Loss of spines in the prelimbic cortex is detrimental to working memory in mice with early-life adversity.

Adverse experiences in early life can shape neuronal structures and synaptic function in multiple brain regions, leading to deficits of distinct cognitive functions later in life. Focusing on the pyramidal cells of the prelimbic cortex (PrL), a main subregion of the medial prefrontal cortex, the impact of early-life adversity (ELA) was investigated in a well-established animal model generated by changing the rearing environment during postnatal days 2 to 9 (P2-P9), a sensitive developmental period. ELA has enduring detrimental impacts on the dendritic spines of PrL pyramidal cells, which is most apparent in a spatially circumscribed region. Specifically, ELA affects both thin and mushroom-type spines, and ELA-provoked loss of spines is observed on selective dendritic segments of PrL pyramidal cells in layers II-III and V-VI. Reduced postsynaptic puncta represented by postsynaptic density protein-95 (PSD-95), but not synaptophysin-labelled presynaptic puncta, in ELA mice supports the selective loss of spines in the PrL. Correlation analysis indicates that loss of spines and postsynaptic puncta in the PrL contributes to the poor spatial working memory of ELA mice, and thin spines may play a major role in working memory performance. To further understand whether loss of spines affects glutamatergic transmission, AMPA- and NMDA-receptor-mediated synaptic currents (EPSCs) were recorded in a group of Thy1-expressing PrL pyramidal cells. ELA mice exhibited a depressed glutamatergic transmission, which is accompanied with a decreased expression of GluR1 and NR1 subunits in the PrL. Finally, upregulating the activation of Thy1-expressing PrL pyramidal cells via excitatory DREADDs can efficiently improve the working memory performance of ELA mice in a T-maze-based task, indicating the potential of a chemogenetic approach in restoring ELA-provoked memory deficits.

Restored presynaptic synaptophysin and cholinergic inputs contribute to the protective effects of physical running on spatial memory in aged mice.

The effects of prolonged physical training on memory performance and underlying presynaptic mechanisms were investigated in old C57BL/6 mice. Training via voluntary running wheels was initiated at 16 months of age and continued for 5 months (1 h per day, 5 days per week), followed by testing of learning and memory functions and counting of presynaptic puncta and cholinergic inputs in the hippocampus. Trained old mice were compared to their age-matched sedentary controls and adult controls. This training strategy improved hippocampal-dependent spatial memory function tested via a novel location task, and enhanced memory was accompanied by restored presynaptic puncta and cholinergic fibers in area CA1 and DG of the hippocampus in old mice. Particularly, the training selectively affected presynaptic vesicle protein synaptophysin but not growth associated protein GAP-43, and the increased number of synaptophysin puncta positively correlates with improved memory performance. To better understand the neurochemical mechanisms by which prolonged physical training protects against aging-related memory deficits, the cholinergic inputs to the hippocampus were compared among the three groups of mice and correlated with memory performance. While the running prevented age-related loss of cholinergic inputs, it has limited impact on the projection source cells in the medial septum-diagonal band (MS-DB). Importantly, cholinergic fibers in area CA1 and DG positively correlated with spatial memory function. These data suggest that the preservation of presynaptic inputs, particularly those involved in the integrity of memory performance, contributes critically to the beneficial effects of physical running initiated at an older age.

Urolithin A attenuates memory impairment and neuroinflammation in APP/PS1 mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by an abnormal accumulation of amyloid-ß (Aß) plaques, neuroinflammation, and impaired neurogenesis. Urolithin A (UA), a gut-microbial metabolite of ellagic acid, has been reported to exert anti-inflammatory effects in the brain. However, it is unknown whether UA exerts its properties of anti-inflammation and neuronal protection in the APPswe/PS1ΔE9 (APP/PS1) mouse model of AD. METHODS: Morris water maze was used to detect the cognitive function. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to detect neuronal apoptosis. Immunohistochemistry analyzed the response of glia, Aß deposition, and neurogenesis. The expression of inflammatory mediators were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The modulating effects of UA on cell signaling pathways were assayed by Western blotting. RESULTS: We demonstrated that UA ameliorated cognitive impairment, prevented neuronal apoptosis, and enhanced neurogenesis in APP/PS1 mice. Furthermore, UA attenuated Aß deposition and peri-plaque microgliosis and astrocytosis in the cortex and hippocampus. We also found that UA affected critical cell signaling pathways, specifically by enhancing cerebral AMPK activation, decreasing the activation of P65NF-κB and P38MAPK, and suppressing Bace1 and APP degradation. CONCLUSIONS: Our results indicated that UA imparted cognitive protection by protecting neurons from death and triggering neurogenesis via anti-inflammatory signaling in APP/PS1 mice, suggesting that UA might be a promising therapeutic drug to treat AD.

