Social isolation reinforces aging-related behavioral inflexibility by promoting neuronal necroptosis in basolateral amygdala.

Aging is characterized with a progressive decline in many cognitive functions, including behavioral flexibility, an important ability to respond appropriately to changing environmental contingencies. However, the underlying mechanisms of impaired behavioral flexibility in aging are not clear. In this study, we reported that necroptosis-induced reduction of neuronal activity in the basolateral amygdala (BLA) plays an important role in behavioral inflexibility in 5-month-old mice of the senescence-accelerated mice prone-8 (SAMP8) line, a well-established model with age-related phenotypes. Application of Nec-1s, a specific inhibitor of necroptosis, reversed the impairment of behavioral flexibility in SAMP8 mice. We further observed that the loss of glycogen synthase kinase 3α (GSK-3α) was strongly correlated with necroptosis in the BLA of aged mice and the amygdala of aged cynomolgus monkeys (Macaca fascicularis). Moreover, genetic deletion or knockdown of GSK-3α led to the activation of necroptosis and impaired behavioral flexibility in wild-type mice, while the restoration of GSK-3α expression in the BLA arrested necroptosis and behavioral inflexibility in aged mice. We further observed that GSK-3α loss resulted in the activation of mTORC1 signaling to promote RIPK3-dependent necroptosis. Importantly, we discovered that social isolation, a prevalent phenomenon in aged people, facilitated necroptosis and behavioral inflexibility in 4-month-old SAMP8 mice. Overall, our study not only revealed the molecular mechanisms of the dysfunction of behavioral flexibility in aged people but also identified a critical lifestyle risk factor and a possible intervention strategy.

Resveratrol suppresses glial activation and alleviates trigeminal neuralgia via activation of AMPK.

BACKGROUND: Glial activation and neuroinflammation in the spinal trigeminal nucleus (STN) play a pivotal role in the genesis and maintenance of trigeminal neuralgia (TN). Resveratrol, a natural compound from grape and red wine, has a potential anti-inflammatory effect. We hypothesized that resveratrol could significantly suppress neuroinflammation in the STN mediated by glial activation and further relieve TN. In this study, we evaluated whether resveratrol could alleviate trigeminal allodynia and explore the mechanism underlying the antinociceptive effect of resveratrol. METHODS: Animals were orally injected with resveratrol after chronic constriction injury (CCI) of the infraorbital nerve. Mechanical thresholds of the affected whisker pad were measured to assess nociceptive behaviors. The STN was harvested to quantify the changing levels of p-NR1, p-PKC, TNF-α, and IL1-ß by western blotting and detect the expression of calcitonin gene-related peptide (CGRP) and c-Fos by immunofluorescence. Glial activation was observed by immunofluorescence and western blotting. Mitogen-activated protein kinase (MAPK) phosphorylation in vivo and in vitro was examined by western blotting. RESULTS: We found that resveratrol significantly attenuated trigeminal allodynia dose-dependently and decreased the increased expression of CGRP and c-Fos in the STN. Additionally, resveratrol showed an inhibitory effect on CCI-evoked astrocyte and microglia activation and reduced production of pro-inflammatory cytokines in the STN. Furthermore, the antinociceptive effect of resveratrol was partially mediated by reduced phosphorylation of MAP kinases via adenosine monophosphate-activated protein kinase (AMPK) activation. CONCLUSIONS: AMPK activation in the STN glia via resveratrol has utility in the treatment of CCI-induced neuroinflammation and further implicates AMPK as a novel target for the attenuation of trigeminal neuralgia.

Senktide blocks aberrant RTN3 interactome to retard memory decline and tau pathology in social isolated Alzheimer's disease mice.

