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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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Exercise ameliorated the active zone protein Bassoon level in aged NMJs.(A) NMJs of trained aged rats (trained-aged) showed higher Bassoon signal intensity compared to the NMJs of un-trained aged rats (aged). Two-year-old rats underwent isometric strength training of tongue muscles for two months. Genioglossus muscles of the tongue were sectioned and stained with antibody against Bassoon (active zone marker), neurofilament and SV2 (nerve morphology, NF+SV2), and α-bungarotoxin (acetylcholine receptors, AChR). Highly magnified Bassoon staining of the area indicated by white dotted boxes are shown in the second column from the left (High Mag. Bassoon). NMJs of young rats (young, postnatal day 56) exhibited higher Bassoon signal intensity than aged rats, similar to our previous mouse study [15]. Scale bar: 10 µm. (B) Average signal intensity of Bassoon was significantly higher in NMJs of young rats and trained-aged rats compared to those of untrained-aged rats (mean ± standard error in arbitrary intensity units: young rats, 8.06±1.27; trained-aged rats, 7.81±1.51; and aged rats, 2.51±0.51; four rats in each group). The red bracket indicates a subgroup of NMJs in aged rats with a minuscule level of Bassoon signal. Young rats and trained-aged rats were not significantly different. (C) The NMJ size did not change significantly by the isometric strength training (trained-aged rats, 261.7±23.2 µm2; untrained aged rats, 264.8±16.5 µm2). Young rats had smaller NMJs (166.5±10.5 µm2) than the aged rats (trained and un-trained), which is consistent with the body and tissue size difference between these two ages. Quantifications in (B, C) are from four rats in each group (a total of 23 – 30 NMJs) and shown by scattered plot with the mean ± standard error by lines. Asterisks indicate significant differences by one-way ANOVA and Bonferroni’s multiple comparison post-test, P<0.05.
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pone-0038029-g003: Exercise ameliorated the active zone protein Bassoon level in aged NMJs.(A) NMJs of trained aged rats (trained-aged) showed higher Bassoon signal intensity compared to the NMJs of un-trained aged rats (aged). Two-year-old rats underwent isometric strength training of tongue muscles for two months. Genioglossus muscles of the tongue were sectioned and stained with antibody against Bassoon (active zone marker), neurofilament and SV2 (nerve morphology, NF+SV2), and α-bungarotoxin (acetylcholine receptors, AChR). Highly magnified Bassoon staining of the area indicated by white dotted boxes are shown in the second column from the left (High Mag. Bassoon). NMJs of young rats (young, postnatal day 56) exhibited higher Bassoon signal intensity than aged rats, similar to our previous mouse study [15]. Scale bar: 10 µm. (B) Average signal intensity of Bassoon was significantly higher in NMJs of young rats and trained-aged rats compared to those of untrained-aged rats (mean ± standard error in arbitrary intensity units: young rats, 8.06±1.27; trained-aged rats, 7.81±1.51; and aged rats, 2.51±0.51; four rats in each group). The red bracket indicates a subgroup of NMJs in aged rats with a minuscule level of Bassoon signal. Young rats and trained-aged rats were not significantly different. (C) The NMJ size did not change significantly by the isometric strength training (trained-aged rats, 261.7±23.2 µm2; untrained aged rats, 264.8±16.5 µm2). Young rats had smaller NMJs (166.5±10.5 µm2) than the aged rats (trained and un-trained), which is consistent with the body and tissue size difference between these two ages. Quantifications in (B, C) are from four rats in each group (a total of 23 – 30 NMJs) and shown by scattered plot with the mean ± standard error by lines. Asterisks indicate significant differences by one-way ANOVA and Bonferroni’s multiple comparison post-test, P<0.05.

Mentions: Next, the tongue muscles were analyzed for the active zone protein Bassoon level in NMJs by fluorescent immunohistochemistry and confocal microscopy, using our previously reported methods [15]. In NMJs of genioglossus muscles of young adult rats at postnatal day 56, the anti-Bassoon antibody stained a punctate staining pattern similar to mouse NMJs (Fig. 3). In the NMJs of aged-rats (two-year-old) genioglossus muscles, the average Bassoon signal intensity was significantly lower than that of young adults (Fig. 3A, B). This result demonstrated that similar decline of the active zone protein occur in NMJs of two mammalian species, rats and mice.


