Fig. 1.

The E3 ubiquitin ligase RNF8 suppresses presynaptic differentiation and synapse formation in the cerebellum in vivo. a Left: a representative image of a cerebellum from a P12 rat pup electroporated with the GFP expression plasmid 8 days earlier. Granule neurons have descended to the internal granule layer (IGL) and axonal parallel fibers reside in the molecular layer (ML). Arrowheads denote varicose structures along the parallel fibers that represent presynaptic boutons. Right: P4 rat pups were electroporated with the RNF8 RNAi or control U6 plasmid together with a GFP expression plasmid and sacrificed 8 days later. The cerebellum was removed, sectioned, and subjected to immunohistochemistry using a GFP antibody. Knockdown of RNF8 increased the density of presynaptic boutons in the cerebellar cortex in vivo (***p < 0.05, t test, n = 5 rats). b P9 RNF8 ^loxP/loxP mice were electroporated with the Cre expression plasmid or control vector together with the GFP expression plasmid and boutons along granule neuron parallel fibers were analyzed as in a. Cre-induced knockout of RNF8 in granule neurons increased presynaptic bouton density (***p < 0.001, t test, n = 6 mice). c P9 RNF8 conditional knockout (RNF8 cKO) and control RNF8 ^loxP/loxP mice were electroporated with the GFP expression plasmid and analyzed at different stages of synapse development as in a. Left: representative axons at P17. Right: quantification of the number of presynaptic boutons at different stages of development. Little or no difference in presynaptic bouton number was observed at P13. By contrast, the number of presynaptic boutons was increased at P17 upon conditional knockout of RNF8 (***p < 0.001, t test, n = 3 mice), and this difference was maintained at P21 (**p < 0.01, t test, n = 4–5 mice). Scale bars represent 10 μm. d The cerebellum from P24 RNF8 cKO and control mice were subjected to electron microscopy analyses. Left: representative electron micrographs of the molecular layer of the cerebellar cortex. Parallel fiber/Purkinje cell synapses are denoted by asterisks. Scale bar = 500 nm. Right: quantification of the density of parallel fiber/Purkinje cell synapses in RNF8 cKO and control mice. The density of parallel fiber/Purkinje cell synapses was increased in RNF8 cKO mice compared to control mice (*p < 0.05, t test, n = 3 mice)