FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Maternal gestational weight gain and adverse pregnancy outcomes in non-diabetic women.

OBJECTIVE: This study investigated the relationship between maternal gestational weight gain (GWG) and the risk of adverse pregnancy outcomes in gestational diabetes mellitus (GDM)-negative pregnant women. METHODS: We did a retrospective cohort study between 1 July 2017, and 1 January 2020, at Women's Hospital, Zhejiang University School of Medicine. Firstly, pregnant women were divided into subgroups according to the entire GWG (inadequate GWG, adequate GWG, and excessive GWG) and GDM status (positive and negative) during pregnancy. Secondly, the whole population of pregnant women with GDM was used as a reference to evaluate the relationship between GWG and adverse pregnancy outcomes in GDM-negative pregnant women. Lastly, subgroup analysis was conducted based on pre-pregnancy body mass index (pp-BMI). RESULTS: A total of 30,910 pregnant women were analysed. Included pregnancy women were divided into three groups based on GWG: 7569 (24.49%) pregnancy women had inadequate GWG, 13088 (42.34%) had adequate GWG, and 10,253 (33.17%) had excessive GWG. In addition to preterm birth and small for gestational age (SGA), the incidence of macrosomia and large for gestational age (LGA) continues to increase from inadequate GWG to excessive GWG groups. Pregnant women without GDM who have excessive GWG are at higher risk of macrosomia and LGA than pregnant women with GDM. Moreover, this risk increased with increasing pp-BMI. Pregnant women without GDM with inadequate GWG were at risk of preterm birth regardless of pp-BMI. Only those with inadequate GWG and pp-BMI < 18.5 kg/m2 had an increased risk of SGA. CONCLUSIONS: In conclusion, inappropriate GWG is strongly associated with adverse pregnancy outcomes, even if they do not have GDM. Therefore, this population should receive attention and management before and during pregnancy.Impact StatementWhat is already known on this subject? Several studies have focused on the GDM population and the risk of adverse pregnancy outcomes, but few have focused on GDM-negative populations. This is because GDM-negative women are perceived to be "safe," leading to less focus on themselves, which can lead to subsequent excessive weight gain during pregnancy. Whether this factor increases the risk of adverse pregnancy outcomes in this population remains unknown.What do the results of this study add? Our study found an inverse relationship between GWG and GDM. Therefore, our study focuses on this group of GDM-negative pregnant women. Their excessive weight gain increases the risk of adverse pregnancy outcomes, even higher than GDM pregnant women.What are the implications of these findings for clinical practice and/or further research? GWG is associated with adverse pregnancy outcomes. Therefore, pregnant women without GDM also need increased attention and management of their weight before and during pregnancy. Prenatal care providers can utilise tools such as diet, exercise counselling, weight tracking, and setting weight gain goals to reduce inappropriate weight gain and mitigate its adverse effects on pregnancy outcomes.

Effect of maternal body mass index on the steroid profile in women with gestational diabetes mellitus.

Objective: To explore the effect of maternal body mass index (BMI) on steroid hormone profiles in women with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT). Methods: We enrolled 79 women with NGT and 80 women with GDM who had a gestational age of 24-28 weeks. The participants were grouped according to their BMI. We quantified 11 steroid hormones profiles by liquid chromatography-tandem mass spectrometry and calculated the product-to-precursor ratios in the steroidogenic pathway. Results: Women with GDM and BMI<25kg/m2 showed higher concentrations of dehydroepiandrosterone (DHEA) (p<0.001), testosterone (T) (p=0.020), estrone (E1) (p=0.010) and estradiol (E2) (p=0.040) and lower Matsuda index and HOMA-ß than women with NGT and BMI<25kg/m2. In women with GDM, concentrations of E1 (p=0.006) and E2 (p=0.009) declined, accompanied by reduced E2/T (p=0.008) and E1/androstenedione (A4) (p=0.010) in the BMI>25 kg/m2 group, when compared to that in the BMI<25 kg/m2 group. The values of E2/T and E1/A4 were used to evaluate the cytochrome P450 aromatase enzyme activity in the steroidogenic pathway. Both aromatase activities negatively correlated with the maternal BMI and positively correlated with the Matsuda index in women with GDM. Conclusions: NGT women and GDM women with normal weight presented with different steroid hormone profiles. Steroidogenic pathway profiling of sex hormones synthesis showed a significant increase in the production of DHEA, T, E1, and E2 in GDM women with normal weight. Additionally, the alteration of steroid hormone metabolism was related to maternal BMI in women with GDM, and GDM women with overweight showed reduced estrogen production and decreased insulin sensitivity compared with GDM women with normal weight.

