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Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina.

Morgans CW, Kensel-Hammes P, Hurley JB, Burton K, Idzerda R, McKnight GS, Bajjalieh SM - PLoS ONE (2009)

Bottom Line: Of the three isoforms expressed in mammals, SV2B is the most divergent.Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health & Sciences University, Portland, OR, USA.

ABSTRACT
The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.

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Generation of SV2B knockouts.A) Targeting construct. A region of the SV2B gene containing the first exon (depicted as a black box) was cloned from a mouse 129/SVJ genomic library. The genomic fragment was used to generate the indicated targeting vector in which exon 1 was replaced with a gene encoding neomycin resistance. A thymidine kinase gene, which makes cells sensitive to gancyclovir was placed at the end of the targeting construct to allow for negative selection of non-homologous recombination events. The mutant gene produces a smaller HindIII (H3) restriction fragment as detected in Southern analyses with a probe indicated by the band above the disrupted gene. Notation key: H2 = HindII restriction site, H3 = HindIII restriction site, RV = EcoRV restriction site, neo = DNA encoding neomycin resistance, TK = DNA encoding thymidine kinase. B) Southern blot of Hind III-digested genomic DNA from littermates resulting from the crossing of heterozygous breeders demonstrating the production of mice homozygous for the SV2B gene disruption. C) SV2 expression in brain of SV2B mutants. 25 ug of a Triton X-100 extract of whole brain was probed with antibodies to the indicated protein. Antibody binding was visualized with HRP-conjugated secondary antibody reacted with enhanced chemiluminescence (ECL) reagent and exposed to film. Disruption of the SV2B gene results in loss of full length SV2B and a decrease in total SV2. Data are representative of experiments from two series of mice.
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pone-0005230-g001: Generation of SV2B knockouts.A) Targeting construct. A region of the SV2B gene containing the first exon (depicted as a black box) was cloned from a mouse 129/SVJ genomic library. The genomic fragment was used to generate the indicated targeting vector in which exon 1 was replaced with a gene encoding neomycin resistance. A thymidine kinase gene, which makes cells sensitive to gancyclovir was placed at the end of the targeting construct to allow for negative selection of non-homologous recombination events. The mutant gene produces a smaller HindIII (H3) restriction fragment as detected in Southern analyses with a probe indicated by the band above the disrupted gene. Notation key: H2 = HindII restriction site, H3 = HindIII restriction site, RV = EcoRV restriction site, neo = DNA encoding neomycin resistance, TK = DNA encoding thymidine kinase. B) Southern blot of Hind III-digested genomic DNA from littermates resulting from the crossing of heterozygous breeders demonstrating the production of mice homozygous for the SV2B gene disruption. C) SV2 expression in brain of SV2B mutants. 25 ug of a Triton X-100 extract of whole brain was probed with antibodies to the indicated protein. Antibody binding was visualized with HRP-conjugated secondary antibody reacted with enhanced chemiluminescence (ECL) reagent and exposed to film. Disruption of the SV2B gene results in loss of full length SV2B and a decrease in total SV2. Data are representative of experiments from two series of mice.

Mentions: A portion of the SV2B gene was isolated from a mouse 129SV genomic library (Stratagene) by screening with a PCR-generated probe encoding bases 60–245 of the rat SV2B cDNA. A fragment of approximately 7.5 kb was subcloned from the genomic library. This fragment contained the exon encoding the translation start site through most of the first transmembrane domain of the SV2A cDNA. A targeting construct was generated in which this exon and surrounding DNA were replaced with a gene encoding neomycin phosphotransferase as illustrated in Figure 1. DNA encoding thymidine kinase was placed at the end of the short arm of the targeting construct to allow for negative selection against non-homologous recombination using the antiviral agent gancyclovir.


Loss of the Synaptic Vesicle Protein SV2B results in reduced neurotransmission and altered synaptic vesicle protein expression in the retina.

