Mutant kri1l causes abnormal retinal development via cell cycle arrest and apoptosis induction.

Damage to the ribosome or an imbalance in protein biosynthesis can lead to some human diseases, such as diabetic retinopathy (DR) and other eye diseases. Here, we reported that the kri1l gene was responsible for retinal development. The kri1l gene encodes an essential component of the rRNA small subunit processome. The retinal structure was disrupted in kri1l mutants, which resulted in small eyes. The boundaries of each layer of cells in the retina were blurred, and each layer of cells was narrowed and decreased. The photoreceptor cells and Müller glia cells almost disappeared in kri1l mutants. The lack of photoreceptor cells caused a fear of light response. The development of the retina started without abnormalities, and the abnormalities began two days after fertilization. In the kri1l mutant, retinal cell differentiation was defective, resulting in the disappearance of cone cells and Müller cells. The proliferation of retinal cells was increased, while apoptosis was also enhanced in kri1l mutants. γ-H2AX upregulation indicated the accumulation of DNA damage, which resulted in cell cycle arrest and apoptosis. The kri1l mutation reduced the expression of some opsin genes and key retinal genes, which are also essential for retinal development.

Astragaloside IV against Alzheimer's disease via microglia-mediated neuroinflammation using network pharmacology and experimental validation.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases in the world. The effective therapeutic methods and drugs are still not clear. Astragaloside IV (AS-IV), a triterpenoid saponin isolated from the root of Huangqi, has a beneficial effect in the treatment of AD. However, whether AS-IV alters microglia in the inflammation of AD is still ambiguous. In our study, 99 common targets were collected between AS-IV and AD. BCL2 apoptosis regulator (Bcl-2), pro-apoptotic BCL-2 protein BAX, epidermal growth factor receptor (EGFR), and receptor tyrosine phosphatase type C (PTPRC) were screened for inflammation and microglia in the above targets by network pharmacology. Interleukin-1ß (IL-1ß) and EGFR both interact with signal transducer and activator of transcription 3 (STAT3) by a protein interaction network, and IL-1ß had a higher affinity for AS-IV based on molecular docking. Enrichment revealed targets involved in the regulation of neuronal cell bodies, growth factor receptor binding, EGFR tyrosine kinase inhibitor resistance., etc. Besides, AS-IV alleviated the reduced cell proliferation in amyloid-beta (Aß)-treated microglial BV2 cells. AS-IV affected BV2 cell morphological changes and decreased cluster of differentiation 11b (CD11b) gene, IL-1ß, and EGFR mRNA levels increment during lipopolysaccharide (LPS) injury in BV2 cell activation. Therefore, AS-IV may regulate microglial activation and inflammation via EGFR-dependent pathways in AD. EGFR and IL-1ß are vital targets that may relate to each other to coregulate downstream molecular functions in the cure of AD. Our study provides a candidate drug and disease target for the treatment of neurodegenerative diseases in the clinic.

Hypoxic Preconditioning Modulates BDNF and Its Signaling through DNA Methylation to Promote Learning and Memory in Mice.

Hypoxic preconditioning (HPC) as an endogenous mechanism can resist hypoxia/ischemia injury and exhibit protective effects on neurological function including learning and memory. Although underlying molecular mechanisms remain unclear, HPC probably regulates the expression of protective molecules by modulating DNA methylation. Brain-derived neurotrophic factor (BDNF) activates its signaling upon binding to the tropomyosin-related kinase B (TrkB) receptor, which is involved in neuronal growth, differentiation, and synaptic plasticity. Therefore, this study focused on the mechanism by which HPC regulates BDNF and BDNF/TrkB signaling through DNA methylation to influence learning and memory. Initially, the HPC model was established by hypoxia stimulations on ICR mice. We found that HPC downregulated the expression of DNA methyltransferase (DNMT) 3A and DNMT3B. Then, the upregulation of BDNF expression in HPC mice was generated from a decrease in DNA methylation of the BDNF gene promoter detected by pyrophosphate sequencing. Subsequently, upregulation of BDNF activated BDNF/TrkB signaling and ultimately improved learning and spatial memory in HPC mice. Moreover, after mice were intracerebroventricularly injected with the DNMT inhibitor, the restraint of DNA methylation accompanied by an increase of BDNF and BDNF/TrkB signaling was also discovered. Finally, we observed that the inhibitor of BDNF/TrkB signaling prevented HPC from ameliorating learning and memory in mice. However, the DNMT inhibitor promoted spatial cognition in mice. Thus, we suggest that HPC may upregulate BDNF by inhibiting DNMTs and decreasing DNA methylation of the BDNF gene and then activate BDNF/TrkB signaling to improve learning and memory in mice. This may provide theoretical guidance for the clinical treatment of cognitive dysfunction caused by ischemia/hypoxia disease.

Global-Scale Profiling of Differential Expressed Lysine-Lactylated Proteins in the Cerebral Endothelium of Cerebral Ischemia-Reperfusion Injury Rats.

Acute ischemic stroke (AIS) is a serious threat to human health. Following AIS, cerebral ischemia-reperfusion injury (CIRI) must be treated to improve prognosis. By combining 4D label-free quantitative proteomics with lactylation modification-specific proteomics analysis, we assessed lysine lactylation (Kla) in cortical proteins of a CIRI rat model. We identified a total of 1003 lactylation sites on 469 proteins in this study, gathering quantitative information (PXD034232) on 660 of 310 proteins, which were further classified by cell composition, molecular function, and biological processes. In addition, we analyzed the metabolic pathways, domains, and protein-protein interaction networks. Lastly, we evaluated differentially expressed lysine lactylation sites, determining 49 upregulated proteins and 99 downregulated proteins with 54 upregulated sites and 54 downregulated sites in the experimental group in comparison with the healthy control group. Moreover, we identified the Kla of Scl25a4 and Slc25a5 in the Ca2+ signaling pathway, but the Kla of Vdac1 was eliminated, as confirmed in vivo. Overall, these results provide new insights into lactylation involved in the underlying mechanism of CIRI because this post-translational modification affects the mitochondrial apoptosis pathway and mediates neuronal death. Therefore, this study may enable us to develop new molecules with therapeutic properties, which have both theoretical significance and broad clinical application prospects. A new model of cerebral ischemia-reperfusion injury (CIRI) induced by lactylation through the regulation of key proteins of the Ca2+ signaling pathway.