Biopsychosocial risk factors of depression during menopause transition in southeast China.

OBJECTIVE: More than 2 billion women are experiencing menopause transition in China and some of them suffered from depression; while the risk factors of depression during menopause transition were still unclearin China. We aimed to investigate the risk factors in mid-life women in Southeast China. METHOD: This study included 1748 Chinese women aged 40-65 years-old who visited gynecology outpatient department of Women's hospital School of Medicine, Zhejiang University during 2010-2018. Demographic information was collected, and the modified Kupperman Menopausal Index (mKMI) and Hamilton Rating Scale for Depression were assessed. Circulating levels of sex hormones were tested. Ordinal logistic regression analysis was performed to identify risk factors for depression. RESULTS: The prevalence of depression symptoms was 47.43%. The majority of women had mild (38.56%) or moderate depressive symptoms (8.00%); only 0.86% had severe depressive symptoms. Compared with perimenopausal women, postmenopausal women had increased risks of more severe depression. The associations between menopausal syndromes and the intensity of depression were strongly positive (OR 6.69, 95% CI 5.39-8.29). Elder age, higher follicle stimulating hormone levels, lower estradiol levels, and fewer parity were positively related with the intensity of depression. Among postmenopausal women, underweight, mKMI > 14, earlier age at menopause, shorter reproductive period, and longer duration after menopause were risk factors for incresed intensity of depression. CONCLUSIONS: The results demonstrated a high proportion of depression in women complaining of menopause. Menopausal symptoms were strongly related to the intensity of depression. In postmenopausal women, estrogen related events are associated with the intensity of depression. Gynecological endocrinologists in China should consider screening for depression in high-risk women.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

BACKGROUND: Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. METHODS: Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. RESULTS: We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. CONCLUSIONS: Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

Overexpression of miR-132-3p contributes to neuronal protection in in vitro and in vivo models of Alzheimer's disease.

One of the neuropathological hallmarks of Alzheimer's disease (AD) is accumulation and deposition of amyloid-beta (Aß1-42) plaques in the hippocampus. Recently, microRNAs (miRNAs), have been demonstrated to play an essential role in AD. We have previously demonstrated that miR-132-3p exerts neuroprotection via regulating histone deacetylase 3 (HDAC3) in a mouse model of AD. In the present study, we further unveiled neuroprotective roles of miR-132-3p in transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice compared with those in age-matched wild-type C57BL/6 mice. Lentiviral-mediated inhibition or overexpression of miR-132-3p in the hippocampus of APP/PS1 mice was used to explore the contributions of hippocampal miR-132-3p in spatial memory, amyloid burden, apoptosis, and the number of hippocampal cells in a mouse model of AD. Overexpression of hippocampal miR-132-3p ameliorated spatial memory deficits in the Morris water maze, reduced both Aß1-42 accumulation and apoptosis, and promoted the numbers of hippocampal cells in the brains of APP/PS1 mice. Furthermore, trichostatin A (TSA) promoted the expression of miR-132-3p in Aß1-42-burdened neurons while increasing the expression levels of synaptic proteins. Taken together, our results suggest that miR-132-3p may represent a promising therapeutic target for the treatment of AD.

Unique Biomarker Characteristics in Gestational Diabetes Mellitus Identified by LC-MS-Based Metabolic Profiling.

