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• W2014474433 abstract "IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm. IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm. Rho GTPases such as Cdc42, Rac, and Rho control key events in neuronal cell biology, including the generation of neuronal polarity and morphology, establishment of dendritic spines, the generation of postsynaptic specializations and synaptic plasticity (1Govek E.E. Newey S.E. Van Aelst L. Genes Dev. 2005; 19: 1-49Crossref PubMed Scopus (764) Google Scholar, 2Tashiro A. Minden A. Yuste R. Cereb. Cortex. 2000; 10: 927-938Crossref PubMed Scopus (348) Google Scholar). Specificity in these processes is thought to arise through control of different downstream targets which are recognized and activated by the active, GTP-bound forms of Rho family members. The insulin receptor substrate of 53 kDa (IRSp53) 3The abbreviations used are: IRSp53; insulin receptor substrate of 53 kDa; fEPSP, field excitatory postsynaptic potential; LTP, long term potentiation; PSD, postsynaptic density; PSD-95, PSD protein of 95 kDa; PDZ, PSD-95/discs large/ZO-1; PPF, paired-pulse facilitation; SH3, Src homology; NMDA, N-methyl-d-aspartate; PBS, phosphate-buffered saline. is an essential mediator between activated Rac or Cdc42 and the formation of lamellipodia or filopodia, respectively. GTPase binding to IRSp53 enables interactions of its SH3 domain with downstream effectors WAVE2, Mena, Eps8, or N-WASP, all of which are known regulators of actin dynamics (3Miki H. Yamaguchi H. Suetsugu S. Takenawa T. Nature. 2000; 408: 732-735Crossref PubMed Scopus (457) Google Scholar, 4Krugmann S. Jordens I. Gevaert K. Driessens M. Vandekerckhove J. Hall A. Curr. Biol. 2001; 11: 1645-1655Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 5Disanza A. Mantoani S. Hertzog M. Gerboth S. Frittoli E. Steffen A. Berhoerster K. Kreienkamp H.J. Milanesi F. Di Fiore P.P. Ciliberto A. Stradal T.E. Scita G. Nat. Cell Biol. 2006; 8: 1337-1347Crossref PubMed Scopus (183) Google Scholar, 6Lim K.B. Bu W. Goh W.I. Koh E. Ong S.H. Pawson T. Sudhaharan T. Ahmed S. J. Biol. Chem. 2008; 283: 20454-20472Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In addition, the N-terminal IRSp53/missing in metastasis homology domain of IRSp53 assists in generating cellular protrusions by bundling actin filaments (5Disanza A. Mantoani S. Hertzog M. Gerboth S. Frittoli E. Steffen A. Berhoerster K. Kreienkamp H.J. Milanesi F. Di Fiore P.P. Ciliberto A. Stradal T.E. Scita G. Nat. Cell Biol. 2006; 8: 1337-1347Crossref PubMed Scopus (183) Google Scholar, 7Yamagishi A. Masuda M. Ohki T. Onishi H. Mochizuki N. J. Biol. Chem. 2004; 279: 14929-14936Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 8Millard T.H. Bompard G. Heung M.Y. Dafforn T.R. Scott D.J. Machesky L.M. Futterer K. EMBO J. 2005; 24: 240-250Crossref PubMed Scopus (193) Google Scholar) and promoting membrane curvature (9Suetsugu S. Kurisu S. Oikawa T. Yamazaki D. Oda A. Takenawa T. J. Cell Biol. 2006; 173: 571-585Crossref PubMed Scopus (134) Google Scholar, 10Mattila P.K. Pykalainen A. Saarikangas J. Paavilainen V.O. Vihinen H. Jokitalo E. Lappalainen P. J. Cell Biol. 2007; 176: 953-964Crossref PubMed Scopus (301) Google Scholar). Expression of IRSp53 is particularly high in the brain, and consequently IRSp53 contributes to the formation of dendritic spines in the cultured hippocampal neuron model (11Choi J. Ko J. Racz B. Burette A. Lee J.R. Kim S. Na M. Lee H.W. Kim K. Weinberg R.J. Kim E. J. Neurosci. 2005; 25: 869-879Crossref PubMed Scopus (176) Google Scholar). Via the SH3 domain and a C-terminal PDZ binding motif, IRSp53 also bridges postsynaptic shank and PSD-95 family members (11Choi J. Ko J. Racz B. Burette A. Lee J.R. Kim S. Na M. Lee H.W. Kim K. Weinberg R.J. Kim E. J. Neurosci. 2005; 25: 869-879Crossref PubMed Scopus (176) Google Scholar, 12Soltau M. Richter D. Kreienkamp H.J. Mol. Cell. Neurosci. 2002; 21: 575-583Crossref PubMed Scopus (79) Google Scholar, 13Bockmann J. Kreutz M.R. Gundelfinger E.D. Bockers T.M. J. Neurochem. 