubulin antibody (loading control). Both the CALEB/NGC band (doublet, arrow) and the proteoglycan variant of CALEB/NGC (*) were stained. (F) Quantification of relative fluorescence intensities of cell bodies of hippocampal neurons transfected with the indicated siRNA constructs at DIV10 and analyzed at DIV10+2 after CALEB/NGC staining; n=30, *P<0.05 and **P<0.01. AU, arbitrary units. (G) Quantification of TNDET in neurons transfected as in (F); n=150, *P<0.05. Scale bar, 20 μm. Download figure Download PowerPoint To describe dendritic phenotypes more precisely, we determined the number of dendritic end tips of apical and basal dendrites and the number of higher order dendrites in the same experimental approach as described above. We restricted this part of analysis to those neurons with clear distinguishable basal and apical dendrites. We found that mCALEBb expression only slightly increased the number of end tips (NDET) of basal dendrites but significantly increased NDET of apical dendrites when compared to EGFP as control (Figure 1F). Expression of mCALEBb significantly increased the number of higher order dendrites (Figure 1G). Together, these results show that overexpression of CALEB/NGC increases dendritic branching of primary hippocampal neurons. Knockdown of CALEB/NGC reduces dendritic tree complexity To examine the impact of endogenous CALEB/NGC on dendritic tree morphogenesis, we used RNA interference with siRNA and shRNA directed to CALEB/NGC. Primary hippocampal neurons were transfected at DIV9 either with shRNA construct CAL3sh (specific for rat and mouse CALEB/NGC) or with control shRNA construct CAL1sh (derived from a part of chicken CALEB/NGC sequence, which is not conserved between chicken and rat or mouse). Three days after transfection, cells were stained for GFP (green) and CALEB/NGC (red) and analyzed. We could observe a correlation of endogenous CALEB/NGC levels and dendritic tree complexity (Figure 2B1–B5 and Supplementary Figure S1) in CAL3sh knockdown neurons. Two CAL1sh control cells are given in Figure 2A1 and A2. We determined TNDET and found it to be significantly reduced in neurons targeted by CAL3sh when compared to neurons transfected with CAL1sh or pCGLH vector as control (Figure 2C). A further control of knockdown efficiency is given in Figure 2D. A Western blot was performed with detergent cell extracts of HEK293 cells co-transfected with mCALEBb and either CAL1sh or CAL3sh and stained for FLAG-tagged CALEB/NGC and β-tubulin as a loading control. We also transfected DIV10 primary hippocampal neurons with siRNA oligonucleotides CAL1 and CAL3 and analyzed the CAL3-induced reduction of endogenous CALEB/NGC by immunoblotting with a monoclonal antibody to CALEB/NGC (Figure 2E and Supplementary Figure S2). β-Tubulin was stained as a loading control. A quantitative analysis of CALEB/NGC-specific immunofluorescence signal in cell bodies of EGFP-expressing neurons corroborated the knockdown of CALEB/NGC expression by CAL3 but not by CAL1 (Figure 2F). Neurons co-transfected with CALEB/NGC-specific siRNA oligonucleotide CAL3 and EGFP-encoding plasmid had decreased CALEB/NGC expression and reduced dendritic arborization (Figure 2F and G and Supplementary Figure S3) when compared to neurons co-transfected with control oligonucleotide CAL1 and EGFP-encoding plasmid (Figure 2F and G and Supplementary Figure S3). The quantification of TNDET showed that the specific knockdown of CALEB/NGC by CAL3 resulted in a reduced number of end tips when compared to CAL1 or EGFP alone (Figure 2G). These findings support the relevance of CALEB/NGC for regulation of dendritic arbor complexity. Figure 3.The EGF-like domain and a specific cytoplasmic peptide segment of CALEB/NGC are important for increasing dendritic tree complexity. (A) Scheme of transfected CALEB/NGC-derived constructs. EGF, EGF-like domain; acidic, acidic peptide segment; TM, transmembrane region. (A) Juxtamembrane cytoplasmic peptide segment of CALEB/NGC shown to bind to the PDZ protein PIST; (B) peptide segment shown to be necessary for CALEB/NGC-induced dendritic branching; (C) peptide segment generated due to alternative splicing; (D) peptide segment of unknown function. (B) Cultured hippocampal neurons were co-transfected at DIV7 with EGFP-encoding plasmid and different CALEB/NGC-derived constructs shown schematically in (A). Neurons were analyzed at DIV7+2 after staining for GFP. (C) Quantification of TNDET