FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080353409> ?p ?o ?g. }

• W2080353409 endingPage "2334" @default.
• W2080353409 startingPage "2323" @default.
• W2080353409 abstract "β-Neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer α-neurexins function at both glutamate and γ-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of α-neurexins to neuroligins was recently reported, but the role of the α-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three α-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. α-Neurexins also induce clustering of the GABAA receptor γ2 subunit. This synapse promoting activity of α-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in β-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, α-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 β-neurexins and, even more selectively, α-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization. β-Neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer α-neurexins function at both glutamate and γ-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of α-neurexins to neuroligins was recently reported, but the role of the α-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three α-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. α-Neurexins also induce clustering of the GABAA receptor γ2 subunit. This synapse promoting activity of α-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in β-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, α-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 β-neurexins and, even more selectively, α-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization. Synaptic connections in the brain require precise alignment of neurotransmitter receptors on dendrites opposite transmitter release sites on axons. Neurexin and neuroligin cell adhesion molecules are thought to function in development and maintenance of the two main synapse types, excitatory glutamatergic and inhibitory GABAergic 3The abbreviations used are: GABAγ-aminobutyric acidLNSlaminin neurexin sex hormone-binding proteinS4splice site 4EGFepidermal growth factorAPV2-amino-5-phosphonopentanoic acidDIVdays in vitroCFPcyan fluorescent proteinmCFPmembrane-associated CFPNMDAN-methyl-d-aspartateE18embryonic day 18P11postnatal day 11GAPDHglyceraldehyde-phosphate dehydrogenaseRTreverse transcriptaseANOVAanalysis of variance. 3The abbreviations used are: GABAγ-aminobutyric acidLNSlaminin neurexin sex hormone-binding proteinS4splice site 4EGFepidermal growth factorAPV2-amino-5-phosphonopentanoic acidDIVdays in vitroCFPcyan fluorescent proteinmCFPmembrane-associated CFPNMDAN-methyl-d-aspartateE18embryonic day 18P11postnatal day 11GAPDHglyceraldehyde-phosphate dehydrogenaseRTreverse transcriptaseANOVAanalysis of variance. synapses (reviewed in Refs. 1Craig A.M. Kang Y. Cur. Opin. Neurosci. 2007; 17: 43-52Crossref PubMed Scopus (431) Google Scholar and 2Dean C. Dresbach T. Trends Neurosci. 2006; 29: 21-29Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Neuroligins, normally present on dendrites, alone are sufficient to induce presynaptic differentiation when presented to axons of cultured neurons (3Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar, 4Fu Z. Washbourne P. Ortinski P. Vicini S. J. Neurophysiol. 2003; 90: 3950-3957Crossref PubMed Scopus (87) Google Scholar, 5Chubykin A.A. Liu X. Comoletti D. Tsigelny I. Taylor P. Sudhof T.C. J. Biol. Chem. 2005; 280: 22365-22374Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Conversely, neurexins, normally present on axons, alone are sufficient to induce postsynaptic differentiation when presented to dendrites of cultured neurons (6Graf E.R. Zhang X. Jin S.X. Linhoff M.W. Craig A.M. Cell. 2004; 119: 1013-1026Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 7Nam C.I. Chen L. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6137-6142Crossref PubMed Scopus (240) Google Scholar). These coculture studies suggest that neurexins and neuroligins play some role in synapse development in vivo, perhaps in protein recruitment and stabilization of synaptic complexes. There are 4-5 neuroligins in mammals. Neuroligin 1 localizes primarily at glutamatergic synapses (8Song J.Y. Ichtchenko K. Sudhof T.C. Brose N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1100-1105Crossref PubMed Scopus (533) Google Scholar), and neuroligin 2 localizes primarily at GABAergic synapses (6Graf E.R. Zhang X. Jin S.X. Linhoff M.W. Craig A.M. Cell. 2004; 119: 1013-1026Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 9Varoqueaux F. Jamain S. Brose N. Eur. J. Cell Biol. 2004; 83: 449-456Crossref PubMed Scopus (382) Google Scholar). Six main neurexin isoforms are derived from three genes (1Craig A.M. Kang Y. Cur. Opin. Neurosci. 2007; 17: 43-52Crossref PubMed Scopus (431) Google Scholar, 2Dean C. Dresbach T. Trends Neurosci. 2006; 29: 21-29Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 3Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar) and two promoters each (α and β) (10Tabuchi K. Sudhof T.C. Genomics. 2002; 79: 849-859Crossref PubMed Scopus (211) Google Scholar). The shorter β-neurexins bind neuroligins via their single LNS domain (11Ichtchenko K. Hata Y. Nguyen T. Ullrich B. Missler M. Moomaw C. Sudhof T.C. Cell. 1995; 81: 435-443Abstract Full Text PDF PubMed Scopus (562) Google Scholar). Alternative splicing at multiple sites also contributes to neurexin and neuroligin diversity. In particular, the absence of the splice site 4 (S4) insert in β-neurexins and presence of the B insert in neuroligin 1 selectively promotes function at glutamatergic synapses (12Boucard A.A. Chubykin A.A. Comoletti D. Taylor P. Sudhof T.C. Neuron. 2005; 48: 229-236Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 13Graf E.R. Kang Y. Hauner A.M. Craig A.M. J. Neurosci. 2006; 26: 4256-4265Crossref PubMed Scopus (131) Google Scholar, 14Chih B. Gollan L. Scheiffele P. Neuron. 2006; 51: 171-178Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 15Comoletti D. Flynn R.E. Boucard A.A. Demeler B. Schirf V. Shi J. Jennings L.L. Newlin H.R. Sudhof T.C. Taylor P. Biochemistry. 2006; 45: 12816-12827Crossref PubMed Scopus (105) Google Scholar). γ-aminobutyric acid laminin neurexin sex hormone-binding protein splice site 4 epidermal growth factor 2-amino-5-phosphonopentanoic acid days in vitro cyan fluorescent protein membrane-associated CFP N-methyl-d-aspartate embryonic day 18 postnatal day 11 glyceraldehyde-phosphate dehydrogenase reverse transcriptase analysis of variance. γ-aminobutyric acid laminin neurexin sex hormone-binding protein splice site 4 epidermal growth factor 2-amino-5-phosphonopentanoic acid days in vitro cyan fluorescent protein membrane-associated CFP N-methyl-d-aspartate embryonic day 18 postnatal day 11 glyceraldehyde-phosphate dehydrogenase reverse transcriptase analysis of variance. The importance of these protein families for human cognition is suggested by the linkage of rare mutations in neuroligins 3 and 4 to autism and mental retardation (16Jamain S. Quach H. Betancur C. Rastam M. Colineaux C. Gillberg I.C. Soderstrom H. Giros B. Leboyer M. Gillberg C. Bourgeron T. Nat. Genet. 2003; 34: 27-29Crossref PubMed Scopus (1343) Google Scholar, 17Laumonnier F. Bonnet-Brilhault F. Gomot M. Blanc R. David A. Moizard M.P. Raynaud M. Ronce N. Lemonnier E. Calvas P. Laudier B. Chelly J. Fryns J.P. Ropers H.H. Hamel B.C. Andres C. Barthelemy C. Moraine C. Briault S. Am. J. Hum. Genet. 