JAK2/STAT3 signaling mediates IL-6-inhibited neurogenesis of neural stem cells through DNA demethylation/methylation.

Neuroinflammation, considered as a pathological hallmark of Alzheimer's disease (AD), has been demonstrated to affect hippocampal neurogenesis and cognitive function. Interleukin-6 (IL-6) is a proinflammatory cytokine known to modulate neurogenesis. However, the mechanisms are still largely unknown. Here, we reported that IL-6 suppressed neurogenesis via a JAK2/STAT3 signaling in neural stem cells (NSCs). Importantly, we found that NeuroD1 (Neurogenic differentiation 1) gene expression, which drives NSCs neurodifferentiation, was regulated by TET3 and DNMT1 in a JAK2/STAT3-dependent manner. We further found that JAK2/STAT3 inhibition enhanced demethylation of NeuroD1 regulatory elements in IL-6-treated cells, which is related to the significant upregulation of TET3 expression as well as the decreased expression of DNMT1. Furthermore, Inhibiting JAK2/STAT3 significantly rescued the memory deficits and hippocampal neurogenesis dysfunction in APP/PS1 mice. Our data suggest that JAK2/STAT3 signaling plays a vital role in suppressing neurogenesis of NSCs exposed to IL-6 at the epigenetic level, by regulating DNA methylation/demethylation.

Netrin-1 attenuates brain injury after middle cerebral artery occlusion via downregulation of astrocyte activation in mice.

BACKGROUND: Netrin-1 functions largely via combined receptors and downstream effectors. Evidence has shown that astrocytes express netrin-1 receptors, including DCC and UNC5H2. However, whether netrin-1 influences the function of astrocytes was previously unknown. METHODS: Lipopolysaccharide was used to stimulate the primary cultured astrocytes; interleukin release was used to track astrocyte activation. In vivo, shRNA and netrin-1 protein were injected in the mouse brain. Infarct volume, astrocyte activation, and interleukin release were used to observe the function of netrin-1 in neuroinflammation and brain injury after middle cerebral artery occlusion. RESULTS: Our results demonstrated that netrin-1 reduced lipopolysaccharide-induced interleukin-1ß and interleukin-12ß release in cultured astrocytes, and blockade of the UNC5H2 receptor with an antibody reversed this effect. Additionally, netrin-1 increased p-AKT and PPAR-γ expression in primary cultured astrocytes. In vivo studies showed that knockdown of netrin-1 increased astrocyte activation in the mouse brain after middle cerebral artery occlusion (p < 0.05). Moreover, injection of netrin-1 attenuated GFAP expression (netrin-1 0.27 ± 0.06 vs. BSA 0.62 ± 0.04, p < 0.001) and the release of interleukins and reduced infarct volume after brain ischemia (netrin-1 0.27 ± 0.06 vs. BSA 0.62 ± 0.04 mm3, p < 0.05). CONCLUSION: Our results indicate that netrin-1 is an important molecule in regulating astrocyte activation and neuroinflammation in cerebral ischemia and provides a potential target for ischemic stroke therapy.

Metformin treatment prevents amyloid plaque deposition and memory impairment in APP/PS1 mice.

