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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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Impairment of GABA release due to reduction of VGAT in μ3B−/−ΔNeo mice. (A and B) Measurement of glutamate (A) and GABA (B) release was performed as described in Materials and methods. Basal (circles) and K+-evoked (squares) release of wild-type (closed symbols) and μ3B−/−ΔNeo mice (open symbols) is shown (n = 3 each). Results are expressed as means ± SEM (* indicates P < 0.05; ** indicates P < 0.01). (C) Western blotting of VGAT, VGLUT1, VGLUT2, synaptophysin, synaptotagmin, rabphilin-3A, Rab3A, and VAMP2 in total (lanes 1, 2, 5, and 6) and synaptosomal (lanes 3 and 4) or LP2 (lanes 7 and 8) lysates from hippocampus (left) and whole brain (right) of wild-type (lanes 1, 3, 5, and 7) and μ3B−/−ΔNeo (lanes 2, 4, 6, and 8) mice. Shown are the representatives of four independent experiments. (D) Quantitative analysis of the amount of VGAT protein in total (left) and synaptosomal (right) hippocampal lysates of wild-type and μ3B−/−ΔNeo mice (n = 4 each) as shown in C. Results are expressed as means ± SD. (* indicates P < 0.05). AU, arbitrary unit.
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fig4: Impairment of GABA release due to reduction of VGAT in μ3B−/−ΔNeo mice. (A and B) Measurement of glutamate (A) and GABA (B) release was performed as described in Materials and methods. Basal (circles) and K+-evoked (squares) release of wild-type (closed symbols) and μ3B−/−ΔNeo mice (open symbols) is shown (n = 3 each). Results are expressed as means ± SEM (* indicates P < 0.05; ** indicates P < 0.01). (C) Western blotting of VGAT, VGLUT1, VGLUT2, synaptophysin, synaptotagmin, rabphilin-3A, Rab3A, and VAMP2 in total (lanes 1, 2, 5, and 6) and synaptosomal (lanes 3 and 4) or LP2 (lanes 7 and 8) lysates from hippocampus (left) and whole brain (right) of wild-type (lanes 1, 3, 5, and 7) and μ3B−/−ΔNeo (lanes 2, 4, 6, and 8) mice. Shown are the representatives of four independent experiments. (D) Quantitative analysis of the amount of VGAT protein in total (left) and synaptosomal (right) hippocampal lysates of wild-type and μ3B−/−ΔNeo mice (n = 4 each) as shown in C. Results are expressed as means ± SD. (* indicates P < 0.05). AU, arbitrary unit.

Mentions: That μ3B−/−ΔNeo mice exhibited morphological abnormalities in synapses led us to ask whether the release of neurotransmitters was impaired in these mice. To this end, the release of glutamate and GABA in the hippocampal minislice was measured. The amounts of basal release were similar between μ3B−/−ΔNeo mice and wild-type mice at all the ages tested (Fig. 4, A and B). However, the K+-evoked release of GABA, but not of glutamate, was impaired in μ3B−/−ΔNeo mice at 8 wk old or over (Fig. 4, A and B). As the contents of these neurotransmitters in the hippocampus were equivalent between wild-type and μ3B−/−ΔNeo mice (unpublished data), the difference in GABA release could not be attributed to the changes in the metabolism of GABA itself. Therefore, we postulated that the accumulation of GABA in synaptic vesicles may be impaired, and examined the amounts of vesicular GABA transporter (VGAT) and VGLUT, transporters responsible for the uptake of GABA and glutamate into the synaptic vesicles, respectively (McIntire et al., 1997; Reimer et al., 1998; Gasnier, 2000; Fremeau et al., 2001). The amount of VGAT protein was decreased significantly in synaptosomal lysates from the hippocampus of μ3B−/−ΔNeo mice (Fig. 4, C and D) despite the fact that the amounts of VGLUT1 and VGLUT2, and other synaptic vesicle proteins such as synaptophysin, synaptotagmin, VAMP2, rabphilin-3A, and Rab3A (Fig. 4 C) were unchanged. This was not due to the decrease or loss of the inhibitory neurons themselves, because there was no difference in the number of neurons immunoreactive for GAD67, a marker for inhibitory neurons (unpublished data). These results suggest that the impairment of GABA release in μ3B−/−ΔNeo mice is attributable, at least in part, to the decrease in the amount of VGAT protein in the hippocampus.


