Principal neurons in the olfactory cortex mediate bidirectional modulation of seizures.

Although the piriform cortex (PC) has been previously implicated as a critical node for seizure generation and propagation, the underlying neural mechanism has remained unclear. Here, we found increased excitability in PC neurons during amygdala kindling acquisition. Optogenetic or chemogenetic activation of PC pyramidal neurons promoted kindling progression, whereas inhibition of these neurons retarded seizure activities induced by electrical kindling in the amygdala. Furthermore, chemogenetic inhibition of PC pyramidal neurons alleviated the severity of kainic acid-induced acute seizures. These results demonstrate that PC pyramidal neurons bidirectionally modulate seizures in temporal lobe epilepsy, providing evidence for the efficacy of PC pyramidal neurons as a potential therapeutic target for epileptogenesis. KEY POINTS: While the piriform cortex (PC) is an important olfactory centre critically involved in olfactory processing and plays a crucial role in epilepsy due to its close connection with the limbic system, how the PC regulates epileptogenesis is largely unknown. In this study, we evaluated the neuronal activity and the role of pyramidal neurons in the PC in the mouse amygdala kindling model of epilepsy. PC pyramidal neurons are hyperexcited during epileptogenesis. Optogenetic and chemogenetic activation of PC pyramidal neurons significantly promoted seizures in the amygdala kindling model, whereas selective inhibition of these neurons produced an anti-epileptic effect for both electrical kindling and kainic acid-induced acute seizures. The results of the present study indicate that PC pyramidal neurons bidirectionally modulate seizure activity.

Binding of Glycerol to Human Galectin-7 Expands Stability and Modulates Its Functions.

Glycerol is seen in biological systems as an intermediate in lipid metabolism. In recent years, glycerol has been reported to act as a chemical chaperone to correct the conformation of proteins. Here, we investigate the role of glycerol in galectin-7 (Gal-7). The thermal shift and CD assays showed that the thermal stability of Gal-7 increased with glycerol concentration but with little secondary structure changes induced by glycerol. In addition, glycerol can inhibit Gal-7-mediated erythrocyte agglutination. We also solved the crystal structures of human Gal-7 in complex with glycerol in two different conditions. Glycerol binds at the carbohydrate-recognition binding sites of Gal-7, which indicates glycerol as a small ligand for Gal-7. Surprisingly, glycerol can bind a new pocket near the N-terminus of Gal-7, which can greatly reduce the flexibility and improve the stability of this region. Moreover, overexpression of Gal-7 decreased the intracellular triglyceride levels and increased mRNA expression of aquaporin-3 (AQP-3) when HeLa cells were incubated with glycerol. These findings indicate that Gal-7 might regulate glycerol metabolism. Overall, our results on human Gal-7 raise the perspective to systematically explore this so far unrecognized phenomenon for Gal-7 in glycerol metabolism.

Self-Enhanced Chemiluminescence of Tris(bipyridine) Ruthenium(II) Derivative Nanohybrids: Mechanism Insight and Application for Sensitive Silver Ions Detection.

In recent years, self-enhanced tris(bipyridine) ruthenium(II)-based luminescence systems have achieved great development in electrochemiluminescence (ECL) but are seldom mentioned in chemiluminescence (CL). Herein, a self-enhanced CL luminophore with excellent CL behavior was synthesized by covalently cross-linking tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) dichloride ([Ru(dcbpy)3]Cl2) with branched polyethylenimine (BPEI) in one molecule (BPEI-Ru(II)), which then self-assembled into nanoparticles (BRuNPs). The nanoparticles exhibited stable and strong CL emission with potassium persulfate (K2S2O8) as the oxidant. After the redox reaction between K2S2O8 and BRuNPs, and the subsequent intramolecular electron-transfer reaction, excited state luminophores were generated to emit light. This self-enhanced CL system shortened the electron transfer distance and reduced energy loss, thus improving the luminous efficiency. In addition, the CL lifetime of BRuNPs/K2S2O8 was longer than classical luminophores such as N-(4-aminobutyl)-N-ethylisoluminol (ABEI), indicating the potential application of this system in CL imaging. Surprisingly, Ag+ was found to greatly improve the CL efficiency of BRuNPs/K2S2O8 by catalyzing the decomposition of K2S2O8 to generate SO4•-. On the basis of the enhancement effect of Ag+, a simple and rapid CL method was proposed for Ag+ detection. The chemosensor showed a wide linear range from 25 to 3000 nM and low detection limit of 9.03 nM, as well as good stability and excellent selectivity. More importantly, this result indicated that Ag+ can be used as a coreaction accelerator to develop a ternary self-enhanced CL system, BRuNPs/K2S2O8/Ag+.

