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• W2106971401 abstract "Article15 February 2007free access Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation Christian Proepper Christian Proepper Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Svenja Johannsen Svenja Johannsen Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Stefan Liebau Stefan Liebau Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Janine Dahl Janine Dahl Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Bianca Vaida Bianca Vaida Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Juergen Bockmann Juergen Bockmann Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Michael R Kreutz Michael R Kreutz Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, IfN, Magdeburg, Germany Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, IfN, Magdeburg, Germany Search for more papers by this author Tobias M Boeckers Corresponding Author Tobias M Boeckers Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Christian Proepper Christian Proepper Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Svenja Johannsen Svenja Johannsen Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Stefan Liebau Stefan Liebau Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Janine Dahl Janine Dahl Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Bianca Vaida Bianca Vaida Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Juergen Bockmann Juergen Bockmann Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Michael R Kreutz Michael R Kreutz Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, IfN, Magdeburg, Germany Search for more papers by this author Eckart D Gundelfinger Eckart D Gundelfinger Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, IfN, Magdeburg, Germany Search for more papers by this author Tobias M Boeckers Corresponding Author Tobias M Boeckers Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Author Information Christian Proepper1, Svenja Johannsen1, Stefan Liebau1, Janine Dahl1, Bianca Vaida1, Juergen Bockmann1, Michael R Kreutz2, Eckart D Gundelfinger2 and Tobias M Boeckers 1 1Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany 2Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, IfN, Magdeburg, Germany *Corresponding author. Institute for Anatomy and Cell Biology, Ulm University, Albert Einstein Allee 11, 89081 Ulm, Germany. Tel.: +49 731 5023220; Fax: +49 731 5023217; E-mail: [email protected] The EMBO Journal (2007)26:1397-1409https://doi.org/10.1038/sj.emboj.7601569 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein-1 (Abi-1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi-1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi-1, Eps8 and Sos-1. Abi-1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi-1 co-immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E-box-regulated gene transcription. Downregulation of Abi-1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi-1 has the opposite effect. Data show that Abi-1 can act as a specific synapto-nuclear messenger and is essentially involved in dendrite and synapse formation. Introduction Communication in the central nervous system is mainly accomplished via highly specialized cellular contact sites called chemical synapses. Spinous excitatory synapses are highly dynamic and the morphology of dendritic spines can be altered within a few minutes (Hering and Sheng, 2001). These morphological alterations can be induced by synaptic activity and are thought to be the structural basis for synaptic plasticity underlying learning and memory formation (Yuste and Bonhoeffer, 2004). It is generally accepted that motility as well as dynamic changes of synaptic contacts is brought about by the reorganization of the actin and microtubule cytoskeleton via small GTPases. These GTPases are organized and regulated in micromolecular or macromolecular complexes, which localize to specific cellular microdomains (Wong et al, 2000; Ichigotani et al, 2002; Innocenti et al, 2003, 2004; Disanza et al, 2004). One signaling complex