2004; 74: 552-557Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar) and the association of variations in neurexin 1 with autism (18Feng J. Schroer R. Yan J. Song W. Yang C. Bockholt A. Cook Jr., E.H. Skinner C. Schwartz C.E. Sommer S.S. Neurosci. Lett. 2006; 409: 10-13Crossref PubMed Scopus (256) Google Scholar, 19Szatmari P. Paterson A.D. Zwaigenbaum L. Roberts W. Brian J. Liu X.Q. Vincent J.B. Skaug J.L. Thompson A.P. Senman L. Feuk L. Qian C. Bryson S.E. Jones M.B. Marshall C.R. Scherer S.W. Vieland V.J. Bartlett C. Mangin L.V. Goedken R. Segre A. Pericak-Vance M.A. Cuccaro M.L. Gilbert J.R. Wright H.H. Abramson R.K. Betancur C. Bourgeron T. Gillberg C. Leboyer M. Buxbaum J.D. Davis K.L. Hollander E. Silverman J.M. Hallmayer J. Lotspeich L. Sutcliffe J.S. Haines J.L. Folstein S.E. Piven J. Wassink T.H. Sheffield V. Geschwind D.H. Bucan M. Brown W.T. Cantor R.M. Constantino J.N. Gilliam T.C. Herbert M. Lajonchere C. Ledbetter D.H. Lese-Martin C. Miller J. Nelson S. Samango-Sprouse C.A. Spence S. State M. Tanzi R.E. Coon H. Dawson G. Devlin B. Estes A. Flodman P. Klei L. McMahon W.M. Minshew N. Munson J. Korvatska E. Rodier P.M. Schellenberg G.D. Smith M. Spence M.A. Stodgell C. Tepper P.G. Wijsman E.M. Yu C.E. Roge B. Mantoulan C. Wittemeyer K. Poustka A. Felder B. Klauck S.M. Schuster C. Poustka F. Bolte S. Feineis-Matthews S. Herbrecht E. Schmotzer G. Tsiantis J. Papanikolaou K. Maestrini E. Bacchelli E. Blasi F. Carone S. Toma C. Van Engeland H. de Jonge M. Kemner C. Koop F. Langemeijer M. Hijimans C. Staal W.G. Baird G. Bolton P.F. Rutter M.L. Weisblatt E. Green J. Aldred C. Wilkinson J.A. Pickles A. Le Couteur A. Berney T. McConachie H. Bailey A.J. Francis K. Honeyman G. Hutchinson A. Parr J.R. Wallace S. Monaco A.P. Barnby G. Kobayashi K. Lamb J.A. Sousa I. Sykes N. Cook E.H. Guter S.J. Leventhal B.L. Salt J. Lord C. Corsello C. Hus V. Weeks D.E. Volkmar F. Tauber M. Fombonne E. Shih A. Nat. Genet. 2007; 39: 319-328Crossref PubMed Scopus (1111) Google Scholar). Mice lacking the major neuroligins 1/2/3 exhibit defects in inhibitory and excitatory synaptic transmission and die within 24 h of birth (20Varoqueaux F. Aramuni G. Rawson R.L. Mohrmann R. Missler M. Gottmann K. Zhang W. Sudhof T.C. Brose N. Neuron. 2006; 51: 741-754Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). Selective loss of neuroligin 1 or 2 results in selective defects in glutamate or GABA synapses, respectively (21Chubykin A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Sudhof T.C. Neuron. 2007; 54: 919-931Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Overexpression or knockdown of neuroligins in cultured neurons also affects synapse development, altering the function and ratio of excitatory and inhibitory synapses (22Prange O. Wong T.P. Gerrow K. Wang Y.T. El-Husseini A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13915-13920Crossref PubMed Scopus (289) Google Scholar, 23Chih B. Engelman H. Scheiffele P. Science. 2005; 307: 1324-1328Crossref PubMed Scopus (548) Google Scholar, 24Levinson J.N. El-Husseini A. Neuron. 2005; 48: 171-174Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 25Futai K. Kim M.J. Hashikawa T. Scheiffele P. Sheng M. Hayashi Y. Nat. Neurosci. 2007; 10: 186-195Crossref PubMed Scopus (218) Google Scholar). A role for neuroligin in development of neuronal cholinergic synapses has also recently been found (26Conroy W.G. Nai Q. Ross B. Naughton G. Berg D.K. Dev. Biol. 2007; 307: 79-91Crossref PubMed Scopus (30) Google Scholar). α-Neurexins terminate in the same LNS domain, transmembrane region, and intracellular region with PDZ domain binding site as β-neurexins but contain additionally five LNS domains and three epidermal growth factor (EGF)-like domains organized into three modules. Analysis of triple knock-out mice for α-neurexin 1/2/3, leaving expression of the β-neurexins intact, revealed a surprising function in coupling presynaptic calcium channels to synaptic vesicle exocytosis (27Missler M. Zhang W. Rohlmann A. Kattenstroth G. Hammer R.E. Gottmann K. Sudhof T.C. Nature. 2003; 424: 939-948Crossref Scopus (504) Google Scholar). α-Neurexins function in transmitter release linked to N- and P/Q-type calcium channels at central nervous system synapses, in calcium-triggered exocytosis of secretory granules in endocrine cells, and to a lesser degree contribute to efficient neuromuscular transmission (28Zhang W. Rohlmann A. Sargsyan V. Aramuni G. Hammer R.E. Sudhof T.C. Missler M. J. Neurosci. 