2002; 83: 1013-1017Crossref PubMed Scopus (105) Google Scholar, 14Soltau M. Berhorster K. Kindler S. Buck F. Richter D. Kreienkamp H.J. J. Neurochem. 2004; 90: 659-665Crossref PubMed Scopus (73) Google Scholar). A significant enrichment in the postsynaptic density (PSD) of excitatory synapses suggests that Rac/Cdc42 signaling via IRSp53 may exert an as yet undefined role in postsynaptic signal transduction. Here we have investigated the physiological role of IRSp53 by analyzing IRSp53-deficient mice. Our data indicate that the cellular and physiological functions of IRSp53 depend on the effector proteins present. Changes in neuronal morphology due to IRSp53 deficiency that were determined in primary developing cultured neurons in vitro are not observed in brains of developing and adult mice in vivo. The interaction of IRSp53 with the postsynaptic shank proteins abrogates the effects of IRSp53 on the actin cytoskeleton while revealing a synaptic function, which is evidenced by the enhanced synaptic plasticity in IRSp53 KO mice as well as their inability to learn in the hippocampus-dependent contextual fear-conditioning task. Generation of IRSp53-deficient Mice-Embryonic stem cell clone XG-757 (Baygenomics; San Francisco, CA) was found by BLAST analysis to carry a transgenic insertion (“exon trap”) within the mouse IRSp53 gene. After injection of XG-757 cells into blastocysts and implantation, numerous chimeric animals were obtained. These gave rise to heterozygous animals carrying the transgene insertion after cross-breeding with C57BL/6 mice. Genotyping was performed using a forward primer in exon 3, one reverse primer in intron 3 for the WT allele, and one reverse primer in the exon trap insertion for the mutant allele. Animals used for most experiments in this study were in a 129P2/C57BL/6 hybrid background, with the exception of CA1 long term potentiation (LTP) and the behavioral experiments, which were performed after back-crossing for seven generations into the C57BL/6 strain. Morphological Analysis of Mouse Brains-Mouse brains were subjected to Golgi staining, sectioning, and embedding according to published procedures (15Glaser E.M. Van der Loos H. J. Neurosci. Methods. 1981; 4: 117-125Crossref PubMed Scopus (321) Google Scholar, 16Khelfaoui M. Denis C. van Galen E. de Bock F. Schmitt A. Houbron C. Morice E. Giros B. Ramakers G. Fagni L. Chelly J. Nosten-Bertrand M. Billuart P. J. Neurosci. 2007; 27: 9439-9450Crossref PubMed Scopus (117) Google Scholar). 150-μm coronal sections were used for all analyses. For spine analysis, sections were viewed using an Axiovert 135 microscope equipped with 100× objective, Hamamatsu camera, and OpenLab software. Images of adjacent focal planes were stacked and analyzed using ImageJ. Length, width, and the number of spines per dendrite length were logged into an Excel spreadsheet using a modified macro from the ImageJ software. Dendrite branching in young animals was analyzed after importing stacks of microscopic images into ImageJ and tracing neurons through different layers of the stack. Because of their strongly increased complexity, analysis of dendrites in the CA1 region of adult hippocampi required the use of a Neurolucida setup at the Institute for Biology, Free University of Berlin (kindly provided by Prof. Dietmar Kuhl, Berlin). Neuronal Primary Culture-Hippocampal neurons were prepared from P0 animals (mice) or E19 animals (rats) and cultivated and transfected as described (17Quitsch A. Berhorster K. Liew C.W. Richter D. Kreienkamp H.J. J. Neurosci. 2005; 25: 479-487Crossref PubMed Scopus (80) Google Scholar) using Neurobasal media supplemented with B-27 (Invitrogen). Neurons were fixed after 14 days in culture with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and stained as described (17Quitsch A. Berhorster K. Liew C.W. Richter D. Kreienkamp H.J. J. Neurosci. 