Proteasome inhibition triggers activity-dependent increase in the size of the recycling vesicle pool in cultured hippocampal neurons.

The ubiquitin proteasome system, generally known for its function in protein degradation, also appears to play an important role in regulating membrane trafficking. A role for the proteasome in regulating presynaptic release and vesicle trafficking has been proposed for invertebrates, but it remains to be tested in mammalian presynaptic terminals. We used the fluorescent styrylpyridinium dye FM4-64 to visualize changes in the recycling pool of vesicles in hippocampal culture under pharmacological inhibition of the proteasome. We found that a 2 h inhibition increases the recycling pool of vesicles by 76%, with no change in the rate or total amount of dye release. Interestingly, enhancement did not depend on protein synthesis but did depend on synaptic activity; blocking action potentials during proteasome inhibition abolished the effect whereas increasing neuronal activity accelerated the effect with an increased recycling pool evident after 15 min. We propose that the proteasome acts as a negative-feedback regulator of synaptic transmission, possibly serving a homeostatic role.