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Impairment of Axonal Development and of Synaptogenesis in Hippocampal Neurons of Synapsin I-Deficient Mice

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Impairment of Axonal Development and of Synaptogenesis in Hippocampal Neurons of Synapsin I-Deficient Mice Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9230-9234, September 1995 Neurobiology Impairment of axonal development and of synaptogenesis in hippocampal neurons of synapsin I-deficient mice LIH-SHEN CHIN*, LIAN LI*t, ADRIANA FERREIRAt, KENNETH S. KOSIKt, AND PAUL GREENGARD* *Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021; and 1Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 Contributed by Paul Greengard, June 13, 1995 ABSTRACT Synapsin I, the most abundant of all neuro- 6-kb Sst I-Sma I genomic fragment containing the 5' flanking nal phosphoproteins, is enriched in synaptic vesicles. It has sequence of exon 1 and a 3-kb BamHI fragment containing the been hypothesized to regulate synaptogenesis and neurotrans- 3' flanking sequence of exon 1 into the pPNT vector, which mitter release from adult nerve terminals. The evidence for contains the thymidine kinase gene and the neomycin-resistance such roles has been highly suggestive but not compelling. To gene (11). E14 ES cells (12) were transfected with 50 ,ug of evaluate the possible involvement of synapsin I in synapto- linearized targeting construct by electroporation using a Bio- genesis and in the function of adult synapses, we have gener- Rad Gene Pulser at 800 V and 3 ,F. Selection with G418 at ated synapsin I-deficient mice by homologous recombination. 150 ,ug/ml and 0.2 ,uM 1-(2-deoxy, 2-fluoro-f3-D-arabino- We report herein that outgrowth of predendritic neurites and furanosyl)-5-iodouracil (FIAU, Bristol-Meyers) was made of axons was severely retarded in the hippocampal neurons of 24 h after electroporation, and double-resistant clones were embryonic synapsin I mutant mice. Furthermore, synapse isolated after 1 week of selection. Genomic DNA from these formation was significantly delayed in these mutant neurons. clones was digested with Nco I and hybridized with a probe These results indicate that synapsin I plays a role in regulation isolated from the 3' flanking sequence of exon 1 (Fig. 1A). of axonogenesis and synaptogenesis. Positive clones were confirmed by hybridization of the same DNA with a probe isolated from the 5' flanking sequence. The nerve terminal is a highly specialized structure where Generation of Synapsin I-Deficient Mice. ES cells from the synaptic transmission occurs by fusion of synaptic vesicles with identified homologous recombinants were injected into the plasma membrane, with consequent release of neurotrans- C57BL/6 blastocysts, which were then implanted in the uteri mitter into the synaptic cleft. In recent years, many synaptic of pseudopregnant recipient females as described (13). Male vesicle proteins have been isolated, cloned, and characterized chimeric mice were backcrossed to C57BL/6 females to screen (1-5). For the most part, the molecular mechanisms by which for germ-line transmission. The genotypes of the resulting F1 these proteins regulate and/or mediate vesicle fusion and agouti mice were determined by genomic Southern blot anal- neurotransmitter release remain to be clarified. The synapsins ysis of DNA isolated from their tails. Mice heterozygous for are a family of proteins that are distinguishable from most the synapsin I mutation were bred to homozygosity. other synaptic vesicle-associated proteins in two ways. (i) They Western Blot Analysis. Brain tissues were homogenized in are peripheral rather than integral membrane proteins; (ii) 1% SDS and subjected to SDS/PAGE. The proteins were they are specific to the nervous system, as there are apparently transferred onto nitrocellulose membranes and probed with no homologous proteins in nonneuronal tissues. affinity-purified primary antibodies, followed by incubation The synapsin family is composed of four homologous pro- with 125I-labeled protein A (Amersham). The blots were teins, synapsins Ia and lb (collectively referred to as synapsin exposed to Kodak XAR-5 autoradiographic film or to Phos- I) and synapsins Ila and Ilb (collectively referred to as synapsin phorlmager screens and quantified by using IMAGEQUANT II), which are products of the alternative splicing of the software (Molecular Dynamics). transcripts from two distinct (synapsin I and synapsin II) genes Preparations of Hippocampal Cell Cultures. Hippocampal (6). Injection of synapsin I into Xenopus blastomeres acceler- cell cultures were prepared and analyzed by investigators ates the structural (7) and functional (8) development of without any knowledge of the genotype of the animal. Ho- neuromuscular synapses. In adult synapses, a variety of cell mozygous synapsin I-deficient and wild-type female litter- biological and electrophysiological studies