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.
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Cabot, J.B., Bushnell, A., Alessi, V. & Mendell, N.R. Postsynaptic gephyrin immunoreactivity exhibits a nearly one-to-one correspondence with gamma-aminobutyric acid-like immunogold-labeled synaptic inputs to sympathetic preganglionic neurons. J. Comp. Neurol. 356, 418–432 (1995).
Craig, A.M., Banker, G., Chang, W., McGrath, M.E. & Serpinskaya, A.S. Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. J. Neurosci. 16, 3166–3177 (1996).
Feng, G. et al. Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity. Science 282, 1321–1324 (1998).
Capecchi, M.R. Altering the genome by homologous recombination. Science 244, 1288–1292 (1989).
McManus, M.T. & Sharp, P.A. Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737–747 (2002).
Hedstrom, K.L., Ogawa, Y. & Rasband, M.N. AnkyrinG is required for maintenance of the axon initial segment and neuronal polarity. J. Cell Biol. 183, 635–640 (2008).
Incontro, S., Asensio, C.S., Edwards, R.H. & Nicoll, R.A. Efficient, complete deletion of synaptic proteins using CRISPR. Neuron 83, 1051–1057 (2014).
Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).
Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat. Struct. Mol. Biol. 19, 117–121 (2012).
Yeh, J.T., Binari, R., Gocha, T., Dasgupta, R. & Perrimon, N. PAPTi: a peptide aptamer interference toolkit for perturbation of protein-protein interaction networks. Sci. Rep. 3, 1156 (2013).
Gross, G.G. et al. Recombinant probes for visualizing endogenous synaptic proteins in living neurons. Neuron 78, 971–985 (2013).
Galbán, S. & Duckett, C.S. XIAP as a ubiquitin ligase in cellular signaling. Cell Death Differ. 17, 54–60 (2010).
Kins, S., Betz, H. & Kirsch, J. Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nat. Neurosci. 3, 22–29 (2000).
Chen, L., Wang, H., Vicini, S. & Olsen, R.W. The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics. Proc. Natl. Acad. Sci. USA 97, 11557–11562 (2000).
Jacob, T.C. et al. Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. J. Neurosci. 25, 10469–10478 (2005).
Wilson, C.J. GABAergic inhibition in the neostriatum. Prog. Brain Res. 160, 91–110 (2007).
Ade, K.K., Janssen, M.J., Ortinski, P.I. & Vicini, S. Differential tonic GABA conductances in striatal medium spiny neurons. J. Neurosci. 28, 1185–1197 (2008).
Santhakumar, V., Jones, R.T. & Mody, I. Developmental regulation and neuroprotective effects of striatal tonic GABAA currents. Neuroscience 167, 644–655 (2010).
Tritsch, N.X., Oh, W.J., Gu, C. & Sabatini, B.L. Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis. eLife 3, e01936 (2014).
Zelenchuk, T.A. & Brusés, J.L. In vivo labeling of zebrafish motor neurons using an mnx1 enhancer and Gal4/UAS. Genesis 49, 546–554 (2011).
Hirata, H., Takahashi, M., Yamada, K. & Ogino, K. The biological role of the glycinergic synapse in early zebrafish motility. Neurosci. Res. 71, 1–11 (2011).
Cui, W.W. et al. The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS. J. Neurosci. 25, 6610–6620 (2005).
Hirata, H. et al. Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor beta-subunit. Proc. Natl. Acad. Sci. USA 102, 8345–8350 (2005).
Hirata, H., Carta, E., Yamanaka, I., Harvey, R.J. & Kuwada, J.Y. Defective glycinergic synaptic transmission in zebrafish motility mutants. Front. Mol. Neurosci. 2, 26 (2009).
Ganser, L.R. et al. Distinct phenotypes in zebrafish models of human startle disease. Neurobiol. Dis. 60, 139–151 (2013).
Yu, W. et al. Gephyrin clustering is required for the stability of GABAergic synapses. Mol. Cell. Neurosci. 36, 484–500 (2007).
Mora, R.J., Roberts, R.W. & Arnold, D.B. Recombinant probes reveal dynamic localization of CaMKIIalpha within somata of cortical neurons. J. Neurosci. 33, 14579–14590 (2013).
