Genetic Arg-304-His substitution in GRK5 protects against sepsis progression by alleviating NF-κB-mediated inflammation.

BACKGROUND: Previous studies have demonstrated that G protein-coupled receptor kinase 5 (GRK5) exerts a pivotal regulatory effect on the inflammation associated with sepsis. The present study aimed to investigate the clinical association of GRK5 genetic variants with sepsis and to further explore the underlying genetic mechanisms involved in regulating sepsis-induced inflammatory responses and the pathogenesis of sepsis. METHODS: This case-control study enrolled 1081 septic patients and 1147 matched controls for genotyping of GRK5 rs2230349 and rs2230345 polymorphisms. The effect of these genetic variants on GRK5-mediated inflammatory responses was analyzed in peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages. A clinically relevant polymicrobial sepsis model was established by subjecting wild-type (WT) and GRK5-knockout mice to cecal ligation and puncture (CLP) to evaluate the role of GRK5 in sepsis. RESULTS: We identified significant differences in the genotype/allele distribution of rs2230349 G > A, but not rs2230345, between the sepsis subtype and septic shock subgroups (GA + AA vs. GG genotype, OR = 0.698, 95% CI = 0.547-0.893, P = 0.004; A vs. G allele, OR = 0.753, 95% CI = 0.620-0.919, P = 0.005) and between the survivor and nonsurvivor subgroups (GA + AA vs. GG genotype, OR = 0.702, 95% CI = 0.531-0.929, P = 0.015; A vs. G allele, OR = 0.753, 95% CI = 0.298-0.949, P = 0.017). PBMCs carrying the sepsis-associated protective A allele produced significantly lower levels of TNF-α and IL-1ß upon LPS stimulation. The results from the in vitro experiment showed that the Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 significantly decreased the LPS-induced production of several proinflammatory cytokines, such as TNF-α, IL-6, IL-1ß and MCP-1, via the IκB-α/NF-κB signaling pathway in THP-1 macrophages. Furthermore, GRK5-knockout mice exhibited a significant decrease in IκB-α phosphorylation/degradation, the p-p65/p65 ratio, the p-p50/p50 ratio, p65 nuclear translocation and downstream cytokine (TNF-α, IL-6, IL-1ß and VCAM-1) production compared to WT mice after CLP surgery. A significant improvement in 7-day survival rate in GRK5-KO septic mice was observed in the presence of antibiotics. CONCLUSIONS: The Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 might disrupt GRK5 function and alleviate IKB-α/NF-κB-mediated inflammatory responses, which ultimately conferred a genetic protective effect against susceptibility to sepsis progression and mortality. These results may, to some extent, explain the heterogeneity of the clinical prognoses of septic patients and provide novel opportunities for individualized approaches for sepsis treatment.

The influence of two functional genetic variants of GRK5 on tau phosphorylation and their association with Alzheimer's disease risk.

Our work explores the relationship between G protein-coupled receptor kinase-5 (GRK5) single nucleotide polymorphisms and Alzheimer's disease risk. We confirmed that GRK5 translocates from the cellular membrane to the cytosol in the hippocampus of Alzheimer's disease mice and that GRK5 deficiency promotes tau hyperphosphorylation, a hallmark of Alzheimer's disease pathology. Our results indicate that one functional variant, or mutant, of GRK5 (GRK5-Gln41Leu) decreased GRK5 translocation from the membrane to the cytoplasm and reduced tau hyperphosphorylation, whereas, another GRK5 mutant (GRK5-Arg304His) increased GRK5 translocation to the cytoplasm and promoted tau hyperphosphorylation. In addition, case-control studies revealed that GRK5-Gln41Leu is associated with a lower risk of late-onset Alzheimer's disease. Our findings suggest that the GRK5-Gln41Leu mutant may resist tau hyperphosphorylation by promoting GRK5 membrane stability and, in effect, may contribute to lower Alzheimer's disease risk.

Intranasal Delivery of miR-146a Mimics Delayed Seizure Onset in the Lithium-Pilocarpine Mouse Model.

Unveiling the key mechanism of temporal lobe epilepsy (TLE) for the development of novel treatments is of increasing interest, and anti-inflammatory miR-146a is now considered a promising molecular target for TLE. In the current study, a C57BL/6 TLE mouse model was established using the lithium-pilocarpine protocol. The seizure degree was evaluated according to the Racine scale, and level 5 was considered the threshold for generalized convulsions. Animals were sacrificed to analyze the hippocampus at three time points (2 h and 4 and 8 weeks after pilocarpine administration to evaluate the acute, latent, and chronic phases, resp.). After intranasal delivery of miR-146a mimics (30 min before pilocarpine injection), the percent of animals with no induced seizures increased by 6.7%, the latency to generalized convulsions was extended, and seizure severity was reduced. Additionally, hippocampal damage was alleviated. While the relative miR-146a levels significantly increased, the expression of its target mRNAs (IRAK-1 and TRAF-6) and typical inflammatory modulators (NF-κB, TNF-α, IL-1ß, and IL-6) decreased, supporting an anti-inflammatory role of miR-146a via the TLR pathway. This study is the first to demonstrate that intranasal delivery of miR-146a mimics can improve seizure onset and hippocampal damage in the acute phase of lithium-pilocarpine-induced seizures, which provides inflammation-based clues for the development of novel TLE treatments.