Ratiometric Electrochemiluminescent/Electrochemical Strategy for Sensitive Detection of MicroRNA Based on Duplex-Specific Nuclease and Multilayer Circuit of Catalytic Hairpin Assembly.

In this study, we proposed a ratiometric electrochemiluminescent (ECL)/electrochemical (EC) biosensor based on duplex-specific nuclease (DSN)-assisted target recycling and multilayer catalytic hairpin assembly (CHA) amplification cascades for the detection of microRNA (miRNA). The DSN-assisted target recycling transformed miRNAs into a large number of ssDNA, which then catalyzed a multilayer CHA amplification cascade to produce numerous long dsDNA duplexes Hn/Hn+1 (n = 2, 4, 6, ...). Then the Hn/Hn+1 displaced the ferrocene (Fc)-labeled ssDNA (Sx+1, x = 1, 3, 5, ...) to hybridize with the Sx sequence on the gold electrode surface. Consequently, a great number of long Sx/Hn/Hn+1 duplexes were immobilized for binding Ru(phen)32+ to obtain an amplified ECL signal. Meanwhile, the EC signal of Fc was reduced, and the quenching effect of Fc to ECL signal also decreased. By measuring the ratio of the ECL signal of Ru(phen)32+ to the EC signal of Fc, quantitative analysis of miRNA-499 with high accuracy and reproducibility was obtained. The ratiometric biosensor shows high sensitivity and a wide linear range of 6 orders of magnitude. With the help of DSN-assisted target recycling, this strategy can be easily extended to detect other miRNAs without redesigning the CHA cascade system. The proposed "hybrid" ratiometric ECL/EC strategy enriches the ratiometric sensors and can find extensive applications in bioanalysis, especially for multiplex detection.

Clathrin-independent but dynamin-dependent mechanisms mediate Ca2+-triggered endocytosis of the glutamate GluK2 receptor upon excitotoxicity.

We first explore the features of GluK2 endocytosis during kainate excitotoxicity and then explore the role of Ca2+ in the regulation of GluK2 endocytosis. The roles of Ca2+ were examined by treating cells with Ca2+ inhibitors or chelators. Surface biotinylation was used to examine the surface localization of GluK2. Immunoprecipitation followed by immunoblotting was used to identify the interaction of GluK2 with the endocytosis regulator protein-interacting with C kinase 1 and dynamin. Dynamin phosphorylation was examined by immunoblotting with the corresponding antibodies. Our results show that GluK2 internalization is blocked by inhibitors of clathrin-independent endocytosis and relies on intracellular Ca2+/calcineurin signaling. Protein-interacting with C kinase 1-GluK2 interaction is regulated by Ca2+/calcineurin signaling. Dynamin participates in the regulation of GluK2 surface localization. Also, calcineurin activation is related to dynamin function during kainate excitotoxicity. In conclusion, GluK2 receptor endocytosis is probably a clathrin-independent and dynamin-dependent process regulated by the peak Ca2+ transient. This work indicates the roles of the Ca2+ network in the regulation of GluK2 endocytosis during kainate excitotoxicity.

Ultrasensitive Immunosensor for Cardiac Troponin I Detection Based on the Electrochemiluminescence of 2D Ru-MOF Nanosheets.