Alzheimer'sdisease(AD) is characterized by deposition of amyloid-ß (Aß)plaques, neurofibrillary tangles, andneuronal loss, accompaniedbyneuroinflammation. Neuroinflammatoryprocesses are thought to contribute toAD pathophysiology. Metformin has been reported to have anti-inflammatory efficacy. However, whether metformin is responsible for the anti-neuroinflammationand neuroprotection on APPswe/PS1ΔE9 (APP/PS1) mice remains unclear. Here we showed that metformin attenuated spatial memory deficit, neuron loss in the hippocampus and enhanced neurogenesis in APP/PS1 mice. In addition, metformin administration decreased amyloid-ß (Aß)plaque load and chronic inflammation (activated microglia and astrocytes as well as pro-inflammatory mediators) in the hippocampus and cortex. Further study demonstrated that treatment with metformin enhanced cerebral AMPK activation. Meanwhile, metformin notably suppressed the activation of P65 NF-κB, mTOR and S6K, reduced Bace1 protein expression. Our data suggest that metformin can exert functional recovery of memory deficits and neuroprotective effect on APP/PS1 mice via triggering neurogenesis and anti-inflammation mediated by regulating AMPK/mTOR/S6K/Bace1 and AMPK/P65 NF-κB signaling pathways in the hippocampus, which may contribute to improvement in neurological deficits.

Mitochondrial FOXO3a is involved in amyloid ß peptide-induced mitochondrial dysfunction.

Mitochondrial dysfunction is a hallmark of amyloid ß peptide (Aß)-induced neuronal toxicity in Alzheimer's disease (AD). However, the precise mechanism(s) of Aß-induced mitochondrial dysfunction has not been fully understood. There is evidence that Forkhead box O3a (FOXO3a) is normally present in neuronal mitochondria. Using HT22 murine hippocampal neuronal cells and primary hippocampal neurons, the present study investigated whether mitochondrial FOXO3a was involved in mitochondrial dysfunction induced by Aß. It was found that Aß induced dephosphorylation and mitochondrial translocation of FOXO3a. In addition, Aß enhanced association of FOXO3a with mitochondrial DNA (mtDNA), causing a decrease in the expression of cytochrome c oxidase subunit 1 (COX1) and the activity of COX. In addition, Aß-induced mitochondrial dysfunction, indicated by the decrease in 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion, mitochondrial adenosine triphosphate (ATP) production and COX activity, could be suppressed by knockdown of FOXO3a (FOXO3a-KD). These results provide new insights into the mechanism underlying Aß-induced neurotoxicity and open up new therapeutic perspectives for AD.

Treatment Efficacy of NGF Nanoparticles Combining Neural Stem Cell Transplantation on Alzheimer's Disease Model Rats.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia. It causes progressive brain disorder involving loss of normal memory and thinking skills. The transplantation of neural stem cells (NSCs) has been reported to improve learning and memory function of AD rats, and protects basal forebrain cholinergic neurons. Nerve growth factor - poly (ethylene glycol) - poly (lactic-co-glycolic acid)-nanoparticles (NGF-PEG-PLGA-NPs) can facilitate the differentiation of NSCs in vitro. This study thus investigated the treatment efficacy of NGF-PEG-PLGA-NPs combining NSC transplantation in AD model rats. MATERIAL AND METHODS: AD rats were prepared by injection of 192IgG-saporin into their lateral ventricles. Embryonic rat NSCs were separated, induced by NGF-PEG-PLGA-NPs in vitro, and were transplanted. The Morris water-maze test was used to evaluate learning and memory function, followed by immunohistochemical staining for basal forebrain cholinergic neurons, hippocampal synaptophysin, and acetylcholine esterase (AchE) fibers. RESULTS: Rats in the combined treatment group had significantly improved spatial learning ability compared to AD model animals (p<0.05). The number of basal forebrain cholinergic neurons, hippocampal synaptophysin, and AchE-positive fibers were all significantly larger than in the NSC-transplantation group, with no difference from control animals. CONCLUSIONS: NGF-PEG-PLGA-NPs plus NSC transplantation can significantly improve learning and memory functions of AD rats, replenish basal forebrain cholinergic neurons, and help form hippocampal synapses and AchE-positive fibers. These findings may offer practical support for and insight into treatment of Alzheimer's disease.

Intromitochondrial IκB/NF-κB signaling pathway is involved in amyloid ß peptide-induced mitochondrial dysfunction.

Mitochondrial dysfunction is a hallmark of amyloid ß peptide (Aß)-induced neuronal toxicity in Alzheimer's disease (AD). However, the underlying mechanism (s) of Aß-induced mitochondrial dysfunction is still not fully understood. There is evidence that nuclear factor-κB (NF-κB) is involved in Aß-induced neurotoxicity and is present in mitochondria. Using HT22 murine hippocampal neuronal cells and isolated mitochondria, the present study investigated whether intramitochondrial inhibitor of NF-κB (IκB)/NF-κB signaling pathway was involved in mitochondrial dysfunction induced by Aß. It was found that Aß impaired mitochondrial function through a NF-κB-dependent signaling pathway. Intramitochondrial IκBα/NF-κB pathway, induced by Aß, decreased the expression of cytochrome c oxidase subunit (COXIII) and inhibited COX activity. These results provide new insights into the mechanism underlying the neurotoxic effect of Aß and open up new therapeutic perspectives for AD.