that has been studied extensively is the multiprotein complex of the ubiquitously expressed non-receptor tyrosine kinase c-Abl, which is localized both at the cellular membrane and in the nucleus (Taagepera et al, 1998; Pendergast, 2002). In brain, non-receptor tyrosine kinases of the c-Abl family could be detected at the presynaptic membrane and in the postsynaptic density (PSD), where they regulate synaptic efficacy and synaptic plasticity, presumably through their ability to locally reorganize the actin-based cytoskeleton (Moresco and Koleske, 2003). One important interacting protein of c-Abl is the abelson tyrosine kinase substrate or abelson interactor 1, abelson interacting protein-1 (Abi-1). Abi-1 was found to be part of several macromolecular complexes, including a trimeric signaling complex, where it closely interacts with Eps8 and Sos-1. Abi-1 is essential, via the control of Rac activity, for the formation and activation of the WAVE2 signaling complex. In turn, the activated WAVE proteins lead to enhanced actin nucleation via the Arp2/3 complex (Ichigotani et al, 2002; Innocenti et al, 2003, 2004). The role and functional importance of the Abi-1 complex has been studied in various cellular systems, showing that Abi-1 proteins play a pivotal role in phosphorylation (Tani et al, 2003) and localization of protein complexes to cellular subcompartments and in actin reorganization, especially through the regulation of Rac-dependent pathways (Stradal et al, 2001; Leng et al, 2005). Recently, it has been reported that Abi-1, like c-Abl, shuttles to the nucleus in NIH 3T3 fibroblasts, pointing to a dual function of the protein in distinct cellular compartments (Echarri et al, 2004). Spines and synapses are neuronal cell membrane protrusions that are characterized by constant remodeling processes. Several molecules can induce or influence the shape and maturation of these highly specialized contact sites (Hering and Sheng, 2001) mostly by binding and/or by regulating small GTPases (Pak et al, 2001; Ma et al, 2003). Interestingly, members of the ProSAP/Shank family of PSD scaffolding molecules can also effectively influence spine shape and size (Sala et al, 2001), and are even able to induce spines in normally aspiny neurons (Roussignol et al, 2005). Recently, it has been shown that multimers of the ProSAP/Shank adapter proteins are organized as huge macromolecular platforms in parallel to the postsynaptic membrane (Baron et al, 2006), which organize higher order receptor clustering, actin binding and reorganization as well as regulated signal transduction upon receptor stimulation (Sheng and Kim, 2000; Boeckers et al, 2002). Here, we report that in the central nervous system Abi-1 is a synaptic molecule that is tightly bound to the PSD matrix by interaction with ProSAP2/Shank3. Interestingly, Abi-1 is not confined to the PSD but shuttles to the nucleus upon NMDA application. Moreover, we found that Abi-1 is part of a Myc/Max complex of transcription factors. The entry into the nucleus is dependent on phosphorylation at tyrosine 53. Downregulation of Abi-1 during synaptogenesis by small interfering RNAs results in increase in the number of dendrites and a decrease in synaptic sites. These synapses display a significant higher percentage of immature morphology. These results point to an essential role of the Abi-1 complex in the establishment and structural reorganization of dendrites, spines and synapses. As Abi-1 proteins that are resistant to tyrosine 53 phosphorylation and unable to enter the nucleus cannot rescue the small interference RNA (RNAi) phenotype, it is tempting to speculate that the regulation of dendritic outgrowth and synapse maturation is at least in part dependent upon the nuclear role of Abi-1 interacting with the Myc/Max transcriptional complex. Results Abi-1 interacts with ProSAP2/Shank3 via the C-terminal SH3 domain In a yeast two-hybrid (YTH) screen using a conserved proline-rich region of ProSAP2/Shank3, we identified Abi-1 as a binding partner. Abi-1 