Electrochemiluminescence (ECL)-functionalized metal-organic frameworks (MOFs) have attracted increasing attention in biosensing in virtue of their diverse and tunable optical properties. A famous ECL luminophore, carboxyl-rich tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) (Ru(dcbpy)32+), possesses the characteristics of good water solubility and excellent ECL performance and also has the potential to be the organic ligand of metal-organic frameworks. Herein, functionalized MOF nanosheets (RuMOFNSs) containing plenty of Ru(dcbpy)32+ in the frameworks were synthesized in aqueous solution by a simple one-pot method. In this protocol, Ru(dcbpy)32+ acted as organic ligand to coordinate with Zn2+ originated from Zn(NO3)2, and polyvinylpyrrolidone (PVP) was used as structure-directing agent to control the formation of sheetlike structure. For practical application, a "signal-on" ECL immunosensor was designed for cardiac troponin I (cTnI) detection by employing RuMOFNSs as ECL probe. The immunosensor exhibited high sensitivity and excellent selectivity for cTnI detection in the range from 1 fg/mL to 10 ng/mL with a detection limit as low as 0.48 fg/mL. Finally, the biosensor was successfully applied for the detection of cTnI in human serum sample with satisfactory results, demonstrating its potential application in bioanalysis and clinical diagnosis.

Highly Luminescent and Self-Enhanced Electrochemiluminescence of Tris(bipyridine) Ruthenium(II) Nanohybrid and Its Sensing Application for Label-Free Detection of MicroRNA.

Inspired by the coreactive activity of carbon nanodots (CDs) and branched polyethylenimine (BPEI) toward electrochemiluminescence (ECL) of Ru(bpy)32+, a highly luminescent and self-enhanced ECL nanohybrid (Ru-BCDs) was synthesized through covalently linking BPEI-coated carbon dots (BCDs) with Tris (4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) dichloride (Ru(dcbpy)32+). The composition and morphological characterization demonstrated that the spherical Ru-BCDs particles with 12.1 ± 1.4 nm diameter were obtained. The enhanced ECL property of Ru-BCDs was proved to originate from the dual coreactive contribution of BPEI and CDs as coreactants as well as the intramolecular electron transfer process, which could shorten the electron transfer path and minimize energy loss. A carbon nitride nanosheet (CNN) was utilized to stabilize the Ru-BCDs-modified glassy carbon electrode, which greatly improved the stability of solid-state ECL. By utilizing the affinity discrepancy of the CNN to single-stranded and double-stranded nucleic acids, a label-free and signal-on ECL biosensor was constructed for the determination of microRNA-133a (miR-133a), a potential biomarker of acute myocardial infarction. The designed biosensor exhibited good performance of miR-133a detection with a detection limit of 60 fM and could be used for the detection of real human serum with satisfactory results. The self-enhanced ECL nanohybrid with distinguished ECL efficiency holds a promising prospect in biosensing and bioimaging applications.

Dual-Wavelength Ratiometric Electrochemiluminescence Immunosensor for Cardiac Troponin I Detection.

Ratiometric electrochemiluminescence (ECL) has attracted special focus in the biological analysis field, because it could eliminate the environmental interference and allow for precise measurement. Herein, a dual-wavelength ratiometric ECL biosensor was designed for the detection of cardiac troponin I (cTnI), where (4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) (Ru(dcbpy)32+) and Au nanoparticle-loaded graphene oxide/polyethylenimine (GPRu-Au) nanomaterial acts as an acceptor, and Au nanoparticle-modified graphitic phase carbon nitride nanosheet composite (Au-CNN) acts as donor. Au-CNN shows a high and steady ECL signal centered at 455 nm, which is well-matched with the adsorption of GPRu-Au; thereby, a highly efficient electrochemiluminescent resonance energy transfer (ECL-RET) sensing platform is designed. AuNPs facilitate the immobilization of antibody on the nanomaterials through a Au-N bond. The high surface area of graphene oxide/polyethylenimine allows a large number of Ru(dcbpy)32+ to be loaded, immensely amplifying the ECL signal. This sensing platform exhibits outstanding analytical performance toward cTnI with a detection limit of 3.94 fg/mL (S/N = 3). The high reliability, selectivity, and sensitivity of this ratiometric ECL biosensor provides a versatile sensing platform for the bioanalysis.

A wavelength-resolved electrochemiluminescence resonance energy transfer ratiometric immunosensor for detection of cardiac troponin I.