2005; 25: 4330-4342Crossref PubMed Scopus (121) Google Scholar, 29Sons M.S. Busche N. Strenzke N. Moser T. Ernsberger U. Mooren F.C. Zhang W. Ahmad M. Steffens H. Schomburg E.D. Plomp J.J. Missler M. Neuroscience. 2006; 138: 433-446Crossref PubMed Scopus (51) Google Scholar, 30Dudanova I. Sedej S. Ahmad M. Masius H. Sargsyan V. Zhang W. Riedel D. Angenstein F. Schild D. Rupnik M. Missler M. J. Neurosci. 2006; 26: 10599-10613Crossref PubMed Scopus (42) Google Scholar). Intracellularly, α- and β-neurexins bind CASK, Mint, syntenin, and synaptotagmin (31Hata Y. Davletov B. Petrenko A.G. Jahn R. Sudhof T.C. Neuron. 1993; 10: 307-315Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 32Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar, 33Biederer T. Sudhof T.C. J. Biol. Chem. 2000; 275: 39803-39806Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 34Grootjans J.J. Reekmans G. Ceulemans H. David G. J. Biol. Chem. 2000; 275: 19933-19941Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Extracellularly, α-neurexins presumably have unique binding partners to explain the calcium channel coupling phenotype that is not shared with β-neurexins. Known extracellular binding partners of α-neurexins include dystroglycan (35Sugita S. Saito F. Tang J. Satz J. Campbell K. Sudhof T.C. J. Cell Biol. 2001; 154: 435-445Crossref PubMed Scopus (363) Google Scholar) and the secreted peptides neurexophilins (36Missler M. Hammer R.E. Sudhof T.C. J. Biol. Chem. 1998; 273: 34716-34723Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), although the significance of these interactions is not well understood. Originally, it was reported that α-neurexins did not bind neuroligins (11Ichtchenko K. Hata Y. Nguyen T. Ullrich B. Missler M. Moomaw C. Sudhof T.C. Cell. 1995; 81: 435-443Abstract Full Text PDF PubMed Scopus (562) Google Scholar), but while the current work was in progress it became clear that α-neurexins also bind some neuroligins (12Boucard A.A. Chubykin A.A. Comoletti D. Taylor P. Sudhof T.C. Neuron. 2005; 48: 229-236Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Neurexin-1α with or without the S4 insert binds to neuroligins 2 and 3 and the minor variant of neuroligin 1 lacking a B insert but not to the major variant of neuroligin 1 containing a B insert (Fig. 3 of Boucard et al. (12Boucard A.A. Chubykin A.A. Comoletti D. Taylor P. Sudhof T.C. Neuron. 2005; 48: 229-236Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar)). Neurexin 1α lacking the S4 insert was also reported to induce clustering of gephyrin and neuroligin 2 but not PSD-95 when presented on COS cells to dendrites of cultured hippocampal neurons (14Chih B. Gollan L. Scheiffele P. Neuron. 2006; 51: 171-178Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). We set out here to characterize the activity of α-neurexins in COS cell neuron coculture assays; that is, to test the ability of α-neurexins to induce glutamatergic and/or GABAergic postsynaptic differentiation in comparison with β-neurexins. We further explore the structural basis for the difference in synapse-promoting activity of α-compared with β-neurexins and the regulated expression patterns of neurexins with an emphasis on regulation at the S4 splice site. Primary Neuronal Culture and COS Cell Coculture—Dissociated primary hippocampal neuronal cultures were prepared from embryonic day 18 (E18) rats as previously described (37Goslin K. Asmussen H. Banker G. Banker G. Goslin K. Culturing Nerve Cells. 2nd Ed. MIT Press, Cambridge, MA1998: 339-370Google Scholar, 38Kaech S. Banker G. Nat. Protoc. 