is a 476-aa proline-rich protein comprising an N-terminal WAB (Wave binding) and SNARE (syntaxin binding) domain, a homeobox homology region (HHR domain) and a C-terminal SH3 domain (Figure 1A–C). In a detailed YTH assay, we identified a proline-rich consensus motif in ProSAP2/Shank3, which is responsible for the interaction with the Abi-1 SH3 domain (Figure 1A and D). A similar motif at a conserved, identical site in the protein is present in ProSAP1/Shank2 but not Shank1 (Figure 1A). The interaction via these domains could be confirmed by cotransfection experiments in Cos-7 cells using ProSAP2/Shank3, ProSAP1/Shank2 and Abi-1 deletion constructs (Figure 1E). The proline-rich segment of ProSAP2/Shank3 was recruited to Abi-1 or Abi-SH3 domain clusters upon coexpression (Figure 1E(II,III and VI)). In contrast, Shank1A coexpression, removal of the proline-rich domain of ProSAP2/Shank3 or the SH3 domain of Abi-1 abolished colocalization (Figure 1E(IV,V and VII)). To test whether the interaction between ProSAP2/Shank3 and Abi-1 is direct and occurs in vivo, we confirmed the interaction in a GST pull-down assay (Figure 1F). Finally, we could demonstrate the co-immunoprecipitation of Abi-1 from rat brain lysate using ProSAP2/Shank3 antibodies and vice versa. Moreover, the Abi-1 precipitate was found to contain ProSAP1/Shank2 and proteins of the Abi-1 complex (i.e. Eps8 and WAVE) but not MAP2 (Figure 1F). Figure 1.Abi-1 interacts with a proline-rich region of ProSAP2/Shank3. (A) Schematic representation of the ProSAP2/Shank3 protein showing the localization and sequence of a proline-rich region (bait) flanking the PDZ domain (conserved in ProSAP1/Shank2 and ProSAP2/Shank3). (B, C) Characteristic Abi-1 protein/protein interaction domains. The C-terminal SH3 domain is marked in gray. The tyrosine phosphorylation site (tyrosine 53) of Abi-1 is marked in black. The 476-aa Abi-1 protein is coding for proline-rich regions (PP), an N-terminal WAB and SNARE domain, a conserved Abi-1 HHR and a C-terminal SH3 domain. (D) Yeast two-hybrid assay showing that point mutations of the ProSAP2/Shank3 bait lead to a partial or complete loss of protein/protein interaction with Abi-1; pPPPxxxP was found as a consensus sequence. (E) GFP- and RFP-fusion proteins coding for ProSAP1,2/Shank2,3 or Abi-1 that are cotransfected in Cos7 cells colocalize in clusters when coding for the above-described interacting domains (I–IV and VIII). Interaction is abolished when the Abi-1 SH3 domain or the ProSAP2 proline-rich domain (PR) is deleted (IV and V); Shank1a does not cocluster with Abi-1 (VII). Permanent nuclear localization of Abi-1 by adding a nuclear localization signal (Abi-1-NLS-GFP) results in the recruitment of the ProSAP2/Shank3 proline-rich domain into the nucleus (VI). (F) Characterization of the monoclonal Abi-1 antibody used in the study. The antibody recognizes the endogenous protein in HeLA cells (lane 2), in hippocampal neurons (lane 3) but not in Cos7 cells (lane 1). Expression of different GFP proteins shows that the antibody is directed against the N-terminal part of the protein (lanes 1 and 5, untransfected Cos7 cells; lanes 2 and 6, Abi-1-GFP transfected; lanes 3 and 7, Abi-1ΔSH3-GFP transfected; lanes 4 and 8, Abi-1-SH3-GFP transfected). Co-immunoprecipitation (Co-IP) and GST pull-down experiments (PD) from rat brain lysate (5 μg of protein has been loaded) were performed. Rat brain extracts (input,1 and 4) were immunoprecipitated with Abi-1 (lanes 6–10), ProSAP2/Shank3 (lane 3) or control (IgG, lanes 2 and 5). Subsequently, immunoprecipitates were blotted for Abi-1 (lane 3) and ProSAP2/Shank3 (lane 6). These Co-IPs experiments from brain lysates show that both proteins interact in rat brain. Moreover, ProSAP1, Wave and Eps8 but not Map2 are detected in the Abi-1 precipitate (lanes 7–10). PD experiments show the direct interaction of the proline-rich region of ProSAP2 with Abi-1 in both directions. Download figure Download PowerPoint Abi-1 and interacting proteins are widely expressed in brain