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Distinct Roles of Synapsin I and Synapsin II during Neuronal Development

Distinct Roles of Synapsin I and Synapsin II during Neuronal Development The synapsins are a family of neuron-specific proteins, Deletion of synapsin II, but not synapsin I, greatly re- associated with the surface of vesi- tarded axon formation. deletion of cytoplasmic synaptic Conversely, synapsin which have been shown to neurotransmit- but not retarded forma- cles, regulate I, synapsin II, greatly synapse ter release in mature and to accelerate devel- tion. the deletion of both led to synapses Remarkably, synapsins of the nervous neuronal cultures partial restoration of the wild The results opment system. Using phenotype. from mice lacking synapsin I, synapsin II, or both syn- suggest that the synapsins play separate but coordinated I and we have now found that I and roles. apsins II, synapsin developmental II distinct roles in neuronal synapsin play development. mitter release, from what appeared to be a re- Introduction serve of after pool vesicles, microinjecting syn- The synapsins represent 1% of total neuronal antibodies apsin into lamprey giant neurons (3). protein. There are two synapsin genes, synapsin In addition, fewer synaptic vesicles were de- I and synapsin II, each of which can be alterna- tected in the reserve pool in synaptic terminals of tively spliced to produce a total of four synapsin I knockout mice when with synapsin compared isoforms (1). The synapsins appear to play a key in those wild-type ones (4-6). The synapsins role in the structural and functional organization in also appear to play a role the formation and of the presynaptic terminal (2). They have been maintenance of synapses. The injection of either in implicated controlling neurotransmitter re- I or II into blas- synapsin synapsin Xenopus lease in by clustering synaptic vesicles a reserve tomeres accelerated synapse formation (7-9) and at the nerve terminal in ref. pool (reviewed 2). the transfection of synapsin II into neuroblasto- for this idea was in Support recently obtained ma/glioma hybrid cells promoted neurite out- studies a loss of vesicle demonstrating synaptic growth and the differentiation of the nerve ter- and a concomitant reduction in trans- clusters, minal (10). Conversely, depletion of either I or II resulted in inhibition of Address correspondence and reprint requests to: Dr. Adri- synapsin synapsin ana Ferreira, Northwestern Institute for Neuroscience, in neurons synaptogenesis hippocampal (1 1-13). Room East Chi- Searle Building 5-474, 320 Superior Street, Furthermore, the suppression of synapsin II after IL U.S.A. Phone: Fax: cago, 60611, (312) 503-0597; (312) E-mail: [email protected] were formed resulted in the loss of most 503-7345; synapses in Neuronal Development 23 A. Ferreira et al.: Role of the Synapsins DM1A); of the synaptic contacts (12). These earlier stud- ies were used: anti a-tubulin (clone 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 IgG rho- synapse formation. fluorescein-conjugated; and anti-rabbit In The aim of the present study was to deter- damine-conjugated (Boehringer Mannheim). rhodamine-labeled mine whether the synapsins play distinct roles some experiments, phalloi- was in- during neuronal development. Using cultures din (Molecular Probes, Eugene, OR) of to visualize prepared from mice with targeted disruptions cluded with the secondary antibody we demonstrate that filamentous actin. the synapsin genes (1 1, 14), roles in synapsins I and II in fact play distinct 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 mice. At embryonic day 16 (E 16), the aver- type age brain weight of synapsin 11-deficient mice and I/Il-de- Synapsin I-, synapsin II-, synapsin ± 1.6 mg; mean ± SEM) was significantly (29.7 re- ficient mice were generated by homologous lower (-17%) than that of wild-type mice of combination (1 1,14). Littermates wild-type (35.5 ± 1.7 mg; mean ± SEM; P < 0.05). This mice were and homozygous synapsin mutant decrease in brain weight in the mutant mice was The brain used to prepare hippocampal cultures. reduced to 12% at El9. No difference was de- and mutant was as- size of wild-type embryos tected in the brain weights of synapsin knock- brain wet at sessed by determining the weight out or synapsin I/II double knockout mice com- different stages of embryonic development. pared with wild-type embryos (data not shown). When in low density culture, wild-type plated of Hippocampal Cultures Preparation E16 hippocampal neurons undergo a series of morphological changes that include the forma- cultures were from the hip- Neuronal prepared tion of lamellipodial veils surrounding the cell of 16 mice as pre- pocampi embryonic day (E16) bodies (stage I; hr after plating), followed by described Briefly, embryos were viously (1 1,1 5). the consolidation of the lamellipodia into shafts removed and their hippocampi dissected and 4-6 of short undifferentiated neurites (stage 