Banaszynski, L.A. & Wandless, T.J. Conditional control of protein function. Chem. Biol. 13, 11–21 (2006).
Sakamoto, K.M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).
Varley, Z.K. et al. Gephyrin regulates GABAergic and glutamatergic synaptic transmission in hippocampal cell cultures. J. Biol. Chem. 286, 20942–20951 (2011).
Fan, X., Jin, W.Y., Lu, J., Wang, J. & Wang, Y.T. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation. Nat. Neurosci. 17, 471–480 (2014).
Dev, K.K. PDZ domain protein-protein interactions: a case study with PICK1. Curr. Top. Med. Chem. 7, 3–20 (2007).
Young Kim, H., Young Yum, S., Jang, G. & Ahn, D.-R. Discovery of a non-cationic cell penetrating peptide derived from membrane-interacting human proteins and its potential as a protein delivery carrier. Sci. Rep. 5, 11719 (2015).
Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).
Hayashi-Takagi, A. et al. Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525, 333–338 (2015).
Olson, C.A. & Roberts, R.W. Design, expression, and stability of a diverse protein library based on the tenth fibronectin type III domain. Protein Sci. 16, 476–484 (2007).
Saunders, A., Johnson, C.A. & Sabatini, B.L. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons. Front. Neural Circuits 6, 47 (2012).
Blobel, G. & Sabatini, D.D. Controlled proteolysis of nascent polypeptides in rat liver cell fractions. J. Cell Biol. 45, 130–145 (1970).
Pang, Z. & Geddes, J.W. Mechanisms of cell death induced by the mitochondrial toxin 3-nitropropionic acid: acute excitotoxic necrosis and delayed apoptosis. J. Neurosci. 17, 3064–3073 (1997).
Hudmon, A. et al. A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association. J. Neurosci. 25, 6971–6983 (2005).
De Koninck, Y. & Mody, I. Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels. J. Neurophysiol. 71, 1318–1335 (1994).
Detrich, H.W., Westerfield, M. & Zon, L.I. (eds) Essential Zebrafish Methods: Cell and Developmental Biology 1st edn (Elsevier, 2009).
Kawakami, K. & Shima, A. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239–244 (1999).
Uchida, D., Yamashita, M., Kitano, T. & Iguchi, T. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J. Exp. Biol. 205, 711–718 (2002).
Dempsey, W.P. et al. In vivo single-cell labeling by confined primed conversion. Nat. Methods 12, 645–648 (2015).
Straub, C., Tritsch, N.X., Hagan, N.A., Gu, C. & Sabatini, B.L. Multiphasic modulation of cholinergic interneurons by nigrostriatal afferents. J. Neurosci. 34, 8557–8569 (2014).
We thank M. Roth for technical support, Y. Davis and C. Tian for assistance in analyzing the data, L. Chen for use of his microplate reader and E. Liman for helpful suggestions on the manuscript. Lentivirus was created by L. Asatryan at the USC School of Pharmacy Lentiviral Core Laboratory. This work was supported by the NIH (grants AI085583 and GM083898 to R.W.R., NS046579 to B.L.S. and NS081687 to D.B.A.), by the Human Frontier Science Program (to Y.D.K. and D.B.A.), by the Canadian Institutes of Health Research (MOP12942 to Y.D.K. and MOP286161 to P.D.K.) and by the McKnight foundation (to D.B.A.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Generation of a synthetic E3 ligase that targets Gephyrin to proteasomes for degradation.