BACKGROUND: Gestational diabetes mellitus (GDM) is a type of glucose intolerance disorder that first occurs during women's pregnancy. The main diagnostic method for GDM is based on the midpregnancy oral glucose tolerance test. The rise of metabolomics has expanded the opportunity to better identify early diagnostic biomarkers and explore possible pathogenesis. METHODS: We collected blood serum from 34 GDM patients and 34 normal controls for a LC-MS-based metabolomics study. RESULTS: 184 metabolites were increased and 86 metabolites were decreased in the positive ion mode, and 65 metabolites were increased and 71 were decreased in the negative ion mode. Also, it was found that the unsaturated fatty acid metabolism was disordered in GDM. Ten metabolites with the most significant differences were selected for follow-up studies. Since the diagnostic specificity and sensitivity of a single differential metabolite are not definitive, we combined these metabolites to prepare a ROC curve. We found a set of metabolite combination with the highest sensitivity and specificity, which included eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, arachidonic acid, citric acid, α-ketoglutaric acid, and genistein. The area under the curves (AUC) value of those metabolites was 0.984 between the GDM and control group. CONCLUSIONS: Our results provide a direction for the mechanism of GDM research and demonstrate the feasibility of developing a diagnostic test that can distinguish between GDM and normal controls clearly. Our findings were helpful to develop novel biomarkers for precision or personalized diagnosis for GDM. In addition, we provide a critical insight into the pathological and biological mechanisms for GDM.

Metabolic profile of women with premature ovarian insufficiency compared with that of age-matched healthy controls.

OBJECTIVE: . To compare the metabolic profile of women with spontaneous premature ovarian insufficiency (POI) with that of age-matched healthy controls. STUDY DESIGN: . A cross-sectional case-control study was conducted using 1:1 matching by age. Women below the age of 40 with spontaneous POI who did not receive any medication (n = 303) and age-matched healthy women (n = 303) were included in this study. MAIN OUTCOME MEASURES: . Metabolic profiles, including serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), glucose, uric acid, urea and creatinine, were compared between women with POI and controls. For women with POI, factors associated with the metabolic profile were analyzed. RESULTS: . Women with POI were more likely to exhibit increased serum levels of TG (ß, 0.155; 95% CI, 0.086, 0.223) and glucose (0.067; 0.052, 0.083), decreased levels of HDL-C (-0.087; -0.123, -0.051), LDL-C (-0.047; -0.091, -0.003) and uric acid (-0.053; -0.090, -0.015), and impaired kidney function (urea [0.070; 0.033, 0.107]; creatinine [0.277; 0.256, 0.299]; eGFR [-0.234; -0.252, -0.216]) compared with controls after adjusting for age and BMI. BMI, parity, gravidity, FSH and E2 levels were independent factors associated with the metabolic profile of women with POI. CONCLUSION: . Women with POI exhibited abnormalities in lipid metabolism, glucose metabolism, and a decrease in kidney function. In women with POI, early detection and lifelong management of metabolic abnormalities are needed.

MiR-30a-5p Regulates GLT-1 Function via a PKCα-Mediated Ubiquitin Degradation Pathway in a Mouse Model of Parkinson's Disease.

Glutamate excitotoxicity is caused by dysfunctional glutamate transporters and plays an important role in the pathogenesis of Parkinson's disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs(miRNA), which are abundant in astrocytes and neurons, have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. In this study, we hypothesized that the miR-30a-5p contributes to the pathogenesis of PD by regulating the ubiquitin-mediated degradation of glutamate transporter 1 (GLT-1). We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice). Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with 1-methyl-4-phenylpyridinium (MPP+) (cell model of PD). Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, we conclude that miR-30a-5p represents a potential therapeutic target for the treatment of PD.

Association of higher HbA1c within the normal range with adverse pregnancy outcomes: a cross-sectional study.