2006; 1: 2406-2415Crossref PubMed Scopus (1114) Google Scholar). Hippocampi were dissociated by trypsinization and trituration. Dissociated neurons were plated onto poly-l-lysine-coated glass coverslips in 60-mm culture dishes at a density of 300,000 cells/dish and cocultured over a monolayer of glia. After 2 days, cytosine arabinoside (5 μm) was added to neuron cultures to prevent the overgrowth of glia. Cultures were maintained in serum-free minimal essential medium with N2 supplements, 0.1% ovalbumin, and 1 mm pyruvate, with replacing ⅓ of the media per dish once per week. Neurons were treated with 100 μm 2-amino-5-phosphonopentanoic acid (APV, Research Biochemicals) beginning on day 7. COS-7 and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected with Lipofectamine 2000 (Invitrogen). The flat shape of COS cells was preferred for coculture compared with the rounder shape of HEK cells preferred for confocal analysis of surface association. Transfected COS cells were trypsinized 24 h after transfection, washed, and plated at 200,000 cells/dish onto neurons pre-grown for 8-12 days in vitro (DIV). After 24 h of coculture, cells were fixed for 15 min in warm phosphate-buffered saline with 4% paraformaldehyde and 4% sucrose and permeabilized with 0.25% Triton X-100. For experiments involving immunocytochemistry for neuroligins in the cocultures, cells were fixed in -20 °C methanol for 10 min. Construction of Expression Vectors—Neurexin-1β-cyan fluorescent protein (CFP) (+S4) was described previously (13Graf E.R. Kang Y. Hauner A.M. Craig A.M. J. Neurosci. 2006; 26: 4256-4265Crossref PubMed Scopus (131) Google Scholar). To generate neurexin-1α-CFP, the N-terminal portion of rat neurexin 1α was cloned by RT-PCR and joined with the C-terminal portion of the mouse cDNA (BC047146; Open Biosystems) at the internal BstEII site and inserted in-frame into the pECFP-N1 vector (Clontech). Neurexin 2α (AK129239) and neurexin 3α (BC060719) were first corrected for apparent errors and then cloned into pECFP-N1. For neurexin 2α, the Stratagene site-directed mutagenesis kit was used to restore CAG in place of a premature TAG at residue 1301. For neurexin 3α, the splice site 1 insert consisting of an apparent duplication of exons 2 and 3 was removed by an overlap PCR method. To generate neurexin 2β′ and 3β′, the LNS domain of neurexin 1β (residues 84-261) was replaced with the equivalent residues of LNS6 of neurexin 2α or 3α, altering the junctional amino acids GT to EF and amino acids EV to ST to facilitate cloning. Neurexin 1αBC contained a deletion of residues 31-473, and neurexin 1αC contained a deletion of residues 31-899 (numbering according to (39Ushkaryov Y.A. Petrenko A.G. Geppert M. Sudhof T.C. Science. 1992; 257: 50-56Crossref PubMed Scopus (542) Google Scholar), each with the addition of an extra LV at the junction. All neurexin variants used in this paper have the insert at the splice site 4 position. Immunocytochemistry and Imaging—Fixed neuron/COS cell cocultures were blocked with 10% bovine serum albumen (30 min; 37 °C) and incubated with appropriate primary antibodies in phosphate-buffered saline with 3% bovine serum albumen (overnight; room temperature) and then with secondary antibodies (45 min; 37 °C). Coverslips were mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol with 2% 1,4-diazabicyclo(2,2,2)octane). For Figs. 1 and 2, cocultures were stained with anti-gephyrin (mAb7a, IgG1, 1:500; Synaptic Systems), anti-PSD-95 (6G6-1C9, IgG2a, 1:500; Affinity Bioreagents), and anti-synapsin (rabbit, 1:1000; Chemicon) followed by secondary antibodies conjugated to Alexa 488, Alexa 568, and Alexa 647 (Molecular Probes), respectively. For Fig. 4, cocultures were labeled for neuroligin-1/3/4 (4F9, IgG2a, 1:1000; Synaptic Systems) or neuroligin-2 (Graf et al. (13Graf E.R. Kang Y. Hauner A.M. Craig A.M. J. Neurosci. 2006; 26: 4256-4265Crossref PubMed Scopus (131) Google Scholar), rabbit, 1:400) in the Alexa 568 channel and anti-synapsin (46.1, IgG1, 1:100; Synaptic Systems) in the Alexa 647 channel. For Fig. 5, cocultures were labeled for GABAA receptor γ2 (rabbit; 1:200; Alomone) in the Alexa 568 channel and glutamic acid decarboxylase (GAD) 65 (GAD6, IgG2A, 1:100; Developmental Studies Hybridoma Bank) in the Alexa 488 channel. For Fig. 6, cocultures were labeled for neuroligin-2, gephyrin, or PSD-95 in the Alexa 568 channel and anti-synapsin in the Alexa 488 channel. For supplemental Fig. S3, cocultures were incubated live with anti-GABAR α2 (kind gift of J. M. Fritschy (40Brunig I. Scotti E. Sidler C. Fritschy J.M. J. Comp. Neurol. 