br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion Endophilin A1 is a membrane-binding protein that is mainly distributed in the central nervous system (Giachino et al., 1997), localizes in the dendritic shafts and spines of mature neurons (Yang et al., 2015), and relates to the function and morphology of synapses (Kaneko et al., 2005; Masuda et al., 2006; Yang et al., 2015). Our study found that endophilin A1 was up-regulated in the human temporal neocortex. Similar expression of endophilin A1 was also observed in PTZ-kindled epileptic mice. However, there is no difference in the localization of endophilin A1 in Necrostatin 1 tissues between the epilepsy group and control group. According to the aberrant expression pattern of endophilin A1 in epilepsy, we proposed that endophilin A1 might be involved in that disease. Behavioral analyses of the hippocampal endophilin A1 knockdown mice verified this involvement. Under the repeated administration of PTZ, the hippocampal endophilin A1 knockdown mice showed reduced seizure susceptibility and severity compared with the control mice. To explore the underlying mechanism of endophilin A1 in epilepsy, we used the whole-cell patch-clamp technique to measure the CA3 pyramidal neurons in brain slices of both endophilin A1 knockdown mice and control mice. After endophilin A1 was knocked down in the mouse hippocampus, the AP frequencies of pyramidal neurons were decreased within the Mg2+-free ACSF. As AP frequency can reflect the excitability of the neuron and can be related to seizure activity (Barker et al., 2017; Zhang et al., 2017), these electrophysiological data support the role of endophilin A1 in the regulation of neural excitability. Further, given the possible effect of endophilin A1 on EPRs via its function on synaptic receptor internalization (Kaneko et al., 2005; Masuda et al., 2006), we speculate that endophilin A1 may mediate postsynaptic currents by regulating the function or expression of EPRs. Subsequent whole-cell patch-clamp recording showed that endophilin A1 knockdown resulted in a decreased amplitude of mEPSCs. Regarding the role of endophilin A1 in regulation of EPR-related currents, AMPA- and NMDA-EPSC recording indicated that the endophilin A1 knockdown produced a decreased amplitude of AMPA- EPSCs, while the amplitude of NMDA-EPSCs showed no significant change. The results of whole-cell patch-clamp recording supported the role of endophilin A1 in the regulation of postsynaptic current through regulating AMPA-specific currents. AMPARs are tetrameric ligand-gated ion channels assembled from the four subunits GluR1-4 (Traynelis et al., 2010); our research revealed that, among the surface and intracellular levels of those four subunits, only the surface expression of AMPAR GluR2 reduced in the endophilin A1 knockdown mice. Endophilin A1 interacted with AMPAR GluR2, which indicates the possibility that endophilin A1 regulates AMPA-specific currents by mediating surface expression of AMPAR GluR2. Given the function of endophilin A1 in the regulation of AMPA-specific currents, we thought that endophilin A1 might mediate seizure activity via regulating AMPARs. CX546 belongs to a category of drugs called AMPAkines, which activate AMPAR and increase AMPAR transmission by binding to a modulatory site on AMPAR to reduce its deactivation and desensitization kinetics (Arai and Kessler, 2007; Lynch, 2006; Montgomery et al., 2009; Su et al., 2016). The synaptic expression of AMPAR is also up-regulated by CX546 (L. Huang et al., 2016). When AMPARs were activated with CX546, endophilin A1 knockdown failed to reduce the seizure activity in a PTZ-kindled mouse model, which further supported the idea that AMPAR might play an important role in the process whereby endophilin A1 mediates seizure activity in epilepsy. Our experimental data strongly suggests that endophilin A1 may mediate seizure activity via an AMPA-dependent process, and AMPAR GluR2 is the most promising subunit associated with the endophilin A1-related mechanism in epilepsy. AMPARs are postsynaptic receptors that are aligned with synaptic release of glutamate to regulate synaptic signaling (Lisman et al., 2007). The activation of AMPARs drives propagation of synaptic impulses via depolarization of the postsynaptic membrane (Leidenheimer, 2017; Traynelis et al., 2010). With low apparent glutamate affinity and rapid diffusion on the membrane, AMPARs need to be trapped at synaptic sites to contribute effectively to synaptic signal transmission (Choquet and Triller, 2013; Roth et al., 2017). Aside from synaptic transmission, AMPARs are also required in synaptic plasticity (i.e., regulating the synaptic strength and the development and morphologic changes of the neuronal axon and dendritic spine) (Chater and Goda, 2014; Lüthi et al., 1999; Nishimune et al., 1998; Watson et al., 2017). The primary subunit of the AMPAR, namely, AMPAR GluR2, regulates AMPA-mediated excitotoxicity (Wang et al., 2012) and synaptic morphogenesis (F. H. F. Lee et al., 2016). AMPAR GluR2 knockout can result in decreased postsynaptic density and postsynaptic function in hippocampal synapses, indicating that the changed expression of AMPAR GluR2 significantly altered synaptic plasticity (Medvedev et al., 2008), thus altering neuronal activity and excitatory transmission in epilepsy (Houser et al., 2012). The basic characteristics of AMPARs suggest their role in epileptogenesis and seizure generation, and previous published studies have confirmed their involvement (Houser et al., 2012; Sommer et al., 2001). Thus far, the role of AMPARs as targets for drug treatment in epilepsy has been studied for more than two decades (K. Lee et al., 2016; Rogawski, 2011, Rogawski, 2013), and AMPARs have already been investigated for their potentially responsible role in seizure activity in a broad range of animal seizure models and in humans (Citraro et al., 2014; De Sarro et al., 2005; Twele et al., 2015). In our research, endophilin A1, as a postsynaptic protein, was found to interact with AMPAR GluR2, and suppression of the expression of endophilin A1 also suppressed AMPAR GluR2 surface expression and AMPA-dependent synaptic function. Regarding this observed relationship, endophilin A1 may influence the trafficking of AMPAR GluR2 in different ways. On one hand, it may function as a membrane-binding protein to mediate trafficking (Bai et al., 2010; Fu et al., 2011), thereby influencing the distribution of AMPAR GluR2. For example, via its function of regulating the endocytosis of receptor tyrosine kinases (RTKs) (Majumdar et al., 2013; Soubeyran et al., 2002), endophilin A1 might alter the abundance of cell surface glutamate receptors and thereby alter neuronal activity in epilepsy (Li et al., 2013; Miyamoto et al., 2016). Alternatively, it may influence AMPARs via regulating the morphogenesis and stability of dendritic spines (Yang et al., 2015). Moreover, endophilin A1 was observed to colocalize with the PSD (Yang et al., 2015), which is a large collection of signaling proteins at the mature spine head (Sala and Segal, 2014). The spine heads contain AMPARs, and endophilin A1 may consequently cause an alteration in the abundance of postsynaptic AMPARs in epilepsy (Sala and Segal, 2014). Given the specific role of AMPARs in epilepsy, the close relationship between endophilin A1 and AMPARs further supports the existence of an endophilin A1-related mechanism in epilepsy.