and are components of the postsynaptic density complex The expression profile of Abi-1 mRNA and protein in rat brain was analyzed by in situ hybridization and immunohistochemistry. During all stages of development, Abi-1 transcripts as well as Abi-1 protein were widely expressed in rat brain. High levels were detected in cortex, hippocampus (HC) and cerebellum (CE) (Figure 2A(I–III)). Higher magnification (Figure 2A(III)) revealed a dendritic and a punctate labeling of neuropil in all brain areas. Immunoelectron microscopy showed that Abi-1 can be detected in dendrites and at PSDs of excitatory synapses (Figure 2A(IV), inset, arrowheads). In addition, the antigen could also be identified in some cells close to the nuclear membrane and in the nucleus (Figure 2A(IV)). Postsynaptic staining of Abi-1 was confirmed by immunofluorescence staining of cultured hippocampal neurons where it completely overlaps with ProSAP2/Shank3 and colocalized with the presynaptic marker protein Bassoon (Figure 2B). Staining with antibodies directed against other components of the Abi-1 complex (Eps8, Sos-1 and WAVE) revealed that these molecules are also enriched in spines and/or PSDs (Figure 2B). After subcellular fractionation of brain tissue, ProSAP2/Shank3, Abi-1, c-Abl and WAVE were enriched within the PSD fraction, whereas Eps8 showed only a weak band in the PSD fraction. Sos-1 is not enriched within the cytoskeletal matrix of the PSD (see Figure 2C). The analysis of the spatial and temporal pattern of Abi-1 expression showed that the mRNA expression only slightly increases between days 0 and 14. Protein expression, however, rises significantly between days 1 and 3. Moreover, additional bands at higher molecular weight, reflecting phosphorylated Abi-1 (Courtney et al, 2000), are seen at later stages of neuronal development starting from day 10 onwards (Figure 2C). Immunohistochemical staining shows Abi-1 enriched in growth cones of young neurons (days 1 and 3; arrowheads). At later developmental stages (days 10 and 14), Abi-1 is finally localized in spines and synapses as shown (Figure 2B and C). Figure 2.Spatial and temporal localization of endogenous Abi-1 in hippocampal neurons and rat brain. (A) In situ hybridization and immunohistochemistry during rat brain development reveals high expression levels of Abi-1, especially in cortex, hippocampus (HC) and cerebellum (CE, I–III). In the hippocampal CA3 region, an intense punctate labeling of the stratum oriens and stratum radiatum is seen. In cerebellum, granule cells are immunopositive and some nuclei are stained (III). Immunoelectron microscopy (magnification × 37 800) revealed that Abi-1 can be detected in dendrites and at PSDs (arrowheads, inset). In some neurons, the DAB/silver/gold precipitate could also be identified close to the nuclear membrane and in the nucleus (star, IV). (B) Abi-1 at synapses in hippocampal neurons. DIV21 staining of Abi-1 and ProSAP2/Shank3 reveals colocalization of these proteins at excitatory synapses. Double staining of Abi-1 (green) and Bassoon (red) shows a pattern of juxtaposed proteins at synapses. Double staining of other Abi-1 complex-forming proteins (Eps8, Sos-1 and WAVE; green) with the presynaptic marker Bassoon (red) shows the localization of these proteins at spines/PSDs. (C) Subcellular fractionation indicates the strong enrichment of c-Abl, ProSAP2/Shank3 and Abi-1 within the PSD preparation, whereas WAVE and Eps8 are only moderately attached to the PSD cytoskeleton. Sos-1 is not found in the PSD matrix. Developmental analysis of Abi-1 mRNA and protein expression in hippocampal culture shows a significant increase of mRNA and protein levels between days 1 and 3 (actin mRNA and protein are detected as internal control). Abi-1 staining of developing neurons reveals the localization of Abi-1 in dendrites and growth cones (arrowheads); from day 10 onwards the protein is localized to spines and synapses. Download figure Download PowerPoint Abi-1 is also a nuclear protein in neurons and shuttles from