2002; 443: 43-55Crossref PubMed Scopus (232) Google Scholar)) or γ2 antibodies for 30 min in the neuronal medium plus 50 mm HEPES, pH 7.4, at room temperature, washed extensively, fixed, permeabilized, and then incubated with anti-synapsin followed by secondary antibodies. Fluorescence and phase contrast images were captured with a Photometrics Sensys-cooled CCD camera mounted on a Zeiss Axioplan microscope with a 63× 1.4 numerical aperture oil objective using MetaMorph imaging software (Molecular Devices) and customized filter sets. Controls lacking specific antibodies confirmed no detectable bleed-through between channels CFP, Alexa 488 (imaged through a yellow fluorescent protein filter set), Alexa 568, and Alexa 647. Images were acquired as grey scale from individual channels, and pseudo-color overlays prepared using Adobe Photoshop software. For quantification, sets of cells were cocultured and stained simultaneously. Images of transfected COS cells showing extensive contact with dendrites were taken in all fluorescence channels using the same exposure time for all constructs. For supplemental Fig. S4, optical sections were captured on an Olympus Fluoview FV500 confocal on a BX61W microscope with a 60× 1.4 numerical aperture oil objective, 442-nm laser, and customized filter set.FIGURE 2β-Neurexins induce clustering of the inhibitory synaptic scaffolding protein gephyrin and the excitatory synaptic scaffolding protein PSD-95. COS cells expressing the neurexin variants tagged intracellularly with CFP were overlaid on hippocampal neurons pre-grown for 8-10 days in culture. After 1 day of coculture, cells were fixed and immunolabeled for PSD-95, gephyrin, and synapsin; full field overlays are shown of PSD-95 (green) plus synapsin (blue) or gephyrin (red) plus synapsin (blue). Endogenous inter-neuronal synapses have apposed clusters of postsynaptic PSD-95 or gephyrin and presynaptic synapsin; thus, clusters appear turquoise or purple, respectively, in the color overlays (arrows). In contrast, clusters of PSD-95 or gephyrin lacking associated synapsin appear green or red, respectively, in the color overlays and were induced by all β-neurexins (arrowheads). By alignment with the phase contrast images, induced clusters of PSD-95 and gephyrin can be seen corresponding to sites of neuronal process contact with the neurexin-expressing COS cells and were frequently but not always localized near the edges of transfected COS cells, presumably where the COS cells come into closest contact with the neighboring neurons. Although the induced clusters of gephyrin and PSD-95 appear to overlap at low resolution, at higher resolution they usually resolve into separate clusters side by side (enlarged regions at the bottom of triple overlay of PSD-95 (green), gephyrin (red), and synapsin (blue)). All neurexin constructs used in this paper contain the splice site 4 insert. See also supplemental Fig. S2 for single channel larger field of view and phase contrast images corresponding to the cropped fields shown here for neurexin 2β-CFP. Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4α-Neurexins induce clustering of neuroligin 2 but not neuroligin 1/3/4. A, COS cells expressing the neurexin variants tagged intracellularly with CFP (blue) were overlaid on hippocampal neurons pre-grown for 10-12 days in culture. After 1 day of coculture, cells were fixed and immunolabeled for neuroligin 1/3/4 (with an antibody that recognizes all three) or neuroligin 2 (red) and synapsin (green). Whereas the β-neurexin constructs induced clustering of neuroligin 1/3/4 and neuroligin 2 in contacting dendrites (red or pink not associated with green), the α-neurexins induced clustering of neuroligin 2 but not neuroligin 1/3/4. Scale bar, 10 μm. B, cocultures were scanned, and the COS cells with the strongest apparent associated clusters were imaged and quantitated. The total integrated intensity of associated neuroligin that did not overlap with synapsin was normalized to a value of 100 for neurexin 1β. Significant differences among constructs were found (p < 0.001 ANOVA; n = 4-11 cells per construct).