Neuroplastin exerts antiepileptic effects through binding to the α1 subunit of GABA type A receptors to inhibit the internalization of the receptors.

BACKGROUND: Seizures are associated with a decrease in γ-aminobutyric type A acid receptors (GABAaRs) on the neuronal surface, which may be regulated by enhanced internalization of GABAaRs. When interactions between GABAaR subunit α-1 (GABRA1) and postsynaptic scaffold proteins are weakened, the α1-containing GABAaRs leave the postsynaptic membrane and are internalized. Previous evidence suggested that neuroplastin (NPTN) promotes the localization of GABRA1 on the postsynaptic membrane. However, the association between NPTN and GABRA1 in seizures and its effect on the internalization of α1-containing GABAaRs on the neuronal surface has not been studied before. METHODS: An in vitro seizure model was constructed using magnesium-free extracellular fluid, and an in vivo model of status epilepticus (SE) was constructed using pentylenetetrazole (PTZ). Additionally, in vitro and in vivo NPTN-overexpression models were constructed. Electrophysiological recordings and internalization assays were performed to evaluate the action potentials and miniature inhibitory postsynaptic currents of neurons, as well as the intracellular accumulation ratio of α1-containing GABAaRs in neurons. Western blot analysis was performed to detect the expression of GABRA1 and NPTN both in vitro and in vivo. Immunofluorescence co-localization analysis and co-immunoprecipitation were performed to evaluate the interaction between GABRA1 and NPTN. RESULTS: The expression of GABRA1 was found to be decreased on the neuronal surface both in vivo and in vitro seizure models. In the in vitro seizure model, α1-containing GABAaRs showed increased internalization. NPTN expression was found to be positively correlated with GABRA1 expression on the neuronal surface both in vivo and in vitro seizure models. In addition, NPTN overexpression alleviated seizures and NPTN was shown to bind to GABRA1 to form protein complexes that can be disrupted during seizures in both in vivo and in vitro models. Furthermore, NPTN was found to inhibit the internalization of α1-containing GABAaRs in the in vitro seizure model. CONCLUSION: Our findings provide evidence that NPTN may exert antiepileptic effects by binding to GABRA1 to inhibit the internalization of α1-containing GABAaRs.

The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A.

Background: Excitation/inhibition imbalance (E/I imbalance), which involves an increase of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors (AMPARs) and decrease of gamma-aminobutyric acid type A (GABA) type A receptors (GABAaRs) on the neuron surface, has been documented in the pathogenesis of seizures. Notably, it has been established that both the glutamate receptor subunit 2 (GluR2) of AMPARs and beta 2/3 subunits of GABAaRs (Gabrb2+3) participate in the recycling mechanism mediated by the kinesin heavy chain isoform 5A (KIF5A), which determines the number of neuron surface receptors. However, it remains unclear whether receptor recycling is involved in the pathogenesis of seizures. Methods: Twelve adult male Sprague-Dawley rats were randomly allocated to the normal control (Ctl) group (n=6) and the pentylenetetrazol (PTZ)-induced seizure (Sez) group (n=6). The rats in the Ctl group were treated with saline. The rats in the Sez group received an intraperitoneal injection of PTZ at an initial dose of 40 mg/kg. Primary cultured neurons were obtained from newborn rats (24-hour-old). The neurons were exposed to magnesium-free (Mg2+-free) extracellular fluid for 3 hours to establish the seizure model in vitro. We detected the electrophysiology of the seizure model, the expression levels of KIF5A, GluR2, and Gabrb2+3, the recycling ratio of GluR2 and Gabrb2+3, the interaction between KIF5A and GluR2, and the interaction between KIF5A and Gabrb2+3. Results: In the Sez group, the expression of GluR2 on the cell surface was increased and the expression of Gabrb2+3 on the cell surface was decreased. The amplitude and frequency of action potentials were significantly increased in the Mg2+-free group. The amplitude and decay time of AMPAR-mediated miniature excitatory postsynaptic currents were increased in the Mg2+-free group. The amplitude and decay time of miniature inhibitory postsynaptic currents were decreased in the Mg2+-free group. The recycling ratio of GluR2 was increased and the recycling ratio of Gabrb2+3 was decreased in the Mg2+-free group. The interaction between KIF5A and GluR2 was increased, and the interaction between KIF5A and Gabrb2+3 was decreased in the seizure model in vivo and in vitro. Conclusions: The recycling of AMPA receptors/GABAa receptors is related to E/I imbalance and may be regulated by KIF5A.

