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Distinct Roles of Synapsin I and Synapsin II During Neuronal Development

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Distinct Roles of Synapsin I and Synapsin II During Neuronal Development Molecular Medicine 4: 22-28, 1998 Molecular Medicine © 1998 The Picower Institute Press Distinct Roles of Synapsin I and Synapsin II during Neuronal Development Adriana Ferreira,"2 Lih-Shen Chin,3 Lian Li,3 Lorene M. Lanier,3 Kenneth S. Kosik,7 and Paul Greengard3 'Department of Cell and Molecular Biology and Institute for Neuroscience, Northwestern University, Chicago, Illinois, U.S.A. 2Center for Neurologic Diseases, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts, U.S.A. 3Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, U.S.A. Communicated by P. Greengard. Accepted November 21, 1997. Abstract The synapsins are a family of neuron-specific proteins, Deletion of synapsin II, but not synapsin I, greatly re- associated with the cytoplasmic surface of synaptic vesi- tarded axon formation. Conversely, deletion of synapsin cles, which have been shown to regulate neurotransmit- I, but not synapsin II, greatly retarded synapse forma- ter release in mature synapses and to accelerate devel- tion. Remarkably, the deletion of both synapsins led to opment of the nervous system. Using neuronal cultures partial restoration of the wild phenotype. The results from mice lacking synapsin I, synapsin II, or both syn- suggest that the synapsins play separate but coordinated apsins I and II, we have now found that synapsin I and developmental roles. synapsin II play distinct roles in neuronal development. Introduction mitter release, from what appeared to be a re- The synapsins represent 1% of total neuronal serve pool of vesicles, after microinjecting syn- protein. There are two synapsin genes, synapsin apsin antibodies into lamprey giant neurons (3). I and synapsin II, each of which can be alterna- In addition, fewer synaptic vesicles were de- tively spliced to produce a total of four synapsin tected in the reserve pool in synaptic terminals of isoforms (1). The synapsins appear to play a key synapsin I knockout mice when compared with role in the structural and functional organization those in wild-type ones (4-6). The synapsins of the presynaptic terminal (2). They have been also appear to play a role in the formation and implicated in controlling neurotransmitter re- maintenance of synapses. The injection of either lease by clustering synaptic vesicles in a reserve synapsin I or synapsin II into Xenopus blas- pool at the nerve terminal (reviewed in ref. 2). tomeres accelerated synapse formation (7-9) and Support for this idea was recently obtained in the transfection of synapsin II into neuroblasto- studies demonstrating a loss of synaptic vesicle ma/glioma hybrid cells promoted neurite out- clusters, and a concomitant reduction in trans- growth and the differentiation of the nerve ter- minal (10). Conversely, depletion of either Address correspondence and reprint requests to: Dr. Adri- synapsin I or synapsin II resulted in inhibition of ana Ferreira, Northwestern Institute for Neuroscience, synaptogenesis in hippocampal neurons (1 1-13). Searle Building Room 5-474, 320 East Superior Street, Chi- cago, IL 60611, U.S.A. Phone: (312) 503-0597; Fax: (312) Furthermore, the suppression of synapsin II after 503-7345; E-mail: [email protected] synapses were formed resulted in the loss of most A. Ferreira et al.: Role of the Synapsins in Neuronal Development 23 of the synaptic contacts (12). These earlier stud- ies were used: anti a-tubulin (clone DM1A); ies suggested that synapsins I and II play quali- polyclonal anti-tubulin (Sigma, St. Louis, MO); tatively similar roles in regulating clustering of anti-synaptophysin (clone SY38, Boehringer synaptic vesicles, neurotransmitter release, and Mannheim, Indianapolis, IN.); anti mouse IgG synapse formation. fluorescein-conjugated; and anti-rabbit IgG rho- The aim of the present study was to deter- damine-conjugated (Boehringer Mannheim). In mine whether the synapsins play distinct roles some experiments, rhodamine-labeled phalloi- during neuronal development. Using cultures din (Molecular Probes, Eugene, OR) was in- prepared from mice with targeted disruptions of cluded with the secondary antibody to visualize the synapsin genes (1 1, 14), we demonstrate that filamentous actin. synapsins I and II in fact play distinct roles in axonogenesis and synaptogenesis. Results and Discussion A significant difference in brain size was detected Experimental Methods in synapsin 11-deficient mice, relative to wild- Generation of Synapsin-Deficient Mice type mice. At embryonic day 16 (E16), the aver- Synapsin I-, synapsin II-, and synapsin I/Il-de- age brain weight of synapsin 11-deficient mice ficient mice were generated by homologous re- (29.7 ± 1.6 mg; mean ± SEM) was significantly combination (1 1,14). Littermates of wild-type lower (-17%) than that