METTL3 confers protection against mitochondrial dysfunction and cognitive impairment in an Alzheimer disease mouse model by upregulating Mfn2 via N6-methyladenosine modification.

Mitofusin 2 (MFN2) has been found to be downregulated in patients with Alzheimer disease (AD) but little is known about its roles in the pathogenesis of AD. We explored the mechanism of N6-methyladenosine (m6A) methylation of Mfn2 in hippocampal mitochondrial dysfunction in an AD mouse model. APP/PS1 transgenic mice underwent stereotaxic injection of adeno-associated viruses and their behaviors were assessed. METTL3 and MFN2 expressions were measured by qRT-PCR and Western blot, accompanied by assessment of mitochondrial morphology, ATP, mitochondrial membrane potential, and amyloid-ß content. Binding between METTL3 and MFN2, the total amount of m6A, and the m6A modification of Mfn2 were also determined. METTL3 and MFN2 were downregulated in hippocampal tissues of the AD model mice; METTL3 enhanced MFN2 expression via m6A modification. Overexpression of METTL3 or MFN2 ameliorated mitochondrial dysfunction indicated by fewer damaged mitochondria, increased ATP and JC-1 levels, and reduced Aß content; improved cognitive impairment in the mice was indicated by the novel object discrimination index and Morris water maze tests. Effects of METTL3 overexpression were abrogated by further knockdown of MFN2. Thus, METTL3 ameliorated mitochondrial dysfunction and cognitive impairment in the AD model mice by increasing MFN2 expression via m6A modification.

Circ_0049472 regulates the damage of Aß-induced SK-N-SH and CHP-212 cells by mediating the miR-107/KIF1B axis.

Alzheimer's disease (AD) is a neurodegenerative disease that seriously affects the life and health of the elderly. Studies have found that circular RNAs (circRNAs) are associated with human diseases, including AD. Hsa_circ_0049472 has been uncovered to be overexpressed in AD, but the role of circ_0049472 remains unclear. AD patients were recruited to collect cerebrospinal fluid (CSF) and serum samples. Amyloid beta (Aß)-induced SK-N-SH and CHP-212 cells were used as the AD cell models in vitro. Quantitative real-time PCR (qRT-PCR) was used to assess the expression of circ_0049472, microRNA-107 (miR-107) and kinesin family member 1B (KIF1B). Cell counting kit-8 assay tested the cell viability, and flow cytometry measured cell apoptosis. The levels of proliferating cell nuclear antigen (PCNA), BCL2 Associated X (Bax) and kinesin family member 1B (KIF1B) protein were examined by western blot. In addition, the relative inflammatory cytokines (TNF-α, IL-6 and IL-1ß) were detected by enzyme-linked immunosorbent assay (ELISA). The malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured by relative kits. Dual-luciferase reporter assays and RNA pull-down assay verified the relationship between miR-107 and circ_0049472 or KIF1B. Circ_0049472 and KIF1B were overexpressed in AD patient-derived cerebrospinal fluid (CSF) and serum samples, as well as Aß-induced SK-N-SH and CHP-212 cells. Silencing circ_0049472 promoted cell proliferation, and inhibited cell apoptosis in Aß-induced SK-N-SH and CHP-212 cells. MiR-107 was a target of circ_0049472. MiR-107 silencing abolished the cell viability and apoptosis affected by down-regulation of circ_0049472 in Aß-induced SK-N-SH and CHP-212 cells. Besides, miR-107 targeted KIF1B, and overexpressed KIF1B reverted miR-107 elevation-mediated effects on cell apoptosis, inflammation, and oxidative stress of Aß-induced SK-N-SH and CHP-212 cells. Circ_0049472 modulated KIF1B by serving as a miR-107 decoy, thereby mediating Aß-induced neurotoxicity, suggesting that circ_0049472 may be involved in AD pathogenesis.

Prior nasal delivery of antagomiR-122 prevents radiation-induced brain injury.