MiR-210-5p promotes the differentiation of human induced pluripotent stem cells into dopaminergic neural precursors by targeting SMAD4 and SUFU and treats parkinsonian rats.

The differentiation of human induced pluripotent stem cells (hiPSCs) into functional dopaminergic neural precursors is the basis of cell therapy for Parkinson's disease (PD). However, the use of small molecule inhibitors/activators in the differentiation of hiPSCs in vitro leads to cell death and low differentiation efficiency. Moreover, the mechanism of differentiation remains unclear. MiR-210-5p was increased during hiPSCs differentiation. Whether it promotes hiPSCs differentiation and transplantation needs further study. Here, we overexpressed miR-210-5p in hiPSCs to study its roles and mechanisms. We found that miR-210-5p promoted the differentiation of hiPSCs into dopaminergic neural precursors and reduced the expression of SMAD4 and SUFU meanwhile. Luciferase assays showed that miR-210-5p binded to SMAD4 and SUFU, which are key molecules in the key signals (TGF-ß and SHH) of hiPSCs differentiation. Furthermore, in the effect evaluation of cell transplantation into parkinsonian rats, the degree of behavioral recovery and the growth of transplanted cells in the group overexpressed miR-210-5p were similar to those in the positive group with all small molecule inhibitors/activators. Therefore, we conclude that miR-210-5p promotes the differentiation of hiPSCs into dopaminergic neural precursors by targeting SMAD4 and SUFU. In the therapeutic evaluation of cell transplantation, miR-210-5p can replace the use of corresponding small molecule inhibitors/activators to reduce cell death. This study provides an experimental basis and a new target for the miRNA-modified differentiation of hiPSCs and cell transplantation in clinical treatment of PD in the future.

Hydrogen sulfide attenuates spatial memory impairment and hippocampal neuroinflammation in ß-amyloid rat model of Alzheimer's disease.

BACKGROUND: Endogenously produced hydrogen sulfide (H(2)S) may have multiple functions in brain. An increasing number of studies have demonstrated its anti-inflammatory effects. In the present study, we investigated the effect of sodium hydrosulfide (NaHS, a H(2)S donor) on cognitive impairment and neuroinflammatory changes induced by injections of Amyloid-ß(1-40) (Aß(1-40)), and explored possible mechanisms of action. METHODS: We injected Aß(1-40) into the hippocampus of rats to mimic rat model of Alzheimer's disease (AD). Morris water maze was used to detect the cognitive function. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to detect neuronal apoptosis. Immunohistochemistry analyzed the response of glia. The expression of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α was measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Aß(1-40), phospho-p38 mitogen-activated protein kinase (MAPK), phospho-p65 Nuclear factor (NF)-κB, and phospho-c-Jun N-terminal Kinase (JNK) was analyzed by western blot. RESULTS: We demonstrated that pretreatment with NaHS ameliorated learning and memory deficits in an Aß(1-40) rat model of AD. NaHS treatment suppressed Aß(1-40)-induced apoptosis in the CA1 subfield of the hippocampus. Moreover, the over-expression in IL-1ß and TNF-α as well as the extensive astrogliosis and microgliosis in the hippocampus induced by Aß(1-40) were significantly reduced following administration of NaHS. Concomitantly, treatment with NaHS alleviated the levels of p38 MAPK and p65 NF-κB phosphorylation but not JNK phosphorylation that occurred in the Aß(1-40)-injected hippocampus. CONCLUSIONS: These results indicate that NaHS could significantly ameliorate Aß(1-40)-induced spatial learning and memory impairment, apoptosis, and neuroinflammation at least in part via the inhibition of p38 MAPK and p65 NF-κB activity, suggesting that administration of NaHS could provide a therapeutic approach for AD.

The Effects of Physical Running on Dendritic Spines and Amyloid-beta Pathology in 3xTg-AD Male Mice.