of wild-type mice and homozygous synapsin mutant mice were (35.5 ± 1.7 mg; mean ± SEM; P < 0.05). This used to prepare hippocampal cultures. The brain decrease in brain weight in the mutant mice was size of wild-type and mutant embryos was as- reduced to 12% at El9. No difference was de- sessed by determining the brain wet weight at tected in the brain weights of synapsin I knock- different stages of embryonic development. out or synapsin I/II double knockout mice com- pared with wild-type embryos (data not shown). When plated in low density culture, wild-type Preparation of Hippocampal Cultures E16 hippocampal neurons undergo a series of Neuronal cultures were prepared from the hip- morphological changes that include the forma- pocampi of embryonic day 16 (E16) mice as pre- tion of lamellipodial veils surrounding the cell viously described (11,1 5). Briefly, embryos were bodies (stage I; 1 hr after plating), followed by removed and their hippocampi dissected and the consolidation of the lamellipodia into shafts freed of meninges. The cells were dissociated by of short undifferentiated neurites (stage 11, 4-6 trypsinization (0.25% for 15 min at 370C) fol- hr after plating), and later the differentiation of lowed by trituration with a fire-polished Pasteur one of these processes into an axon (stage III, 24 pipette and plated onto poly-L-lysine coated cov- hr after plating) (1 1,17). In synapsin 11-deficient erslips (100,000 cells/60 mm dish) in MEM with neurons, this timed sequence of developmental 10% horse serum. After 4 hr the coverslips were changes was altered dramatically and the mor- transferred to dishes containing an astroglial phology of the neurons was abnormal. After 1 monolayer and maintained in MEM containing day in culture, the majority of the wild-type N2 supplements (16) plus ovalbumin (0.1%) and neurons were at stage Ill (Fig. IA, B; Table 1). In sodium pyruvate (0.1 mM). contrast, the majority of the synapsin II-defi- cient neurons remained at stage II (Fig. 1E, F; Table 1), their neurites appeared broad and flat- Immunocytochemical Procedures tened, and the distribution of actin filaments was Cultures were fixed for 20 min with 4% parafor- aberrant (Fig. IE, F). In wild-type neurons, actin maldehyde-0.12 M sucrose in phosphate-buff- filaments were most prominent at the tips of ered saline (PBS). They were then permeabilized developing processes; however, in the synapsin in 0.3% triton in PBS for 5 min and rinsed twice II mutants, actin filaments completely sur- in PBS. The cells were preincubated in 10% BSA rounded the cell bodies in stage I and the devel- in PBS for 1 hr at 370C and exposed to the oping processes in stage 11. A similarly abnormal primary antibodies (diluted in 1% BSA in PBS) distribution of actin filaments was observed overnight at 40C. Finally, the cultures were when synapsin 11 expression was acutely sup- rinsed in PBS and incubated with secondary an- pressed using antisense oligonucleotides (18). In tibodies for 1 hr at 370C. The following antibod- contrast, deletion of synapsin I did not affect the 24 Molecular Medicine, Volume 4, Number 1, January 1998 Fig. 1. Phenotype of hippocampal neurons out mice were cultured for 24 hr, stained with a from wild-type mice, and from synapsin I, syn- monoclonal antibody against tubulin (A, C, E, G) apsin II, and synapsin I/II double knockout and counterstained with rhodamine-tagged phalloi- mice. Hippocampal neurons obtained from embry- din (B, D, F, H). Note the aberrant morphology of onic E16 wild-type (A, B), synapsin I (C, D), synap- the synapsin II-deficient neurons. Scale bar: 20 ,um. sin II (E, F), and synapsin 1/11 double (G, H) knock- timing of neurite formation or axon differentia- was normal. Remarkably, when both synapsins tion (stages II and III, respectively; Fig. 1C, D, were deleted, the wild-type phenotype was Table 1), but it did affect subsequent develop- largely restored; hippocampal neurons from syn- ment; axons from synapsin I-deficient neurons apsin I/MI double knockout mice developed with elongated at a slower rate and were shorter and the same time course as did wild-type neurons less branched than their wild-type counterparts (Table 1) and the distribution of actin appeared (1 1). Moreover, in the synapsin I-deficient neu- much more normal than in the synapsin II rons, the distribution of actin filaments (Fig. ID) knockout neurons (Fig. 1G, H). Table 1. Quantitative analysis of cell morphology in wild-type and in synapsin I, synapsin II, and synapsin I/II double knockout (k.o.) mice Neuronal development Synapsin I Synapsin II Synapsin I/II (stage) Wild-type k.o. k.o. k.o. I 3± 1 4 1 7 1* 3 1 II 30 2 32 2 85 +6* 37 3 III 67 4 64 5 8 1* 60 4 Embryonic E 16 hippocampal cultures were prepared as described (1 1,17). Twenty-four hr after plating, cells were fixed and stained with a tubulin antibody and their morphology analyzed in 20 fields from three different experiments.
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