In this study, a wavelength-resolved electrochemiluminescence resonance energy transfer (ECL-RET) ratiometric immunosensor from Au nanoparticle functionalized graphite-like carbon nitride nanosheets (Au-g-C3N4) to Au nanoclusters (Au NCs) has been constructed for the first time. At a working voltage of 0 to -1.2 V, Au-g-C3N4 showed a strong cathodic ECL emission with a peak at 460 nm, which overlapped well with the absorption spectra of Au NCs thus stimulating the fluorescence emission of Au NCs at 610 nm. Moreover, within this voltage range, the Au NCs showed no ECL signal; therefore, they would not interfere with the detection of the system. We used cardiac troponin I (cTnI) as an analytical model to construct a sandwich immunosensor based on the ECL-RET ratiometric strategy. By measuring the responses of the ECL460 nm/FL610 nm ratio at different cTnI concentrations, the sensitive detection of cTnI with a wide range of 50 fg mL-1 to 50 ng mL-1 and a low detection limit of 9.73 fg mL-1 can be achieved. This work enriches the wavelength-resolved ECL-RET system and provides an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.

Application of MALDI-TOF MS to rapid identification of anaerobic bacteria.

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been rapidly developed and widely used as an analytical technique in clinical laboratories with high accuracy in microorganism identification. OBJECTIVE: To validate the efficacy of MALDI-TOF MS in identification of clinical pathogenic anaerobes. METHODS: Twenty-eight studies covering 6685 strains of anaerobic bacteria were included in this meta-analysis. Fixed-effects models based on the P-value and the I-squared were used for meta-analysis to consider the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 12.0. RESULTS: The identification accuracy of MALDI-TOF MS was 84% for species (I2 = 98.0%, P < 0.1), and 92% for genus (I2 = 96.6%, P < 0.1). Thereinto, the identification accuracy of Bacteroides was the highest at 96% with a 95% CI of 95-97%, followed by Lactobacillus spp., Parabacteroides spp., Clostridium spp., Propionibacterium spp., Prevotella spp., Veillonella spp. and Peptostreptococcus spp., and their correct identification rates were all above 90%, while the accuracy of rare anaerobic bacteria was relatively low. Meanwhile, the overall capabilities of two MALDI-TOF MS systems were different. The identification accuracy rate was 90% for VITEK MS vs. 86% for MALDI biotyper system. CONCLUSIONS: Our research showed that MALDI-TOF-MS was satisfactory in genus identification of clinical pathogenic anaerobic bacteria. However, this method still suffers from different drawbacks in precise identification of rare anaerobe and species levels of common anaerobic bacteria.

Highly Chemiluminescent Magnetic Beads for Label-Free Sensing of 2,4,6-Trinitrotoluene.

Until now, despite the great success acquired in scientific research and commercial applications, magnetic beads (MBs) have been used for nothing more than a carrier in most cases in bioassays. In this work, highly chemiluminescent magnetic beads containing N-(4-aminobutyl)-N-ethyl isoluminol (ABEI) and Co2+ (Co2+/ABEI/MBs) were first synthesized via a facile strategy. ABEI and Co2+ were grafted onto the surface of carboxylated MBs by virtue of a carboxyl group and electrostatic interaction. The as-prepared Co2+/ABEI/MBs exhibited good paramagnetic properties, satisfactory stability, and intense chemiluminescence (CL) emission when reacted with H2O2, which was more than 150 times that of ABEI functionalized MBs. Furthermore, it was found that 2,4,6-trinitrotoluene (TNT) aptamer could attach to the surface of Co2+/ABEI/MBs via electrostatic interaction and coordination interaction between TNT aptamer and Co2+, leading to a decrease in CL intensity due to the catalytic site Co2+ being blocked by the aptamer. In the presence of TNT, TNT would bind strongly with TNT aptamer and detach from the surface of Co2+/ABEI/MBs, resulting in partial restoration of the CL signal. Accordingly, label-free aptasensor was developed for the determination of TNT in the range of 0.05-25 ng/mL with a detection limit of 17 pg/mL. This work demonstrates that Co2+/ABEI/MBs are easily connected with recognition biomolecules, which are not only magnetic carriers but also direct sensing interfaces with excellent CL activity. It provides a novel CL interface with a magnetic property which easily separates analytes from the sample matrix to construct label-free bioassays.

Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia.