the PSD to the nucleus upon NMDA application Endogenous Abi-1 was found in the nucleus after treatment with the nuclear export inhibitor leptomycinB in NIH cells as well as in hippocampal neurons (Figure 3A), indicating that Abi-1 shuttles between cytoplasm and nucleus. As Abi-1 is concentrated in PSDs, we tested whether a translocation of Abi-1 can be induced by NMDA application. NMDA treatment for 3 min resulted in nuclear staining and a decrease of dendritic Abi-1 labeling that could already be observed 3 min after changing the media (Figure 3B(I)). Nuclear staining was substantiated by confocal analysis (Figure 3B(II)) and significantly increased with highest levels after 60 min. Moreover, it was found to be completely reversible, as after 2 h the neuronal staining pattern was identical to nontreated controls (Figure 3B(I)). The NMDA-induced translocation of Abi-1 could also be observed in acute slice preparations from rat brain. In contrast to controls, neurons of the NMDA-treated slices exhibited an obvious nuclear staining for Abi-1 (Figure 3C). The accumulation of Abi-1 in the nucleus could be verified by Western blot analysis of the cytoplasmic and the nuclear/pellet fraction of NMDA-stimulated neurons (Figure 3C). Moreover, arrowheads indicate the appearance of slightly larger Abi-1 bands in the nuclear/pellet fractions that have already been characterized as phosphorylated Abi-1 proteins (Juang and Hoffmann, 1999; Courtney et al, 2000). In vitro phosphorylation experiments of wild-type and mutated Abi-1-GFP proteins with a recombinant abelson kinase revealed that the mutation at tyrosine 53 results in a nearly complete loss of abelson kinase-mediated tyrosine phosphorylation. Figure 3.Abi-1 translocates from the PSD to the nucleus after NMDA treatment. (A) Treatment of NIH cells as well as hippocampal neurons with the nuclear export inhibitor leptomycinB results in the nuclear accumulation of Abi-1. (B) Application of NMDA (100 μM) to DIV21 neurons for 3 min leads to nuclear translocation of Abi-1. The most intense and significant nuclear staining of Abi-1 was observed after 60 min; in parallel, the dendritic compartment becomes more and more devoid of Abi-1 protein. Redistribution of Abi-1 into the dendrites can be observed from 90 min up to 2 h. The synaptic depletion of Abi-1 does not influence the gross morphology and number of synaptic contacts as shown by Bassoon staining (red) and Abi-1 staining (green) at time points 0 and 120 min. For quantification of nuclear Abi-1 staining, the total fluorescence as nuclear gray levels (linear scale of 0–256) was measured (I). Confocal analysis showed nuclear localization of Abi-1 in an NMDA-treated hippocampal neuron (II). (C) Acute rat brain slices were incubated with NMDA for 30 min, followed by Abi-1 DAB immunostaining. More than 90% of the pyramidal neurons in the stimulated slices exhibited nuclear staining (arrowheads). For Western blot analysis of Abi-1 protein, lysates of these neurons were separated into a nuclear/pellet (P) and a cytoplasmic fraction (C). Histone-H2B antibodies were used as a nuclear marker. Note the slight shift of Abi-1 protein from the cytoplasm to the nuclear pellet fraction. Arrowheads indicate the appearance of slightly larger Abi-1 bands that have already been characterized as phosphorylated Abi-1 protein. For an in vitro phosphorylation assay, Abi-1-GFP (lane 3) and the mutated Abi-1(Y53A)-GFP protein (lane 5) were transfected into Cos7 cells (lane 1 as untransfected control), immunoprecipitated on a column and incubated with a recombinant abelson tyrosine kinase using radioactive 32P-labeled ATP. After gel separation of the proteins, blotting and exposure to X-ray film, it could be shown in independent experiments that the WT-Abi-1-GFP was heavily phosphorylated by the abelson kinase, whereas the mutated form (Abi-1(Y53A)-GFP) showed nearly no signal on the X-ray film (loaded Abi-1 protein amounts are shown by immunodetection, lanes 4 and 6). Significances: *0.01, **0.001, ***0.0001. Download