Modulation of cortical GABAergic currents by anti-gephyrin intrabodies (scFv-geph NLSC)

The efficiency of synaptic transmission relies on the temporally and spatially regulated expression of postsynaptic receptors localized in precise apposition to presynaptic release sites. At synapses, receptors are organized in clusters which are formed by highly regulated events, dynamically controlled by a number of proteins including scaffolds, adhesion molecules and active transport processes along the cytoskeleton (Kneussel and Loebrich, 2007). At inhibitory synapses, GABAergic signaling controls dendritic integration, neural excitability, circuit reorganization and fine tuning of network activity. Among different players, the tubulin-binding protein gephyrin plays a key role in anchoring GABAA receptors to synaptic membranes (Tyagarajan and Fritschy, 2014). Moreover, gephyrin is instrumental in establishing and maintaining a proper excitatory (E)/inhibitory (I) balance necessary for the correct functioning of neuronal networks (Pizzarelli and Cherubini, 2011). A disruption of the E/I balance is thought to be at the origin of several neuropsychiatric disorders including epilepsy, schizophrenia and autism (Penzes et al. 2013; Cellot and Cherubini, 2014; Nelson and Valakh, 2015). Cortical neurons were infected at DIV 7 with lentivirus driving the expression of the single chain Fv fragments (scFv) intrabody against aminoacids 153-348 of gephyrin protein (scFv-geph) fused with a specific nuclear localization signal (scFv-geph NLSC). Four days after infection (at DIV 11) miniature inhibitory postsynaptic currents (mIPSCs) were recorded from cortical pyramidal neurons expressing EGFP. Miniature currents were reversibly blocked by bicuculline (10 μM) indicating that they were GABAA receptor-mediated. The mean mIPSCs frequency was 1.5 ± 0.1 Hz in control (n=35) and 1.1 ± 0.1 Hz in the presence of scFV-geph NLSC (n=20). The mean mIPSCs amplitude was 53.2 ± 2.8 pA in control and 37.9 ± 1.7 pA in the presence of scFv-geph NLSC. Significant differences were found between controls and scFv-geph NLSC in amplitude and frequency (p=0.037 and 0.00028, respectively). These results suggest that scFv-geph NLSC (which removes gephyrin from the synapses, retargeting it away into the nucleus) significantly impaired inhibitory GABAergic neurotransmission. Cellot G, Cherubini E (2014) GABAergic signaling as therapeutic target for autism spectrum disorders. Front Pediatr 2:70. doi: 10.3389/fped.2014.00070. Kneussel M, Loebrich S (2007) Trafficking and synaptic anchoring of ionotropic inhibitory neurotransmitter receptors. Biol Cell 99: 297-309. Nelson SB, Valakh V (2015) Excitatory/Inhibitory Balance and Circuit Homeostasis in Autism Spectrum Disorders. Neuron 87: 684-698 Penzes P, Buonanno A, Passafaro M, Sala C, Sweet RA (2013) Developmental vulnerability of synapses and circuits associated with neuropsychiatric disorders. J Neurochem 126:165-82. Pizzarelli R, Cherubini E (2011) Alterations of GABAergic signaling in autism spectrum disorders. Neural Plast 297153. doi: 10.1155/2011/297153 Tyagarajan SK, Fritschy JM (2014) Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci 15: 141-156.