Morgans CW, Kensel-Hammes P, Hurley JB, Burton K, Idzerda R, McKnight GS, Bajjalieh SM - PLoS ONE (2009)

Generation of SV2B knockouts.A) Targeting construct. A region of the SV2B gene containing the first exon (depicted as a black box) was cloned from a mouse 129/SVJ genomic library. The genomic fragment was used to generate the indicated targeting vector in which exon 1 was replaced with a gene encoding neomycin resistance. A thymidine kinase gene, which makes cells sensitive to gancyclovir was placed at the end of the targeting construct to allow for negative selection of non-homologous recombination events. The mutant gene produces a smaller HindIII (H3) restriction fragment as detected in Southern analyses with a probe indicated by the band above the disrupted gene. Notation key: H2 = HindII restriction site, H3 = HindIII restriction site, RV = EcoRV restriction site, neo = DNA encoding neomycin resistance, TK = DNA encoding thymidine kinase. B) Southern blot of Hind III-digested genomic DNA from littermates resulting from the crossing of heterozygous breeders demonstrating the production of mice homozygous for the SV2B gene disruption. C) SV2 expression in brain of SV2B mutants. 25 ug of a Triton X-100 extract of whole brain was probed with antibodies to the indicated protein. Antibody binding was visualized with HRP-conjugated secondary antibody reacted with enhanced chemiluminescence (ECL) reagent and exposed to film. Disruption of the SV2B gene results in loss of full length SV2B and a decrease in total SV2. Data are representative of experiments from two series of mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667261&req=5

pone-0005230-g001: Generation of SV2B knockouts.A) Targeting construct. A region of the SV2B gene containing the first exon (depicted as a black box) was cloned from a mouse 129/SVJ genomic library. The genomic fragment was used to generate the indicated targeting vector in which exon 1 was replaced with a gene encoding neomycin resistance. A thymidine kinase gene, which makes cells sensitive to gancyclovir was placed at the end of the targeting construct to allow for negative selection of non-homologous recombination events. The mutant gene produces a smaller HindIII (H3) restriction fragment as detected in Southern analyses with a probe indicated by the band above the disrupted gene. Notation key: H2 = HindII restriction site, H3 = HindIII restriction site, RV = EcoRV restriction site, neo = DNA encoding neomycin resistance, TK = DNA encoding thymidine kinase. B) Southern blot of Hind III-digested genomic DNA from littermates resulting from the crossing of heterozygous breeders demonstrating the production of mice homozygous for the SV2B gene disruption. C) SV2 expression in brain of SV2B mutants. 25 ug of a Triton X-100 extract of whole brain was probed with antibodies to the indicated protein. Antibody binding was visualized with HRP-conjugated secondary antibody reacted with enhanced chemiluminescence (ECL) reagent and exposed to film. Disruption of the SV2B gene results in loss of full length SV2B and a decrease in total SV2. Data are representative of experiments from two series of mice.
Mentions: A portion of the SV2B gene was isolated from a mouse 129SV genomic library (Stratagene) by screening with a PCR-generated probe encoding bases 60–245 of the rat SV2B cDNA. A fragment of approximately 7.5 kb was subcloned from the genomic library. This fragment contained the exon encoding the translation start site through most of the first transmembrane domain of the SV2A cDNA. A targeting construct was generated in which this exon and surrounding DNA were replaced with a gene encoding neomycin phosphotransferase as illustrated in Figure 1. DNA encoding thymidine kinase was placed at the end of the short arm of the targeting construct to allow for negative selection against non-homologous recombination using the antiviral agent gancyclovir.

Bottom Line: Of the three isoforms expressed in mammals, SV2B is the most divergent.Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform.

View Article: PubMed Central - PubMed

Affiliation: Casey Eye Institute, Oregon Health & Sciences University, Portland, OR, USA.

ABSTRACT
The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content.

Show MeSH
Related in: MedlinePlus