OBJECTIVE: The aim of this study was to investigate the association between a relatively high HbA1c level within the normal range and the risk of adverse pregnancy outcomes. METHODS: This retrospective cohort study was conducted between March 2018 and March 2019 at Women's Hospital, School of Medicine, Zhejiang University. Multiple logistic regression models after adjusting for plausible confounders were implemented to assess the relationships between the level of HbA1c and adverse pregnancy outcomes. RESULTS: A total of 8585 women were included in our study. The rates of preterm birth, macrosomia and preeclampsia were 4.4% (380/8585), 5.3% (457/8585) and 1.7% (149/8585), respectively. After adjusting for potential confounding variables, an HbA1c range of 5.5-5.9% (37-41 mmol/mol) remained significantly associated with an increased risk of preterm delivery (a-OR 2.27; 95% CI, 1.50-3.43), macrosomia (a-OR 1.97; 95% CI, 1.32-2.94) and preeclampsia (a-OR 3.70; 95% CI, 2.07-6.60). GDM-negative pregnant women with an HbA1c level in the range of 5.5-5.9% (37-41 mmol/mol) had an increased risk of preterm delivery (a-OR 2.84; 95% CI, 1.71-4.71) and preeclampsia (a-OR 3.82; 95% CI, 1.81-8.04). However, GDM-positive pregnant women had an increased risk of macrosomia (a-OR 2.12; 95% CI, 1.13-3.97) and preeclampsia (a-OR 2.62; 95% CI, 1.01-6.81). CONCLUSION: A higher HbA1c level within the normal range is an independent risk factor for preterm delivery and preeclampsia, especially among GDM-negative women. Therefore, relevant medical staff should enhance the awareness of risk and prevention to strengthen pregnancy monitoring.

Combination of metformin and cold atmospheric plasma induces glioma cell death to associate with c-Fos.

Glioma is the most common type of brain cancer. Chemotherapy combination with surgery and radiotherapy is a standard treatment for patients. Although there are many advances in glioma therapy, the prognosis of glioma patients has not significantly been improved over the past decades. Hence, there is still an urgent need to develop a new therapy to treat glioma. Cell viability was assessed by CellTiter Blue assay; flow cytometry (FCM) was used for detecting cell apoptosis; ROS detection was detected by ROS Assay; H2O2 detection was performed by hydrogen peroxide detection kits; real-time PCR and WB were used to determine gene expression. Using the glioma cell line U251 and U87, we investigated a possible combination inhibitory effect includes metformin and cold atmospheric plasma (CAP). The combination treatment showed a synergistic inhibitory effect on cell viability, significantly inducing cell apoptosis. Furthermore, we also found H2O2 produced by CAP has an important role in the synergistic inhibitory effect, eliminating H2O2 with catalase reversed the synergistic inhibitory effect. In addition, the transcript and protein levels of c-FOS were robustly increased after co-treated with metformin and CAP. Taken together, we propose that pre-treatment of glioma cells with metformin sensitize tumor cells to CAP, which may serve as a potential therapeutic strategy for glioma.

Ticagrelor prevents tumor metastasis via inhibiting cell proliferation and promoting platelet apoptosis.

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. Platelets inhibition or blocking platelet-tumor cell interactions has become a strategy to suppress tumor progression. In this study, we investigated the effect of ticagrelor, a new antiplatelet drug, on tumor cell proliferation and metastasis. Our results show that ticagrelor not only inhibits the proliferation, migration, and invasion of B16F10 and Lewis lung carcinoma cells but also induces platelet apoptosis. In addition, we find that apoptosis of the platelet cells is dose dependent. Further, the result of in-vivo experiments proved that ticagrelor treatment decreased the tumor metastasis. The results of this study demonstrate that ticagrelor may be a potential anti-tumor agent for tumor metastasis.

Generation of a Novel Mouse Model of Parkinson's Disease via Targeted Knockdown of Glutamate Transporter GLT-1 in the Substantia Nigra.

Parkinson's disease (PD) is a common neurodegenerative disease that is characterized by pathological dopaminergic (DA) neuronal death and α-synuclein aggregation. Glutamate excitotoxicity is a well-established pathogenesis of PD that involves dysfunctional expression of glutamate transporters. Glutamate transporter-1 (GLT-1) is mainly responsible for clearance of glutamate at synapses, including DA synapses. However, the role of GLT-1 in the aberrant synaptic transmission in PD remains elusive. In the present study, we generated small-interfering RNAs (siRNAs) to knockdown GLT-1 expression in primary astrocytes, and we report that siRNA knockdown of astrocytic GLT-1 decreased postsynaptic density-95 (PSD-95) expression in neuron-astrocyte cocultures in vitro. Using adeno-associated viruses (AAVs) targeting GLT-1 short-hairpin RNA (shRNA) sequences with a glial fibrillary acidic protein (GFAP) promoter, we abolished astrocytic GLT-1 expression in the substantia nigra pars compacta (SNpc) of mice. We found that GLT-1 deficiency in the SNpc induced parkinsonian phenotypes in terms of progressive motor deficits and nigral DA neuronal death in mice. We also found that there were reactive astrocytes and microglia in the SNpc upon GLT-1 knockdown. Furthermore, we used RNA sequencing to determine altered gene expression patterns upon GLT-1 knockdown in the SNpc, which revealed that disrupted calcium signaling pathways may be responsible for GLT-1 deficiency-mediated DA neuronal death in the SNpc. Taken together, our findings provide evidence for a novel role of GLT-1 in parkinsonian phenotypes in mice, which may contribute to further elucidation of the mechanisms of PD pathogenesis.