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5α-Neurexins induce clustering of GABAA receptor γ2 subunit. A COS cell expressing neurexin-2α-CFP (blue) is shown here in contact with two major hippocampal neuron dendrites. Clustering of the GABAA receptor γ2 subunit (red) in the absence of glutamic acid decarboxylase (GAD; green) is prominent where the dendrites contact the neurexin-2α-CFP-expressing COS cell (pink). In contrast, the endogenous synaptic clusters of GABARγ2 at axon-dendrite contacts are associated with glutamic acid decarboxylase-positive GABAergic input (yellow). Similar induced clustering of GABARγ2 in COS cell-neuron coculture was observed for the other α and β neurexin constructs (not shown). Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Postsynaptic protein clustering by neurexin-1α requires LNS6 and is negatively modulated by upstream sequences. A, neurexin 1β, 1α, and derivatives of 1α mutated in a predicted calcium binding residue in LNS6 (1αD1176A), lacking module A (1αBC), or lacking modules A and B (1αC) were tagged intracellularly with CFP (blue) and tested for postsynaptic protein clustering activity in the COS cell-neuron coculture assay. Activity was considered positive if the contacting dendrites exhibited any clusters of PSD-95, gephyrin, or neuroligin 2 (red) lacking synapsin (green). Neurexin 1αD1176A had no detectable clustering activity. For PSD-95, all neurexin-1α derivatives also had no detectable clustering activity; PSD-95 clustering was only observed in response to neurexin 1β (compare these images with Figs. 1, 2, and 4). For gephyrin and neuroligin 2, the clustering activity of neurexin 1αBC was indistinguishable from that of 1α. In contrast, the clustering activity of neurexin 1αC was higher than that of 1α and intermediate between that of 1α and 1β. Scale bar, 10 μm. B, quantitation of the percentage of expressing COS cells exhibiting non-synaptic clusters in contacting dendrites confirmed these differences in relative synaptogenic activity (p < 0.001 by ANOVA; *, p < 0.001 by t test compared with neurexin 1α; n = 4 cultures with 10-78 cells scored each). PDZBD, PDZ binding domain; TM, transmembrane domain. CH, glycosylated region.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Image Analysis—For quantification for Figs. 3 and 4, images were randomized so that the experimenter was blind to the transfection group. The area for measuring was defined by the perimeter of the transfected COS cell. Images of the presynaptic and postsynaptic proteins were thresholded. For each postsynaptic protein cluster, a region was drawn around each cluster, and the area and total gray value was measured. Thresholded synapsin was measured through postsynaptic protein regions to determine which clusters were synaptic. Postsynaptic protein clusters that were apposed to synapsin were excluded from the final quantification. Analysis was performed using MetaMorph and Microsoft Excel. All data are reported as mean ± S.E. For quantification for Fig. 6, cocultures were assessed blind to the transfection group. On" @default.
• W2080353409 created "2016-06-24" @default.
• W2080353409 creator A5008833193 @default.
• W2080353409 creator A5021147856 @default.
• W2080353409 creator A5063001738 @default.
• W2080353409 creator A5063642770 @default.
• W2080353409 creator A5067985944 @default.
• W2080353409 date "2008-01-01" @default.
• W2080353409 modified "2023-10-03" @default.
• W2080353409 title "Induction of GABAergic Postsynaptic Differentiation by α-Neurexins" @default.
• W2080353409 cites W1546762987 @default.
• W2080353409 cites W1584583500 @default.
• W2080353409 cites W1590098244 @default.
• W2080353409 cites W1919311176 @default.
• W2080353409 cites W1966727055 @default.
• W2080353409 cites W1974312192 @default.
• W2080353409 cites W1975861627 @default.
