GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

Discovery of sesquiterpene from Youngia japonica with antitumor effect.

Fourteen sesquiterpenes, including one undescribed sesquiterpene lactone, were isolated from Youngia japonica, and their structures were identified by NMR, HRESIMS, ECD and calculated ECD. Cytotoxic activities of all isolates against A549, HeLa, and 4 T1 cell lines were detected by CCK8 assay. Among them, 2 showed obvious cytotoxic activity against A549 cells. Subsequently, the production of ROS, and apoptosis of A549 cells treated with 2 were evaluated. The result showed that 2 distinctly increased the ROS level, and induced the apoptosis of A549 cells. Further anticancer mechanism studies showed that 2 increased the expression of cleaved caspase 3. Taken together, our results demonstrated that 2 might become potential leading compounds for the treatment of lung cancer.

miR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought.

Anthocyanins are the primary color components of grapevine berries and wines. In cultivation practices, a moderate water deficit can promote anthocyanin accumulation in red grape skins. Our previous study showed that abscisic acid (ABA) plays a key role in this process. Herein, we identified a microRNA, vv-miR156b, that is generated in grapevine berries in response to drought stress, along with increasing anthocyanin content and biosynthetic structural gene transcripts. In contrast, vv-miR156b short tandem target mimic (STTM) function-loss callus exhibits the opposite phenotype. Results from in vivo and in vitro experiments revealed that the ABA-signaling-regulated transcription factor VvAREB2 binds directly to the ABA-responsive element (ABRE) of the MIR156b promoter and activates miR156b expression. Furthermore, two miR156b downstream targets, VvSBP8 and VvSBP13, exhibited reduced grape anthocyanin content in their overexpressors but there was a contrary result in their CRISPR-edited lines, the decrease in anthocyanin content was rescued in miR156b and SBP8/13 double overexpressors. We further demonstrated that both VvSBP8 and VvSBP13, encoding transcriptional repressors, displayed sufficient ability to interact with VvMYC1 and VvMYBA1, thereby interfering with MYB-bHLH-WD (MBW) repeat transcriptional complex formation, resulting in the repression of anthocyanin biosynthesis. Our findings demonstrate a direct functional relationship between ABA signaling and the miR156-SBP-MBW complex regulatory module in driving drought-induced anthocyanin accumulation in grape berries.

Neuroprotective Effect of Polyphenol Extracts from Terminalia chebula Retz. against Cerebral Ischemia-Reperfusion Injury.

Current therapies for ischemic stroke are insufficient due to the lack of specific drugs. This study aimed to investigate the protective activity of polyphenol extracts from Terminalia chebula against cerebral ischemia-reperfusion induced damage. Polyphenols of ethyl acetate and n-butanol fractions were extracted from T. chebula. BV2 microglial cells exposed to oxygen-glucose deprivation/reoxygenation and mice subjected to middle cerebral artery occlusion/reperfusion were treated by TPE and TPB. Cell viability, cell morphology, apoptosis, mitochondrial membrane potential, enzyme activity and signaling pathway related to oxidative stress were observed. We found that TPE and TPB showed strong antioxidant activity in vitro. The protective effects of TPE and TPB on cerebral ischemia-reperfusion injury were demonstrated by enhanced antioxidant enzyme activities, elevated level of the nucleus transportation of nuclear factor erythroid 2-related factor 2 and expressions of antioxidant proteins, with a simultaneous reduction in cell apoptosis and reactive oxygen species level. In conclusion, TPE and TPB exert neuroprotective effects by stimulating the Nrf2 signaling pathway, thereby inhibiting apoptosis.

Reprogramming astrocytes to motor neurons by activation of endogenous Ngn2 and Isl1.

