SPARC triggers a cell-autonomous program of synapse elimination.

Elimination of the excess synaptic contacts established in the early stages of neuronal development is required to refine the function of neuronal circuits. Here we investigate whether secreted protein acidic and rich in cysteine (SPARC), a molecule produced by glial cells, is involved in synapse removal. SPARC production peaks when innervation of the rat superior cervical ganglion and the tail of Xenopus tropicalis tadpoles are remodeled. The formation of new cholinergic synapses in autaptic single-cell microcultures is inhibited by SPARC. The effect resides in the C-terminal domain, which is also responsible for triggering a concentration- and time-dependent disassembly of stable cholinergic synapses. The loss of synaptic contacts is associated with the formation of retracted axon terminals containing multivesicular bodies and secondary lysosomes. The biological relevance of in vitro results was supported by injecting the tail of Xenopus tropicalis tadpoles with peptide 4.2, a 20-aa sequence derived from SPARC that mimics full-length protein effects. Swimming was severely impaired at ∼5 h after peptide application, caused by the massive elimination of neuromuscular junctions and pruning of axonal branches. Effects revert by 6 d after injection, as motor innervation reforms. In conclusion, SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons that likely occurs when protein production peaks during normal development.

Presynaptic clathrin levels are a limiting factor for synaptic transmission.

To maintain communication, neurons must recycle their synaptic vesicles with high efficiency. This process places a huge burden on the clathrin-mediated endocytic machinery, but the consequences of this are poorly understood. We found that the amount of clathrin in a presynaptic terminal is not fixed. During stimulation, clathrin moves out of synapses as a function of stimulus strength and neurotransmitter release probability, which, together with membrane coat formation, transiently reduces the available pool of free clathrin triskelia. Correlative functional and morphological experiments in cholinergic autapses established by superior cervical ganglion neurons in culture show that presynaptic terminal function is compromised if clathrin levels fall by 20% after clathrin heavy chain knock down using RNAi. Synaptic transmission is depressed due to a reduction of cytoplasmic and readily releasable pools of vesicles. However, synaptic depression reverts after dialysis of exogenous clathrin, thus compensating RNAi-induced depletion. Lowering clathrin levels also reduces quantal size, which occurs concomitantly with a decrease in the size of synaptic vesicles. Large dense-core vesicles are unaffected by clathrin knock down. Together, our results show that clathrin levels are a dynamic property of presynaptic terminals that can influence short-term plasticity in a stimulus-dependent manner.