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Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

Nishimune H, Numata T, Chen J, Aoki Y, Wang Y, Starr MP, Mori Y, Stanford JA - PLoS ONE (2012)

Bottom Line: However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon.Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months.However, Bassoon level in aged NMJs can be improved by muscle exercise.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical School, Kansas City, Kansas, United States of America. hnishimune@kumc.edu

ABSTRACT
The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

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Related in: MedlinePlus

P/Q-type VDCCs localize at the NMJ active zones.(A) P/Q-type VDCCs stained with an antibody against the α subunit (Cav2.1, green) in NMJs of sternomastoid muscle from postnatal day 15 wild-type mice stained with Alexa Fluor 594-labeled α-bungarotoxin to label the acetylcholine receptors (AChR, red). The immunoreactivity is absent in the NMJ area of the littermate P/Q-type VDCC knockout mice (Cacna1a−/−) demonstrating the specificity of the immunohistochemical signals. The same result was confirmed in four independent litters. (B) P/Q-type VDCCs were detected inside the primary gutter of endplates labeled with α-bungarotoxin, where motor nerve terminals reside (arrowheads). An YZ-orthogonal view (a single optical plane) of a region indicated by an orange line in A is shown. The nerve is placed toward the top of the Z-axis and the muscle toward the bottom. Some P/Q-type VDCC signals were also detected in the muscle side. (C) P/Q-type VDCCs aligned with the active zone protein Bassoon in NMJs (arrowheads). Sternomastoid muscle of postnatal day 21 wild-type mice was stained using an anti-P/Q-type VDCC α subunit antibody (red), an anti-Bassoon antibody (green), and Alexa Fluor 647-labeled α-bungarotoxin (blue). The maximal projected XY-view of confocal Z-stack is shown in C1. Panels C2 and C3 show YZ- and XZ-orthogonal views (a single optical plane) at the positions indicated by the orange lines in C1. Panels C4 and C5 show a magnified region of C1 indicated by the dotted-box (C4) and orange line inside the dotted box (C5). Many Bassoon and P/Q-type VDCCs signals (white arrowheads) align in the XY-views (C4) and YZ-orthogonal views (C5, single optical plane). In C5, the nerve is placed towards the top, and muscle is placed towards the bottom. (C6) Colocalization analysis of Bassoon and P/Q-type VDCC within NMJ presynaptic terminals of postnatal day 21 wild-type mice by the Manders’ coefficients (M). Some degree of colocalization of these proteins was indicated by the significantly higher Manders’ coefficients M values for Bassoon overlapping with P/Q-type VDCC (0.42±0.04, 3 NMJs, Bsn on PQ) and P/Q-type VDCC overlapping with Bassoon (0.39±0.02, 3 NMJs, PQ on Bsn) compared to the M value for acetylcholine receptor overlapping minimally with neurofilament (0.11±0.03; 6 NMJs, AChR on NF). A significant difference was detected using one-way ANOVA (P = 0.0002). Asterisks indicate significant difference against AChR on NF by Bonferroni post-test. Scale bars: A, 10 µm; B, C, 1 µm.
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pone-0038029-g001: P/Q-type VDCCs localize at the NMJ active zones.(A) P/Q-type VDCCs stained with an antibody against the α subunit (Cav2.1, green) in NMJs of sternomastoid muscle from postnatal day 15 wild-type mice stained with Alexa Fluor 594-labeled α-bungarotoxin to label the acetylcholine receptors (AChR, red). The immunoreactivity is absent in the NMJ area of the littermate P/Q-type VDCC knockout mice (Cacna1a−/−) demonstrating the specificity of the immunohistochemical signals. The same result was confirmed in four independent litters. (B) P/Q-type VDCCs were detected inside the primary gutter of endplates labeled with α-bungarotoxin, where motor nerve terminals reside (arrowheads). An YZ-orthogonal view (a single optical plane) of a region indicated by an orange line in A is shown. The nerve is placed toward the top of the Z-axis and the muscle toward the bottom. Some P/Q-type VDCC signals were also detected in the muscle side. (C) P/Q-type VDCCs aligned with the active zone protein Bassoon in NMJs (arrowheads). Sternomastoid muscle of postnatal day 21 wild-type mice was stained using an anti-P/Q-type VDCC α subunit antibody (red), an anti-Bassoon antibody (green), and Alexa Fluor 647-labeled α-bungarotoxin (blue). The maximal projected XY-view of confocal Z-stack is shown in C1. Panels C2 and C3 show YZ- and XZ-orthogonal views (a single optical plane) at the positions indicated by the orange lines in C1. Panels C4 and C5 show a magnified region of C1 indicated by the dotted-box (C4) and orange line inside the dotted box (C5). Many Bassoon and P/Q-type VDCCs signals (white arrowheads) align in the XY-views (C4) and YZ-orthogonal views (C5, single optical plane). In C5, the nerve is placed towards the top, and muscle is placed towards the bottom. (C6) Colocalization analysis of Bassoon and P/Q-type VDCC within NMJ presynaptic terminals of postnatal day 21 wild-type mice by the Manders’ coefficients (M). Some degree of colocalization of these proteins was indicated by the significantly higher Manders’ coefficients M values for Bassoon overlapping with P/Q-type VDCC (0.42±0.04, 3 NMJs, Bsn on PQ) and P/Q-type VDCC overlapping with Bassoon (0.39±0.02, 3 NMJs, PQ on Bsn) compared to the M value for acetylcholine receptor overlapping minimally with neurofilament (0.11±0.03; 6 NMJs, AChR on NF). A significant difference was detected using one-way ANOVA (P = 0.0002). Asterisks indicate significant difference against AChR on NF by Bonferroni post-test. Scale bars: A, 10 µm; B, C, 1 µm.

