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1.  Quantitative proteomic analysis of age-related subventricular zone proteins associated with neurodegenerative disease 
Scientific Reports  2016;6:37443.
Aging is characterized by a progressive decline in the function of adult tissues which can lead to neurodegenerative disorders. However, little is known about the correlation between protein changes in the subventricular zone (SVZ) and neurodegenerative diseases with age. In the present study, neural stem cells (NSCs) were derived from the SVZ on postnatal 7 d, 1 m, and 12 m-old mice. With age, NSCs exhibited increased SA-β-gal activity and decreased proliferation and pool size in the SVZ zone, and were associated with elevated inflammatory chemokines and cytokines. Furthermore, quantitative proteomics and ingenuity pathway analysis were used to evaluate the significant age-related alterations in proteins and their functions. Some downregulated proteins such as DPYSL2, TPI1, ALDH, and UCHL1 were found to play critical roles in the neurological disease and PSMA1, PSMA3, PSMC2, PSMD11, and UCHL1 in protein homeostasis. Taken together, we have provided valuable insight into the cellular and molecular processes that underlie aging-associated declines in SVZ neurogenesis for the early detection of differences in gene expression and the potential risk of neurological disease, which is beneficial in the prevention of the diseases.
doi:10.1038/srep37443
PMCID: PMC5114652  PMID: 27857231
2.  α-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals 
The Journal of Neuroscience  2016;36(16):4408-4414.
α-Synuclein (α-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, α-syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how α-syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human α-syn A53T (h-α-synA53T) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h-α-synA53T, either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h-α-synA53T in calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how α-syn mutations impair neurotransmission before neurodegeneration.
SIGNIFICANCE STATEMENT α-Synuclein (α-syn) missense or multiplication mutations may cause neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. The initial impact of α-syn mutations before neuronal loss is impairment of exocytosis, which may contribute to eventual neurodegeneration. The mechanism underlying impairment of exocytosis is poorly understood. Here we report that an α-syn mutant, the human α-syn A53T, inhibited two of the most commonly observed forms of endocytosis, slow and rapid endocytosis, at a mammalian central synapse. We also found that α-syn A53T inhibited vesicle replenishment to the readily releasable pool. These results may contribute to accounting for the widely observed early synaptic impairment caused by α-syn mutations in the progression toward neurodegeneration.
doi:10.1523/JNEUROSCI.3627-15.2016
PMCID: PMC4837680  PMID: 27098685
α-synuclein; endocytosis; nerve terminal; Parkinson's disease; transmitter; vesicle
3.  Tumor endothelial cells promote metastasis and cancer stem cell-like phenotype through elevated Epiregulin in esophageal cancer 
American Journal of Cancer Research  2016;6(10):2277-2288.
Tumor endothelial cells have been found to be associated with metastasis and cancer progression. In this study, we reported that human esophageal cancer endothelial cells (HECEC), unlike corresponding human esophageal normal endothelial cells (HENEC) displayed several distinct feature couple with unique gene expression profile. Further studies showed that HECEC can enhance migration, invasion and self-renewal properties of esophageal carcinoma cell in vitro by a direct cell-cell interaction. In vivo assay demonstrated that HECEC could significantly enhance the invasion and lung metastasis of esophageal cancer cells. To elucidate the molecular mechanisms of HECEC in esophageal carcinoma progression, we employed the microarray to analyze the gene expression profiles before and after treating with HECEC, HENEC or conditioned meium from HECEC. Among the highly expressed HECEC-regulated genes, we focused on Epiregulin (EREG). Further studies demonstrated that overexpression of EREG in EC9706 or Kyse30 cells can induce actin reorganization, sphere formation ability and a significantly enrichment of CD44+ cancer stem-like cells. Moreover, up-regulation of EREG in esophageal cancer cells could enhance lung metastasis and decrease the survival time in vivo. Further study indicated that EREG could induce activation of the Src and FAK. In addition, all these effects could also be inhibited by the function-blocking anti-EREG antibody in a dose dependent manner. Immunohistochemical analysis revealed that high level of EREG was significantly correlated with lymph node metastases and poor prognosis. In summary, HECEC play key roles in enhancing the invasion, migration, cancer stem cell phenotype and metastatic potential of esophageal cancer cells through Epiregulin.