Environmental enrichment prevents Aß oligomer-induced synaptic dysfunction through mirna-132 and hdac3 signaling pathways.

As the most common cause of progressive cognitive decline in humans, Alzheimer's disease (AD) has been intensively studied, but the mechanisms underlying its profound synaptic dysfunction remain unclear. Here we confirm that exposing wild-type mice to an enriched environment (EE) facilitates signaling in the hippocampus that promotes long-term potentiation (LTP). Exposing the hippocampus of mice kept in standard housing to soluble Aß oligomers impairs LTP, but EE can fully prevent this. Mechanistically, the key molecular features of the EE benefit are an upregulation of miRNA-132 and an inhibition of histone deacetylase (HDAC) signaling. Specifically, soluble Aß oligomers decreased miR-132 expression and increased HDAC3 levels in cultured primary neurons. Further, we provide evidence that HDAC3 is a direct target of miR-132. Overexpressing miR-132 or injecting an HDAC3 inhibitor into mice in standard housing mimics the benefits of EE in enhancing hippocampal LTP and preventing hippocampal impairment by Aß oligomers in vivo. We conclude that EE enhances hippocampal synaptic plasticity by upregulating miRNA-132 and reducing HDAC3 signaling in a way that counteracts the synaptotoxicity of human Aß oligomers. Our findings provide a rationale for prolonged exposure to cognitive novelty and/or epigenetic modulation to lessen the progressive effects of Aß accumulation during human brain aging.

Ginsenoside Rb1 prevents MPTP-induced changes in hippocampal memory via regulation of the α-synuclein/PSD-95 pathway.

Memory deficiency is a common non-motor symptom of Parkinson's disease (PD), and conventionally, α-synuclein is considered to be an important biomarker for both motor and cognitive characteristics attributed to PD. However, the role of physiological α-synuclein in cognitive impairment remains undetermined. Ginsenoside Rb1 has been shown to protect dopaminergic neurons (DA) from death and inhibit α-synuclein fibrillation and toxicity in vitro. Our recent study also revealed that ginsenoside Rb1 ameliorates motor deficits and prevents DA neuron death via upregulating glutamate transporter GLT-1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Whether Rb1 can improve memory deficiency and the underlying mechanism is still unknown. In this study, we found that Rb1 can prevent the spatial learning and memory deficits, increase long-term potentiation (LTP) and hippocampal glutamatergic transmission in the MPTP mouse model. The underlying neuroprotective mechanism of Rb1-improved synaptic plasticity involves Rb1 promoting hippocampal CA3 α-synuclein expression, restoring the glutamate in the CA3-schaffer collateral-CA1 pathway, and sequentially increasing postsynaptic density-95 (PSD-95) expression. Thus, we provide evidence that Rb1 modulates memory function, synaptic plasticity, and excitatory transmission via the trans-synaptic α-synuclein/PSD-95 pathway. Our findings suggest that Rb1 may serve as a functional drug in treating the memory deficiency in PD.