Mentions: A commercial antibody against P/Q-type VDCCs stained NMJs of wild-type mice at postnatal day 15 in a punctate pattern (Fig. 1A). Importantly, these signals were absent in the NMJs of littermate P/Q-type VDCC knockout mice (Cacna1a−/−), demonstrating the specificity of the immunohistochemistry signals (Fig. 1A). Three-dimensional reconstruction using confocal microscope z-stacks revealed that P/Q-type VDCCs distributed in a discrete punctate pattern within the NMJs labeled by α-bungarotoxin that binds specifically to acetylcholine receptors (Fig. 1B, C). In the orthogonal cross section view of the NMJ, P/Q-type VDCCs were detected at the bottom of the primary gutter of endplates, where motor nerve terminals reside (Fig. 1B).


Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

Nishimune H, Numata T, Chen J, Aoki Y, Wang Y, Starr MP, Mori Y, Stanford JA - PLoS ONE (2012)

P/Q-type VDCCs localize at the NMJ active zones.(A) P/Q-type VDCCs stained with an antibody against the α subunit (Cav2.1, green) in NMJs of sternomastoid muscle from postnatal day 15 wild-type mice stained with Alexa Fluor 594-labeled α-bungarotoxin to label the acetylcholine receptors (AChR, red). The immunoreactivity is absent in the NMJ area of the littermate P/Q-type VDCC knockout mice (Cacna1a−/−) demonstrating the specificity of the immunohistochemical signals. The same result was confirmed in four independent litters. (B) P/Q-type VDCCs were detected inside the primary gutter of endplates labeled with α-bungarotoxin, where motor nerve terminals reside (arrowheads). An YZ-orthogonal view (a single optical plane) of a region indicated by an orange line in A is shown. The nerve is placed toward the top of the Z-axis and the muscle toward the bottom. Some P/Q-type VDCC signals were also detected in the muscle side. (C) P/Q-type VDCCs aligned with the active zone protein Bassoon in NMJs (arrowheads). Sternomastoid muscle of postnatal day 21 wild-type mice was stained using an anti-P/Q-type VDCC α subunit antibody (red), an anti-Bassoon antibody (green), and Alexa Fluor 647-labeled α-bungarotoxin (blue). The maximal projected XY-view of confocal Z-stack is shown in C1. Panels C2 and C3 show YZ- and XZ-orthogonal views (a single optical plane) at the positions indicated by the orange lines in C1. Panels C4 and C5 show a magnified region of C1 indicated by the dotted-box (C4) and orange line inside the dotted box (C5). Many Bassoon and P/Q-type VDCCs signals (white arrowheads) align in the XY-views (C4) and YZ-orthogonal views (C5, single optical plane). In C5, the nerve is placed towards the top, and muscle is placed towards the bottom. (C6) Colocalization analysis of Bassoon and P/Q-type VDCC within NMJ presynaptic terminals of postnatal day 21 wild-type mice by the Manders’ coefficients (M). Some degree of colocalization of these proteins was indicated by the significantly higher Manders’ coefficients M values for Bassoon overlapping with P/Q-type VDCC (0.42±0.04, 3 NMJs, Bsn on PQ) and P/Q-type VDCC overlapping with Bassoon (0.39±0.02, 3 NMJs, PQ on Bsn) compared to the M value for acetylcholine receptor overlapping minimally with neurofilament (0.11±0.03; 6 NMJs, AChR on NF). A significant difference was detected using one-way ANOVA (P = 0.0002). Asterisks indicate significant difference against AChR on NF by Bonferroni post-test. Scale bars: A, 10 µm; B, C, 1 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368936&req=5