2005; 25: 479-487Crossref PubMed Scopus (80) Google Scholar). Mouse anti-MAP2 (1:2000; Sigma) was used as the primary antibody followed by Cy3-labeled secondary antibody. Immunostaining was visualized using a Zeiss Axiovert 135 microscope equipped with a Hamamatsu camera or a confocal microscope (Zeiss LSM 510). Openlab 2.2.5 software (Improvision) was used for image analysis. Dendritic branching was quantified as described (17Quitsch A. Berhorster K. Liew C.W. Richter D. Kreienkamp H.J. J. Neurosci. 2005; 25: 479-487Crossref PubMed Scopus (80) Google Scholar). The number of branch points per dendrite was quantified and logged into an Excel spreadsheet. Statistical significance was in each case determined by Student's t test. Biochemical Analyses-Fusion proteins (full-length His6-tagged IRSp53; GST-Cdc42; GST-IRSp53-SH3; GST-Eps8-N-PPP, residues 201–250; GST-shank3-PPP, residues 877–938; GST-shank1-PPP, residues 858–970) were generated using standard methods. For actin bundling, monomeric G-actin was polymerized at room temperature in F-actin buffer (5 mm Tris-HCl, pH 7.8, 0.2 mm ATP, 1 mm dithiothreitol, 0.1 mm CaCl2, 1 mm MgCl2, and 100 mm KCl). F-actin was mixed with varying concentrations of recombinant proteins and incubated at room temperature for 30 min as described (5Disanza A. Mantoani S. Hertzog M. Gerboth S. Frittoli E. Steffen A. Berhoerster K. Kreienkamp H.J. Milanesi F. Di Fiore P.P. Ciliberto A. Stradal T.E. Scita G. Nat. Cell Biol. 2006; 8: 1337-1347Crossref PubMed Scopus (183) Google Scholar). Actin was then labeled with rhodamine-phalloidin, and 0.1% 1,4-diazabicyclo(2.2.2)octane and 0.1% methyl cellulose were added to the mixture. Samples were mounted between a slide and a coverslip coated with poly-lysine and imaged by fluorescence microscopy. The number of actin bundles in at least 10 randomly chosen microscopic fields was counted blindly by at least two investigators. PSDs were prepared as described (12Soltau M. Richter D. Kreienkamp H.J. Mol. Cell. Neurosci. 2002; 21: 575-583Crossref PubMed Scopus (79) Google Scholar) from forebrains of 2-week- and 2-month-old mice. For immunoprecipitation analysis, hippocampi were prepared from rats at different developmental stages. After lysis in immunoprecipitation buffer (50 mm Tris/HCl, pH 7.5, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, plus protease inhibitors pepstatin, leupeptin, and trasylol), samples were cleared by centrifugation and subjected to immunoprecipitation using immobilized anti-IRSp53. Material insoluble in immunoprecipitations buffer was solubilized in 1% SDS and again cleared by centrifugation. Samples were analyzed by Western blotting. Electron Microscopy-Anesthetized animals were perfusion fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer with 1% sucrose, pH 7.3. Brains were post-fixed with 1% osmium tetroxide for 4 h. Small hippocampal tissue pieces were dehydrated through an ethanol series, stained with 2% uranyl acetate, and embedded in epoxy resin (Epon 812, Fluka, Germany). Ultrathin sections (70–80 nm) were cut with a diamond knife and collected on 300-mesh grids. Sections were stained with lead citrate and examined at a voltage of 80 kV using an EM10 transmission electron microscope (Carl Zeiss, Germany). Images were scanned at 600 dots/inch; PSDs were identified as dense membrane-associated structures in apposition to nerve terminals. For each animal at least 20 sections were analyzed. All PSDs from each section were scored for length, width, and area using ImageJ in combination with Excel. For all morphological analyses, the person evaluating the microscopic images was blind to the experimental conditions. Immunohistochemistry-For pre-embedding immuno light microscopy and electron microscopy, animals were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4. Brains were cut into 30- and 150-μm-thick Vibratome sections, respectively, and immersed in 2.3 mol/liter sucrose in PBS overnight at 4 °C for cryoprotection. Thereafter, they were subjected to two freeze-thaw cycles in liquid nitrogen to aid penetration of immunoreagents, incubated with 10% horse serum containing 0.2% bovine serum albumin (BSA) (blocker) for 15 min, and left overnight at 4 °C in IRSp53 antibody (1:25) in PBS containing 1% PS and 0.2% BSA (Carrier). After