• W2080353409 cites W1990122129 @default.
• W2080353409 cites W1992257474 @default.
• W2080353409 cites W1993589817 @default.
• W2080353409 cites W1996092810 @default.
• W2080353409 cites W2002025388 @default.
• W2080353409 cites W2003217331 @default.
• W2080353409 cites W2007087849 @default.
• W2080353409 cites W2012981686 @default.
• W2080353409 cites W2013895597 @default.
• W2080353409 cites W2014907593 @default.
• W2080353409 cites W2015262258 @default.
• W2080353409 cites W2018414679 @default.
• W2080353409 cites W2023179463 @default.
• W2080353409 cites W2036879415 @default.
• W2080353409 cites W2038872732 @default.
• W2080353409 cites W2043002682 @default.
• W2080353409 cites W2043929916 @default.
• W2080353409 cites W2045997854 @default.
• W2080353409 cites W2046165746 @default.
• W2080353409 cites W2050243692 @default.
• W2080353409 cites W2051468191 @default.
• W2080353409 cites W2054235364 @default.
• W2080353409 cites W2054643844 @default.
• W2080353409 cites W2055720472 @default.
• W2080353409 cites W2056042967 @default.
• W2080353409 cites W2057132526 @default.
• W2080353409 cites W2058544036 @default.
• W2080353409 cites W2061463538 @default.
• W2080353409 cites W2061655868 @default.
• W2080353409 cites W2063777298 @default.
• W2080353409 cites W2064352628 @default.
• W2080353409 cites W2080324921 @default.
• W2080353409 cites W2093884718 @default.
• W2080353409 cites W2100414555 @default.
• W2080353409 cites W2104376441 @default.
• W2080353409 cites W2104580322 @default.
• W2080353409 cites W2105603707 @default.
• W2080353409 cites W2106951973 @default.
• W2080353409 cites W2116835533 @default.
• W2080353409 cites W2120598509 @default.
• W2080353409 cites W2125147395 @default.
• W2080353409 cites W2134236497 @default.
• W2080353409 cites W2138530343 @default.
• W2080353409 cites W2138788537 @default.
• W2080353409 cites W2146526204 @default.
• W2080353409 cites W2147152912 @default.
• W2080353409 cites W2147450423 @default.
• W2080353409 cites W2151566815 @default.
• W2080353409 cites W2158524945 @default.
• W2080353409 cites W2167768159 @default.
• W2080353409 cites W2171263596 @default.
• W2080353409 cites W4230194875 @default.
• W2080353409 cites W4361960923 @default.
• W2080353409 doi "https://doi.org/10.1074/jbc.m703957200" @default.
• W2080353409 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2811689" @default.
• W2080353409 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18006501" @default.
• W2080353409 hasPublicationYear "2008" @default.
• W2080353409 type Work @default.
• W2080353409 sameAs 2080353409 @default.
• W2080353409 citedByCount "120" @default.
• W2080353409 countsByYear W20803534092012 @default.
• W2080353409 countsByYear W20803534092013 @default.
• W2080353409 countsByYear W20803534092014 @default.
• W2080353409 countsByYear W20803534092015 @default.
• W2080353409 countsByYear W20803534092016 @default.
• W2080353409 countsByYear W20803534092017 @default.
• W2080353409 countsByYear W20803534092018 @default.
• W2080353409 countsByYear W20803534092019 @default.
• W2080353409 countsByYear W20803534092020 @default.
• W2080353409 countsByYear W20803534092021 @default.
• W2080353409 countsByYear W20803534092022 @default.
• W2080353409 countsByYear W20803534092023 @default.
• W2080353409 crossrefType "journal-article" @default.
• W2080353409 hasAuthorship W2080353409A5008833193 @default.
• W2080353409 hasAuthorship W2080353409A5021147856 @default.
• W2080353409 hasAuthorship W2080353409A5063001738 @default.
• W2080353409 hasAuthorship W2080353409A5063642770 @default.
• W2080353409 hasAuthorship W2080353409A5067985944 @default.
• W2080353409 hasBestOaLocation W20803534092 @default.
• W2080353409 hasConcept C169760540 @default.
• W2080353409 hasConcept C170493617 @default.

• next