Although kainate receptors play important roles in ischemic stroke, the molecular mechanisms underlying postischemic regulation of kainate receptors remain unclear. In this study we demonstrate that Src family kinases contribute to the potentiation of kainate receptor function. Brain ischemia and reperfusion induce rapid and sustained phosphorylation of the kainate receptor subunit GluK2 by Src in the rat hippocampus, implicating a critical role for Src-mediated GluK2 phosphorylation in ischemic brain injury. The NMDA and kainate receptors are involved in the tyrosine phosphorylation of GluK2. GluK2 binds to Src, and the tyrosine residue at position 590 (Y590) on GluK2 is a major site of phosphorylation by Src kinases. GluK2 phosphorylation at Y590 is responsible for increases in whole-cell currents and calcium influx in response to transient kainate stimulation. In addition, GluK2 phosphorylation at Y590 facilitates the endocytosis of GluK2 subunits, and the activation of JNK3 and its substrate c-Jun after long-term kainate treatment. Thus, Src phosphorylation of GluK2 plays an important role in the opening of kainate receptor channels and downstream proapoptosis signaling after brain ischemia. The present study reveals an additional mechanism for the regulation of GluK2-containing kainate receptors by Src family kinases, which may be of pathological significance in ischemic stroke.

PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity.

Globally, stroke is one of the leading causes of death and significant contributors to disability. Gaining a thorough comprehension of the underlying pathogenic processes is essential for stroke treatment and prevention. In this study, we investigated the role of p21-activated kinase 1 (PAK1) in stroke by using oxygen-glucose deprivation (OGD) and transient middle cerebral artery occlusion and reperfusion (tMCAO/R) models. We reported that focal ischemia and reperfusion affect the PAK1 expression and activity levels. We further demonstrated that PAK1 is responsible for the endothelial hyperpermeability that occurs in the early stages of ischemia and reperfusion. Additionally, inhibition of PAK1 was discovered to alleviate blood-brain barrier disruption and protect against brain injury induced by tMCAO/R. Mechanistically, we provide the evidence that PAK1 regulates the formation of stress fibers and expression of surface junctional proteins. Together, our findings reveal a pathogenic function of PAK1 in stroke.

Cu2+ enhanced chemiluminescence of carbon dots-H2O2 system in alkaline solution.

Herein, Chemiluminescence (CL) behaviour of carbon dots (CDs) was investigated by using H2O2 as co-reactant in alkaline solution. The study demonstrated O2-, OH and 1O2 involved in the CL reaction processes. These reactive oxygen species reacted with CDs to produce CD•+ and CD•-, then the CD•+ reacted with CD•-to generate energy release in the form of CL emission. Importantly, we found that Cu2+ ion could greatly enhanced CL intensity (7.5 times) of CDs-H2O2 system, which was ascribed to Cu2+-catalysed H2O2 decomposition. Based on this system, the CL assay of ascorbic acid (AA) was developed. The limit of detection was as low as 0.03 µM, and the linear range was from 0.1 µM to 100 µM. The proposed method has been successfully utilized to determinate AA in beverage samples with satisfactory results.

Activity-Induced SUMOylation of Neuronal Nitric Oxide Synthase Is Associated with Plasticity of Synaptic Transmission and Extracellular Signal-Regulated Kinase 1/2 Signaling.

Aims: Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) signaling have been implicated in learning, memory, and underlying long-lasting synaptic plasticity. In this study, we aimed at detecting whether nNOS is a target protein of SUMOylation in the hippocampus and its contributions to hippocampal long-term potentiation (LTP) of synaptic transmission. Results: We showed that N-methyl-d-aspartate receptor-dependent neuronal activity enhancement induced the attachment of small ubiquitin-like modifier 1 (SUMO1) to nNOS. Protein inhibitor of activated STAT3 (PIAS3) promoted SUMO1 conjugation at K725 and K739 on nNOS, which upregulated NO production and nNOS S1412 phosphorylation (activation). In addition, the N-terminus (amino acids 43-86) of PIAS3 bound nNOS directly. Tat-tagged PIAS3 segment representing amino acids 43-86, a cell-permeable peptide containing PIAS3 residues 43-86, suppressed activity-induced nNOS SUMOylation by disrupting PIAS3-nNOS association. It also decreased LTP-related expression of Arc and brain-derived neurotrophic factor and blocked signaling via extracellular signal-regulated kinase (ERK) 1/2 and Elk-1 in the hippocampus. More importantly, PIAS3-mediated nNOS SUMOylation was required for activity-regulated ERK1/2 activation in nNOS-positive neurons and hippocampal LTP induction. Innovation and Conclusion: These findings indicated that network activity-regulated nNOS SUMOylation underlies excitatory synaptic LTP by facilitating nNOS-NO-ERK1/2 signal cascades.