washing in PBS, sections were incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:1000 in Carrier for 90 min. After rinsing, they were incubated with ABC (Vector Laboratories) diluted 1:100 in PBS for 90 min. The sections were washed in PBS and reacted in diaminobenzidine-H202 solution (Sigma) for 10 min. The 30-μm sections were mounted on glass coverslips for light microscopic observation, and the 100-μm-thick sections were further fixed with 1% osmium tetroxide (w/v), dehydrated in an ascending series of ethanol, and embedded in Epon (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Ultrathin sections were examined with a Zeiss EM 902. Electrophysiological Recordings-For in vivo recordings in the dentate gyrus, mice were anesthetized with urethane (Sigma, 1.5 g/kg body weight), and tracheotomies were performed as described (18Stoenica L. Senkov O. Gerardy-Schahn R. Weinhold B. Schachner M. Dityatev A. Eur. J. Neurosci. 2006; 23: 2255-2264Crossref PubMed Scopus (74) Google Scholar). The recording electrode was placed 2 mm posterior and 1.5 mm lateral to bregma. The stimulation electrode was inserted under an angle of 70 degrees 0.5 mm anterior and 1.5 mm lateral to lambda. An additional hole was drilled on the surface of the contralateral frontal cortex for the ground electrode made of chlorided silver wire. A stimulating electrode was made of two 125-μm Teflon-insulated stainless steel wires, whereas a recording electrode was constructed of 75-μm Teflon-insulated tungsten wire. Electrodes were advanced until large positive field excitatory postsynaptic potentials (fEPSPs) with superimposed negative population spikes were evoked. The z-coordinate was adjusted to maximize the field responses to a biphasic current pulse of 100 μs and 200–400 μA elicited using an isolated pulse stimulator (Model 2100; A-M Systems, Everett, WA). The stimulation strength for recordings before and after induction of LTP was selected to provide a population spike of 2–3 mV. The responses were collected every 30 s, and averages of five consecutive sweeps were used for analysis. LTP was induced by 6 trains (inter-train interval of 20 s) of 6 bursts (inter-burst interval of 200 ms) of 6 pulses applied at 400 Hz. The stimulation intensity used for induction of LTP was two times higher than the one used for base-line recordings. Only experiments without rundown of fEPSPs (>90% of base line) were included in the analysis. The recorded signals were amplified ×100 and filtered (0.1–5 kHz bandpass) using a differential AC amplifier (Model 1700; A-M Systems) and digitized using a Pico ADC board (18Stoenica L. Senkov O. Gerardy-Schahn R. Weinhold B. Schachner M. Dityatev A. Eur. J. Neurosci. 2006; 23: 2255-2264Crossref PubMed Scopus (74) Google Scholar). The signals were acquired and measured using the LTP Program (19Anderson W.W. Collingridge G.L. J. Neurosci. Methods. 2001; 108: 71-83Crossref PubMed Scopus (348) Google Scholar). For measurements of the slope of fEPSPs, only the initial part (∼0.5 ms) of the responses was used, as shown by arrows in Fig. 4D. Acute hippocampal slices were prepared from adult (5–6-month-old) mice. IRSp53 KO animals and age-matched WT animals from the same line were used. In brief, mice were anesthetized and decapitated, and the brains were quickly removed and placed into ice-cold constantly aerated (95% O2, 5%CO2) artificial cerebrospinal fluid (125 mm NaCl, 2.5 mm KCl, 2 mm MgCl2, 26 mm NaHCO3, 1.25 mm NaH2PO4, 2 CaCl2, 25 glucose) for 3 min. Hippocampi were isolated and cut into 400-μm-thick transversal slices with a Vibratome (VT 1000S; Leica, Nussloch, Germany), and sections were kept submerged in a storage chamber in aerated artificial cerebrospinal fluid. Before electrical recording slices were left in the storage chamber for ≥1 h at room temperature so that neuronal activity could recover to base line. For the extracellular recordings, slices were transferred to a recording chamber and kept submerged at 32 °C while constantly being perfused (1.5 ml/min) with aerated recording-artificial cerebrospinal fluid (125 