mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines

Abstract

Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gαq/PLC/Ca(2+)/PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

mTOR is essential for corticosteroid effects on hippocampal AMPA receptor function and fear memory

“…Primary hippocampal neurons were prepared from E18 pregnant Wistar rats as previously described (Loriol et al 2013(Loriol et al , 2014. Neurons were plated in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamax, and penicillin/streptomycin on 12-mm glass coverslips precoated with 0.1 mg/ mL poly-L-lysine.…”

Tracking the activity-dependent diffusion of synaptic proteins using restricted photoconversion of Dendra2

“…This first section reports the step-by-step procedure to prepare primary hippocampal neurons from embryonic day 18 (E18) Wistar rats (Kaech and Banker, 2006 ; Loriol et al, 2013 , 2014 ).…”

“…Note : It is essential to verify the functionality of the tagged protein and to show that the selected tag does not impair the subcellular localization (Figure 2 ) and the overall activity of the studied protein (Loriol et al, 2014 ).…”

“…The proper functioning of such modifications requires a rigorous spatial and temporal control of the associated enzymatic machinery. Recently, using restricted GFP-photobleaching and Dendra2-photoconversion, we demonstrated that the activation of glutamatergic mGlu5 receptors (mGlu5R) transiently promotes the transient synaptic trapping of Ubc9 leading to an increased sumoylation in spines and consequently, to the modulation of neuronal excitability (Loriol et al, 2014 ).…”

The entangled ER-mitochondrial axis as a potential therapeutic strategy in neurodegeneration: A tangled duo unchained

“…However, in these reports, calcium did not directly regulate SUMOylation, but instead, mediated a switch from SUMOylation to acetylation through calcineurin-dependent dephosphorylation (184). A calcium-dependent PKC signaling pathway was recently found to regulate trapping of Ubc9, a SUMOylating enzyme, to dendritic spines in cultured hippocampal neurons (185). Ubc9-dependent SUMOylation of Drp1 is associated in greater accumulation of Drp1 at the mitochondrial membrane (186), yet increased mitochondrial fission at dendritic spines due to calcium-dependent Ubc9 localization has not been reported.…”