11, freed of meninges. The cells were dissociated by of hr after plating), and later the differentiation for 15 min at 370C) fol- trypsinization (0.25% axon 24 one of these processes into an (stage III, lowed by trituration with a fire-polished Pasteur hr In 11-deficient after plating) (1 1,17). synapsin pipette and plated onto poly-L-lysine coated cov- of neurons, this timed sequence developmental erslips (100,000 cells/60 mm dish) in MEM with changes was altered dramatically and the mor- 10% horse serum. After 4 hr the coverslips were phology of the neurons was abnormal. After transferred to dishes containing an astroglial in of the day culture, the majority wild-type monolayer and maintained in MEM containing Ill In neurons were at stage (Fig. IA, B; Table 1). N2 plus ovalbumin (0.1%) and supplements (16) contrast, the majority of the synapsin II-defi- sodium pyruvate (0.1 mM). II cient neurons remained at stage (Fig. 1E, F; Table their neurites appeared broad and flat- 1), Procedures Immunocytochemical and the distribution of actin filaments was tened, fixed for 20 4% aberrant In actin Cultures were min with parafor- (Fig. IE, F). wild-type neurons, M sucrose in phosphate-buff- filaments were most at the tips of maldehyde-0.12 prominent ered saline They were then permeabilized in the synapsin (PBS). developing processes; however, 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 in stage 11. A similarly abnormal oping processes in in primary antibodies (diluted 1% BSA PBS) distribution of actin filaments was observed 40C. the cultures were 11 overnight at Finally, when synapsin expression was acutely sup- rinsed in PBS and incubated with secondary an- using antisense oligonucleotides (18). In pressed tibodies for 1 hr at 370C. The antibod- deletion of I did not affect the following contrast, synapsin 24 Molecular Medicine, Volume 4, Number 1, January 1998 Fig. 1. Phenotype of hippocampal neurons out mice were cultured for 24 stained with a hr, from wild-type mice, and from synapsin I, syn- monoclonal tubulin antibody against (A, C, E, G) and double knockout and counterstained with apsin II, synapsin I/II rhodamine-tagged phalloi- mice. Hippocampal neurons obtained from embry- din Note the aberrant of (B, D, F, H). morphology onic E16 wild-type (A, B), synapsin I (C, D), synap- the II-deficient neurons. Scale bar: 20 synapsin ,um. sin II (E, F), and synapsin 1/11 double (G, H) knock- of neurite formation or when both timing axon differentia- was normal. Remarkably, synapsins tion II and were the was (stages III, respectively; Fig. 1C, D, deleted, wild-type phenotype Table 1), but it did affect subsequent develop- neurons from largely restored; hippocampal syn- ment; axons from synapsin I-deficient neurons I/MI double knockout mice with apsin developed elongated at a slower rate and were shorter and the same time course as did neurons wild-type less branched than their (Table and the distribution of actin wild-type counterparts 1) appeared (1 1). Moreover, in the synapsin I-deficient neu- much more normal than in the II synapsin rons, the distribution of actin filaments knockout neurons (Fig. ID) (Fig. 1G, H). Table 1. of cell in and in and Quantitative analysis morphology wild-type synapsin I, synapsin II, double knockout mice synapsin I/II (k.o.) Neuronal development Synapsin I Synapsin II Synapsin I/II (stage) Wild-type k.o. k.o. k.o. I 3± 1* 3 1 1 4 1 7 II 30 2 32 2 85 +6* 37 3 III 4 64 8 1* 4 67 5 60 E 16 cultures were as (1 hr were fixed and Embryonic hippocampal prepared described 1,17). Twenty-four after plating, cells stained with a tubulin in antibody and their morphology analyzed 20 fields from three different experiments. Values are as of total. Each number the mean + *Differs from P < 0.001. given percent represents SEM. wild-type et al.: Role of the Synapsins in Neuronal Development 25 A. Ferreira it XFg=3 >IS-iS;, _. I t;. _s 2. detection of syn- in culture. The cells were double stained with anti- Fig. Immunocytochemical in hippocampal neurons from wild-type bodies against tubulin (A, C, E, G, I, K) and synapto- apses of mice and from II and synapsin I/II (B, D, F, H, J, L). Note the absence synap- synapsin physin double knockout mice. neurons ob- immunoreactive spots at 4 days in neurons Hippocampal tophysin tained from (A-D), synapsin II (E-H), and from synapsin II and synapsin I/II double knockout wild-type double knockout (I-L) mice were fixed mice. Scale bar: 20 ,um. synapsin I/II after 4 or 7 H, K, L) days (A, B, E, F, I, J) (C, D, G, The effect of synapsin deletion on synapto- These results are in agreement with previous II is was examined using synaptophysin, an that suggested that synapsin genesis experiments and stabilization integral protein of synaptic vesicles, as a synaptic involved in synapse formation I in a marker (12,19). Synaptophysin immunoreactive (12). Deletion of synapsin resulted greater in could be detected in cultured wild-type in formation than that observed spots delay synapse