Radiation-induced brain injury is a major adverse event in head and neck tumor treatment, influencing the quality of life for the more than 50% of patients who undergo radiation therapy and experience long-term survival. However, no effective treatments are available for these patients, and preventative drugs and effective drug-delivery methods must be developed. Based on our results, miR-122-5p was upregulated in the mouse radiation-induced brain injury (RBI) model and patients with nasopharyngeal carcinoma (NPC) who received radiation therapy. Intranasal administration of a single antagomiR-122-5p dose before irradiation effectively alleviated radiation-induced cognitive impairment, neuronal injury, and neuroinflammation in the mouse RBI model. Results further indicated that miR-122-5p inhibition in microglia reduced the levels of proinflammatory cytokines and enhanced the phagocytic function to protect against radiation-induced neuronal injury in cell models. Further, we profiled transcriptome data and verified that Tensin 1 (TNS1) may be the target of miR-122-5p in RBI. In summary, our results reveal a distinct role for miR-122-5p in regulating neuroinflammation in RBI, indicating that a non-invasive strategy for intranasal miR-122-5p administration may be an attractive therapeutic target in RBI, providing new insights for clinical trials. Further systematic safety assessment, optimization of drug administration, and clarity of mechanism will accelerate the process into clinical practice.

Zafirlukast protects blood-brain barrier integrity from ischemic brain injury.

Stroke has been considered the second leading cause of death worldwide, and ischemic stroke accounts for the vast majority of stroke cases. Some of the main features of ischemic stroke are increased brain permeability, ischemia/reperfusion injury, oxidative stress, and acute inflammation. Antagonism of cysLT1R has been shown to provide cardiovascular and neural benefits. In the present study, we investigated the effects of the cysLT1R antagonist zafirlukast both in vivo and in vitro using a middle cerebral artery occlusion (MCAO) mouse model and human brain microvascular endothelial cells (HBMVECs). In vivo, we found that zafirlukast pretreatment could reduce MCAO-induced increased brain permeability by rescuing the expression levels of the tight junction proteins occludin and ZO-1. In vitro, we found that zafirlukast could suppress the increase in endothelial monolayer permeability induced by OGD/R via rescue of occludin and ZO-1 expression; additionally, we found that zafirlukast prevented OGD/R-induced degradation of the extracellular matrix via inhibition of MMP-2 and MMP-9 expression. Finally, we found that zafirlukast could also inhibit OGD/R-induced activation of the critical proinflammatory regulator NF-κB by preventing phosphorylation and nuclear translocation of p65 protein. Together, our findings demonstrate a promising role for zafirlukast in preventing damage induced by ischemic stroke and reperfusion injury.

HMGB1 contributes to the irradiation-induced endothelial barrier injury through receptor for advanced glycation endproducts (RAGE).

This study aimed to investigate whether HMGB1 (high mobility group box-1 protein) and receptor for advanced glycation end products (RAGE) were involved in the irradiation-induced endothelial barrier damage and their mechanism. We constructed the damage model of endothelium barrier model with bEnd.3 cells. The permeability of endothelial barrier was detected by sodium fluorescein (Na-F) permeation test, and the irradiation dose which could induce permeability transition was determined by being exposed to different irradiation doses (5, 10, 15, 20 Gy). MTT assay was applied to detect cell viability under different concentrations of HMGB1, glycyrrhizic acid (GA, a specific inhibitor of HMGB1), and FPS-ZM1 (a blood-brain-barrier permeant blocker of RAGE V domain-mediated ligand binding). The expression of HMGB1, RAGE, and related molecules involved in MAPK signaling pathway, MMP-2, MMP-9, ZO-1, and claudin 5 of differently treated groups were measured by qRT-PCR, western blot, and immunofluorescence. Cells possessed stable endothelial barrier function on 4-7 days after seeded on transwell plates. The permeability of endothelial barrier would change under at least 10 Gy radiation. Both radiation and HMGB1 treatment alone could improve the permeability. After irradiation, the expressions of HMGB1 and RAGE increased and MAPK signal pathway was activated. Meanwhile, MMP-2 and MMP-9 were overexpressed, while the expression of tight junction proteins ZO-1 and claudin 5 was decreased. Radiation could activate MAPK signaling pathway through promoting the expression of HMGB1 and RAGE, which further led to endothelial barrier injury and changed its permeability.

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.

3,6-Dibromo-7-ethyl-amino-4-methyl-2H-chromen-2-one.

In title compound, C(12)H(11)Br(2)NO(2), the coumarin ring system is almost planar, the two rings being inclined to one another by 1.40 (15)°. There are two short intra-molecular inter-actions (N-H⋯Br and C-H⋯Br) involving the Br atoms. In the crystal, mol-ecules stack along the a-axis direction via π-π inter-actions; the centroid-centroid distances vary from 3.6484 (19) to 3.7942 (19) Å.