figure Download PowerPoint Targeting of Abi-1 to the PSD is SH3 domain dependent, whereas the nuclear accumulation is regulated by the phosphorylation of tyrosine 53 To investigate the targeting mechanisms for the synaptic and nuclear compartment, we employed Abi-1-GFP deletion constructs. The full-length protein as well as the SH3 domain alone was perfectly targeted to synaptic contacts as revealed by Bassoon staining. In contrast, constructs missing the SH3 domain were evenly distributed in the cytoplasm and neurites (Figure 4A). Figure 4.Determinants of Abi-1 targeting to the nuclear and synaptic compartments. (A) Full-length Abi-1-GFP protein and the Abi-1 SH3 domain colocalize with the presynaptic marker protein Bassoon (red, insets). Deletion of the SH3 domain results in the loss of synaptic targeting. (B) Phosphorylation of Abi-1 tyrosine 53 is a prerequisite for nuclear import leptomycinB treatment of Abi-1-(53Y-A)-Myc-transfected HeLA cells (localized in the cytoplasm under control conditions, I) leads to accumulation of the protein in the perinuclear area (II). Wild-type Abi-1-Myc (III) is enriched in the nucleus after leptomycinB treatment. (C) After NMDA application to hippocampal neurons, full-length Abi-1-GFP as well as the endogenous Abi-1 (red) readily translocates into the nucleus (yellow staining, I). Transfected Abi-1 protein, mutated at tyrosine 53 (Abi-1-(53Y-A)-GFP), accumulates after NMDA application in the perinuclear area. Endogenous Abi-1 (red) is enriched in the nucleus (II). (D) Inhibition of c-Abl by the specific compound STI571 for 24 h before NMDA treatment results in a perinuclear accumulation and no nuclear enrichment of Abi-1 (compare B and C). Nuclear translocation can also be prevented by the disturbance of cytoskeletal components with colchicin or cytochalasinD. The application of anisomycin or brefeldinA does not influence nuclear accumulation. Download figure Download PowerPoint In order to analyze whether the phosphorylation of tyrosine 53 influences synaptic or nuclear targeting, we designed Abi-1-GFP and Myc-tagged fusion proteins with a mutated tyrosine at position 53(Y-A). After application of leptomycinB for 4 h, the mutated Abi-1(53Y-A) fusion protein did not accumulate in the nucleus but accumulated in the perinuclear area (Figure 4B(I and II)). Abi-1-Myc, however, readily entered the nucleus (Figure 4B(III)). Next, we transfected hippocampal neurons with the described Abi-1-GFP chimeras, applied NMDA, fixed and additionally counterstained with the Abi-1 antibody to detect the wild-type protein simultaneously as an internal control. Abi-1-GFP as well as Abi-1(53Y-A)-GFP was targeted to synaptic sites. After NMDA treatment, Abi-1-GFP accumulated in the nucleus, whereas the mutated Abi-1(53Y-A)-GFP was enriched in the perinuclear area (Figure 4C(I and II)), indicating that the tyrosine 53 phosphorylation is not important for synaptic targeting but a prerequisite for nuclear entry. To substantiate these findings, we applied the small-molecule inhibitor of abl family tyrosine kinases, STI571 (Buchdunger et al, 1996), for 24 h before NMDA application. Specific inhibition of c-Abl prevented the translocation of wild-type Abi-1 into the nucleus but led to an accumulation of the protein in the perinuclear area, similar to the mutated Abi-1(53Y-A)-GFP chimera (Figure 4D). To differentiate between the proposed synapto-nuclear transport of Abi-1 and novel protein synthesis, we inhibited the translation machinery and the Golgi apparatus with anisomycin and brefeldinA. Both treatments did not affect nuclear accumulation of Abi-1. The treatment of hippocampal neurons with colchicin or cytochalasinD, however, prevented nuclear translocation, suggesting that Abi-1 nuclear transport is directly or indirectly dependent on a functional microtubular and microfilament system (Figure 4D). Abi-1 protein expression in developing neurons regulates dendritic outgrowth and branching and determines the shape and number of synaptic contacts According to