Strik- hippocampal neurons as early as 4 days after neurons (Table 2). synapsin II-deficient were syn- (Fig. 2; Table 2). In synapsin 11-deficient ingly, when both synapsins deleted, plating same rate as was neurons, synapses were first detected at 7 days in apse formation occurred at the neurons culture 2G, H); in these neurons, synapse observed with the synapsin II-deficient (Fig. rate than observed with formation was delayed by 2-3 days (Table 2). and at a much greater 26 Molecular Medicine, Volume 4, Number 1, January 1998 Table 2. Synapse formation in wild-type, synapsin I, synapsin H, and synapsin I/U double knockout (k.o.) mice Number of synapses/cell Days in Synapsm Ia Synapsin U Synapsins I/U culture Wild-type k.o. k.o. k.o. 4 18.4 2 0** 0** °** 7 ± ± ± ± 40.8 3.9 0.5 0.2** 29.9 4.6* 31.1 2.62* ± ± ± ± 10 64.2 3.9 37.5 4.9** 57.6 2.58* 63.8 5.8 14 ± ± ± 77.5 ± 6.2 75.5 6.5 70.6 6.0 73.2 4.8 E16 in 14 fields were for each time Embryonic hippocampal neurons were grown culture for up to days. Twenty analyzed point and from Values are as number of Each number experimental condition three different experiments. given synapses/cell. repre- sents the mean ± SEM. Differs wild *P < and **P < from type: 0.05 0.001. aData from Chin et al., 1995 (11). the synapsin I-deficient neurons. The levels of inent role than synapsin II in synapse formation synaptophysin are reduced to a comparable ex- (Tables 2 and 3). tent in synapsin I knockout mice (17% reduc- In the synapsin II-deficient neurons, the de- tion) and in synapsin I/II double knockout mice lay in the early stages of development (Table 1) (19% reduction), relative to wild-type controls can account at least in part for the delay in (5). Therefore, the greater impairment in synap- synapse formation (Table 2). However, treat- tic development observed in the synapsin I ment of cultured wild-type hippocampal neu- knockout mice cannot be attributed to decreased rons with synapsin II antisense oligonucleotides synaptophysin immunoreactivity per se. causes a reversible disappearance of synapses, The results of the present investigation indi- indicating that synapsin II may also be involved in In cate that synapsin I and synapsin II each make synapse formation and/or stabilization (12). in unique contributions to neuronal differentiation the synapsin I-deficient neurons, the delay an and synapse formation. Synapsin plays a much synaptogenesis cannot be accounted for by more I in prominent role than synapsin the early effect on axonal differentiation and/or elonga- of Two lines of this stages lamellipodial formation, neurite forma- tion. evidence support view. and axon 1 and in the we demonstrated tion, differentiation (Tables 3). First, present study, that I a much more 11-deficient neurons failed to Conversely, synapsin plays prom- although synapsin Table 3. of of Comparison the phenotype developing neurons from synapsin I, synapsin U, and I/U knockout synapsin (k.o.) mice Synapsin I Synapsin U Synapsin I/U k.o. k.o. k.o. Lamellipodia formation Normal Impaired Normal Neurite formation Normal Impaired Normal Axon differentiation Normal Normal Impaired formation Synapse Impaired Impaireda Impaireda Normal Intermediate Cytoskeletal organization Impaired 'A 2- in in a in axon to 3-day delay synapse formation could be attributable, part, to corresponding delay formation. Role of the Synapsins in Neuronal Development 27 A. Ferreira et al.: P. (1989) Synapsins: Mo- De Camilli Greengard elongate their axons for more than 48 hr, they P, and individual domains in a family saic of shared formed synapses earlier than the synapsin I mu- of synaptic vesicle phosphoproteins. Science 245: tant neurons, which differentiated axons with 1474-1480. the same time course as did their wild-type Valtorta F, Czernik AJ, Benfenati F. 2. Greengard P, counterparts (Table 1-3). Second, synapsin I- phosphoproteins and reg- (1993) Synaptic vesicle deficient neurons are delayed in their ability to Science 259: 780-785. ulation of synaptic function. form synapses even after making contact with VA, Shupliakov 0, Brodin L, Hilfiker- 3. Pieribone other cells (13). Czernik AJ, Greengard P. (1995) Rothenfluh S, It is of particular interest that the synapsin of synaptic vesicles in neurotrans- Distinct pools I/II double deletion largely eliminated the dele- release. Nature 375: 493-497. mitter 4. Li Chin L, Shupliakov 0, Brodin L, Sihra T, terious effects both of the synapsin II deletion on L, V, Zieng D, McNamara JO, Hvalby 0, Jensen the early stages of neuronal differentiation and Andersen P. (1995) Impairment of Greengard P, of the synapsin I deletion on synapse formation synaptic vesicle clustering and of synaptic trans- the synapsins (Tables 1-3). If, as suggested (5), and increased seizure propensity, in syn- mission, might expect to have redundant functions, one I-deficient mice. Proc. Natl. Acad. Sci. U.S.A. apsin defects in observe more severe developmental 92: 9235-9239. in knock- the double knockout than either single 5. Rosahl