Central nervous system injury and neurodegenerative diseases cause irreversible loss of neurons. Overexpression of exogenous specific transcription factors can reprogram somatic cells into functional neurons for regeneration and functional reconstruction. However, these practices are potentially problematic due to the integration of vectors into the host genome. Here, we showed that the activation of endogenous genes Ngn2 and Isl1 by CRISPRa enabled reprogramming of mouse spinal astrocytes and embryonic fibroblasts to motor neurons. These induced neurons showed motor neuronal morphology and exhibited electrophysiological activities. Furthermore, astrocytes in the spinal cord of the adult mouse can be converted into motor neurons by this approach with high efficiency. These results demonstrate that the activation of endogenous genes is sufficient to induce astrocytes into functional motor neurons in vitro and in vivo. This direct neuronal reprogramming approach may provide a novel potential therapeutic strategy for treating neurodegenerative diseases and spinal cord injury.

Tau inhibits PKA by nuclear proteasome-dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation.

Intraneuronal accumulation of wild-type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full-length wild-type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse- and memory-associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP-response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ-20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau-induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau-induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ-20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.

Development of Depotentiation in Adult-Born Dentate Granule Cells.

Activity-dependent synaptic plasticity, i.e., long-term potentiation (LTP), long-term depression (LTD) and LTP reversal, is generally thought to make up the cellular mechanism underlying learning and memory in the mature brain, in which N-methyl-D-aspartate subtype of glutamate (NMDA) receptors and neurogenesis play important roles. LTP reversal may be the mechanism of forgetting and may mediate many psychiatric disorders, such as schizophrenia, but the specific mechanisms underlying these disorders remain unclear. In addition, LTP reversal during the development of adult-born dentate granule cells (DGCs) remains unknown. We found that the expression of the NMDA receptor subunits NR2A and NR2B displayed dynamic changes during the development of postnatal individuals and the maturation of adult-born neurons and was coupled with the change in LTP reversal. The susceptibility of LTP reversal progressively increases with the rise in the expression of NR2A during the development of postnatal individual and adult-born neurons. In addition, NMDA receptor subunits NR2A, but not NR2B, mediated LTP reversal in the DGCs of the mouse hippocampus.

Memory Susceptibility to Retroactive Interference Is Developmentally Regulated by NMDA Receptors.

Retroactive interference (RI) occurs when new incoming information impairs an existing memory, which is one of the primary sources of forgetting. Although long-term potentiation (LTP) reversal shows promise as the underlying neural correlate, the key molecules that control the sensitivity of memory circuits to RI are unknown, and the developmental trajectory of RI effects is unclear. Here we found that depotentiation in the hippocampal dentate gyrus (DG) depends on GluN2A-containing NMDA receptors (NMDARs). The susceptibility of LTP to disruption progressively increases with the rise in the GluN2A/GluN2B ratio during development. The vulnerability of hippocampus-dependent memory to interference from post-learning novelty exploration is subject to similar developmental regulation by NMDARs. Both GluN2A overexpression and GluN2B downregulation in the DG promote RI-induced forgetting. Altogether, our results suggest that a switch in GluN2 subunit predominance may confer age-related differences to depotentiation and underlie the developmental decline in memory resistance to RI.

Difference of acute dissociation and 1-day culture on the electrophysiological properties of rat dorsal root ganglion neurons

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6-1.9 and Cav2.1-2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6-1.9 and Cav2.1-2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences

Difference of acute dissociation and 1-day culture on the electrophysiological properties of rat dorsal root ganglion neurons.

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6-1.9 and Cav2.1-2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6-1.9 and Cav2.1-2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences.

A Single-Cell-Type Real-Time PCR Method Based on a Modified Patch-Pipette Cell Harvesting System.

Real-time PCR is a powerful tool for quantifying nucleic acid expression. Real-time PCR is conventionally performed at the tissue level to guarantee an abundance of nucleic acid for detection. The precision and reliability of this method, however, is limited by usually being composed of a mixture of different cell types. Single-cell PCR, in contrast, eliminates the purity problem of the cell source. However, use of this method is usually impeded by difficulties in cell harvesting and stringent requirements for processing of very small quantities of nucleic acids. In this study, we combined the advantages of the high purity of selected cells in single-cell PCR with the greater nucleic acid quantities and thus greater ease of tissue-level PCR. The key aspect of our method is to use a modified patch-clamp pipette to harvest several selected cells of the same type. This method is therefore especially useful for cells that can be morphologically or histologically identified such as primary sensory neurons, striated muscle fibers and cells labeled with fluorescent makers.

