Bertone NI, Groisman AI, Mazzone GL, et al. Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction. Synapse (New York, N.Y.). 2017 Dec;71(12). DOI: 10.1002/syn.22009. PMID: 28873252.

Resumen:

Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine.
La acetazolamida (AZ), una molécula utilizada frecuentemente para tratar diferentes síndromes neurológicos, es un inhibidor de la anhidrasa carbónica (AC), una enzima que regula el pH dentro y fuera de las células. Combinamos colorantes fluorescentes FM de tipo estiril y técnicas electrofisiológicas en uniones neuromusculares (UNM) del músculo levator auris longus de ratones ex vivo para investigar la modulación de la transmisión sináptica y el reciclaje de vesículas por parte de la AZ.

La liberación de neurotransmisores se vio mínimamente afectada por la AZ, como lo evidenciaron las mediciones de los potenciales de placa terminal evocados y espontáneos. Sin embargo, la evaluación óptica con colorantes FM de la exocitosis vesicular inducida por estímulos de 50 Hz mostró una fuerte reducción en la pérdida de fluorescencia en las UNM tratadas con AZ, un efecto que se abolió al bañar las UNM en Hepes. El colorante residual fue apagado por bromofenol, una molécula pequeña capaz de difundirse dentro de las vesículas.

Además, en ratones transgénicos que expresan Synaptophysin-pHluorin (SypHy), las respuestas de fluorescencia de las terminales nerviosas motoras a un tren de estímulos de 50 Hz se redujeron al 50 % de los controles en presencia de AZ. Experimentos de inmunohistoquímica para evaluar el estado de la quinasa de la cadena ligera de miosina (MLCK), una enzima involucrada en el reciclaje de vesículas, demostraron que la fosforilación de la MLCK fue mucho más fuerte en presencia de AZ que en su ausencia en UNM estimuladas a 50 Hz.

Postulamos que la AZ, a través de la acidificación del citosol y la activación de la MLCK, desvía el reciclaje de las vesículas sinápticas hacia un modo rápido (kiss-and-run), lo que altera el rendimiento sináptico. Estos cambios podrían contribuir a la acción terapéutica reportada en varios síndromes neurológicos como la ataxia, la epilepsia y la migraña.

Abstract:

Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decreased to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes such as ataxia, epilepsy, and migraine.
Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combine styryl-type fluorescent FM dyes and electrophysiological techniques in neuromuscular junctions (NMJs) of the levator auris longus muscle of mice ex vivo to investigate the modulation of synaptic transmission and vesicle recycling by the AZ.

Neurotransmitter release was minimally affected by the AZ, as evidenced by the measurements of evoked and spontaneous endplate potentials. However, optical assessment with FM dyes of 50 Hz stimulus-induced vesicular exocytosis showed a strong reduction in fluorescence loss in AZ-treated NMJs, an effect that is abolished by bathing the UNM in Hepes. The residual dye was quenched by bromophenol, a small molecule capable of diffusing into vesicles.

Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz stimulus train were reduced to 50% of controls in the presence of AZ. Immunohistochemistry experiments to assess the status of myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence of AZ than in its absence at NMJs stimulated at 50 Hz.

We postulate that AZ, through cytosol acidification and MLCK activation, diverts synaptic vesicle recycling toward a fast (kiss) mode. -and-run), which alters synaptic performance. These changes could contribute to the therapeutic action reported in several neurological syndromes such as ataxia, epilepsy and migraine.