Possible Involvement of DNA Methylation in TSC1 Gene Expression in Neuroprotection Induced by Hypoxic Preconditioning.

Background: It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods: Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results: HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion: TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.

Overview of Traditional Mongolian Medical Warm Acupuncture.

Mongolian medical warm acupuncture is a traditional therapy of Mongolian medicine and was developed by people living on the Mongolian Plateau. This kind of traditional oriental medicine has a long history. The main characteristics of Mongolian medical warm acupuncture are the acupoints and the needles used. Its theory is based on the human anatomical structure and the distinct local culture. Mongolian medical warm acupuncture has been practiced for centuries and proved to be very effective in the treatment of age-related diseases, including the musculoskeletal and nervous diseases. This paper aims to briefly introduce the history and scope of Mongolian medical warm acupuncture, with a particular focus on age-related diseases, where Mongolian medical warm acupuncture has shown significant beneficial effects.

Exosomal MicroRNA-126 from RIPC Serum Is Involved in Hypoxia Tolerance in SH-SY5Y Cells by Downregulating DNMT3B.

Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.

Nd2O3 Nanoparticles Induce Toxicity and Cardiac/Cerebrovascular Abnormality in Zebrafish Embryos via the Apoptosis Pathway.

INTRODUCTION: Rare-earth nanoparticles in the environment and human body pose a potential threat to human health. Although toxic effects of rare-earth nanoparticles have been extensively studied, the effects on the early development are not well understood. In this study, we attempted to explain the toxic effects of neodymium oxide (Nd2O3) nanoparticles on early development. METHODS: We added the Nd2O3 nanoparticles at different concentrations and recorded the mortality and malformation rate per 24 hrs under a microscope. The live embryos treated with Nd2O3 nanoparticles were imaged as movies and Z step lapses with a confocal microscope, and heart rates were counted for 30 s to measure the cardiac function. The live Tg (Flk1:EGFP) transgenic embryos exposed to Nd2O3 nanoparticles were observed under confocal microscope to measure the cerebrovascular development. Subsequently, we extracted the total protein for Western blot at 5 days post-fertilisation (dpf). Embryos were collected to undergo TUNEL staining for apoptosis detection. RESULTS: Nd2O3 nanoparticles disturbed embryo development at high concentrations (>200 µg/mL). The mortality and malformation rate gradually increased in a dose-dependent manner by morphological observation, while the Nd2O3 median lethal concentration (LD50) was 203.4 µg/mL at 120 hrs post-fertilisation (hpf). Furthermore, the Nd2O3-treated embryos showed severe arrhythmia and reduced heart rate. We also observed the markedly cerebrovascular disappearance at middle concentration (100 and 200 µg/mL). The downregulated autophagy flux in brain blood vessels and increased apoptosis level in neurons might affect vessels sprouting and contribute to the vanished cerebrovascular. CONCLUSION: The results suggested that the embryos exposed to Nd2O3 activated the apoptosis pathway and induced toxicity and abnormal cardiac/cerebrovascular development.

5-Aza-2'-deoxycytidine increases hypoxia tolerance-dependent autophagy in mouse neuronal cells by initiating the TSC1/mTOR pathway.

BACKGROUND: Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. METHODS: 5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. RESULTS: 5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. CONCLUSIONS: 5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.

Effects of 5-Aza on p-Y1472 NR2B related to learning and memory in the mouse hippocampus.

BACKGROUND: We have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. METHODS: 5-Aza-cdR (10 µM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 µM 5-Aza-cdR compared with an untreated control group. RESULTS: After injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 µM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. CONCLUSIONS: Our results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.

Absence of RstA results in delayed initiation of DNA replication in Escherichia coli.

RstB/RstA is an uncharacterized Escherichia coli two-component system, the regulatory effects of which on the E. coli cell cycle remain unclear. We found that the doubling time and average number of replication origins per cell in an ΔrstB mutant were the same as the wild-type, and the average number of replication origins in an ΔrstA mutant was 18.2% lower than in wild-type cells. The doubling times were 34 min, 35 min, and 40 min for the wild-type, ΔrstB, and ΔrstA strains, respectively. Ectopic expression of RstA from plasmid pACYC-rstA partly reversed the ΔrstA mutant phenotypes. The amount of initiator protein DnaA per cell was reduced by 40% in the ΔrstA mutant compared with the wild-type, but the concentration of DnaA did not change as the total amount of cellular protein was also reduced in these cells. Deletion or overproduction of RstA does not change the temperature sensitivity of dnaA46, dnaB252 and dnaC2. The expression of hupA was decreased by 0.53-fold in ΔrstA. RstA interacted with Topoisomerase I weakly in vivo and increased its activity of relaxing the negative supercoiled plasmid. Our data suggest that deletion of RstA leads to delayed initiation of DNA replication, and RstA may affect initiation of replication by controlling expression of dnaA or hupA. Furthermore, the delayed initiation may by caused by the decreased activity of topoisomerase I in RstA mutant.

5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, induces cytotoxicity, cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells.

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 µmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.