Modulation of hippocampal GABAergic currents by anti-gephyrin intrabodies (scFv-geph NLS)

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). In cultured hippocampal neurons, spontaneous inhibitory postsynaptic currents, IPSCs, were recorded from EGFP and EGFP plus scFv-geph NLS transfected pyramidal neurons. Gephyrin-specific single chain antibody fragments (scFv-gephyrin) were expressed as a fusion protein containing a nuclear localization signal (scFv-geph NLS), to efficiently and selectively remove endogenous gephyrin from synapses and retarget it away into the nucleus. Spontaneus events were reversibly blocked by bicuculline (10 μM), indicating that they were GABAA receptor-mediated currents (n=6). The mean sIPSCs frequency was 1.4 ± 0.1 Hz in controls (n=10) and 0.6 ± 0.2 Hz in the presence of scFV-gephyrin NLS (n=7). The mean sIPSCs amplitude was 156.4 ± 30 pA in control and 75.8 ± 19.2 pA in the presence of scFV-gephyrin NLS. Both sIPSC frequency and amplitude were significantly different between control and scFV-gephyrin NLS conditions (p=0.005 and p=0.036, respectively). These data suggest that hampering gephyrin function by the relocalization of the protein into the nucleus alters GABAergic neurotransmission. Andjus, P R, Stevic‐Marinkovic, Z and Cherubini E (1997) Immunoglobulins from motoneurone disease patients enhance glutamate release from rat hippocampal neurones in culture. The Journal of Physiology, 504: 103-112 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