Bioaccessibility and health risk of heavy metals in ash from the incineration of different e-waste residues.

Ash from incinerated e-waste dismantling residues (EDR) may cause significant health risks to people through ingestion, inhalation, and dermal contact exposure pathways. Ashes of four classified e-waste types generated by an incineration plant in Zhejiang, China were collected. Total contents and the bioaccessibilities of Cd, Cu, Ni, Pb, and Zn in ashes were measured to provide crucial information to evaluate the health risks for incinerator workers and children living in vicinity. Compared to raw e-waste in mixture, ash was metal-enriched by category incinerated. However, the physiologically based extraction test (PBET) indicates the bioaccessibilities of Ni, Pb, and Zn were less than 50 %. Obviously, bioaccessibilities need to be considered in noncancer risk estimate. Total and PBET-extractable contents of metal, except for Pb, were significantly correlated with the pH of the ash. Noncancer risks of ash from different incinerator parts decreased in the order bag filter ash (BFA) > cyclone separator ash (CFA) > bottom ash (BA). The hazard quotient for exposure to ash were decreased as ingestion > dermal contact > inhalation. Pb in ingested ash dominated (>80 %) noncancer risks, and children had high chronic risks from Pb (hazard index >10). Carcinogenic risks from exposure to ash were under the acceptable level (<10(-6)) both for children and workers. Exposure to ash increased workers' cancer risks and children's noncancer risks. Given the risk estimate is complex including toxicity/bioaccessibility of metals, the ways of exposure, and many uncertainties, further researches are required before any definite decisions on mitigating health risks caused by exposure to EDR incinerated ash are made.

Enhanced reductive dechlorination of polychlorinated biphenyl-contaminated soil by in-vessel anaerobic composting with zero-valent iron.

Anaerobic dechlorination is an effective degradation pathway for higher chlorinated polychlorinated biphenyls (PCBs). The enhanced reductive dechlorination of PCB-contaminated soil by anaerobic composting with zero-valent iron (ZVI) was studied, and preliminary reasons for the enhanced reductive dechlorination with ZVI were investigated. The results show that the addition of nanoscale ZVI can enhance dechlorination during in-vessel anaerobic composting. After 140 days, the average number of removed Cl per biphenyl with 10 mg g(-1) of added nanoscale ZVI was 0.63, enhancing the dechlorination by 34 % and improving the initial dechlorination speed. The ZVI enhances dechlorination by providing a suitable acid base environment, reducing volatile fatty acid inhibition and stimulating the microorganisms. The C/N ratios for treatments with the highest rate of ZVI addition were smaller than for the control, indicating that ZVI addition can promote compost maturity.

Dechlorination of polychlorinated biphenyl-contaminated soil via anaerobic composting with pig manure.

Anaerobic dechlorination is an effective degradation pathway of higher chlorinated polychlorinated biphenyls (PCBs). The efficiency of anaerobic composting remediation of PCB-contaminated soil using pig manure was determined. The results show that the dechlorination of PCB-contaminated soil via anaerobic composting with pig manure is feasible. PCB concentration is the most critical factor. Elevated PCB concentrations can inhibit dechlorination but does not disrupt the anaerobic fermentation process. At 1 mg kg(-1) PCBs, the degradation rate of five or more chlorinated biphenyls is 43.8%. The highest dechlorination performance in this experiment was obtained when the soil-to-organic waste ratio, carbon-to-nitrogen ratio, moisture content, and PCB concentration were 2:3, 20, 60%, and 1 mg kg(-1), respectively.