of transfected cells (performed as described in Figure 1; n=42; ***P<0.0001 and **P<0.005). (D) Quantification of TNDET of neurons co-transfected as described in (B); n=45, **P<0.01. (E) Neurons were transfected at DIV12 to express EGFP or co-express EGFP and CALEB/NGC-derived construct '396', and analyzed 2 days later after staining for GFP. (F) Quantification of TNDET of of neurons co-transfected as described in (B); n=90, ***P<0.001. (G) Hippocampal neurons were co-transfected with the indicated constructs and analyzed as described in (B). (H) Quantification of TNDET; n=40, ***P<0.0001. Scale bar, 25 μm. Download figure Download PowerPoint The EGF-like domain and a specific cytoplasmic peptide segment of CALEB/NGC are important for increasing dendritic tree complexity To gain insight into the intracellular regions of CALEB/NGC necessary for signal transduction to the cytoskeleton, we transfected several CALEB/NGC-derived constructs into hippocampal neurons in culture. The cytoplasmic part of CALEB/NGC can be subdivided into four regions, A–D (Figure 3A). Construct '388', which has only cytoplasmic region A, and construct '400', that contains region B in addition, were transfected into hippocampal neurons at DIV7 and analyzed at DIV7+2. The quantification of TNDET (Figure 3C) showed that construct '400', like mCALEBb, was able to increase the complexity of dendritic arbors. To exclude the possibility that any of the CALEB/NGC-derived constructs was not correctly targeted to all dendrites, we co-transfected these constructs together with EGFP-encoding plasmid into DIV7 hippocampal cells, which were analyzed at DIV7+2 (Figure 3B). Quantification of TNDET (Figure 3D) confirmed the result indicated above. We next analyzed the CALEB/NGC-derived construct '396', which lacks the cytoplasmic region (Figure 3A). We hypothesized that it might uncouple extracellular events (e.g. binding of a putative ligand) from intracellular signal transduction. Indeed, we found that construct '396' led to a reduction in TNDET (Figure 3E and F). These findings suggest that CALEB/NGC-derived construct '396' may work in a dominant-negative manner to inhibit endogenous CALEB/NGC action. To determine which region of the extracellular part of CALEB/NGC is necessary for promoting dendritic tree complexity, we tested several deletion constructs. We found that the construct EGFshedding1 (Figure 3A), which only contains the EGF-like domain outside the cell, was sufficient to drive dendritic branching like mCALEBb (Figure 3G). To examine whether the EGF-like domain is necessary for stimulating dendrite morphogenesis, we analyzed the construct EGFmutant1 (Figure 3A), which is identical to mCALEBb with the exception of four-point mutations in the EGF-like domain (see Materials and methods). This construct did not promote dendritic branching above EGFP control (Figure 3G). Quantification of TNDET confirmed these results (Figure 3H). Thus, the EGF-like domain of CALEB/NGC drives dendritic branching. CALEB/NGC stimulates dendritic tree complexity in mouse cortex To explore whether CALEB/NGC is important for dendritic tree development in vivo, we performed analysis of mouse brain pyramidal neurons of neocortex targeted by in utero electroporation. This technique was chosen because it allows a selective manipulation of CALEB/NGC function in a subset of cells in otherwise normal tissue. It further allows a temporally discrete interference with endogenously expressed CALEB/NGC protein. In this way, the caveat of expression upregulation of genes that could compensate for functional loss of CALEB/NGC can be circumvented. This compensatory expression upregulation of genes might be a problem in classical genetic knockout strategies (Deuel et al, 2006; Koizumi et al, 2006). We used the in utero electroporation protocol to transfect embryonic day 15.5 (E15.5) cortical neurons, mostly pyramidal neurons of cortical layers II and III in vivo with the constructs mCALEBb and '396' (Figure 3A) cloned into the pCLEG vector. In addition, the shRNA constructs CAL3sh and CAL1sh as control cloned into the pCGLH vector (Chen et al, 2005) were used. With both vectors, expression of GFP is translationally driven by an internal ribosomal entry site (IRES). Electroporated animals were examined at postnatal day 7 (P7). Examples of coronal sections of these animals stained for GFP are given in Figure 4A1–E1. In each case, cortical cells in one