Sporadic or late-onset Alzheimer's disease (LOAD) accounts for more than 95% of Alzheimer's disease (AD) cases without any family history. Although genome-wide association studies have identified associated risk genes and loci for LOAD, numerous studies suggest that many adverse environmental factors, such as social isolation, are associated with an increased risk of dementia. However, the underlying mechanisms of social isolation in AD progression remain elusive. In the current study, we found that 7 days of social isolation could trigger pattern separation impairments and presynaptic abnormalities of the mossy fibre-CA3 circuit in AD mice. We also revealed that social isolation disrupted histone acetylation and resulted in the downregulation of 2 dentate gyrus (DG)-enriched miRNAs, which simultaneously target reticulon 3 (RTN3), an endoplasmic reticulum protein that aggregates in presynaptic regions to disturb the formation of functional mossy fibre boutons (MFBs) by recruiting multiple mitochondrial and vesicle-related proteins. Interestingly, the aggregation of RTN3 also recruits the PP2A B subunits to suppress PP2A activity and induce tau hyperphosphorylation, which, in turn, further elevates RTN3 and forms a vicious cycle. Finally, using an artificial intelligence-assisted molecular docking approach, we determined that senktide, a selective agonist of neurokinin3 receptors (NK3R), could reduce the binding of RTN3 with its partners. Moreover, application of senktide in vivo effectively restored DG circuit disorders in socially isolated AD mice. Taken together, our findings not only demonstrate the epigenetic regulatory mechanism underlying mossy fibre synaptic disorders orchestrated by social isolation and tau pathology but also reveal a novel potential therapeutic strategy for AD.

Ghrelin alleviates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells.

Ghrelin is a neuropeptide that has various physiological functions and has been demonstrated to be neuroprotective in a number of neurological disease models. However, the underlying mechanisms of ghrelin in Parkinson's disease remain largely unexplored. The current study aimed to study the effects of ghrelin in a 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease model and evaluate the potential underlying mechanisms. In the present study, we treated an SH-SY5Y cell model with 6-OHDA, and observed that pretreatment with different concentrations of ghrelin (1, 10, and 100 nM) for 30 minutes relieved the neurotoxic effects of 6-OHDA, as revealed by Cell Counting Kit-8 and Annexin V/propidium iodide (PI) apoptosis assays. Reverse transcription quantitative polymerase chain reaction and western blot assay results demonstrated that 6-OHDA treatment upregulated α-synuclein and lincRNA-p21 and downregulated TG-interacting factor 1 (TGIF1), which was predicted as a potential transcription regulator of the gene encoding α-synuclein (SNCA). Ghrelin pretreatment was able to reverse the trends caused by 6-OHDA. The Annexin V/PI apoptosis assay results revealed that inhibiting either α-synuclein or lincRNA-p21 expression with small interfering RNA (siRNA) relieved 6-OHDA-induced cell apoptosis. Furthermore, inhibiting lincRNA-p21 also partially upregulated TGIF1. By retrieving information from a bioinformatics database and performing both double luciferase and RNA immunoprecipitation assays, we found that lincRNA-p21 and TGIF1 were able to form a double-stranded RNA-binding protein Staufen homolog 1 (STAU1) binding site and further activate the STAU1-mediated mRNA decay pathway. In addition, TGIF1 was able to transcriptionally regulate α-synuclein expression by binding to the promoter of SNCA. The Annexin V/PI apoptosis assay results showed that either knockdown of TGIF1 or overexpression of lincRNA-p21 notably abolished the neuroprotective effects of ghrelin against 6-OHDA-induced neurotoxicity. Collectively, these findings suggest that ghrelin exerts neuroprotective effects against 6-OHDA-induced neurotoxicity via the lincRNA-p21/TGIF1/α-synuclein pathway.

Signaling transduction regulated by 5-hydroxytryptamine 1A receptor and orexin receptor 2 heterodimers.

As G-protein-coupled receptors (GPCRs), 5-hydroxytryptamine 1A receptor (5-HT1AR) and orexin receptor 2 (OX2R) regulate the levels of the cellular downstream molecules. The heterodimers of different GPCRs play important roles in various of neurological diseases. Moreover, 5-HT1AR and OX2R are involved in the pathogenesis of neurological diseases such as depression with deficiency of hippocampus plasticity. However, the direct interaction of the two receptors remains elusive. In the present study, we firstly demonstrated the heterodimer formation of 5-HT1AR and OX2R. Exchange protein directly activated by cAMP (Epac) cAMP bioluminescence resonance energy transfer (BRET) biosensor analysis revealed that the expression levels of cellular cAMP significantly increased in HEK293T cells transfected with the two receptors compared with the 5-HT1AR group. Additionally, the cellular level of calcium was upregulated robustly in HEK293T cells co-transfected with 5-HT1AR and OX2R group after agonist treatment. Furthermore, western blotting data showed that 5-HT1AR and OX2R heterodimer decreased the levels of phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element-binding protein (CREB). These results not only unraveled the formation of 5-HT1AR and OX2R heterodimer but also suggested that the heterodimer affected the downstream signaling pathway, which will provide new insights into the function of the two receptors in the brain.