TW, Spillane D, Missler M, Herz J, Selig D, data indicate that the out. On the contrary, our Wolff Hammer RE, Malenka RC, Sudhof TC. JR, developmental defects were less severe in the Essential functions of synapsin I and II in (1995) in either knockout. double knockout than single vesicle Nature 375: 488-493. synaptic regulation. the During normal neuronal development, 6. Takei Harada A, Takeda Kobayashi K, Terada Y, S, actin filaments drives the Takahashi Hirokawa N. (1995) Syn- protrusive activity of Noda T, T, S, I deficiency results in the structural change apsin formation of lamellipodial structures; process in the presynaptic terminals in the murine ner- formation and directional elongation require vous J. Cell Biol. 131: 1789-1800. system. that this protrusive activity be suppressed along 7. Lu Greengard Poo MM. (1992) Exogenous B, P, that this the axon shaft. It has been proposed synapsin I promotes functional maturation of de- unidentified suppression is mediated by proteins neuromuscular synapses. Neuron 8: 521- veloping of microtubules that are capable cross-linking There is substantial and actin filaments (20). now 8. E, Alder J, Greengard Poo MM. Schaeffer P, and even more evidence that synapsin I, po- Exogenous synapsin I promotes functional (1994) actin tently, synapsin II, nucleates polymeriza- maturation of developing neuromuscular syn- filaments tion and bundles actin (21). Moreover, Proc. Natl. Acad. Sci. U.S.A. 91: 3882-3886. apses. I and bundles microtubules 9. Valtorta Iezzi N, Benfenati F, Lu B, Poo M-M, synapsin binds F, P. Accelerated structural matu- to Greengard (1995) through a domain with high homology syn- ration induced by synapsin I at developing neuro- II Our data as a con- apsin (22,23). suggest that, muscular synapses of Xenopus Iaevis. Eur. J. Neuro- of the loss of actin sequence synapsin II, protru- sci. 7: 261-270. sive remain unsuppressed, resulting activity may 10. Han HQ, Nichols RA, Rubin MR, Bahler M, in the formation of actin veils that surround the P. Induction of formation of Greengard (1991) neuritic processes. It will be of interest to deter- presynaptic terminals in neuroblastoma cells by mine the extent to which the distinct effects of synapsin Ib. Nature 349: 697-700. I and synapsin II on neuronal develop- synapsin 11. Chin LS, Li L, Ferreira A, Kosik KS, Greengard P. ment are attributable to differences in their abil- (1995) Impairment of axonal development and of ity to organize the cytoskeleton. in hippocampal neurons of synap- synaptogenesis sin I-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 92: 9230-9234. 12. Ferreira A, Han HQ, Greengard Kosik KS. P, Acknowledgments (1995) Suppression of synapsin II inhibits the for- in work was by NIH grant MH39327 mation and maintenance of synapses hip- This supported Acad. Sci. U.S.A. 92: pocampal culture. Proc. Natl. and by Northwestern University grant (P.G.) 9225-9229. 100510330U (A.F.). 13. Ferreira Li Chin LS, Greengard Kosik KS. A, L, P, Postsynaptic element contributes to the (1996) in synaptogenesis in synapsin I deficient delay References neurons. Mol. Cell. Neurosci. 8: 286-299. Li L, Chin LS, 1. Sudhof TC, Czernik AJ, Kao H, Takei K, Johnston 14. Shupliakov 0, Magarinos AM, Gustafsson McEwen Pieribone PA, Horiuchi A, Wagner M, Kanazir SD, Perin MS, J, BS, V, Greengard 28 Molecular Medicine, Volume 4, Number 1, January 1998 Brodin L. Structural alterations in nerve termi- 19. Fletcher TP, Cameron De Camilli P, Banker GA. P, P, nals of synapsin-deficient mice. J. Cell Biol. (in press). (1991) The distribution of synapsin I and synap- 15. Goslin K, Banker GA. (1991) Rat hippocampal in tophysin in hippocampal neurons culture. neurons in low-density culture. In: Banker GA, J. Neurosci. 11: 1617-1632. Goslin K (eds). Culturing nerve cells. MIT Press, 20. Mitchison T, and Kirschner M. (1988) Cytoskel- Cambridge, MA, pp. 251-283. etal dynamics and nerve growth. Neuron 1: 761- 16. Bottenstein JE, Sato GH. (1979) Growth of a rat neuroblastoma cell line in serum-free supple- 21. Chilcote TJ, Siow YL, Schaeffer E, Greengard P, mented media. Proc. Natl. Acad. Sci. U.S.A. 76: 514- Thiel G. (1994) Synapsin IIa bundles actin fila- ments. J. Neurochem. 63: 1568-1571. 17. Dotti CG, Sullivan CA, Banker GA. (1988) The 22. Baines AJ, Bennett V. (1986) Synapsin I is a establishment of polarity by hippocampal neurons microtubule-bundling protein. Nature 319: 145- in culture. J. Neurosci. 8: 1454-1468. 18. Ferreira A, Kosik KS, Greengard Han HQ. P, 23. Petrucci TC, Morrow JF. (1991) Synapsin I: an (1994) Aberrant neurites and synaptic vesicle pro- tein in deficiency synapsin II-depleted neurons. actin-bundling protein under phosphorylation Science 977-979. 264: control. 30: 413-422. Biochemistry http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Medicine Springer Journals

Distinct Roles of Synapsin I and Synapsin II during Neuronal Development

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Springer Journals