Memory loss is the key symptom of Alzheimer's disease (AD). As successful drug treatments have not yet been identified, non-pharmaceutical interventions such as physical exercise and training have been employed to improve the memory function of people with dementia. We investigated the effect of prolonged physical running on hippocampal-dependent spatial memory and its underlying mechanisms using a well-established rodent model of AD. 3xTg-AD transgenic mice and non-transgenic mice were subjected to voluntary wheel running for 5 months (1 hour per day, 5 days per week), followed by spatial memory testing. After the behavioral testing, dendritic spines, synapses, and synaptic proteins as well as amyloid-beta (Aß) pathology were analyzed in the dorsal hippocampi. Running improved hippocampal-dependent spatial memory in 3xTg-AD mice. This running strategy prevented both thin and mushroom-type spines on CA1 pyramidal cells in 3xTg-AD mice, whereas the effects of running in non-transgenic mice were limited to thin spines. The enormous effects of running on spines were accompanied by an increased number of synapses and upregulated expression of synaptic proteins. Notably, running downregulated the processing of amyloid precursor protein, decreasing intracellular APP expression and extracellular Aß accumulation, and spatial memory performance correlated with levels of Aß peptides Aß1-40 and Aß1-42. These data suggest that prolonged running may improve memory in preclinical AD via slowing down the amyloid pathology and preventing the loss of synaptic contacts.

Early-life adversity selectively interrupts the dendritic differentiation of dorsolateral striatal neurons in male mice.

The effects of early-life adversity (ELA) on dendritic differentiation of striatal neurons were investigated in the dorsal striatum including the dorsomedial striatum and dorsolateral striatum (DMS and DLS, respectively). An animal model of ELA was created by changing the growth environment of newborn mouse pups by giving limited bedding and nesting materials from postnatal day 2 to day 9 (P2-P9). One week after the stress paradigm (P16), the dendritic branches and spines of striatal spiny neurons as well as the synapses represented by postsynaptic density protein-95 (PSD-95) in DMS and DLS were stereologically analyzed. Adverse experience in early life selectively affected the spiny neurons in DLS, leading to abundant proximal dendritic branches and an increased number of filopodia-like protrusions, but a reduced number of dendritic spines. The selective effects of stress on neurons in DLS were further identified by reduced expression of PSD-95, including a reduced optical density of PSD-95 immunoreactivity and fewer individual PSD-95 immunoreactive synapses in this region. Notably, stress in early life affected either D1 or D2 dopamine receptor-expressing DLS neurons. These findings suggest that adverse early-life experience delayed the maturation of dendritic spines on neurons in the dorsolateral striatum. Altered dendritic differentiation provoked by stress in early life may contribute critically to the formation of proper neuronal circuits in the dorsal striatum and, therefore, affect striatum-dependent habitual behavior and emotional function later in life.

Neural stem cell transplantation improves learning and memory by protecting cholinergic neurons and restoring synaptic impairment in an amyloid precursor protein/presenilin 1 transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent age­related neurodegenerative disorder. It is featured by the progressive accumulation of ß­amyloid (Aß) plaques and neurofibrillary tangles. This can eventually lead to a decrease of cholinergic neurons in the basal forebrain. Stem cell transplantation is an effective treatment for neurodegenerative diseases. Previous studies have revealed that different types of stem or progenitor cells can mitigate cognition impairment in different Alzheimer's disease mouse models. However, understanding the underlying mechanisms of neural stem cell (NSC) therapies for AD requires further investigation. In the present study, the effects and the underlying mechanisms of the treatment of AD by NSCs are reported. The latter were labelled with the enhanced green fluorescent protein (EGFP) prior to implantation into the bilateral hippocampus of an amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (Tg) mouse model of AD. It was observed that the number of basal forebrain cholinergic neurons was restored and the expression of choline acetyltransferase (ChAT) protein was increased. Moreover, the levels of synaptophysin (SYP), postsynaptic density protein 95 (PSD­95) and microtubule­associated protein (MAP­2) were significantly increased in the hippocampus of NSC­treated AD mice. Notably, spatial learning and memory were both improved after transplantation of NSCs. In conclusion, the present study revealed that NSC transplantation improved learning and memory functions in an AD mouse model. This treatment allowed repairing of basal forebrain cholinergic neurons and increased the expression of the cognition­related proteins SYP, PSD­95 and MAP­2 in the hippocampus.

