Nedd8 wires the brain, drives synaptic development and stabilize adult synapses protecting from cognitive deterioration: first mechanistic insights

VOGL AM; GIUSTI SA; VERCELLI CA; REFOJO D

Ciudad Autónoma de Bs As

Conferencia; EMBO Meeting: Ubiquitin & ubiquitin-like proteins: At the crossroads from chromatin to protein; 2014

Nedd8 (neural precursor cell development-ally regulated gene 8) was originally described as a brain-expressed transcript downregulated along development. Further studies uncovered a critical role of neddylation controlling cell cycle and cellular proliferation in replicating cells by covalently conjugating Nedd8 to Cullins and more recently to other non-cullin targets. Although Nedd8 was originally cloned from brain tissue, its role in non-replicating postmitotic neurons remains unexplored. Here we report that Nedd8 conjugation increases during postnatal brain development and controls axonal and dendritic growth at early developmental stages and synaptic formation later on development. Comprehensive studies using Cullins-DN, Rbx-siRNAi constructs and MLN4924, suggest that Cullins participate of early stages of neurite development and neuronal migration. Conversely Cullins do not seem to significantly affect later stages of neuronal development such as synaptogenesis or influence synaptic stability during adulthood. We validated these results in vivo by developing different Nael and Csn5 conditional KO mouse lines where the either the neddylation pathway or only the Cullin system were respectively ablated in different neuronal population during development or only in adult animals. During the course of the experiments we observed that Nedd8 and its conjugating enzyme Ubc12 are enriched in synapses where many neddylated substrates were found, revealing that neddylation is an active post-translational modification within the synaptic compartment. Using genetic and pharmacological tools we found that neddylation controls dendritic spine development during neuronal maturation and spine stability in mature neurons both in cell cultures systems and in vivo. The synapse is structurally and functionally organized by scaffolding complexes. We screened all members of the four families of postsynaptic scaffolding proteins. Using a new mass spectrometry variant to specifically detected neddylated proteins in combination with pharmacological tools and the Nedpl (DEN1) CKO mouse line here described for the first time we found that PSD-95 is neddylated and that Lys 202 is the most consistently neddylated residue. The mutation of this Lys 202 residue in PSD-95 rendered the scaffold dominant negative and the sole expression of PSD-95 K202 blocked spine development and maturation and impaired AMPA-mediated neurotransmission, the two main roles of PSD-95 in the dendritic spine. So far, all genetic animal models of the neddylation pathway have been found to be lethal. In order to validate our results in vivo, we developed another mouse line, Nae1CamKIICre-ERT2 which renders a specific deficiency in neddylation only in the anterior forebrain. With these animals we demonstrated that this pathway is critical to keep synaptic networks in place thus avoiding cognitive deterioration.