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Copyright © Picower Institute Press 1998
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10.1007/bf03401726
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Abstract

The synapsins are a family of neuron-specific proteins, Deletion of synapsin II, but not synapsin I, greatly re- associated with the surface of vesi- tarded axon formation. deletion of cytoplasmic synaptic Conversely, synapsin which have been shown to neurotransmit- but not retarded forma- cles, regulate I, synapsin II, greatly synapse ter release in mature and to accelerate devel- tion. the deletion of both led to synapses Remarkably, synapsins of the nervous neuronal cultures partial restoration of the wild The results opment system. Using phenotype. from mice lacking synapsin I, synapsin II, or both syn- suggest that the synapsins play separate but coordinated I and we have now found that I and roles. apsins II, synapsin developmental II distinct roles in neuronal synapsin play development. mitter release, from what appeared to be a re- Introduction serve of after pool vesicles, microinjecting syn- The synapsins represent 1% of total neuronal antibodies apsin into lamprey giant neurons (3). protein. There are two synapsin genes, synapsin In addition, fewer synaptic vesicles were de- I and synapsin II, each of which can be alterna- tected in the reserve pool in synaptic terminals of tively spliced to produce a total of four synapsin I knockout mice when with synapsin compared isoforms (1). The synapsins appear to play a key in those wild-type ones (4-6). The synapsins role in the structural and functional organization in also appear to play a role the formation and of the presynaptic terminal (2). They have been maintenance of synapses. The injection of either in implicated controlling neurotransmitter re- I or II into blas- synapsin synapsin Xenopus lease in by clustering synaptic vesicles a reserve tomeres accelerated synapse formation (7-9) and at the nerve terminal in ref. pool (reviewed 2). the transfection of synapsin II into neuroblasto- for this idea was in Support recently obtained ma/glioma hybrid cells promoted neurite out- studies a loss of vesicle demonstrating synaptic growth and the differentiation of the nerve ter- and a concomitant reduction in trans- clusters, minal (10). Conversely, depletion of either I or II resulted in inhibition of Address correspondence and reprint requests to: Dr. Adri- synapsin synapsin ana Ferreira, Northwestern Institute for Neuroscience, in neurons synaptogenesis hippocampal (1 1-13). Room East Chi- Searle Building 5-474, 320 Superior Street, Furthermore, the suppression of synapsin II after IL U.S.A. Phone: Fax: cago, 60611, (312) 503-0597; (312) E-mail: [email protected] were formed resulted in the loss of most 503-7345; synapses in Neuronal Development 23 A. Ferreira et al.: Role of the Synapsins DM1A); of the synaptic contacts (12). These earlier stud- ies were used: anti a-tubulin (clone 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 IgG rho- synapse formation. fluorescein-conjugated; and anti-rabbit In The aim of the present study was to deter- damine-conjugated (Boehringer Mannheim). rhodamine-labeled mine whether the synapsins play distinct roles some experiments, phalloi- was in- during neuronal development. Using cultures din (Molecular Probes, Eugene, OR) of to visualize prepared from mice with targeted disruptions cluded with the secondary antibody we demonstrate that filamentous actin. the synapsin genes (1 1, 14), roles in synapsins I and II in fact play distinct 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 mice. At embryonic day 16 (E 16), the aver- type age brain weight of synapsin 11-deficient mice and I/Il-de- Synapsin I-, synapsin II-, synapsin ± 1.6 mg; mean ± SEM) was significantly (29.7 re- ficient mice were generated by homologous lower (-17%) than that of wild-type mice of combination (1 1,14). Littermates wild-type (35.5 ± 1.7 mg; mean ± SEM; P < 0.05). This mice were and homozygous synapsin mutant decrease in brain weight in the mutant mice was The brain used to prepare hippocampal cultures. reduced to 12% at El9. No difference was de- and mutant was as- size of wild-type embryos tected in the brain weights of synapsin knock- brain wet at sessed by determining the weight out or synapsin I/II double knockout mice com- different stages of embryonic development. pared with wild-type embryos (data not shown). When in low density culture, wild-type plated of Hippocampal Cultures Preparation E16 hippocampal neurons undergo a series of morphological changes that include the forma- cultures were from the hip- Neuronal prepared tion of lamellipodial veils surrounding the cell of 16 mice as pre- pocampi embryonic day (E16) bodies (stage I; hr after plating), followed by described Briefly, embryos were viously (1 1,1 5). the consolidation of the lamellipodia into shafts removed and their hippocampi dissected and 4-6 of short undifferentiated neurites (stage 11, freed of meninges. The cells were dissociated by of hr after plating), and later the differentiation for 15 min at 370C) fol- trypsinization (0.25% axon 24 one of these processes into an (stage III, lowed by trituration with a fire-polished Pasteur hr In 11-deficient after plating) (1 1,17). synapsin pipette and plated onto poly-L-lysine coated cov- of neurons, this timed sequence developmental erslips (100,000 cells/60 mm dish) in MEM with changes was altered dramatically and the mor- 10% horse serum. After 4 hr the coverslips were phology of the neurons was abnormal. After transferred to dishes containing an astroglial in of the day culture, the majority wild-type monolayer and maintained in MEM containing Ill In neurons were at stage (Fig. IA, B; Table 1). N2 plus ovalbumin (0.1%) and supplements (16) contrast, the majority of the synapsin II-defi- sodium pyruvate (0.1 mM). II cient neurons remained at stage (Fig. 1E, F; Table their neurites appeared broad and flat- 1), Procedures Immunocytochemical and the distribution of actin filaments was tened, fixed for 20 4% aberrant In actin Cultures were min with parafor- (Fig. IE, F). wild-type neurons, M sucrose in phosphate-buff- filaments were most at the tips of maldehyde-0.12 prominent ered saline They were then permeabilized in the synapsin (PBS). developing processes; however, 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 in stage 11. A similarly abnormal oping processes in in primary antibodies (diluted 1% BSA PBS) distribution of actin filaments was observed 40C. the cultures were 11 overnight at Finally, when synapsin expression was acutely sup- rinsed in PBS and incubated with secondary an- using antisense oligonucleotides (18). In pressed tibodies for 1 hr at 370C. The antibod- deletion of I did not affect the following contrast, synapsin 24 Molecular Medicine, Volume 4, Number 1, January 1998 Fig. 1. Phenotype of hippocampal neurons out mice were cultured for 24 stained with a hr, from wild-type mice, and from synapsin I, syn- monoclonal tubulin antibody against (A, C, E, G) and double knockout and counterstained with apsin II, synapsin I/II rhodamine-tagged phalloi- mice. Hippocampal neurons obtained from embry- din Note the aberrant of (B, D, F, H). morphology onic E16 wild-type (A, B), synapsin I (C, D), synap- the II-deficient neurons. Scale bar: 20 synapsin ,um. sin II (E, F), and synapsin 1/11 double (G, H) knock- of neurite formation or when both timing axon differentia- was normal. Remarkably, synapsins tion II and were the was (stages III, respectively; Fig. 1C, D, deleted, wild-type phenotype Table 1), but it did affect subsequent develop- neurons from largely restored; hippocampal syn- ment; axons from synapsin I-deficient neurons I/MI double knockout mice with apsin developed elongated at a slower rate and were shorter and the same time course as did neurons wild-type less branched than their (Table and the distribution of actin wild-type counterparts 1) appeared (1 1). Moreover, in the synapsin I-deficient neu- much more normal than in the II synapsin rons, the distribution of actin filaments knockout neurons (Fig. ID) (Fig. 1G, H). Table 1. of cell in and in and Quantitative analysis morphology wild-type synapsin I, synapsin II, double knockout mice synapsin I/II (k.o.) Neuronal development Synapsin I Synapsin II Synapsin I/II (stage) Wild-type k.o. k.o. k.o. I 3± 1* 3 1 1 4 1 7 II 30 2 32 2 85 +6* 37 3 III 4 64 8 1* 4 67 5 60 E 16 cultures were as (1 hr were fixed and Embryonic hippocampal prepared described 1,17). Twenty-four after plating, cells stained with a tubulin in antibody and their morphology analyzed 20 fields from three different experiments. Values are as of total. Each number the mean + *Differs from P < 0.001. given percent represents SEM. wild-type et al.: Role of the Synapsins in Neuronal Development 25 A. Ferreira it XFg=3 >IS-iS;, _. I t;. _s 2. detection of syn- in culture. The cells were double stained with anti- Fig. Immunocytochemical in hippocampal neurons from wild-type bodies against tubulin (A, C, E, G, I, K) and synapto- apses of mice and from II and synapsin I/II (B, D, F, H, J, L). Note the absence synap- synapsin physin double knockout mice. neurons ob- immunoreactive spots at 4 days in neurons Hippocampal tophysin tained from (A-D), synapsin II (E-H), and from synapsin II and synapsin I/II double knockout wild-type double knockout (I-L) mice were fixed mice. Scale bar: 20 ,um. synapsin I/II after 4 or 7 H, K, L) days (A, B, E, F, I, J) (C, D, G, The effect of synapsin deletion on synapto- These results are in agreement with previous II is was examined using synaptophysin, an that suggested that synapsin genesis experiments and stabilization integral protein of synaptic vesicles, as a synaptic involved in synapse formation I in a marker (12,19). Synaptophysin immunoreactive (12). Deletion of synapsin resulted greater in could be detected in cultured wild-type in formation than that observed spots delay synapse