PMCID: PMC5088291  PMID: 27822417
Esophageal cancer; EREG; cancer stem cell; CD44; tumor endothelial cells; metastasis
4.  Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression 
Human Molecular Genetics  2015;24(18):5299-5312.
Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 (LRRK2) G2019S missense mutation (LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release.
doi:10.1093/hmg/ddv249
PMCID: PMC4550828  PMID: 26123485
5.  Fast-track surgery improves postoperative clinical recovery and cellular and humoral immunity after esophagectomy for esophageal cancer 
BMC Cancer  2016;16:449.
Background
Our aim was to investigate the influence of FTS on human cellular and humoral immunity using a randomized controlled clinical study in esophageal cancer patients.
Methods
Between October 2013 and December 2014, 276 patients with esophageal cancer in our department were enrolled in the study. The patients were randomized into two groups: FTS pathway group and conventional pathway group. The postoperative hospital stay, hospitalization expenditure, and postoperative complications were recorded. The markers of inflammatory and immune function were measured before operation as well as on the 1st, 3rd, and 7th postoperative days (POD), including serum level of interleukin-6 (IL-6), C-reactive protein (CRP), serum globulin, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) and lymphocyte subpopulations (CD3 lymphocytes, CD4 lymphocytes, CD8 lymphocytes and the CD4/CD8 ratio) in the patients between the two groups.
Results
In all, 260 patients completed the study: 128 in the FTS group and 132 in the conventional group. We found implementation of FTS pathway decreases postoperative length of stay and hospital charges (P < 0.05). In addition, inflammatory reactions, based on IL-6 and CRP levels, were less intense following FTS pathway compared to conventional pathway on POD1 and POD3 (P < 0.05). On POD1 and POD3, the levels of IgG, IgA, CD3 lymphocytes, CD4 lymphocytes and the CD4/CD8 ratio in FTS group were significantly higher than those in control group (All P < 0.05). However, there were no differences in the level of IgM and CD8 lymphocytes between the two groups.
Conclusions
FTS improves postoperative clinical recovery and effectively inhibited release of inflammatory factors via the immune system after esophagectomy for esophageal cancer.
Trial registration
ChiCTR-TRC-13003562, the date of registration: August 29, 2013.
doi:10.1186/s12885-016-2506-8
PMCID: PMC4940721  PMID: 27401305
Esophageal cancer; Fast-track surgery; Cellular immunity; Humoral immunity
6.  Effect of Adoptive Transfer or Depletion of Regulatory T Cells on Triptolide-induced Liver Injury 
Objective: The aim of this study is to clarify the role of regulatory T cell (Treg) in triptolide (TP)-induced hepatotoxicity.
Methods: Female C57BL/6 mice received either adoptive transfer of Tregs or depletion of Tregs, then underwent TP administration and were sacrificed 24 h after TP administration. Liver injury was determined according to alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum and histopathological change in liver tissue. Hepatic frequencies of Treg cells and the mRNA expression levels of transcription factor Forkhead box P3 and retinoid orphan nuclear receptor γt (RORγt), interleukin-10 (IL-10), suppressor of cytokine signaling (SOCS), and Notch/Notch ligand were investigated.
Results: During TP-induced liver injury, hepatic Treg and IL-10 decreased, while T helper 17 cells cell-transcription factor RORγt, SOCS and Notch signaling increased, accompanied with liver inflammation. Adoptive transfer of Tregs ameliorated the severity of TP-induced liver injury, accompanied with increased levels of hepatic Treg and IL-10. Adoptive transfer of Tregs remarkably inhibited the expression of RORγt, SOCS3, Notch1, and Notch3. On the contrary, depletion of Treg cells in TP-administered mice resulted in a notable increase of RORγt, SOCS1, SOCS3, and Notch3, while the Treg and IL-10 of liver decreased. Consistent with the exacerbation of liver injury, higher serum levels of ALT and AST were detected in Treg-depleted mice.