A Novel, Multi-Target Natural Drug Candidate, Matrine, Improves Cognitive Deficits in Alzheimer's Disease Transgenic Mice by Inhibiting Aß Aggregation and Blocking the RAGE/Aß Axis.

The treatment of AD is a topic that has puzzled researchers for many years. Current mainstream theories still consider Aß to be the most important target for the cure of AD. In this study, we attempted to explore multiple targets for AD treatments with the aim of identifying a qualified compound that could both inhibit the aggregation of Aß and block the RAGE/Aß axis. We believed that a compound that targets both Aß and RAGE may be a feasible strategy for AD treatment. A novel and small natural compound, Matrine (Mat), was identified by high-throughput screening of the main components of traditional Chinese herbs used to treat dementia. Various experimental techniques were used to evaluate the effect of Mat on these two targets both in vitro and in AD mouse model. Mat could inhibit Aß42-induced cytotoxicity and suppress the Aß/RAGE signaling pathway in vitro. Additionally, the results of in vivo evaluations of the effects of Mat on the two targets were consistent with the results of our in vitro studies. Furthermore, Mat reduced proinflammatory cytokines and Aß deposition and attenuated the memory deficits of AD transgenic mice. We believe that this novel, multi-target strategy to inhibit both Aß and RAGE, is worthy of further exploration. Therefore, our future studies will focus on identifying even more effective multi-target compounds for the treatment of AD based on the molecular structure of Mat.

Association Study Between Promoter Polymorphisms of ADAM17 and Progression of Sepsis.

BACKGROUND: A disintegrin and metalloproteinase 17 (ADAM17) has been confirmed to play a significant role in the pathogenesis of sepsis. However, little is known about the clinical relevance of ADAM17 polymorphisms to sepsis onset and development. METHODS: This study analyzed the associations of five ADAM17 promoter polymorphisms (rs55790676, rs12692386, rs11684747, rs1524668 and rs11689958) with sepsis (370 sepsis cases and 400 controls). Genotyping was performed using pyrosequencing and polymerase chain reaction-length polymorphism method. The ADAM17 expression was measured using the real-time PCR method and the concentrations of related cytokines were detected using enzyme-linked immunosorbent assay. RESULTS: No associations were observed between these polymorphisms and sepsis susceptibility, while the rs12692386GA/GG genotypes were overrepresented among the patients with severe sepsis (P=0.002) or septic shock (P=0.0147) compared to those with sepsis subtype, suggesting a susceptible role of rs12692386A>G in the progression of sepsis. Moreover, ADAM17 expression was increased in the sepsis patients with the rs12692386GA/GG genotypes, accompanied by up-regulation of expression of the ADAM17 substrates (TNF-α, IL-6R and CX3CL1) and pro-inflammatory cytokines (IL-1ß and IL-6). CONCLUSION: The present study has provided potentially valuable clinical evidence that the ADAM17 rs12692386 polymorphism is a functional variant that might be used as a relevant risk estimate for the progression of sepsis.

A Highly Selective Inhibitor of Glycine Transporter-1 Elevates the Threshold for Maximal Electroshock-Induced Tonic Seizure in Mice.

Many anti-epileptic drugs (AEDs) that mainly target ion channels or post-synaptic receptors are in clinical use, but a proportion of patients are resistant to these traditional AEDs and experience repeated severe break-out seizures. Given its involvement in the etiology of epilepsy, the neurotransmitter glycine may serve as a novel target for epilepsy treatment. Increasing evidence suggests that inhibitors of glycine transporter 1 (GlyT1) exhibit anti-seizure properties in mouse models and show potential as anti-convulsions drugs. In the present study, we investigated the effect of a highly selective GlyT1 inhibitor (named M22) on glycine transport kinetics using a radioactive substrate uptake assay and investigated the anti-seizure effects of M22 on the maximal electroshock seizure threshold (MEST) test and the timed intravenous (i.v.) pentylenetetrazole (PTZ) intravenous test. Our results demonstrate that M22 was capable of elevating the seizure threshold in the MEST test but did not alter the seizure threshold in the PTZ i.v. test. Strychnine, an inhibitor of glycine receptor activity, reversed the threshold elevation at a subconvulsive dosage (0.1 mg/kg subcutaneously) in the MEST test and did not affect M22 plasma levels in mice, suggesting that the anti-seizure effect in this model may be mediated by increased glycine receptor activity. Moreover, M22 administration did not influence motor function and coordination in mice. In combination with the previously reported excellent pharmacokinetic features of M22, our present results suggested that M22 has the potential to serve as a new anti-convulsive drug or as a lead compound for the development of AEDs.