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Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor.

Nakatsu F, Okada M, Mori F, Kumazawa N, Iwasa H, Zhu G, Kasagi Y, Kamiya H, Harada A, Nishimura K, Takeuchi A, Miyazaki T, Watanabe M, Yuasa S, Manabe T, Wakabayashi K, Kaneko S, Saito T, Ohno H - J. Cell Biol. (2004)

Bottom Line: Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved.This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway.Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.

ABSTRACT
AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

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Related in: MedlinePlus

Targeted disruption of μ3B gene by homologous recombination. (A) Structure of mouse μ3B gene including the exon encoding ATG start codon (wild type); the targeting vector containing 5′ (3.0 kb) and 3′ (1.5 kb) homologous regions, the EGFP gene, the Neo gene flanked with two loxP sites, and the herpes simplex virus-thymidine kinase (HSV-tk) gene for positive and negative selections (targeting vector); the resultant mutant allele generated by homologous recombination (mutant); and the mutant allele lacking the Neo gene after crossing with Cre-transgenic mice (ΔNeo). The following restriction enzyme sites are indicated: H, HindIII; Sc, SacI; Se, SpeI; and Sh, SphI. An additional HindIII site, shown by (H)*, exists only in the genome from C57BL/6 mice and not in that from 129 mice. (B–D) Southern blotting using 5′ (probe L) and 3′ (probe S) probes as depicted in A. Digestion of genome DNA with HindIII and SpeI yielded a 3.6-kb wild-type band and a 2.5-kb band generated by homologous recombination using probe S (B). Similarly, digestion of genome DNA with SacI and SphI yielded a 6.4-kb wild-type band and a 5.5-kb band generated by homologous recombination using probe L (C). The mutant allele digested with HindIII and SacI became 1.2 kb shorter (from 6.5 to 5.3 kb) using probe L after deletion of the Neo gene by crossing with Cre-transgenic mice (D, compare first and second lanes). Data shown in D were obtained using mice with C57BL/6 background. Note that the additional HindIII site ((H)*, A) in C57BL/6 genome gave a 2-kb band corresponding the C57BL/6 wild-type allele (third lane). (E) RT-PCR analysis using total RNA from brain (left) or spinal cord (right) as a template for PCR. (F) Whole brain lysates from wild-type, μ3B+/−ΔNeo and μ3B−/−ΔNeo mice were subjected to immunoblotting with anti-μ3 (top left) and anti-β3B (top right) antibodies. After stripping off the antibodies, both membranes were reblotted with anti–α adaptin (bottom) as an internal control for the amount of protein in each lane.
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fig1: Targeted disruption of μ3B gene by homologous recombination. (A) Structure of mouse μ3B gene including the exon encoding ATG start codon (wild type); the targeting vector containing 5′ (3.0 kb) and 3′ (1.5 kb) homologous regions, the EGFP gene, the Neo gene flanked with two loxP sites, and the herpes simplex virus-thymidine kinase (HSV-tk) gene for positive and negative selections (targeting vector); the resultant mutant allele generated by homologous recombination (mutant); and the mutant allele lacking the Neo gene after crossing with Cre-transgenic mice (ΔNeo). The following restriction enzyme sites are indicated: H, HindIII; Sc, SacI; Se, SpeI; and Sh, SphI. An additional HindIII site, shown by (H)*, exists only in the genome from C57BL/6 mice and not in that from 129 mice. (B–D) Southern blotting using 5′ (probe L) and 3′ (probe S) probes as depicted in A. Digestion of genome DNA with HindIII and SpeI yielded a 3.6-kb wild-type band and a 2.5-kb band generated by homologous recombination using probe S (B). Similarly, digestion of genome DNA with SacI and SphI yielded a 6.4-kb wild-type band and a 5.5-kb band generated by homologous recombination using probe L (C). The mutant allele digested with HindIII and SacI became 1.2 kb shorter (from 6.5 to 5.3 kb) using probe L after deletion of the Neo gene by crossing with Cre-transgenic mice (D, compare first and second lanes). Data shown in D were obtained using mice with C57BL/6 background. Note that the additional HindIII site ((H)*, A) in C57BL/6 genome gave a 2-kb band corresponding the C57BL/6 wild-type allele (third lane). (E) RT-PCR analysis using total RNA from brain (left) or spinal cord (right) as a template for PCR. (F) Whole brain lysates from wild-type, μ3B+/−ΔNeo and μ3B−/−ΔNeo mice were subjected to immunoblotting with anti-μ3 (top left) and anti-β3B (top right) antibodies. After stripping off the antibodies, both membranes were reblotted with anti–α adaptin (bottom) as an internal control for the amount of protein in each lane.

Mentions: To disrupt the μ3B locus in E14.1 embryonic stem (ES) cells, the downstream of the start codon of μ3B exon 2 was replaced with EGFP cDNA and neomycin (Neo) resistance gene flanked with loxP sequences by homologous recombination (Fig. 1 A). ES cell lines with the mutant allele were injected into blastocysts from C57BL/6 mice to obtain chimeric offspring. After crossing these chimeras with C57BL/6 mice, heterozygous animals were identified by Southern blotting of tail DNA using 5′ and 3′ probes (Fig. 1, B and C). It has been reported that Neo gene inserted into the genome may perturb the expression of adjacent genes in several knockout mice (Olson et al., 1996). To avoid this possibility, we removed Neo gene by crossing the μ3B−/− mice with Cre-transgenic mice (Sakai and Miyazaki, 1997) to establish μ3B−/−ΔNeo mice (Fig. 1 D). We further backcrossed μ3B−/−ΔNeo mice with C57BL/6 mice to avoid a possible variation of the phenotype(s) with the mixed genotype of C57BL/6 × 129.


Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor.

