Evidence for the Role of Camkinase II and Synapsin I in the Restoration of Neurotransmission in Botulinum Neurotoxin a Intoxicated Nerve Terminals

Abstract

At the active zones in the nerve terminals, presynaptic Ca2+ is recognized as an important second messenger that regulates neurotransmission. However, the precise role of Ca2+ in the regulation of exocytosis still remains to be elusive. Synapsin I (SYN1) is a member of the synapsin family (SYN1/2/3) of synaptic vesicle phosphoproteins that is phosphorylated by Ca2+/calmodulin dependent protein kinase II (CaMKII) at the Serine 603. At the motor nerve terminals (MNT), SYN I regulates synaptic transmission and the depolarization signal mediated rise in Ca2+ influx stimulates CaMKII dependent phosphorylation of SYN1. Botulinum neurotoxin A (BoNT/A) selectively proteolyzes SNAP-25 to inhibit acetylcholine (ACh) release and causes life-threatening neuroparalysis. In our efforts to develop therapeutic strategies to counteract BoNT/A at its primary site of action, the MNT, we discovered that mouse MNT express transient receptor potential vanilloid 1 (TRPV1) channel protein and that capsaicin (TRPV1 agonist) antagonized the neuroparalytic effects of BoNT/A. Capsaicin, when injected post BoNT/A exposure, significantly accelerated recovery from neuroparalysis by restoring ACh release and muscle functions. Capsaicin treatment also decreased the total duration of paralysis by 50% and synergistically increased the effects of 3,4 diaminopyridine (blocks K+ channels and prolongs action potential) on ACh release measured by stimulus evoked twitch tension in BoNT/A poisoned nerve muscle preparations. Further, BoNT/A treatment, in vitro, decreased the expression of CaMKII and SYN1 and the phosphorylation of SYN1 in cholinergic Neuro 2a cells. Capsaicin treatment, post BoNT/A, reversed this. We hypothesize that at the MNT, capsaicin stimulated Ca2+ influx via TRPV1 triggers exocytosis by facilitating synaptic transmission via CaMKII mediated phosphorylation of SYN1.