Strik- hippocampal neurons as early as 4 days after neurons (Table 2). synapsin II-deficient were syn- (Fig. 2; Table 2). In synapsin 11-deficient ingly, when both synapsins deleted, plating same rate as was neurons, synapses were first detected at 7 days in apse formation occurred at the neurons culture 2G, H); in these neurons, synapse observed with the synapsin II-deficient (Fig. rate than observed with formation was delayed by 2-3 days (Table 2). and at a much greater 26 Molecular Medicine, Volume 4, Number 1, January 1998 Table 2. Synapse formation in wild-type, synapsin I, synapsin H, and synapsin I/U double knockout (k.o.) mice Number of synapses/cell Days in Synapsm Ia Synapsin U Synapsins I/U culture Wild-type k.o. k.o. k.o. 4 18.4 2 0** 0** °** 7 ± ± ± ± 40.8 3.9 0.5 0.2** 29.9 4.6* 31.1 2.62* ± ± ± ± 10 64.2 3.9 37.5 4.9** 57.6 2.58* 63.8 5.8 14 ± ± ± 77.5 ± 6.2 75.5 6.5 70.6 6.0 73.2 4.8 E16 in 14 fields were for each time Embryonic hippocampal neurons were grown culture for up to days. Twenty analyzed point and from Values are as number of Each number experimental condition three different experiments. given synapses/cell. repre- sents the mean ± SEM. Differs wild *P < and **P < from type: 0.05 0.001. aData from Chin et al., 1995 (11). the synapsin I-deficient neurons. The levels of inent role than synapsin II in synapse formation synaptophysin are reduced to a comparable ex- (Tables 2 and 3). tent in synapsin I knockout mice (17% reduc- In the synapsin II-deficient neurons, the de- tion) and in synapsin I/II double knockout mice lay in the early stages of development (Table 1) (19% reduction), relative to wild-type controls can account at least in part for the delay in (5). Therefore, the greater impairment in synap- synapse formation (Table 2). However, treat- tic development observed in the synapsin I ment of cultured wild-type hippocampal neu- knockout mice cannot be attributed to decreased rons with synapsin II antisense oligonucleotides synaptophysin immunoreactivity per se. causes a reversible disappearance of synapses, The results of the present investigation indi- indicating that synapsin II may also be involved in In cate that synapsin I and synapsin II each make synapse formation and/or stabilization (12). in unique contributions to neuronal differentiation the synapsin I-deficient neurons, the delay an and synapse formation. Synapsin plays a much synaptogenesis cannot be accounted for by more I in prominent role than synapsin the early effect on axonal differentiation and/or elonga- of Two lines of this stages lamellipodial formation, neurite forma- tion. evidence support view. and axon 1 and in the we demonstrated tion, differentiation (Tables 3). First, present study, that I a much more 11-deficient neurons failed to Conversely, synapsin plays prom- although synapsin Table 3. of of Comparison the phenotype developing neurons from synapsin I, synapsin U, and I/U knockout synapsin (k.o.) mice Synapsin I Synapsin U Synapsin I/U k.o. k.o. k.o. Lamellipodia formation Normal Impaired Normal Neurite formation Normal Impaired Normal Axon differentiation Normal Normal Impaired formation Synapse Impaired Impaireda Impaireda Normal Intermediate Cytoskeletal organization Impaired 'A 2- in in a in axon to 3-day delay synapse formation could be attributable, part, to corresponding delay formation. Role of the Synapsins in Neuronal Development 27 A. Ferreira et al.: P. (1989) Synapsins: Mo- De Camilli Greengard elongate their axons for more than 48 hr, they P, and individual domains in a family saic of shared formed synapses earlier than the synapsin I mu- of synaptic vesicle phosphoproteins. Science 245: tant neurons, which differentiated axons with 1474-1480. the same time course as did their wild-type Valtorta F, Czernik AJ, Benfenati F. 2. Greengard P, counterparts (Table 1-3). Second, synapsin I- phosphoproteins and reg- (1993) Synaptic vesicle deficient neurons are delayed in their ability to Science 259: 780-785. ulation of synaptic function. form synapses even after making contact with VA, Shupliakov 0, Brodin L, Hilfiker- 3. Pieribone other cells (13). Czernik AJ, Greengard P. (1995) Rothenfluh S, It is of particular interest that the synapsin of synaptic vesicles in neurotrans- Distinct pools I/II double deletion largely eliminated the dele- release. Nature 375: 493-497. mitter 4. Li Chin L, Shupliakov 0, Brodin L, Sihra T, terious effects both of the synapsin II deletion on L, V, Zieng D, McNamara JO, Hvalby 0, Jensen the early stages of neuronal differentiation and Andersen P. (1995) Impairment of Greengard P, of the synapsin I deletion on synapse formation synaptic vesicle clustering and of synaptic trans- the synapsins (Tables 1-3). If, as suggested (5), and increased seizure propensity, in syn- mission, might expect to have redundant functions, one I-deficient mice. Proc. Natl. Acad. Sci. U.S.A. apsin defects in observe more severe developmental 92: 9235-9239. in knock- the double knockout than either single 