Klotho alleviates NLRP3 inflammasome-mediated neuroinflammation in a temporal lobe epilepsy rat model by activating the Nrf2 signaling pathway.

Neuroinflammation not only contributes to epileptogenesis and neurodegeneration, but is also associated with cognitive impairment. Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation is positively correlated with progression of temporal lobe epilepsy (TLE) and cognitive impairment. Recent studies have shown that the anti-aging protein, klotho, exerts anti-neuroinflammation effects and enhances cognition in neurodegenerative disorders. In the present study, we investigated the role and underlying mechanism of klotho action in NLRP3 inflammasome-mediated neuroinflammation in a TLE model. Specifically, we first injected an adeno-associated viral (AAV)-mediated overexpression of klotho (AAV-KL) into the bilateral hippocampus of rats. After 3 weeks, rats were intraperitoneally injected with lithium-chloride pilocarpine (LiCl-Pilo) to generate a TLE model. Results showed that klotho was significantly downregulated six weeks after TLE, while AAV-mediated klotho overexpression substantially attenuated TLE-induced hippocampal neuronal injury and cognitive impairment. Interestingly, klotho overexpression significantly alleviated expression of NLRP3, IL-1ß, and caspase-1 proteins, but up-regulated activation of nuclear factor erythroid 2-related factor 2 (Nrf2). However, treatment with Nrf2 inhibitor ML385 significantly reversed klotho's beneficial effects, including alleviated neuroinflammation, attenuated neuronal injury, and improved cognitive function. Taken together, these results indicated that klotho alleviated NLRP3 inflammasome-mediated neuroinflammation by activating the Nrf2 signaling pathway in the TLE rat model, suggesting that this the anti-aging protein could be a novel and promising therapeutic agent for managing TLE-associated cognitive impairment.

Identification of significant immune-related genes for epilepsy via bioinformatics analysis.

BACKGROUND: Epilepsy is one of the most common neurological disorders, but its underlying mechanism has remained obscure, and the role of immune-related genes (IRGs) in epilepsy have not yet been investigated. Therefore, in this study, we explored the association between IRGs and epilepsy. METHODS: An IRG list was collected from the ImmPort database. The gene expression profiles of GSE143272 were collected from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Differentially expressed genes (DEGs) between epilepsy and normal samples were analyzed, and the intersections between IRGs and DEGs were identified using the VennDiagram package, with the intersected genes subjected to further analysis. Enrichment function for intersected genes were performed, constructed a protein-protein interaction (PPI) network via the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the hub genes (top 10) of the PPI network were calculated by the cytoHubba plug-in in Cytoscape. The top correlated genes were selected to perform correlation analysis with immune cells infiltration and expression levels. Finally, we performed validation of the top correlated genes transcriptional expression levels using an animal model. RESULTS: There were a total of 245 DEGs detected in GSE143272, among which 143 were upregulated and 102 downregulated genes in epilepsy. A total of 44 differential IRGs were obtained via intersection of DEGs and IRGs. Enrichment function analysis of DEGs showed that they played a significant role in immune response. The gene CXCL1 was the most correlated with other differentially expressed IRGs via the PPI network. The results of immune cell infiltration analysis indicated that epilepsy patients had higher activated mast cells infiltration (P=0.021), but lower activated CD4 memory T cells (P=0.001), resting CD4 memory T cells (P=0.011), and gamma delta T cells (P=0.038) infiltration. It was revealed that CXCL1 and activated mast cells (R=0.25, P=0.019) and neutrophils (R=0.3, P=0.0043), and a negative correlation with T cells gamma delta (R=-0.25, P=0.018). The levels of CXCL1 expression were significantly lower in epilepsy patients than those in normal samples. CONCLUSIONS: In this study, the results showed that IRGs such as CXCL1 have a significant influence on epilepsy via regulation of immune cells infiltration.