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

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, 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). scFv geph was engineered without specific localization signals (scFv-gephcyto) , thus binding gephyrin at its normal subcellular location. Four days after infection (at DIV 11) miniature inhibitory postsynaptic currents (mIPSCs) were recorded from cortical pyramidal neurons expressing scFv-gephcyto with EGFP or EGFP alone. 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 1.8 ± 0.2 Hz in the presence of scFv-gephcyto (n=24). The mean mIPSCs amplitude was 53.2 ± 2.8 pA in control and 48.1 ± 3.5 pA in the presence of scFv-gephcyto. No significant differences in both frequency and amplitude were found between controls and scFV-gephcyto (p=0.37 and 0.19, respectively). These data suggest that the cytosolic targeting of gephyrin by scFv-gephcyto is not effective to alter 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

突触传递的效率依赖于突触后受体的时空特异性表达，此类受体定位于与突触前释放位点精确对合的区域。在突触结构中，受体以簇状形式组装，这一过程受严格调控，并由多种蛋白动态管控，包括支架蛋白、黏附分子以及沿细胞骨架开展的主动转运过程（Kneussel与Loebrich，2007）。
在抑制性突触中，γ-氨基丁酸能（GABAergic）信号传导调控树突整合、神经兴奋性、环路重塑以及网络活动的精细调控。在诸多调控因子中，微管结合蛋白盖菲林蛋白（gephyrin）可将γ-氨基丁酸A型受体（GABAA receptors）锚定至突触膜上，发挥关键作用（Tyagarajan与Fritschy，2014）。此外，盖菲林蛋白（gephyrin）对于建立并维持恰当的兴奋性/抑制性（excitatory/inhibitory, E/I）平衡不可或缺，该平衡是神经元网络正常行使功能的必要条件（Pizzarelli与Cherubini，2011）。兴奋性/抑制性（E/I）平衡的紊乱被认为是癫痫、精神分裂症、孤独症谱系障碍等多种神经精神疾病的发病根源（Penzes等，2013；Cellot与Cherubini，2014；Nelson与Valakh，2015）。
本研究于体外培养第7天（days in vitro 7, DIV7）对皮层神经元进行慢病毒（lentivirus）感染，该病毒可表达针对盖菲林蛋白（gephyrin）氨基酸153-348位点的单链可变片段（single chain Fv fragments, scFv）胞内抗体（intrabody），即scFv-geph。其中，scFv-geph未经过特异性定位信号改造（命名为scFv-gephcyto，即胞质型scFv-geph），因此可在其正常亚细胞位置与盖菲林蛋白（gephyrin）结合。
感染后4天（即体外培养第11天，DIV11），研究人员对共表达scFv-gephcyto与增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP），以及仅表达EGFP的皮层锥体神经元，记录了微小抑制性突触后电流（miniature inhibitory postsynaptic currents, mIPSCs）。该微小电流可被10 μM的荷包牡丹碱（bicuculline）可逆性阻断，证实其为GABAA受体（GABAA receptors）介导的电流。
对照组（n=35）的平均mIPSCs频率为1.5 ± 0.1 Hz，scFv-gephcyto处理组（n=24）的平均频率为1.8 ± 0.2 Hz；对照组的平均mIPSCs振幅为53.2 ± 2.8 pA，处理组的平均振幅为48.1 ± 3.5 pA。对照组与scFv-gephcyto处理组在mIPSCs的频率与振幅上均未检测到显著差异（分别为p=0.37与p=0.19）。上述实验数据表明，通过scFv-gephcyto靶向胞质中的盖菲林蛋白（gephyrin），并未有效改变γ-氨基丁酸能神经传递。
参考文献：
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