Nakatsu F, Okada M, Mori F, Kumazawa N, Iwasa H, Zhu G, Kasagi Y, Kamiya H, Harada A, Nishimura K, Takeuchi A, Miyazaki T, Watanabe M, Yuasa S, Manabe T, Wakabayashi K, Kaneko S, Saito T, Ohno H - J. Cell Biol. (2004)

Targeted disruption of μ3B gene by homologous recombination. (A) Structure of mouse μ3B gene including the exon encoding ATG start codon (wild type); the targeting vector containing 5′ (3.0 kb) and 3′ (1.5 kb) homologous regions, the EGFP gene, the Neo gene flanked with two loxP sites, and the herpes simplex virus-thymidine kinase (HSV-tk) gene for positive and negative selections (targeting vector); the resultant mutant allele generated by homologous recombination (mutant); and the mutant allele lacking the Neo gene after crossing with Cre-transgenic mice (ΔNeo). The following restriction enzyme sites are indicated: H, HindIII; Sc, SacI; Se, SpeI; and Sh, SphI. An additional HindIII site, shown by (H)*, exists only in the genome from C57BL/6 mice and not in that from 129 mice. (B–D) Southern blotting using 5′ (probe L) and 3′ (probe S) probes as depicted in A. Digestion of genome DNA with HindIII and SpeI yielded a 3.6-kb wild-type band and a 2.5-kb band generated by homologous recombination using probe S (B). Similarly, digestion of genome DNA with SacI and SphI yielded a 6.4-kb wild-type band and a 5.5-kb band generated by homologous recombination using probe L (C). The mutant allele digested with HindIII and SacI became 1.2 kb shorter (from 6.5 to 5.3 kb) using probe L after deletion of the Neo gene by crossing with Cre-transgenic mice (D, compare first and second lanes). Data shown in D were obtained using mice with C57BL/6 background. Note that the additional HindIII site ((H)*, A) in C57BL/6 genome gave a 2-kb band corresponding the C57BL/6 wild-type allele (third lane). (E) RT-PCR analysis using total RNA from brain (left) or spinal cord (right) as a template for PCR. (F) Whole brain lysates from wild-type, μ3B+/−ΔNeo and μ3B−/−ΔNeo mice were subjected to immunoblotting with anti-μ3 (top left) and anti-β3B (top right) antibodies. After stripping off the antibodies, both membranes were reblotted with anti–α adaptin (bottom) as an internal control for the amount of protein in each lane.
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Related In: Results  -  Collection

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fig1: Targeted disruption of μ3B gene by homologous recombination. (A) Structure of mouse μ3B gene including the exon encoding ATG start codon (wild type); the targeting vector containing 5′ (3.0 kb) and 3′ (1.5 kb) homologous regions, the EGFP gene, the Neo gene flanked with two loxP sites, and the herpes simplex virus-thymidine kinase (HSV-tk) gene for positive and negative selections (targeting vector); the resultant mutant allele generated by homologous recombination (mutant); and the mutant allele lacking the Neo gene after crossing with Cre-transgenic mice (ΔNeo). The following restriction enzyme sites are indicated: H, HindIII; Sc, SacI; Se, SpeI; and Sh, SphI. An additional HindIII site, shown by (H)*, exists only in the genome from C57BL/6 mice and not in that from 129 mice. (B–D) Southern blotting using 5′ (probe L) and 3′ (probe S) probes as depicted in A. Digestion of genome DNA with HindIII and SpeI yielded a 3.6-kb wild-type band and a 2.5-kb band generated by homologous recombination using probe S (B). Similarly, digestion of genome DNA with SacI and SphI yielded a 6.4-kb wild-type band and a 5.5-kb band generated by homologous recombination using probe L (C). The mutant allele digested with HindIII and SacI became 1.2 kb shorter (from 6.5 to 5.3 kb) using probe L after deletion of the Neo gene by crossing with Cre-transgenic mice (D, compare first and second lanes). Data shown in D were obtained using mice with C57BL/6 background. Note that the additional HindIII site ((H)*, A) in C57BL/6 genome gave a 2-kb band corresponding the C57BL/6 wild-type allele (third lane). (E) RT-PCR analysis using total RNA from brain (left) or spinal cord (right) as a template for PCR. (F) Whole brain lysates from wild-type, μ3B+/−ΔNeo and μ3B−/−ΔNeo mice were subjected to immunoblotting with anti-μ3 (top left) and anti-β3B (top right) antibodies. After stripping off the antibodies, both membranes were reblotted with anti–α adaptin (bottom) as an internal control for the amount of protein in each lane.
Mentions: To disrupt the μ3B locus in E14.1 embryonic stem (ES) cells, the downstream of the start codon of μ3B exon 2 was replaced with EGFP cDNA and neomycin (Neo) resistance gene flanked with loxP sequences by homologous recombination (Fig. 1 A). ES cell lines with the mutant allele were injected into blastocysts from C57BL/6 mice to obtain chimeric offspring. After crossing these chimeras with C57BL/6 mice, heterozygous animals were identified by Southern blotting of tail DNA using 5′ and 3′ probes (Fig. 1, B and C). It has been reported that Neo gene inserted into the genome may perturb the expression of adjacent genes in several knockout mice (Olson et al., 1996). To avoid this possibility, we removed Neo gene by crossing the μ3B−/− mice with Cre-transgenic mice (Sakai and Miyazaki, 1997) to establish μ3B−/−ΔNeo mice (Fig. 1 D). We further backcrossed μ3B−/−ΔNeo mice with C57BL/6 mice to avoid a possible variation of the phenotype(s) with the mixed genotype of C57BL/6 × 129.

Bottom Line: Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved.This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway.Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.

ABSTRACT
AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Show MeSH
Related in: MedlinePlus