Abstract
Dravet syndrome (DS) is a refractory epilepsy typically caused by heterozygous mutations of the Scn1a gene, which encodes the voltage-gated sodium channel Nav1.1. Glucagon-like peptide-1 (GLP-1) analogues, effective therapeutic agents for the treatment of diabetes, have recently become attractive treatment modalities for patients with nervous system disease; however, the impact of GLP-1 analogues on DS remains unknown. This study aimed to determine the neuroprotective role of liraglutide in mouse and cell models of Scn1a KO-induced epilepsy. Epileptic susceptibility, behavioral changes, and behavioral seizures were assessed using electroencephalography (EEG), IntelliCage (TSE Systems, Bad Homburg, Germany), and the open field task. Morphological changes in brain tissues were observed using hematoxylin and eosin (HE) and Nissl staining. Expression of apoptosis-related proteins and the mammalian target of rapamycin (mTOR) signaling pathway were determined using immunofluorescence and western blotting in Scn1a KO-induced epileptic mice in vitro. Scn1a KO model cell proliferation was evaluated using the Cell Counting Kit-8 assay, and the effect of liraglutide on cellular apoptosis levels was examined using Annexin V-FITC/PI flow cytometry. Apoptotic signal proteins and mTOR were assessed using reverse transcription - quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that liraglutide significantly increased mRNA ((0.31 ± 0.04) *10-3 vs. (1.07 ± 0.08) * 10-3, P = 0.0004) and protein (0.10 ± 0.02 vs. 0.27 ± 0.02, P = 0.0006) expression of Scn1a in Scn1a KO-induced epileptic mice. In addition, liraglutide significantly alleviated electroencephalographic seizures, the severity of responses to epileptic seizures (96.53 ± 0.45 % vs. 85.98 ± 1.24 %, P = 0.0003), cognitive dysfunction, and epileptic-related necrotic neurons (9.76 ± 0.91 % vs. 19.65 ± 2.64 %, P = 0.0005) in Scn1a KO-induced epileptic mice. Moreover, liraglutide protected against Scn1a KO-induced apoptosis, which was manifested in the phosphorylation of mTOR (KO+NS: 1.99 ± 0.31 vs. KO+Lira: 0.97 ± 0.18, P = 0.0004), as well as the downregulation of cleaved caspase-3 (KO+NS: 0.49 ± 0.04 vs. KO+Lira: 0.30 ± 0.01, P = 0.0003) and restoration of the imbalance between BAX (KO+NS: 0.90 ± 0.02 vs. KO+Lira: 0.75 ± 0.04, P = 0.0005) and BCL-2 (KO+NS: 0.46 ± 0.02 vs. KO+Lira: 0.61 ± 0.02, P = 0.0006). Collectively, these results show that liraglutide reduces seizure susceptibility and cognitive dysfunction in the mouse model of Dravet syndrome, and exerts anti-apoptotic and neuroprotective effects in Scn1a KO mice and cells.
Highlights
Dravet syndrome (DS), known as severe myoclonic epilepsy in infants, is a rare childhood epileptic encephalopathy characterized by early onset seizures, multiple types of seizures, anxiety-like behavior, severe cognitive deficits, and resistance to antiepileptic drug treatment (Brunklaus et al, 2012; Dutton et al, 2017; Knupp and Wirrell, 2018)
The F1 heterozygotes (4 weeks, male) and their Wild Type (WT) littermates were used in all experiments
Scn1a KO mice demonstrated no evidence of NaV1.1 sodium channel (SCN1A) protein production, as the band corresponding to SCN1A was almost totally absent (WT: 1.02 ± 0.02, KO: 0.10 ± 0.01, P < 0.001, Figures 1C, D)
Summary
Dravet syndrome (DS), known as severe myoclonic epilepsy in infants, is a rare childhood epileptic encephalopathy characterized by early onset seizures, multiple types of seizures, anxiety-like behavior, severe cognitive deficits, and resistance to antiepileptic drug treatment (Brunklaus et al, 2012; Dutton et al, 2017; Knupp and Wirrell, 2018). GLP-1 binds to and activates its receptor (GLP-1R) in the pancreatic islets, stimulates insulin secretion, and inhibits glucagon release in a glucose-dependent manner and improves glycemic control (Katsurada and Yada, 2016). GLP-1 analogues such as liraglutide are currently used as a second-line therapy to treat type 2 diabetes. The GLP-1 receptor (GLP-1R) is widely expressed in neurons of several brain subregions such as hypothalamus, hippocampus, and cortex (Cork et al, 2015; Yoshino et al, 2015), suggesting the GLP-1 signaling could modulate a variety of neuronal functions (Korol et al, 2015; Prashant et al, 2018). Previous studies have shown that different apoptosis signaling pathways including BCL-2 associated X protein (BAX)/B cell lymphoma 2 (BCL-2) proteins are involved in seizure-induced neuronal death
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