VAChT overexpression increases acetylcholine at the synaptic cleft and accelerates aging of neuromuscular junctions.

BACKGROUND: Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS).

METHODS: Chat-ChR2-EYFP (VAChT(Hyp)) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1(G93A)), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles.

RESULTS: We show that VAChT is elevated in the spinal cord and at NMJs of VAChT(Hyp) mice. We also show that the amplitude of MEPPs is significantly higher in VAChT(Hyp) muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChT(Hyp) mice compared to control mice. While the development of NMJs was not affected in VAChT(Hyp) mice, NMJs prematurely acquired age-related structural alterations in adult VAChT(Hyp) mice. These structural changes at NMJs were accompanied by motor deficits in VAChT(Hyp) mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChT(Hyp) mice. In the SOD1(G93A) mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice.

CONCLUSIONS: The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.