Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction

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 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.

A new tool to sense pH changes at the neuromuscular junction synaptic cleft

“…Animal protocols. Experiments were carried out on the left Levator auris longus (LAL) muscle of male C57BL/6 J mice (https ://www.jax.org/strai n/00066 4) as previously described 25 . Briefly, animals were supplied by the animal house of the FCEN-UBA.…”

“…Animals were anesthetized with an overdose of 2% tribromoethanol (0.15 mL/10 g body weight) injected in the peritoneal cavity and exsanguinated immediately. The muscle with its nerve supply was excised and dissected on a Sylgard-coated Petri dish containing physiological saline solution of the following composition (in mM): 137 NaCl, 5 KCl, 2 CaCl 2 , 1 MgSO 4 , 12 NaHCO 3 , 1 Na 2 HPO 4 and 11 glucose; continuously bubbled with 95% O 2 /5% CO 2 ; pH 7.3 as previously described 25 .…”

Treatment of migraine in patients with CADASIL

“…20 Recent evidence suggests that acetazolamide leads to increased phosphorylation of MLCK, increasing its inherent activity leading to vasodilation. 21 We postulate that if acetazolamide is truly effective in CADASIL migraine, it may be bypassing the defective NOTCH signaling cascade and immature vascular endothelium to produce vasodilation through modulation of an intrinsically preserved MLCK. As with acetazolamide, sodium valproate is of particular interest, given its pleiotropic effects and purported cytoprotective effect on VSMCs.…”

A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating

“…Our data suggest that therapeutic agents that augment synaptic efficacy should be considered instead. One such compound, the carbonic anhydrase inhibitor acetazolamide, has been successful in blunting attacks of episodic ataxia 2 by increasing cycling of synaptic vesicles (Chen and Chesler, 1992; Bertone et al, 2017). However, the patient carrying the Ca V 2.1 R1673P mutation has been reported to be nonresponsive to this regimen (Luo et al, 2017).…”