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)

Impairment of GABA release due to reduction of VGAT in μ3B−/−ΔNeo mice. (A and B) Measurement of glutamate (A) and GABA (B) release was performed as described in Materials and methods. Basal (circles) and K+-evoked (squares) release of wild-type (closed symbols) and μ3B−/−ΔNeo mice (open symbols) is shown (n = 3 each). Results are expressed as means ± SEM (* indicates P < 0.05; ** indicates P < 0.01). (C) Western blotting of VGAT, VGLUT1, VGLUT2, synaptophysin, synaptotagmin, rabphilin-3A, Rab3A, and VAMP2 in total (lanes 1, 2, 5, and 6) and synaptosomal (lanes 3 and 4) or LP2 (lanes 7 and 8) lysates from hippocampus (left) and whole brain (right) of wild-type (lanes 1, 3, 5, and 7) and μ3B−/−ΔNeo (lanes 2, 4, 6, and 8) mice. Shown are the representatives of four independent experiments. (D) Quantitative analysis of the amount of VGAT protein in total (left) and synaptosomal (right) hippocampal lysates of wild-type and μ3B−/−ΔNeo mice (n = 4 each) as shown in C. Results are expressed as means ± SD. (* indicates P < 0.05). AU, arbitrary unit.
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Related In: Results  -  Collection

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fig4: Impairment of GABA release due to reduction of VGAT in μ3B−/−ΔNeo mice. (A and B) Measurement of glutamate (A) and GABA (B) release was performed as described in Materials and methods. Basal (circles) and K+-evoked (squares) release of wild-type (closed symbols) and μ3B−/−ΔNeo mice (open symbols) is shown (n = 3 each). Results are expressed as means ± SEM (* indicates P < 0.05; ** indicates P < 0.01). (C) Western blotting of VGAT, VGLUT1, VGLUT2, synaptophysin, synaptotagmin, rabphilin-3A, Rab3A, and VAMP2 in total (lanes 1, 2, 5, and 6) and synaptosomal (lanes 3 and 4) or LP2 (lanes 7 and 8) lysates from hippocampus (left) and whole brain (right) of wild-type (lanes 1, 3, 5, and 7) and μ3B−/−ΔNeo (lanes 2, 4, 6, and 8) mice. Shown are the representatives of four independent experiments. (D) Quantitative analysis of the amount of VGAT protein in total (left) and synaptosomal (right) hippocampal lysates of wild-type and μ3B−/−ΔNeo mice (n = 4 each) as shown in C. Results are expressed as means ± SD. (* indicates P < 0.05). AU, arbitrary unit.
Mentions: That μ3B−/−ΔNeo mice exhibited morphological abnormalities in synapses led us to ask whether the release of neurotransmitters was impaired in these mice. To this end, the release of glutamate and GABA in the hippocampal minislice was measured. The amounts of basal release were similar between μ3B−/−ΔNeo mice and wild-type mice at all the ages tested (Fig. 4, A and B). However, the K+-evoked release of GABA, but not of glutamate, was impaired in μ3B−/−ΔNeo mice at 8 wk old or over (Fig. 4, A and B). As the contents of these neurotransmitters in the hippocampus were equivalent between wild-type and μ3B−/−ΔNeo mice (unpublished data), the difference in GABA release could not be attributed to the changes in the metabolism of GABA itself. Therefore, we postulated that the accumulation of GABA in synaptic vesicles may be impaired, and examined the amounts of vesicular GABA transporter (VGAT) and VGLUT, transporters responsible for the uptake of GABA and glutamate into the synaptic vesicles, respectively (McIntire et al., 1997; Reimer et al., 1998; Gasnier, 2000; Fremeau et al., 2001). The amount of VGAT protein was decreased significantly in synaptosomal lysates from the hippocampus of μ3B−/−ΔNeo mice (Fig. 4, C and D) despite the fact that the amounts of VGLUT1 and VGLUT2, and other synaptic vesicle proteins such as synaptophysin, synaptotagmin, VAMP2, rabphilin-3A, and Rab3A (Fig. 4 C) were unchanged. This was not due to the decrease or loss of the inhibitory neurons themselves, because there was no difference in the number of neurons immunoreactive for GAD67, a marker for inhibitory neurons (unpublished data). These results suggest that the impairment of GABA release in μ3B−/−ΔNeo mice is attributable, at least in part, to the decrease in the amount of VGAT protein in the hippocampus.

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