are consistent with mates were mated with homozygous synapsin I-deficient and the possibility that synapsin I tethers synaptic vesicles to the wild-type male littermates, respectively. The hippocampi of actin cytoskeleton in a phosphorylation-state-dependent man- embryonic day 16 fetuses were dissected in parallel, freed of ner and thereby determines the proportion of vesicles in the meninges, and cultured as described for the embryonic day 18 nerve terminal that are available for release (2). In this report, rat (14). The cells were dissociated by trypsinization (0.25% for and in the accompanying paper (9), we have undertaken a 15 min at 37°C) followed by trituration with a fire-polished detailed examination of the development and function of Pasteur pipette and plated onto poly(L-lysine)-coated cover- synapses in synapsin I-deficient mice. slips (100,000 cells per 60-mm dish) in minimum essential medium (MEM) with 10% (vol/vol) horse serum. After 4 h the coverslips were transferred to dishes containing an astroglial MATERIALS AND METHODS monolayer and maintained in MEM containing N2 supple- Targeting Construct and Embryonic Stem (ES) Homolo- ments (15), 0.1% ovalbumin, and 0.1 mM sodium pyruvate. gous Recombinants. Genomic clones containing exon 1 of the Immunocytochemical Procedures. Cultures were fixed for murine synapsin I gene were isolated from a 129/Sv mouse 20 min with 4% (wt/vol) paraformaldehyde in phosphate- genomic library (Stratagene) by using a rat synapsin I cDNA buffered saline (PBS) containing 0.12 M sucrose. They were probe (10). The targeting construct was derived by inserting a then permeabilized in 0.3% Triton X-100 in PBS for 5 min and rinsed twice in PBS. The cells were preincubated in 10% The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: ES cell, embryonic stem cell. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 9230 Downloaded by guest on September 29, 2021 Neurobiology: Chin et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9231 - :V. tz A , e - e :z a A cn A A :Li-,. Targeting vector 4.. hav-tk I 88:3 Wild type allele sT T T Exon 1 -- iI I08 :% A a~~eIn Mutant allele ~~ ~ ~ ~ *' I II 5' probe 3' probe 1 kb B 5' probe 3' probe 3' probe d" Cf ef o-v +O 0vv0 O+ v O+ 0+ FIG. 1. Targeted disruption of the synapsin I - + +/+ + - 4' gene by homologous recombination. (A) Restric- tion map of the targeting vector, the 5' part of the I and the structure Wild Type - -13 kb - Wild Type - -l*|13 kb synapsin gene, predicted of the disrupted synapsin I gene after homologous Mutant- - 8.5kb recombination. Horizontal arrows indicate the - Mutant * _ - kb direction of transcription of the cassettes con- taining the neomycin-resistance gene under the control of the phosphoglycerate kinase 1 gene (pgk-neo) and herpes simplex virus thymidine ES Cells Mice kinase (hsv-tk). The positions of the probes used for screening of ES cell clones and mice are indicated. (B) Southern blot analysis of Nco C I-digested genomic DNA from ES cell clones and Cerebral Cortex Hippocampus Cerebellum Striatum from mouse tails. Hybridization was done with I - - +1+ +1- -I 1 or 1c f the 5' probe the 3' probe shown in A. Restric- (y, y C C tion fragment lengths of the wild-type and mu- + - +4 +1? -I- dF - y1 y/ _/ tant alleles are indicated. The sex and genotype of each clone and each mouse are indicated. (C) Western blot analysis of brain tissues from wild- * ~ ..-_ Syn Ia and A Odo; L type, heterozygous, homozygous mutant 14.t 13;..-v;;.; 4spi a..i4UW.:.: ~~Syn lb AWAX& :, :~-:;ii"-ib.$YI.:60_ _ * Syn:IIa mice. The sex and the genotype of each mouse are 1.111:111!-i indicated. Tissue extracts were prepared from cerebral cortex, hippocampus, cerebellum, and Syn IIb striatum of each mouse and analyzed by using an antibody that recognizes all four synapsin iso- forms. (wt/vol) bovine serum albumin in PBS for 1 h at 37°C and 1A). The targeting vector was electroporated into E14 ES cells exposed to the primary antibodies (diluted in 1% bovine serum and selected by using G418 and 1-(2-deoxy, 2-fluoro-f3-D- albumin in PBS) overnight at 4°C. Finally, the cultures were arabinofuranosyl)-5-iodouracil. Double-resistant clones were rinsed in PBS and incubated with secondary antibodies for 1 screened for homologous recombination by Southern blot h at 37°C. The following antibodies were used: anti-a-tubulin analysis with a 3' flanking probe and a 5' internal probe. Since (clone DM1A) and polyclonal anti-tubulin (Sigma); anti- the synapsin I gene is localized on the X chromosome (17) and synaptophysin (clone SY38), fluorescein-conjugated anti- since the E14 ES cells were derived from male embryos, a mouse IgG, and rhodamine-conjugated anti-rabbit IgG homologous recombinant clone would be expected to contain (Boehringer Mannheim); anti-synapsin I (clone 18.1) (6); only a mutant allele and no wild-type allele (Fig. IB). Of 672 anti-MAP2 (clone AP-14) (16).
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