Bone mesenchymal stem cells for gene transfer of NGF to the adult rat brain: rescue the NGFR p75 positive neurons from fimbria-fornix lesion-induced degeneration.

The study was to evaluate the therapeutic benefit of transplanted bone mesenchymal stem cells (BMSCs) transfected never growth factor (NGF) gene and GFP gene (as a reporter gene), in treating the rat with fimbria-fornix lesion. After transduction of NGF gene via recombinant retroviral vectors into the rat BMSCs, BMSCs were therefore transformed into the GFP-NGF positive BMSCs, nearly 100% of BMSCs expressed NGF, and then transplanted into basal forebrain of rat with fimbria-fornix lesion. After 2 weeks post-transplantation, the GFP-NGF positive BMSCs survive and fuse in vivo with astroglia or NGFR p75 positive neurons in the basal forebrain, no evidence of transdifferentiation was observed in this study. The number of NGFR p75 positive neurons in basal forebrain of NGF group was significantly higher than those of the void plasmid group (p < 0.05) or the PBS group (p < 0.01). These results indicate that the GFP-NGF positive BMSCs provide, by way of paracrine, NGF that effectively perform the functions of neuroprotection, which cell fusion may be also contribute to.

Loss of thin spines and small synapses contributes to defective hippocampal function in aged mice.

Aging is a normal physiological process associated with impairments in cognitive function, including learning and memory. Here, the underlying synaptic mechanisms by which aging leads to the decline of spatial learning and memory function were investigated in 25-month-old aged mice versus 2-month-old young mice. Deficits of spatial learning and memory, as well as selective loss of thin spines, but not mushroom-type spines on apical dendrites of CA1 pyramidal cells were found in aged mice. Specifically, loss of thin spines in aged mice with memory deficits was primarily found on dendritic segments located in the Schaffer pathway, and the density of thin spines significantly correlated with spatial memory performance. The loss of thin spines was evidenced by a decrease in small synapses that express diminutive amounts of postsynaptic density protein-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR1. Furthermore, mushroom-type spines and GluR1-expressed large synapses were not affected in aged mice with impaired memory. Taken together, these data suggest that the selective loss of those highly plastic thin spines with sparse postsynaptic density protein-95 and GluR1 receptors may significantly contribute to cognitive deficits in aged individuals.

Effect of leukocyte inhibitory factor on neuron differentiation from human induced pluripotent stem cell-derived neural precursor cells.

Direct derivation of human induced pluripotent stem cells into neural precursor cells and differentiation of these into neurons holds great promise in the cell therapy of neurodegenerative diseases. However, the availability and survival rate of neurons requires improvement. In the present study, it was found that the addition of 5 ng/ml leukocyte inhibitory factor (LIF) during the process of differentiation significantly improved the expression of neuron­specific class III ß­tubulin (TUJ1) and microtubule­associated protein 2 (MAP2), as detected by immunofluorescence and western blotting. In addition, LIF improved the cell viability, increased the expression of phosphorylated­protein kinase B (AKT), downregulated the expression of proinflammatory cytokines, including interleukin­1α (IL­1α) and tumor necrosis factor­α (TNF-α), and upregulated the expression of anti­inflammatory cytokines, including interleukin­10 (IL­10) and transforming growth factor­ß (TGF-ß). After adding the phosphatidylinositol 3-kinase (PI3K)/AKT signaling inhibitor LY294002 or wortmannin to the LIF differentiation group, LIF-induced changes in the protein expression of TUJ1 and MAP2 were reversed, but this effect could not be prevented by rapamycin, a mechanistic target of rapamycin signaling inhibitor. The expression of cytokines associated with inflammation and cell viability was reversed by LY294002 and wortmannin, but not by rapamycin. In conclusion, LIF could improve neuronal differentiation and survival through the activation of PI3K/AKT signaling and the anti­inflammatory effect. The anti­inflammatory effect may be mediated by the activation of PI3K/AKT.

miR­146b­5p promotes the neural conversion of pluripotent stem cells by targeting Smad4.