Regulatory Mechanism of miR-543-3p on GLT-1 in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) features the degeneration and death of dopamine neurons in the substantia nigra pars compacta and the formation of Lewy bodies that contain α-synuclein. Among the numerous PD etiologies, glutamate excitotoxicity is a research hot spot, and glutamate transporters play key roles in this theory. It has been shown that the expression of the glutamate transporter is regulated by microRNAs. In this study, we found that the levels of expression and function of glutamate transporter type 1 (GLT-1) were significantly reduced and miR-543-3p was upregulated during the development of PD. Furthermore, our results indicated that GLT-1 plays an important role in the pathomechanism of PD. We found that miR-543-3p can suppress the expression and function of GLT-1 in MPP+-treated astrocytes and MPTP-treated mice. Inhibition of miR-543-3p can rescue the expression and function of GLT-1 and relieve dyskinesia in the PD model, which suggests that inhibition of miR-543-3p could serve as a potential therapeutic target for PD.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Simvastatin Inhibits Activation of NADPH Oxidase/p38 MAPK Pathway and Enhances Expression of Antioxidant Protein in Parkinson Disease Models.

Evidence suggests that oxidative stress is involved in the pathogenesis of Parkinson disease (PD). Simvastatin has been suggested to protect against oxidative stress in several diseases. However, the molecular mechanisms by which simvastatin protects against neuropathology and oxidative damage in PD are poorly elucidated. In this study, we aimed to investigate the potential neuroprotective effects of simvastatin owing to its anti-oxidative properties in 6-hydroxydopamine (6-OHDA)-treated SH-SY5Y cells and mice. The results of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence and CCK-8 assay demonstrated that simvastatin reduced intracellular reactive oxygen species (ROS) levels and reversed apoptosis in 6-OHDA-treated SH-SY5Y cells. Mechanistic studies revealed that 6-OHDA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/p38 mitogen-activated protein kinase (MAPK) pathway was inhibited and nuclear factor-κB (NF-κB) nuclear transcription decreased in SH-SY5Y cells after simvastatin treatment. Enhanced expression levels of superoxide dismutase (SOD), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and glutamate-cysteine ligase modifier subunit (GCLM) were observed after simvastatin treatment in 6-OHDA-treated SH-SY5Y cells. In vivo studies revealed that administration of simvastatin by gavage decreased limb-use asymmetry and apomorphine-induced rotations in 6-OHDA-lesioned mice. Simvastatin increased dopaminergic neurons and reduced protein tyrosine nitration and gliosis in the midbrain of PD mice. An inhibitory effect on activation of the NADPH oxidase/p38 MAPK was observed, and increased antioxidant protein expression in the midbrain were seen in the simvastatin plus 6-OHDA group compared with the 6-OHDA-lesioned group. Taken together, these results demonstrate that simvastatin might inhibit the activation of NADPH oxidase/p38 MAPK pathway, enhance antioxidant protein expression and protect against oxidative stress, thereby providing a novel antioxidant mechanism that has therapeutic validity.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Amyloid-beta peptide decreases expression and function of glutamate transporters in nervous system cells.

Glutamate is an essential excitatory neurotransmitter that regulates brain functions, and its activity is tightly regulated by glutamate transporters. Excess glutamate in the synaptic cleft and dysfunction of excitatory amino acid transporters have been shown to be involved in development of Alzheimer's disease, but the precise regulatory mechanism is poorly understood. Using a D-[3H]-aspartic acid uptake assay, we found that Aß1-42 oligomers impaired glutamate uptake in astrocytes and neurons. In astrocytes, this process was accompanied by reduced expression of GLT-1 and GLAST as detected by Western blot and immunocytofluorescence. However, mRNA levels of EAATs detected by qPCR in astrocytes and neurons were not altered, which suggests that this process is post-translational. Co-localization analysis using immunocytofluorescence showed that ubiquitylation of GLT-1 significantly increased. Therefore, we hypothesized that Aß1-42 oligomers-induced endocytosis of astrocytic GLT-1 may be involved in ubiquitylation. In addition, Aß1-42 oligomers enhanced secretion of IL-1ß, TNF-α, and IL-6 into culture supernatant, which may be correlated with an inflammatory response and altered EAATs expression or function in Alzheimer's disease. These findings support the idea that dysregulation of the glutamatergic system may play a significant role in pathogenesis of Alzheimer's disease. Furthermore, enhancing expression or function of EAATs in astrocytes and neurons might be a new therapeutic approach in treatment of Alzheimer's disease.