Cholesterol plays a modulatory role in lipid membrane fusion, a crucial step in neuronal exocytosis. However, it is still debated how cholesterol does it, and what the consequences of pharmacological lowering of cholesterol might be for exocytosis. Here, we ask how cholesterol alters the properties of synaptic vesicle membranes, and how that affects membrane fusion. Both in single-component bilayers (SB) and in bilayers mimicking synaptic vesicle compositions (SVM), fluorescent probes (FlipTR, Nile Red, and Prodan) showed the expected increase in membrane order with cholesterol. Strikingly, nano-indentation by atomic force microscopy (AFM) revealed divergent changes; in SVM bilayers, cholesterol decreased the membrane breakthrough-force, suggesting reduced mechanical resistance to membrane damage, while the opposite was observed in SB bilayers. Importantly, these breakthrough-forces directly correlated with vesicle fusion kinetics measured by total internal reflection fluorescence (TIRF) microscopy, revealing that cholesterol enhanced (reduced) the rate of fusion in SVM (SB). We infer that AFM breakthrough-force provides a more reliable predictor of fusion propensity than conventional membrane-order measurements. Significantly, when we depleted cholesterol in RN46A neuronal cells using methyl-β-cyclodextrin (mβCD), it impaired exocytosis. This is consistent with our in vitro observations. To confirm whether the effect is due to change in the mechanical properties of the membrane, we attempted to reverse this effect with small molecules that were tested in vitro for their ability to reduce the membrane breakthrough-force. For example, ANA-12, a Trk-B receptor antagonist, reduced the breakthrough force by 18.9%. ANA-12 increased the exocytosis in control cells by 33.5% and also rescued fusion defects in cholesterol-depleted cells. Together, these results highlight that membrane mechanical properties, and not lipid order, govern fusion competence. Also, small molecules that can modulate membrane properties can mitigate unintended negative effects of cholesterol reduction on neuronal exocytosis.
Ankur et al. (Sun,) studied this question.