5. Rosahl TW, Spillane D, Missler M, Herz J, Selig D, data indicate that the out. On the contrary, our Wolff Hammer RE, Malenka RC, Sudhof TC. JR, developmental defects were less severe in the Essential functions of synapsin I and II in (1995) in either knockout. double knockout than single vesicle Nature 375: 488-493. synaptic regulation. the During normal neuronal development, 6. Takei Harada A, Takeda Kobayashi K, Terada Y, S, actin filaments drives the Takahashi Hirokawa N. (1995) Syn- protrusive activity of Noda T, T, S, I deficiency results in the structural change apsin formation of lamellipodial structures; process in the presynaptic terminals in the murine ner- formation and directional elongation require vous J. Cell Biol. 131: 1789-1800. system. that this protrusive activity be suppressed along 7. Lu Greengard Poo MM. (1992) Exogenous B, P, that this the axon shaft. It has been proposed synapsin I promotes functional maturation of de- unidentified suppression is mediated by proteins neuromuscular synapses. Neuron 8: 521- veloping of microtubules that are capable cross-linking There is substantial and actin filaments (20). now 8. E, Alder J, Greengard Poo MM. Schaeffer P, and even more evidence that synapsin I, po- Exogenous synapsin I promotes functional (1994) actin tently, synapsin II, nucleates polymeriza- maturation of developing neuromuscular syn- filaments tion and bundles actin (21). Moreover, Proc. Natl. Acad. Sci. U.S.A. 91: 3882-3886. apses. I and bundles microtubules 9. Valtorta Iezzi N, Benfenati F, Lu B, Poo M-M, synapsin binds F, P. Accelerated structural matu- to Greengard (1995) through a domain with high homology syn- ration induced by synapsin I at developing neuro- II Our data as a con- apsin (22,23). suggest that, muscular synapses of Xenopus Iaevis. Eur. J. Neuro- of the loss of actin sequence synapsin II, protru- sci. 7: 261-270. sive remain unsuppressed, resulting activity may 10. Han HQ, Nichols RA, Rubin MR, Bahler M, in the formation of actin veils that surround the P. Induction of formation of Greengard (1991) neuritic processes. It will be of interest to deter- presynaptic terminals in neuroblastoma cells by mine the extent to which the distinct effects of synapsin Ib. Nature 349: 697-700. I and synapsin II on neuronal develop- synapsin 11. Chin LS, Li L, Ferreira A, Kosik KS, Greengard P. ment are attributable to differences in their abil- (1995) Impairment of axonal development and of ity to organize the cytoskeleton. in hippocampal neurons of synap- synaptogenesis sin I-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 92: 9230-9234. 12. Ferreira A, Han HQ, Greengard Kosik KS. P, Acknowledgments (1995) Suppression of synapsin II inhibits the for- in work was by NIH grant MH39327 mation and maintenance of synapses hip- This supported Acad. Sci. U.S.A. 92: pocampal culture. Proc. Natl. and by Northwestern University grant (P.G.) 9225-9229. 100510330U (A.F.). 13. Ferreira Li Chin LS, Greengard Kosik KS. A, L, P, Postsynaptic element contributes to the (1996) in synaptogenesis in synapsin I deficient delay References neurons. Mol. Cell. Neurosci. 8: 286-299. Li L, Chin LS, 1. Sudhof TC, Czernik AJ, Kao H, Takei K, Johnston 14. Shupliakov 0, Magarinos AM, Gustafsson McEwen Pieribone PA, Horiuchi A, Wagner M, Kanazir SD, Perin MS, J, BS, V, Greengard 28 Molecular Medicine, Volume 4, Number 1, January 1998 Brodin L. Structural alterations in nerve termi- 19. Fletcher TP, Cameron De Camilli P, Banker GA. P, P, nals of synapsin-deficient mice. J. Cell Biol. (in press). (1991) The distribution of synapsin I and synap- 15. Goslin K, Banker GA. (1991) Rat hippocampal in tophysin in hippocampal neurons culture. neurons in low-density culture. In: Banker GA, J. Neurosci. 11: 1617-1632. Goslin K (eds). Culturing nerve cells. MIT Press, 20. Mitchison T, and Kirschner M. (1988) Cytoskel- Cambridge, MA, pp. 251-283. etal dynamics and nerve growth. Neuron 1: 761- 16. Bottenstein JE, Sato GH. (1979) Growth of a rat neuroblastoma cell line in serum-free supple- 21. Chilcote TJ, Siow YL, Schaeffer E, Greengard P, mented media. Proc. Natl. Acad. Sci. U.S.A. 76: 514- Thiel G. (1994) Synapsin IIa bundles actin fila- ments. J. Neurochem. 63: 1568-1571. 17. Dotti CG, Sullivan CA, Banker GA. (1988) The 22. Baines AJ, Bennett V. (1986) Synapsin I is a establishment of polarity by hippocampal neurons microtubule-bundling protein. Nature 319: 145- in culture. J. Neurosci. 8: 1454-1468. 18. Ferreira A, Kosik KS, Greengard Han HQ. P, 23. Petrucci TC, Morrow JF. (1991) Synapsin I: an (1994) Aberrant neurites and synaptic vesicle pro- tein in deficiency synapsin II-depleted neurons. actin-bundling protein under phosphorylation Science 977-979. 264: control. 30: 413-422. Biochemistry

Journal

Molecular Medicine – Springer Journals

Published: Jan 1, 1998

Keywords: Synapse Formation; Synaptic Vesicle Clustering; Synapsin Gene; Wild-type Neurons; Axon Differentiation

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