Orexin-A protects SH-SY5Y cells against H2O2-induced oxidative damage via the PI3K/MEK1/2/ERK1/2 signaling pathway.

Orexin-A elicits multiple potent effects on a variety of tumor cells via different signaling pathways. However, it is unknown whether it has a neuroprotective effect on SH-SY5Y human neuroblastoma cells. This study investigated the neuroprotective effect of Orexin-A against hydrogen peroxide (H2O2)-induced oxidative damage in SH-SY5Y cells and the underlying mechanism. H2O2 treatment decreased the viability of SH-SY5Y cells, induced apoptosis, and decreased superoxide dismutase activity. Orexin-A attenuated these effects, indicating that it protects SH-SY5Y cells against H2O2-induced oxidative damage. Pre-treatment with Orexin-A also attenuated H2O2-induced increases in phosphorylation of MEK1/2 and ERK1/2. Moreover, these effects of Orexin-A were reduced in the presence of the PI3K inhibitor LY294002. Finally, pre-treatment with LY294002 abrogated attenuation of the H2O2-induced decrease in cell viability and increase in caspase-3/7 activity by Orexin-A. These results show that the PI3K/MEK1/2/ERK1/2 signaling pathway is involved in the neuroprotective effects of Orexin-A against H2O2-induced oxidative damage in SH-SY5Y cells. Our findings provide insight into the neuroprotective effects of Orexin-A and the underlying mechanism, which will be useful for the treatment of nervous system diseases.

Anti-tumour research of recombinant BCG using BZLF1 and hGM-CSF fusion genes.

The random primer Oligo(dT)15 was used in RT-PCR to obtain cDNA from the human granulocyte macrophage colony stimulating factor (hGM-CSF) gene and the Epstein-Barr virus (EBV) gene BZLF1. Then, the sequence splicing overlap extension method was used to obtain a GCBF fusion gene containing a linker sequence that encoded the polypeptide (Gly4Ser)3. The GCBF fusion gene was inserted into pMV261, which was then transformed into competent E. coli DH5 alpha cells, and positive cells were selected based on kanamycin resistance on LB plates. The recombinant plasmid pMVBZLF1 was extracted from E. coli, and BCG (Bacillus Calmette-Guérin) was transformed into competent cells. According to the RT-PCR results, the target genes hGM-CSF and BZLF1 were 461bp and 788bp in size, which was in agreement with the expected values. Construction of the recombinant plasmid by double enzyme digestion, amplification, sequencing and Western blotting confirmed that the GCBF fusion gene (1204bp) was correctly inserted into pMV261, successfully transformed into BCG competent cells, and properly expressed. After mice were injected with rBCG (recombinant BCG), antibody levels were detected using ELISA, and spleen cells were obtained and the killing rates of specific CTLs by rBCG were detected using a CTL assay kit. Then, the influence of rBCG on tumour cells was analysed in C57BL/6 mice. We found that rBCG-secreting cytokines hybridized with hGM-CSF and BZLF1 antibodies and that the rBCG vaccine stimulated antibody production in C57BL/6 mice. The specific cytotoxic effects of the spleen cells from the rBCG group on EB virus-positive tumour cells was significantly different from the cytotoxic effects of the control group cells (P<0.01). CD8+ T and CD4+ T lymphocytes were detected in the tumour tissues of the rBCG group mice by flow cytometry, indicating that CD8+ T and CD4+ T lymphocytes infiltrated into the tumour tissue in the mice. Morphological observations of the tumour sections from the rBCG-immunized mice showed the infiltration of lymphocytes into the tumour tissues. The average rBCG tumour volume was less than the average tumour volume of the control group. Thus, rBCG may inhibit the growth of EB virus-positive tumour cells in mice.

[Studies on cultivation measures of Angelica dahurica].

OBJECTIVE: To evaluate the effect of seedingtime, density of crop and fertilization on the yield of Angelica dahurica. METHOD: Use weighing method to measure the output of A. dahurica. RESULT: The highest yield of seeding-time is 8373 kg/hm' on April 20, which is considerably different compared with April 5 and May 5; the highest yield of the density is 9300 kg/hm2 on 330,000 plants/hm2; the yield of fertilization tests all are considerable higher than that of the contrast. CONCLUSION: The appropriate seeingtime of A. dahurica is the first or second ten days of April, the appropriate density is 330,000 plants/hm2, and the appropriate amount of fertilization is N24P20, i.e pure N 360 kg and P20, 300 kg per hectare.