Pluripotent stem cells (PSCs) are regarded as potential sources that provide specific neural cells for cell therapy in some nervous system diseases. However, the mechanisms underlying the neural differentiation of PSCs remain largely unknown. MicroRNAs (miRNAs or miRs) are a class of small non­pro-tein-coding RNAs that act as critical regulatory molecules in many cellular processes. In this study, we found that miR­146b­5p expression was markedly increased following the neural induction of mouse embryonic stem cells (ESCs) or induced PSCs (iPSCs). In this study, to further identify the role of miR­146b­5p, we generated stable miR­146b­5p-overexpressing ESC and iPSC cell lines, and induced the differentiation of these cells by the adherent monolayer culture method. In the miR­146b­5p-overexpressing ESC- or iPSC-derived cultures, RT-qPCR analysis revealed that the mRNA expression levels of neuroectoderm markers, such as Sox1, Nestin and Pax6, were markedly increased, and flow cytometric analysis verified that the number of Nestin­positive cells was higher in the miR­146b­5p-overexpressing compared with the control cells. Mechanistically, the miR­146b­5p-overexpressing ESCs or iPSCs exhibited a significant reduction in Oct4 expression, which may be an explanation for these cells having a tendency to differentiate towards the neural lineage. Moreover, we confirmed that miR­146b­5p directly targeted Smad4 and negatively regulated the transforming growth factor (TGF)-ß signaling pathway, which contributed to the neural commitment of PSCs. Collectively, our findings uncover the essential role of miR­146b­5p in the neural conversion of PSCs.

NF-κB pathway link with ER stress-induced autophagy and apoptosis in cervical tumor cells.

Targeting endoplasmic reticulum (ER) stress is being investigated for its anticancer effect in various cancers, including cervical cancer. However, the molecular pathways whereby ER stress mediates cell death remain to be fully elucidated. In this study, we confirmed that ER stress triggered by compounds such as brefeldin A (BFA), tunicamycin (TM), and thapsigargin (TG) leads to the induction of the unfolded protein response (UPR) in cervical cancer cell lines, which is characterized by elevated levels of inositol-requiring kinase 1α, glucose-regulated protein-78, and C/EBP homologous protein, and swelling of the ER observed by transmission electron microscope (TEM). We found that BFA significantly increased autophagy in tumor cells and induced TC-1 tumor cell death in a dose-dependent manner. BFA increased punctate staining of LC3 and the number of autophagosomes observed by TEM in TC-1 and HeLa cells. The autophagic flux was also assessed. Bafilomycin, which blocked degradation of LC3 in lysosomes, caused both LC3I and LC3II accumulation. BFA initiated apoptosis of TC-1 tumor cells through activation of the caspase-12/caspase-3 pathway. At the same time, BFA enhanced the phosphorylation of IκBα protein and translocation into the nucleus of NF-κB p65. Quinazolinediamine, an NF-κB inhibitor, attenuated both autophagy and apoptosis induced by BFA; meanwhile, it partly enhances survival of cervical cancer cells following BFA treatment. In conclusion, our results indicate that the cross-talk between ER stress, autophagy, apoptosis, and the NF-κB pathways controls the fate of cervical cancer cells. Careful evaluation should be given to the addition of an NF-κB pathway inhibitor to treat cervical cancer in combination with drugs that induce ER stress-mediated cell death.

PI3K/AKT/mTOR Signaling Mediates Valproic Acid-Induced Neuronal Differentiation of Neural Stem Cells through Epigenetic Modifications.

Although valproic acid (VPA), has been shown to induce neuronal differentiation of neural stem cells (NSCs), the underlying mechanisms remain poorly understood. Here we investigated if and how mammalian target of rapamycin (mTOR) signaling is involved in the neuronal differentiation of VPA-induced NSCs. Our data demonstrated that mTOR activation not only promoted but also was necessary for the neuronal differentiation of NSCs induced by VPA. We further found that inhibition of mTOR signaling blocked demethylation of neuron-specific gene neurogenin 1 (Ngn1) regulatory element in induced cells. These are correlated with the significant alterations of passive DNA demethylation and the active DNA demethylation pathway in the Ngn1 promoter, but not the suppression of lysine-specific histone methylation and acetylation in the promoter region of Ngn1. These findings highlight a potentially important role for mTOR signaling, by working together with DNA demethylation, to influence the fate of NSCs via regulating the expression of Ngn1 in VPA-induced neuronal differentiation of NSCs.