(a) RING domain of X-linked Inhibitor of Apoptosis Protein (XIAP) is fused to the C-terminus of GPHN.FingR with a triplicated HA-tag (3HA), and GFP or a red fluorescent protein (TagRFP) is fused to the GPHN.FingR N-terminus through a GGGS linker (4GGGS) to give GFE3. A FingR containing randomized residues within its binding interface (Random FingR) is fused to the RING domain of XIAP to give RandE3. GFP-GPHN.FingR is used as a control. (b) Gephyrin and PSD-95 immunostaining in cortical neurons indicates a 93 ± 2% (P < 0.0001, Kruskal-Wallis) reduction in Gephyrin levels in the presence of GFE3 versus RandE3 or GPHN.FingR, and no change in PSD-95 levels (P > 0.97, Kruskal-Wallis). n = 10 neurons per condition. (c) Lysates from COS-7 cells expressing GFE3, HA-tagged Ubiquitin (HA-Ubiquitin), and Gephyrin-GFP for 24 hours labeled with an anti-Gephyrin antibody. GFE3 strongly reduced Gephyrin-GFP (80 ± 2%, P = 0.01, Kruskal-Wallis) compared to RandE3 or GPHN.FingR, but not in the presence of 10 µM Lactacystin (P > 0.78, Kruskal-Wallis). Beta-tubulin is the loading control. The band below Gephyrin-GFP represents an endogenous COS-7 cell protein recognized by the anti-Gephyrin antibody, but not by GPHN.FingR. n = 4 replicate blots. Bars indicate means ± SEM. (d) Immunoprecipitation of Gephyrin-GFP from COS cell lysate using an anti-Gephyrin antibody, and staining for HA-Ubiquitin. High molecular weight bands (>150 kD) in the GFE3 lane likely correspond to poly-ubiquitinated Gephyrin that accumulates due to proteasome inhibition by Lactacystin. Elevated HA-Ubiquitin in lysate from cells expressing GFE3 or RandE3 vs. GPHN.FingR can be attributed to auto-ubiquitination by the overexpressed FingR-E3 ligase.
(a-l) Cultured cortical neurons infected with approximately 4 infectious units (IFU) of lentivirus per cell. Cells were fixed and stained for GFP (green), Gephyrin (red) and DAPI (to label nuclei, blue). Scale bars, 10 µm. (m) Quantification of DAPI labeled, GFP positive, and negative, cells suggests a transduction efficiency of 84 ± 2% of GFE3, 85 ± 4% of RandE3 and 84 ± 2% of GPHN.FingR (P > 0.93, Kruskal-Wallis). Bars indicate means ± SEM.
Supplementary Figure 3 Expression of GFE3 in cortical neurons results in specific and rapid degradation of endogenous Gephyrin.
(a-i) Western blotting for endogenous Gephyrin (a, arrow), GABAA receptor alpha1 (b, arrow), the Gephyrin binding protein Collybistin II (c, arrow), GluA1 (d, arrow), GluN2B (e, arrow), PSD-95 (f, arrow), and the Gephyrin and GABAA receptor binding protein GABARAP (h, arrow) in cells expressing GFE3, RandE3, GPHN.FingR or in untransduced (Control) cells. (g) Beta-tubulin loading control for a-f. (i) Beta-tubulin loading control for (h).
Supplementary Figure 4 Quantification of endogenous synaptic protein levels in cortical neurons expressing GFE3.
(a-g) Western blotting for synaptic proteins in dissociated cortical neurons transduced with GFE3. A marked decrease in Gephyrin (a; 80 ± 3%, P = 0.01, Kruskal-Wallis) occurs in neurons expressing GFE3 compared to neurons not transduced (Control) or transduced with RandE3 or GPHN.FingR. No significant difference is observed for the steady-state levels of the alpha1 subunit of the GABAA receptor, PSD-95, the GluA1 subunit of the AMPA receptor, the GluN2B subunit of the NMDA receptor, or the Gephyrin interacting proteins Collybistin II and GABARAP (b-g; P > 0.8, Kruskal-Wallis). n = 4 replicate blots. (h, i) Quantification of endogenous Gephyrin and GABAA receptor immunostaining in cortical neurons showed a 93 ± 1% (P < 0.0001, Mann-Whitney) reduction in Gephyrin levels in cells expressing GFE3 compared to cells expressing RandE3, and no change in GABAA receptor levels (P > 0.99, Mann-Whitney). Sample size is n = 10 neurons per condition. Bars indicate means ± SEM.
Supplementary Figure 5 Expression of GFE3 in cortical neurons does not affect cell viability or proteasome activity.