pone-0038029-g001: P/Q-type VDCCs localize at the NMJ active zones.(A) P/Q-type VDCCs stained with an antibody against the α subunit (Cav2.1, green) in NMJs of sternomastoid muscle from postnatal day 15 wild-type mice stained with Alexa Fluor 594-labeled α-bungarotoxin to label the acetylcholine receptors (AChR, red). The immunoreactivity is absent in the NMJ area of the littermate P/Q-type VDCC knockout mice (Cacna1a−/−) demonstrating the specificity of the immunohistochemical signals. The same result was confirmed in four independent litters. (B) P/Q-type VDCCs were detected inside the primary gutter of endplates labeled with α-bungarotoxin, where motor nerve terminals reside (arrowheads). An YZ-orthogonal view (a single optical plane) of a region indicated by an orange line in A is shown. The nerve is placed toward the top of the Z-axis and the muscle toward the bottom. Some P/Q-type VDCC signals were also detected in the muscle side. (C) P/Q-type VDCCs aligned with the active zone protein Bassoon in NMJs (arrowheads). Sternomastoid muscle of postnatal day 21 wild-type mice was stained using an anti-P/Q-type VDCC α subunit antibody (red), an anti-Bassoon antibody (green), and Alexa Fluor 647-labeled α-bungarotoxin (blue). The maximal projected XY-view of confocal Z-stack is shown in C1. Panels C2 and C3 show YZ- and XZ-orthogonal views (a single optical plane) at the positions indicated by the orange lines in C1. Panels C4 and C5 show a magnified region of C1 indicated by the dotted-box (C4) and orange line inside the dotted box (C5). Many Bassoon and P/Q-type VDCCs signals (white arrowheads) align in the XY-views (C4) and YZ-orthogonal views (C5, single optical plane). In C5, the nerve is placed towards the top, and muscle is placed towards the bottom. (C6) Colocalization analysis of Bassoon and P/Q-type VDCC within NMJ presynaptic terminals of postnatal day 21 wild-type mice by the Manders’ coefficients (M). Some degree of colocalization of these proteins was indicated by the significantly higher Manders’ coefficients M values for Bassoon overlapping with P/Q-type VDCC (0.42±0.04, 3 NMJs, Bsn on PQ) and P/Q-type VDCC overlapping with Bassoon (0.39±0.02, 3 NMJs, PQ on Bsn) compared to the M value for acetylcholine receptor overlapping minimally with neurofilament (0.11±0.03; 6 NMJs, AChR on NF). A significant difference was detected using one-way ANOVA (P = 0.0002). Asterisks indicate significant difference against AChR on NF by Bonferroni post-test. Scale bars: A, 10 µm; B, C, 1 µm.
Mentions: A commercial antibody against P/Q-type VDCCs stained NMJs of wild-type mice at postnatal day 15 in a punctate pattern (Fig. 1A). Importantly, these signals were absent in the NMJs of littermate P/Q-type VDCC knockout mice (Cacna1a−/−), demonstrating the specificity of the immunohistochemistry signals (Fig. 1A). Three-dimensional reconstruction using confocal microscope z-stacks revealed that P/Q-type VDCCs distributed in a discrete punctate pattern within the NMJs labeled by α-bungarotoxin that binds specifically to acetylcholine receptors (Fig. 1B, C). In the orthogonal cross section view of the NMJ, P/Q-type VDCCs were detected at the bottom of the primary gutter of endplates, where motor nerve terminals reside (Fig. 1B).

Bottom Line: However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon.Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months.However, Bassoon level in aged NMJs can be improved by muscle exercise.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical School, Kansas City, Kansas, United States of America. hnishimune@kumc.edu

ABSTRACT
The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

Show MeSH
Related in: MedlinePlus