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)

Exercise ameliorated the active zone protein Bassoon level in aged NMJs.(A) NMJs of trained aged rats (trained-aged) showed higher Bassoon signal intensity compared to the NMJs of un-trained aged rats (aged). Two-year-old rats underwent isometric strength training of tongue muscles for two months. Genioglossus muscles of the tongue were sectioned and stained with antibody against Bassoon (active zone marker), neurofilament and SV2 (nerve morphology, NF+SV2), and α-bungarotoxin (acetylcholine receptors, AChR). Highly magnified Bassoon staining of the area indicated by white dotted boxes are shown in the second column from the left (High Mag. Bassoon). NMJs of young rats (young, postnatal day 56) exhibited higher Bassoon signal intensity than aged rats, similar to our previous mouse study [15]. Scale bar: 10 µm. (B) Average signal intensity of Bassoon was significantly higher in NMJs of young rats and trained-aged rats compared to those of untrained-aged rats (mean ± standard error in arbitrary intensity units: young rats, 8.06±1.27; trained-aged rats, 7.81±1.51; and aged rats, 2.51±0.51; four rats in each group). The red bracket indicates a subgroup of NMJs in aged rats with a minuscule level of Bassoon signal. Young rats and trained-aged rats were not significantly different. (C) The NMJ size did not change significantly by the isometric strength training (trained-aged rats, 261.7±23.2 µm2; untrained aged rats, 264.8±16.5 µm2). Young rats had smaller NMJs (166.5±10.5 µm2) than the aged rats (trained and un-trained), which is consistent with the body and tissue size difference between these two ages. Quantifications in (B, C) are from four rats in each group (a total of 23 – 30 NMJs) and shown by scattered plot with the mean ± standard error by lines. Asterisks indicate significant differences by one-way ANOVA and Bonferroni’s multiple comparison post-test, P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368936&req=5

pone-0038029-g003: Exercise ameliorated the active zone protein Bassoon level in aged NMJs.(A) NMJs of trained aged rats (trained-aged) showed higher Bassoon signal intensity compared to the NMJs of un-trained aged rats (aged). Two-year-old rats underwent isometric strength training of tongue muscles for two months. Genioglossus muscles of the tongue were sectioned and stained with antibody against Bassoon (active zone marker), neurofilament and SV2 (nerve morphology, NF+SV2), and α-bungarotoxin (acetylcholine receptors, AChR). Highly magnified Bassoon staining of the area indicated by white dotted boxes are shown in the second column from the left (High Mag. Bassoon). NMJs of young rats (young, postnatal day 56) exhibited higher Bassoon signal intensity than aged rats, similar to our previous mouse study [15]. Scale bar: 10 µm. (B) Average signal intensity of Bassoon was significantly higher in NMJs of young rats and trained-aged rats compared to those of untrained-aged rats (mean ± standard error in arbitrary intensity units: young rats, 8.06±1.27; trained-aged rats, 7.81±1.51; and aged rats, 2.51±0.51; four rats in each group). The red bracket indicates a subgroup of NMJs in aged rats with a minuscule level of Bassoon signal. Young rats and trained-aged rats were not significantly different. (C) The NMJ size did not change significantly by the isometric strength training (trained-aged rats, 261.7±23.2 µm2; untrained aged rats, 264.8±16.5 µm2). Young rats had smaller NMJs (166.5±10.5 µm2) than the aged rats (trained and un-trained), which is consistent with the body and tissue size difference between these two ages. Quantifications in (B, C) are from four rats in each group (a total of 23 – 30 NMJs) and shown by scattered plot with the mean ± standard error by lines. Asterisks indicate significant differences by one-way ANOVA and Bonferroni’s multiple comparison post-test, P<0.05.
Mentions: Next, the tongue muscles were analyzed for the active zone protein Bassoon level in NMJs by fluorescent immunohistochemistry and confocal microscopy, using our previously reported methods [15]. In NMJs of genioglossus muscles of young adult rats at postnatal day 56, the anti-Bassoon antibody stained a punctate staining pattern similar to mouse NMJs (Fig. 3). In the NMJs of aged-rats (two-year-old) genioglossus muscles, the average Bassoon signal intensity was significantly lower than that of young adults (Fig. 3A, B). This result demonstrated that similar decline of the active zone protein occur in NMJs of two mammalian species, rats and mice.

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