An E3-ligase-based method for ablating inhibitory synapses

Abstract

Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to Gephyrin. Expression of GFE3 leads to a strong and specific reduction of Gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.

Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

“…DLGE3, a Dlg1-ablating FingR designed in a fashion similar to that used in our previous work ( Fig. 3A) (33), answers the need for a rapid knockdown of endogenous proteins (33), avoiding the challenges of applying RNAi or CRISPR approaches (36,37) in older chicken embryos.…”

“…Ablating FingRs is a nascent technology, and it is important to experimentally test whether total ablation (33) or functional ablation, as in our case, is achieved with any particular FingR. As regards the FingR/ubiquitin ligase combination, it is clear that ubiquitination of proteins does not universally lead to the ubiquitinated protein's degradation (52)(53)(54)(55); therefore, confirmation of ablation will need to be performed on a target-by-target and FingR-by-FingR basis.…”

“…To perturb Dlg1 function in chicken embryos, which are not accessible via conventional genetic approaches, we constructed a synthetic E3 ubiquitin ligase for viral delivery. Recently, an ablating FingR capable of eliminating Gephyrin in neurons (GFE3) was created by fusing the RING domain of X-linked inhibitor of apoptosis protein (XIAP) to Gephyrin FingR (33). Experiments using GFE3 to degrade the inhibitory postsynaptic protein Gephyrin elucidated the importance of GABA receptor scaffolding at inhibitory synapses.…”

GABA type a receptor trafficking and the architecture of synaptic inhibition

“…Recent proteomic strategies have vastly expanded the protein repertoire of GABAergic synapses to nearly 200 proteins by targeted purification of GABA A Rs, NL2, or gephyrin (collybistin and InSyn1 to a lesser extent) and their respective interactomes (Kang et al, ; Loh et al, ; Nakamura et al, ; Uezu et al, ). Microscopy‐based approaches encompass the fastest growing means of discovery in vitro and in vivo , including optogenetically controlled GABA A Rs (Lin et al, ), two‐photon based GABA photolysis (Oh et al, ), single‐particle‐tracking of receptor diffusion (Petrini and Barberis, ), quantitative super‐resolution imaging of gephyrin (Pennacchietti et al, ) and gephyrin recombinant antibody‐like proteins (Gross et al, ) or fluorescent super‐binding peptides (Maric et al, ). Additionally, proximity ligation assays allow for researchers to detect native protein interactions utilizing widely‐available primary antibodies (Smith et al, ; Tseng et al, ), while emerging single‐molecule fluorescence resonance energy transfer methods (smFRET) have yet to be applied.…”

Falling apart

“…For example, adding a transcriptional repressor domain to certain fusion proteins creates a negative feedback loop, whereby excess fusion protein represses its own production (Gross et al, 2013). This system has recently been extended to fusion proteins that include E3 ubiquitin ligases and used to degrade target proteins (Gross et al, 2016). …”