the discrete distribution of Abi-1 during developmental maturation (Figure 2C), we analyzed the functional importance of Abi-1 for neuronal cell morphology and synapse formation by altered Abi-1 protein concentrations. To that end, we elevated Abi-1 protein levels by Abi-1 (over)expression, or applied small RNAi technology to selectively knock down Abi-1. Rescue experiments were performed with mutated RNAi-resistant Abi-1-Myc fusion proteins that carry seven conservative nucleotide exchanges localized at the RNAi targeting area. In contrast to the control vector, Abi-1 protein levels and staining intensity were markedly reduced in RNAi-transfected HeLa and neuronal cells (Figure 5A and B). Hippocampal neurons displayed a highly branched dendritic tree when transfected with the RNAi construct. The increased branching of MAP2-positive dendrites, as determined by the total number of dendrites or branching points (Quitsch et al, 2005), was highly significant at both time points investigated (Figure 5C–G). Interestingly, early reduction of Abi-1 (day 3) had a strong effect on primary dendrites, and transfection at day 7 resulted in a significant increase of secondary and tertiary dendrites (Figure 5F). The analysis of the total number of branching points (TNBPs) in neurons transfected at day 7 accordingly showed a significant increase (Figure 5G). Overexpression of Abi-1 had an opposite effect and reduced the TNBPs (Figure 5E and G); the Abi-1(53Y-A) fusion protein was less effective in reducing TNBPs compared with the wild-type protein. The RNAi-resistant Mut-Abi-1(53Y-A) was unable to rescue the RNAi-induced phenotype in neurons depleted of wild-type Abi-1 protein. When the RNAi construct was transfected at later stages of neuronal development, no obvious dendritic phenotype could be observed (data not shown). Figure 5.Abi-1 protein levels influence dendritogenesis and synaptic maturation. (A, B) The transfection of HeLa cells and hippocampal neurons with the Abi-1-RNAi construct reduces the level of endogenous Abi-1 protein to barely detectable levels. (C, D) In neurons transfected at day 3, the downregulation of Abi-1 leads to a highly branched MAP2-positive dendritic tree. The TNBPs is significantly elevated in RNAi-transfected neurons (E–G). This phenotype is also observed after transfection on day 7, showing especially high numbers of secondary and tertiary dendrites. Overexpression of Abi-1 protein results in a simplified dendritic tree, with a reduction of the TNBPs compared with controls. Significances compared with the empty vector controls are indicated above the bars. Note that RNAi-resistant wild-type Abi-1 cotransfected with RNAi (RNAiAbi-1+Mut Abi-1) reduces TNBPs significantly, whereas the RNAi-resistant Abi-1-(53Y-A) construct is not able to compensate (or overcompensate) the loss of endogenous Abi-1 (RNAiAbi-1+Mut-Abi-1-(53Y-A)) (H–J). Analysis of synaptic contacts reveals a significant reduction of Bassoon-positive boutons in Abi-1-RNAi-transfected neurons. Moreover, RNAi-transfected neurons show a high percentage of immature synaptic contacts on thin filopodia-like protrusions with apical Bassoon-positive presynaptic terminals (arrow heads). In contrast, the overexpression of Abi-1-GFP results in a significant increase of mature synaptic contacts at the dendritic shaft. Note that RNAi-resistant Abi-1 construct cotransfected with RNAi (RNAiAbi-1+Mut Abi-1) also significantly increases the synapse number as well as maturity. In contrast, the RNAi-resistant Abi-1-(53Y-A) construct alone or cotransfected with RNAiAbi-1 is not able to increase synapse number or change synapse morphology. Significances compared with the empty vector controls pSuper and pSuper+pEGFP are" @default.
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• W2106971401 date "2007-02-15" @default.
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• W2106971401 title "Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation" @default.
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