br Materials and methods br Results

Materials and methods
Results
Discussion Our study demonstrates that human GIP(3-30)NH2 is a selective GIPR antagonist that inhibits both GIP-mediated cAMP signaling, β-arrestin recruitment, and GIPR internalization. Furthermore, we demonstrate that human GIP(3-30)NH2 binds with high affinity to human and non-human primate GIP receptors, but not to rodent GIP receptors.
Acknowledgement Mette M. Rosenkilde acknowledges financial support from the Novo Nordisk Foundation. Hans Bräuner-Osborne acknowledges financial support from the Independent Research Fund Denmark Medical Sciences and the Carlsberg Foundation. Asuka Inoue was funded by the Japan Science and Technology, PRESTO (grant number JPMJPR1331), the Japan Society for the Promotion of Science, KAKENHI (grant number 17K08264) and the PRIME from the Japan Agency for Medical Research and Development.
Introduction Epilepsy is one of the most common neurological disorders worldwide, which affects up to 1% of the world population (Mendez-Armenta et al., 2014). The median prevalence of lifetime epilepsy is 5.8 per 1,000 for developed countries and 10.3 per 1,000 for developing countries (Ngugi et al., 2010). With diverse etiology, epilepsy is characterized by spontaneous recurrent seizures (SRS) due to hyperexcitability and hypersynchrony of neurons in the brain. As the most grievous form of an epileptic seizure, status epilepticus (SE) is a major neurological and medical emergency that is associated with significant morbidity and mortality (AM and SR, 1980; Trinka et al., 2015). However, epidemiological studies shown that antiepileptic drugs (AEDs) are ineffective or give rise to unacceptable adverse reactions in approximately one third of epilepsy patients (Fisher et al., 2005; Si et al., 2016). At present, most studies on the effects of AEDs focus on neurons, ion channels and transporters, as well as excitatory and inhibitory neurotransmission, which seems to only influence the acute process of ictogenesis (i.e., induction of an acute seizure), but not modify the underlying SRS (Devinsky et al., 2013). In addition, they do not address the neurodegenerative processes that are initiated by SE. Clearly, there is an impending need to explore novel therapeutic strategies for prevent the emergence of SRS. Epileptogenesis is defined as the latent period before which SRS occur (Biagini et al., 2013), which is a process whereby a CHAPS sale becomes progressively epileptic due to diverse initial destabilizing events such as brain injury, stroke, infection, or prolonged seizures (Reddy, 2013). The traditional molecular mechanisms of AEDs may be more connected with ictogenesis than epileptogenesis, the latter reflecting a variable process leading to a continual state of SRS (Loscher and Brandt, 2010). Therefore, we explore experimental interventions designed to arrest or modify the epileptogenic process. More specifically, the alterations during the epileptogenetic process in animal models include glial cell activation, neuroinflammation, neuronal injury and cell death, axonal and dendritic plasticity, presynaptic and postsynaptic modifications, neurogenesis, vascular damage and angiogenesis, disruption of extracellular matrix integrity, as well as structural (i.e., subunit) and functional changes in ion channels properties (Kobow et al., 2012). In general, one or more therapeutic interventions based on such changes could potentially have protective effects on epileptogenesis (Table 1). Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones that could promote glucose-dependent insulin secretion and inhibit glucagon secretion (Baggio and Drucker, 2007; Campbell and Drucker, 2013; Finan et al., 2013). Mimetics of GLP-1 and GIP can cross the blood-brain barrier (BBB) and respectively activate GLP-1R and GIPR that widely expressed in the brain to promote of nerve cell growth, proliferation, differentiation and repair as well as inhibit glial cell activation, neuroinflammation, oxidative stress and apoptosis (D-i and Park, 2015; Holscher, 2014a; Darsalia et al., 2012; Faivre and Hölscher, 2013). GLP-1R and GIPR single agonists have shown neuroprotective effects in animal models or patients of central nervous system (CNS) disease, such as Alzheimer's disease (AD), Parkinson’s disease (PD) and stroke (Faivre and Hölscher, 2013; Duffy and Hölscher, 2013; Faivre and Holscher, 2013; McClean et al., 2011; Ji et al., 2016; Bertilsson et al., 2008; Harkavyi et al., 2008; Li et al., 2009; Liu et al., 2015; Zhang et al., 2015; Li et al., 2016; Holscher, 2014b; Michael et al., 2016; Hölscher, 2016; Aviles-Olmos et al., 2013; Aviles-Olmos et al., 2014; Athauda et al., 2017; Hölscher, 2018). We have tested the GLP-1 analogue liraglutide in the SE rat model previously and it showed good neuroprotective effects. Chronic inflammation and apoptosis in the brain was much reduced (Wang et al., 2018). We furthermore demonstrated that novel dual-GLP-1/GIP receptor agonists have greater neuroprotective effects compared with single GLP-1 analogues. In a first study, we tested a novel dual GLP-1/GIP receptor agonist that was more protective against functional decline than the GLP-1 analogue Val(8)-GLP-1-(glu-PAL) in the transient focal cerebral ischemia rat model by reducing the chronic inflammatory response and cellular apoptosis. (Han et al., 2016). We furthermore tested a dual GLP-1/GIP receptor agonist (DA-JC4) that alleviates learning and memory deficits by decreasing phosphorylated tau protein levels, chronic inflammation response and apoptosis as well as re-sensitizing insulin signaling in the Alzheimer icv. streptozotocin (STZ) rat model (Shi et al., 2017). In addition, we tested the GLP-1/GIP dual agonist DA3-CH in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP) mouse model of PD, which showed enhanced neuroprotective properties over the single GLP-1 receptor agonist liraglutide in Inhibiting the chronic inflammation response, increasing the expression levels of the neuroprotective growth factor Glial Derived Neurotrophic Factor (GDNF), protecting mitochondria, rescuing impaired dopamine synthesis, and improving dyskinesia (Hölscher, 2018; Lovshin and Drucker, 2009; Yuan et al., 2017). DA3-CH had clear neuroprotective effects in a mouse model of Alzheimer’s disease. Memory formation was improved, the amyloid plaque load reduced, and autophagy normalised (Panagaki et al., 2018). Other dual receptor agonists also had neuroprotective effects in animal models of Parkinson’s or Alzheimer’s disease (Feng et al., 2018; Cao et al., 2018). From these studies, we learned that GLP-1/GIP dual agonists can inhibit neuroinflammation mediated by glial cells and neuronal death induced by oxidative stress that contribute to epileptogenesis (Mendez-Armenta et al., 2014; Devinsky et al., 2013; Wang et al., 2015).