mm NaCl, 2.5 mm KCl, 1 mm MgCl2, 26 mm NaHCO3, 1.25 mm NaH2PO4, 2 mm CaCl2, 25 mm glucose). Schaffer collaterals in area CA3 were stimulated by monopolar tungsten electrodes (9–10 megaohms), and fEPSPs were recorded by a glass electrode (4–15 megaohms) filled with 3 m NaCl and placed in the stratum radiatum of CA1. Synaptic field potentials were elicited with a frequency of 0.1 Hz, and LTP was induced with a theta burst stimulation of 10 bursts with four pulses each (100 Hz, 100-μs pulse duration, 200-ms inter-burst interval) which was repeated 3 times with an interval of 10 s. Data were collected, and slopes of the fEPSPs were calculated with a LabView (National Instruments, Munich, Germany)-based program. Paired-pulse facilitation was calculated as the second fEPSP slope divided by the first fEPSP slope a as percentage for inter-stimulus interval of 10, 20, 40, 80, 160 ms. Behavioral Analysis-Adult male mice (10 for each genotype) were studied in the open field (to analyze locomotion, exploration, and anxiety), accelerated Rotarod (motor coordination) and contextual fear conditioning (learning and memory) tests, as described (20Freitag S. Schachner M. Morellini F. Behav. Brain Res. 2003; 145: 189-207Crossref PubMed Scopus (68) Google Scholar). The open field test was performed in a box (50 × 50 cm and 40 cm high) illuminated with white light (100 lux). Mice were started from one corner of the box, and their behavior was analyzed for 15 min. Distance moved, mean velocity, and time spent in the center (an imaginary 25 × 25-cm square in the middle of the arena) were analyzed with the software EthoVision (Noldus, Wageningen, The Netherlands). In the accelerated Rotarod test mice had to walk on a turning, corrugated rod (3.2 cm in diameter) (Acceler Rotarod for mice Jones & Roberts, TSE Systems, Bad Homburg, Germany). Mice underwent 2 familiarization trials followed by 3 trials (the intertrial interval was about 45 min). The familiarization trials were performed at low (4 rpm) constant speed for a maximum duration of 3 min. Trials 1–3 were performed with the accelerating rod, starting with 4 rpm up to 40 rpm within 4 min, with a maximum duration of 5 min. On the following day, a fourth trial on the accelerating rod was carried out. The performance was evaluated by scoring the latency to fall. In the contextual fear conditioning test mice had to learn the association between the unconditioned (electric footshock) and conditioned (context) stimuli. Mice were conditioned in the context, a chamber (23.5 × 23.5 cm and 19.5 cm high) with Plexiglas walls and ceiling and a stainless grid floor from which an electric shock could be elicited. The chamber was illuminated by white light (10 lux). Mice were placed in the center of the cage and received three electric footshocks (0.25 mA, 1 s) at 120, 160, and 200 s. At 240 s the recoding ended, and the mouse was immediately returned into its home cage. Twenty-four hours after conditioning, mice were placed again in the context for 3 min. The conditioned response was analyzed by quantifying the percentage of time spent freezing (defined as absence of body movements for at least 1 s). Freezing behavior was automatically analyzed using a modified version of the system Mouse-E-Motion (Infra-e-motion, Hamburg, Germany). All behavioral data were analyzed by two-way analysis of variance for repeated measurements having a genotype as between group factor and time interval (for the open field test) and trial (for the accelerated Rotarod and contextual fear conditioning tests) as within group factor. When appropriate, Newman-Keuls post-hoc analyses were performed. Generation of IRSp53-deficient Mice-We generated IRSp53-deficient animals to determine the functional relevance of the protein in the nervous system. For this we used an ES cell line which harbored an insertion of an exon trapping construct (coding for a neomycin phosphoribosyl transferase/β-galactosidase fusion protein) within intron 3 of the mouse IRSp53 gene (Fig. 1, A and B). Injection into blastocysts yielded offspring with efficient