Dual amplification ratiometric biosensor based on a DNA tetrahedron nanostructure and hybridization chain reaction for the ultrasensitive detection of microRNA-133a.

A novel ratiometric electrochemiluminescence (ECL)-electrochemical (EC) hybrid biosensor with a high accuracy and reproducibility was fabricated for the ultrasensitive detection of miRNA-133a. With the help of a DNA tetrahedron nanostructure and hybridization chain reaction dual amplification strategy, the detection limit of 12.17 aM for miRNA-133a was obtained.

Electrochemiluminescence Immunosensor Based on Au Nanocluster and Hybridization Chain Reaction Signal Amplification for Ultrasensitive Detection of Cardiac Troponin I.

Measurement of cardiac troponin I in the blood is crucial for the early diagnosis of acute myocardial infarction. Herein, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor has been developed for determination of cardiac troponin I (cTnI) by using Au nanoclusters and hybridization chain reaction (HCR) signal amplification. In this ECL immunosensor, Au nanoclusters were dual-labeled at each end of hairpin DNA (H1 and H2) and acted as the luminophore. DNA initiator strands (T1) and secondary antibody (Ab2) were conjugated on Au nanoparticles (AuNPs) to obtain a smart probe (Ab2-AuNP-T1). In the presence of target cTnI, the sandwiched immunocomplex composed of cTnI, Ab1, and Ab2-AuNP-T1 was formed. Then the initiator strands T1 of Ab2-AuNP-T1 opened the hairpin DNA structures and triggered a cascade of hybridization events. Consequently, a large number of Au NCs were indirectly modified on the surface of the electrode, which could react with the coreactant (K2S2O8) and emit a strong ECL signal. Under the optimal conditions, the immunosensor exhibited a wide detection range for cTnI from 5 fg/mL to 50 ng/mL and a low detection limit of 1.01 fg/mL (S/N = 3). Because of the excellent specificity, stability, and reproducibility of the proposed ECL-HCR sensor, it has a great application prospect for cTnI detection in clinical diagnosis.

N-(Aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles on cobalt disulfide nanowire hybrids for the non-enzymatic chemiluminescence detection of H2O2.

N-(Aminobutyl)-N-(ethylisoluminol) (ABEI)-functionalized gold nanoparticles (AuNPs) on cobalt disulfide nanowires (ABEI/AuNPs/CoS2 NWs) are rapidly synthesized through a microwave-assisted reduction of chloroauric acid (HAuCl4) on CoS2 NWs with ABEI. The obtained nanohybrids with enhanced chemiluminescence are exploited for the non-enzymatic detection of H2O2.

Novel highly emissive H-aggregates with aggregate fluorescence change in a phenylbenzoxazole-based system.

Fibrous nanoaggregates of a new benzoxazole-based derivative have been reported. This derivative exhibits not only H-aggregates but also strong yellow fluorescence, which is different from the traditional understanding of H-aggregates.

SUMOylation of the kainate receptor subunit GluK2 contributes to the activation of the MLK3-JNK3 pathway following kainate stimulation.

Protein SUMOylation has been implicated in the pathogenesis of ischemic stroke. However, the underlying mechanisms remain unclear. Here, we found that global brain ischemia evokes a sustained elevation of GluK2 SUMOylation in the rat hippocampal CA1 region. Over-expression of wild-type GluK2, but not SUMOylation-deficient mutant, significantly increased the activity of MLK3 and JNK3 after kainate stimulation. SUMOylation deficiency attenuated the kainate-stimulated interaction between MLK3 and GluK2. In addition, inhibition of kainate-evoked GluK2 endocytosis decreased the activation of MLK3-JNK3 signaling and the binding of MLK3-GluK2 in cultured cortical neurons. These results suggest that the internalization of GluK2 following SUMO modification promotes its binding with MLK3, thereby activating the MLK3-JNK3 pathway, which may be responsible for ischemic neuronal cell death.