Effects of cholesterol oxidase on neurotransmission and acetylcholine levels at the mice neuromuscular junctions.

Membrane cholesterol oxidation is a hallmark of redox and metabolic imbalance, and it may accompany neurodegenerative disorders. Using microelectrode recordings of postsynaptic responses as well as fluorescent dyes for monitoring synaptic vesicle cycling and membrane properties, the action of enzymatic cholesterol oxidation on neuromuscular transmission was studied in the mice diaphragms. Cholesterol oxidase (ChO) at low concentration disturbed lipid-ordering specifically in the synaptic membranes, but it did not change markedly spontaneous exocytosis and evoked release in response to single stimuli. At low external Ca2+ conditions, analysis of single exocytotic events revealed a decrease in minimal synaptic delay and the probability of exocytosis upon plasmalemmal cholesterol oxidation. At moderate- and high-frequency activity, ChO treatment enhanced both neurotransmitter and FM-dye release. Furthermore, it precluded a change in exocytotic mode from full-fusion to kiss-and-run during high-frequency stimulation. Accumulation of extracellular acetylcholine (without stimulation) dependent on vesamicol-sensitive transporters was suppressed by ChO. The effects of plasmalemmal cholesterol oxidation on both neurotransmitter/dye release at intense activity and external acetylcholine levels were reversed when synaptic vesicle membranes were also exposed to ChO (i.e., the enzyme treatment was combined with induction of exo-endocytotic cycling). Thus, we suggest that plasmalemmal cholesterol oxidation affects exocytotic machinery functioning, enhances synaptic vesicle recruitment to the exocytosis and decreases extracellular neurotransmitter levels at rest, whereas ChO acting on synaptic vesicle membranes suppresses the participation of the vesicles in the subsequent exocytosis and increases the neurotransmitter leakage. The mechanisms underlying ChO action can be related to the lipid raft disruption.