germ line transmission, leading to mice heterozygous and eventually homozygous for the mutant IRSp53 gene. Two independent lines of mice were obtained. The exon trapping cassette leads to a IRSp53/neo/β-galactosidase fusion protein which contains only 73 (of 521) amino acids of IRSp53, leading to a complete functional loss of the protein. Accordingly, IRSp53 immunoreactivity is lost from brains of homozygous mutant mice in immunohistochemical as well as Western blot analyses using a C terminus-directed antibody (Fig. 1, C and D). Genotyping of offspring from heterozygous crosses showed that IRSp53 KO mice were born at a reduced mendelian ratio (6.6% and 7.2% in the 2 lines of mice we generated with respect to the expected 25%). Survival rates improved to 12% for one of the lines after back-crossing into a C57Bl6 background. Surviving KO animals appeared healthy and displayed no gross abnormalities. Analysis of the brains of adult heterozygous animals by staining for β-galactosidase activity (which arises due to the expression of the IRSp53/neo/β-galactosidase fusion protein) revealed that cortex, striatum, and hippocampus exhibit high levels of β-galactosidase (and by inference IRSp53) expression (Fig. 1E), in agreement with a previous in situ hybridization analysis (21Thomas E.A. Foye P.E. Alvarez C.E. Usui H. Sutcliffe J.G. Neurosci. Lett. 2001; 309: 145-148Crossref PubMed Scopus (16) Google Scholar). Notably, in mouse embryos (shown here for day E15) the central nervous system is almost completely devoid of IRSp53 expression, as analyzed by β-galactosidase staining (Fig. 1F), indicating that expression is switched on postnatally (also see below). Strong expression of IRSp53 in mouse embryos is, however, observed in many peripheral tissues, including thymus, submandibular gland, pituitary, choroid plexus, olfactory epithelium, and the kidney (Fig. 1F). IRSp53 has been suggested to mediate the signaling from activated (GTP-bound) Rho GTPases leading to the generation of cellular protrusions (3Miki H. Yamaguchi H. Suetsugu S. Takenawa T. Nature. 2000; 408: 732-735Crossref PubMed Scopus (457) Google Scholar, 5Disanza A. Mantoani S. Hertzog M. Gerboth S. Frittoli E. Steffen A. Berhoerster K. Kreienkamp H.J. Milanesi F. Di Fiore P.P. Ciliberto A. Stradal T.E. Scita G. Nat. Cell Biol. 2006; 8: 1337-1347Crossref PubMed Scopus (183) Google Scholar, 6Lim K.B. Bu W. Goh W.I. Koh E. Ong S.H. Pawson T. Sudhaharan T. Ahmed S. J. Biol. Chem. 2008; 283: 20454-20472Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Because of the high expression of the protein in the hippocampus, we expected changes in dendritic or spine morphology in this brain region in KO mice. Therefore, we performed a thorough morphological analysis of hippocampi from WT and KO mice at the light microscopic level. The overall morphology appeared unchanged, as analyzed by Nissl staining (Fig. 2A). In addition, Golgi staining revealed that the numbers of primary dendrites and of dendritic branches were not changed in the hippocampus of IRSp53 KO mice (Fig. 2, B–D). To rule out a compensatory effect during development, we also analyzed dendritic branching in young animals (age 9 days); again no differences between WT and KO animals were observed in the CA1 area of the hippocampus (Fig. 2E). Earlier (i.e. prenatal) stages were not analyzed as IRSp53 is not expressed in the embryonic brain (see above, Fig. 1F). Because IRSp53 was previously shown to enhance the formation and maturation of dendritic spines in cultured neurons (11Choi J. Ko J. Racz B. Burette A. Lee J.R. Kim S. Na M. Lee H.W. Kim K. Weinberg R.J. Kim E. J. Neurosci. 2005; 25: 869-879Crossref PubMed Scopus (176) Google Scholar), we also analyzed spine morphology. When comparing Golgi-stained brain sections of WT and KO mice, we could not detect any significant changes in the length, width, frequency, and form (determined as length/width ratio) of dendritic spines in CA1 and dentate gyrus (DG) regions (Fig. 2, F and G). Thus, in the hippocampus, IRSp53 KO animals do not display any significant structural changes when examined at the light microscopic level either during early postnatal development or in the adult stage. IRSp53-containing Complexes Differentially Regulate Actin Dynamics-Our findings contradict previous evidence implying IRSp53 as a key regulator of cellular morphology (3Miki H. Yamaguchi H. Suetsugu S. Takenawa T. Nature. 