hemisphere were targeted. Two examples of individual pyramidal neurons for each transfection condition are presented in Figure 4A2–E3. Dendritic arbors of neurons expressing mCALEBb (Figure 4B2 and B3) were more complex, and dendritic arbors of neurons expressing construct '396' (Figure 4C2 and C3) were less complex than those of control neurons (Figure 4A2 and A3). Expression of different CALEB/NGC-derived constructs in these pyramidal neurons was shown by anti-FLAG epitope staining (Figure 4F). Analysis of mice cortices electroporated with the CAL3sh-knockdown construct specific to CALEB/NGC confirmed these results. The NDET of electroporated neurons in cortex was reduced as shown by two representative neurons (Figure 4D2 and D3) when compared to the knockdown control CAL1sh. The quantification of TNDET (Figure 4G) confirmed that mCALEBb increased, constructs '396' and CAL3sh decreased dendritic tree complexity. The outcome of these experiments is that CALEB/NGC is functionally critical for establishing dendritic tree complexity of mouse pyramidal neurons in vivo. Figure 4.CALEB/NGC stimulates dendritic tree complexity in mouse cortex. (A) E15.5 mouse embryos were electroporated in utero with the pCLEG vector driving GFP expression. Overview of a coronal section (70 μm thick) stained with an antibody to GFP (A1), and two examples of individual neurons of these electroporated animals (A2, A3). (B) In utero electroporation was performed with a construct driving mCALEBb and GFP expression. Pictures of this animal corresponding to A1–A3 are presented in B1–B3. (C) In utero electroporation was done with the CALEB/NGC-derived construct '396' cloned into the pCLEG vector to drive expression of construct '396' and GFP. Pictures of this animal corresponding to A1–A3 are presented in C1–C3. (D) The CAL3sh knockdown construct specific to CALEB/NGC cloned into the pCGLH vector that drives GFP expression was electroporated in utero into cortical layer II and III neurons. Pictures of this animal corresponding to A1–A3 are presented in D1–D3. (E) Pictures derived from an animal electroporated with shRNA control construct CAL1sh corresponding to A1–A3 are shown in E1–E3. (F) Expression control of mCALEBb and construct '396' with an antibody to the FLAG epitope. (G) Quantification of TNDET of pyramidal neurons in tissue sections of in utero electroporated animals. End tips of dendritic branches longer than 8 μm were counted; n=40, **P<0.01, ***P<0.001. Arrows in A1, B1, C1, D1, and E1 indicate cortical layers 1–3; asterisks in A1, B1, C1, D1, and E1 mark the corpus callosum, arrowheads in A2–E3 point to representative neurons. Scale bars, 80 μm (A1, B1, C1, D1, and E1), 15 μm (A2–E3). Download figure Download PowerPoint The PI3K-Akt-mTOR pathway is important for CALEB/NGC-induced increase in dendritic tree complexity To get more insight into the molecular mechanisms of CALEB/NGC function with respect to dendritic arbor elaboration, we focused on the PI3K-Akt-mTOR and the MEK-MAPK signaling pathways which have recently been shown to be important for the control of dendritic arborization (Wu et al, 2001; Vaillant et al, 2002; Jaworski et al, 2005; Kumar et al, 2005). We used established inhibitors for specific kinases of these signaling pathways to determine the relevance of these proteins for CALEB/NGC-induced dendritic branching. Hippocampal neurons were transfected at DIV7 either with EGFP- or mCALEBb-encoding constructs, treated with these specific inhibitors and analyzed at DIV9 (Figure 5A, left and right panels, respectively). All inhibitors of the PI3K-Akt-mTOR pathway blocked CALEB/NGC-increased dendritic tree complexity, whereas U0126, a MEK inhibitor, did not (Figure 5A). In more detail, the PI3K inhibitor LY294002 fully suppressed CALEB/NGC-induced increase in TNDET (Figure 5B). It also reduced dendritic branching in the control as has been published (Jaworski et al, 2005; Kumar et al, 2005). Figure 5.The PI3K-Akt-mTOR pathway is important for CALEB/NGC-induced increase in dendritic tree complexity. (A) Overall view of DIV9 hippocampal neurons transfected at DIV7 either with EGFP- (left panels) or mCALEBb-encoding plasmid (right panels) and treated with indicated concentrations of inhibitors which were added 3 h after transfection. (B) Quantification of TNDET of neurons treated with or without 20 μM LY294002; n=81, ***P<0.0001. (C) Quantifi
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