(a, b) Lentiviral mediated expression of GFE3, RandE3 or GPHN.FingR in cortical neurons for 16 hours had a similar effect on cell viability (P > 0.96, Kruskal-Wallis) and cytotoxicity (P > 0.98, Kruskal-Wallis). In contrast, addition of the mitochondrial toxin 3-nitropropionic acid (3-NP, 15 mM) to the medium for 48 hours resulted in an 80 ± 1% decrease in cell viability (P < 0.0001) and 5 ± 0.1-fold increase in cytotoxicity (P < 0.0001, Kruskal-Wallis). (c) ATP levels in cells expressing GFE3, RandE3 or GPHN.FingR for 16 hours were unchanged (P > 0.96, Kruskal-Wallis). Cells treated with 3-NP for 48 hours exhibited an 80 ± 1% decrease in ATP levels (P < 0.0001, Kruskal-Wallis). (d-f) Measurement of proteasome caspase- (d; P > 0.96, Kruskal-Wallis), chymotrypsin- (e; P > 0.87, Kruskal-Wallis) and trypsin-like (f; P > 0.86, Kruskal-Wallis) activity in cells transduced with GFE3, RandE3 or GPHN.FingR for 16 hours. Addition of the proteasome inhibitor clasto-Lactacystin-[beta]-lactone (Lactacystin, 10 µM) to the medium of cells for 3 hours suppressed proteasome activity (P < 0.0001). Data is shown as experimental (transduced or drug-treated) cell measurements normalized to the mean of Control (not transduced or vehicle-treated) cell measurements. Bars indicate means ± SEM. Sample size is n = 24 wells of cells in a 96 well plate per condition.
(a, b) GFE3-mediated ablation of Gephyrin does not affect mEPSCs. Representative traces of mEPSCs obtained from dissociated hippocampal neurons expressing GFE3 or non-transfected controls (a). No significant change in mEPSC amplitude or frequency was observed in GFE3 expressing cells compared to controls (b; P > 0.75, Mann-Whitney). n = 5 independent experiments for controls and GFE3. Bars indicate means ± SEM.
(a) Power spectrum of a continuous recording after GABA (1 mM) application was fitted with the sum (solid lines) of three Laurentzian functions (dotted lines). Spectral analysis of GABA-induced noise in control (non-transfected) cells revealed three average corner frequencies at 3.2 ± 0.6, 31.6 ± 4.2, 380.0 ± 75.4 Hz (n = 5) corresponding to time constants of 53.7, 4.9, 0.3 ms, respectively. (b) In GFE3 expressing cells the three corner frequencies obtained were 1.7 ± 0.3, 36.7 ± 10.6, 316.9 ± 129.7 Hz (n = 6), corresponding to time constants of 105.4, 6.9, 0.9 ms, respectively. (c) No statistical difference was observed between these measurements (P > 0.05, Mann-Whitney). Data are expressed as means ± SEM.
Supplementary Figure 8 GFE3 expression in spinal cord neurons of zebrafish impairs tail flick behavior.
(a) Images of 5 different zebrafish age 24 hours post fertilization with mosaic expression of RandE3 in spinal motoneurons. (b) Age-matched zebrafish that express GFE3 in spinal motoneurons. Scale bars, 1 mm.
(a) A rat cortical neuron transfected with doxycycline (dox)-inducible GFE3-TagRFP, before addition of dox to the culture medium (t = 0 h). (b) GFE3-TagRFP following incubation with 1 µg/mL of dox for 5 hours (t = 5 h). (c) A rat cortical neuron transfected with dox-inducible GFE3-TagRFP prior to incubation with dox (t = 0 h). (d) GFE3-TagRFP in the same neuron as (c) 24 hours after adding 1 µg/mL of dox (t = 24 h). (e) GFE3-TagRFP expression 48 hours after removal of dox. Scale bars, 10 µm.
Supplementary Figures 1–9 (PDF 1382 kb)
List of amino acid and DNA sequences assembled and used in this study. (DOCX 21 kb)
Zebrafish expressing GFE3-GFP in motoneurons in the spinal cord displays an abnormal tail flick. (MOV 23 kb)
Zebrafish expressing GFE3-GFP in motoneurons in the spinal cord displays an abnormal tail flick. (MOV 24 kb)
Zebrafish expressing GFE3-GFP in motoneurons in the spinal cord displays a normal tail flick. (MOV 16 kb)
Zebrafish expressing GFE3-GFP in motoneurons in the spinal cord displays a normal tail flick. (MOV 30 kb)
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Gross, G., Straub, C., Perez-Sanchez, J. et al. An E3-ligase-based method for ablating inhibitory synapses. Nat Methods 13, 673–678 (2016) doi:10.1038/nmeth.3894
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