2000; 408: 732-735Crossref PubMed Scopus (457) Google Scholar, 5Disanza A. Mantoani S. Hertzog M. Gerboth S. Frittoli E. Steffen A. Berhoerster K. Kreienkamp H.J. Milanesi F. Di Fiore P.P. Ciliberto A. Stradal T.E. Scita G. Nat. Cell Biol. 2006; 8: 1337-1347Crossref PubMed Scopus (183) Google Scholar, 11Choi J. Ko J. Racz B. Burette A. Lee J.R. Kim S. Na M. Lee H.W. Kim K. Weinberg R.J. Kim E. J. Neurosci. 2005; 25: 869-879Crossref PubMed Scopus (176) Google Scholar). As this evidence was based mostly on cultured cells in vitro, we also analyzed morphological parameters in cultured neurons derived from WT and KO mice. Under these conditions, we observed indeed a significant reduction in primary dendrites and dendritic branches (Fig. 3, A and B). These defects could be rescued by ectopic expression of the protein (supplemental Fig. S1). This is mirrored by the finding that the overexpression of IRSp53, but not of mutant lacking the SH3 domain (ΔSH3 in Fig. 3C) in cultured rat hippocampal neurons, increased dendritic branching. Collectively, these observations indicate that IRSp53 regulates morphogenesis of primary processes in neurons in vitro through interaction partners of its SH3 domain. We reasoned that the differences between in vivo and in vitro conditions may be due to the dynamic formation of different IRSp53-based complexes, whose activities may become limiting and, thus, functionally relevant only under controlled in vitro conditions but dispensable in vivo. To determine which of these complexes are relevant in the hippocampus (in particular, which interaction partners of the IRSp53 SH3 domain), we performed a biochemical analysis to identify the major IRSp53 binding proteins in the hippocampus during various developmental stages. Analysis of detergent soluble and insoluble fractions from hipp" @default.
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• W2014474433 date "2009-04-01" @default.
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• W2014474433 title "The Insulin Receptor Substrate of 53 kDa (IRSp53) Limits Hippocampal Synaptic Plasticity" @default.
• W2014474433 cites W1519786933 @default.
• W2014474433 cites W1571919210 @default.
• W2014474433 cites W1624178692 @default.
• W2014474433 cites W1730677387 @default.
• W2014474433 cites W1964772923 @default.
• W2014474433 cites W1967716675 @default.
• W2014474433 cites W1969781703 @default.
• W2014474433 cites W1975899151 @default.
• W2014474433 cites W1979324769 @default.
• W2014474433 cites W1984854704 @default.
• W2014474433 cites W1989652455 @default.
• W2014474433 cites W1994936546 @default.
• W2014474433 cites W1997863481 @default.
• W2014474433 cites W2000763843 @default.
• W2014474433 cites W2003931473 @default.
• W2014474433 cites W2005047484 @default.
• W2014474433 cites W2005641938 @default.
• W2014474433 cites W2009484631 @default.
• W2014474433 cites W2014907593 @default.
• W2014474433 cites W2014913899 @default.
• W2014474433 cites W2022548531 @default.
• W2014474433 cites W2027585354 @default.
• W2014474433 cites W2028363992 @default.
• W2014474433 cites W2039033198 @default.
• W2014474433 cites W2054628503 @default.
• W2014474433 cites W2066190900 @default.
• W2014474433 cites W2072424961 @default.
• W2014474433 cites W2082411508 @default.
• W2014474433 cites W2087938143 @default.
• W2014474433 cites W2091053629 @default.
• W2014474433 cites W2093573816 @default.
• W2014474433 cites W2109067831 @default.
• W2014474433 cites W2133000126 @default.
• W2014474433 cites W2134413471 @default.
• W2014474433 cites W2139784023 @default.
• W2014474433 cites W2141998004 @default.
• W2014474433 cites W2153951916 @default.
• W2014474433 cites W2160207967 @default.
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