Conclusion: These results showed that adoptive transfer or depletion of Tregs attenuated or aggravated TP-induced liver injury, suggesting that Tregs could play important roles in the progression of liver injury. SOCS proteins and Notch signaling affected Tregs, which may contribute to the pathogenesis of TP-induced hepatotoxicity.
doi:10.3389/fphar.2016.00099
PMCID: PMC4840269  PMID: 27148057
regulatory T cell (Treg); triptolide; interleukin-10; hepatotoxicity; adoptive transfer of Tregs; depletion of Tregs; suppressor of cytokine signaling (SOCS); notch signaling
7.  Sex-Related Differences of Lipid Metabolism Induced by Triptolide: The Possible Role of the LXRα/SREBP-1 Signaling Pathway 
Triptolide, a diterpenoid isolated from the plant Tripterygium wilfordii Hook. f., exerts a unique bioactive spectrum of anti-inflammatory and anticancer activities. However, triptolide’s clinical applications are limited due to its severe toxicities. Fatty liver toxicity occurs in response to triptolide, and this toxic response significantly differs between males and females. This report investigated the pathogenesis underlying the sex-related differences in the dyslipidosis induced by triptolide in rats. Wistar rats were administered 0, 150, 300, or 450 μg triptolide/kg/day by gavage for 28 days. Ultrastructural examination revealed that more lipid droplets were present in female triptolide-treated rats than in male triptolide-treated rats. Furthermore, liver triglyceride, total bile acid and free fatty acid levels were significantly increased in female rats in the 300 and 450 μg/kg dose groups. The expression of liver X receptor α (LXRα) and its target genes, cholesterol 7α-hydroxylase (CYP7A1) and Sterol regulatory element-binding transcription factor 1(SREBP-1), increased following triptolide treatment in both male and female rats; however, the female rats were more sensitive to triptolide than the male rats. In addition, the expression of acetyl-CoA carboxylase 1(ACC1), a target gene of SREBP-1, increased in the female rats treated with 450 μg triptolide/kg/day, and ACC1 expression contributed to the sex-related differences in the triptolide-induced dysfunction of lipid metabolism. Our results demonstrate that the sex-related differences in LXR/SREBP-1-mediated regulation of gene expression in rats are responsible for the sex-related differences in lipid metabolism induced by triptolide, which likely underlie the sex-related differences in triptolide hepatotoxicity. This study will be important for predicting sex-related effects on the pharmacokinetics and toxicity of triptolide and for improving its safety.
doi:10.3389/fphar.2016.00087
PMCID: PMC4814849  PMID: 27065871
triptolide; hepatotoxicity; lipid metabolism; liver X receptor α; Sterol regulatory element-binding transcription factor 1
8.  Toll-Like Receptors Promote Mitochondrial Translocation of Nuclear Transcription Factor Nuclear Factor of Activated T-Cells in Prolonged Microglial Activation 
The Journal of Neuroscience  2015;35(30):10799-10814.
Microglia are resident macrophages in the CNS that scavenge pathogens, dying cells, and molecules using pattern recognition Toll-like receptors (TLRs). Nuclear factor of activated T-cells (NFAT) family transcription factors also regulate inflammatory responses in microglia. However, whether there exists cross talk between TLR and NFAT signaling is unclear. Here we show that chronic activation of murine microglia by prolonged stimulation of Toll-like receptor 4 (TLR4) ligand lipopolysaccharides (LPSs) leads to unexpected translocation of NFAT1 into mitochondria. This mitochondrial import of NFAT1 is independent of calcium/calcineurin signaling. Instead, inhibition of Toll/interleukin 1 receptor domain-containing adapter-inducing interferon-β (TRIF) pathway blocks the mitochondrial translocation of NFAT1. Functionally, inhibition of NFAT1 reduces the TRIF-mediated expression of interferon-β and compromises the production of ATP and reactive oxygen species in LPS-treated microglia. Therefore, our findings reveal a new inflammatory signaling pathway that links TLR with NFAT in regulating cytokine production and mitochondrial activity during chronic microglial activation.
SIGNIFICANCE STATEMENT Nuclear factor of activated T-cells (NFAT) family transcription factors are known to undergo nuclear translocation in response to inflammatory stimulation. In this study, we uncovered a surprise transportation of NFATs into mitochondria in microglia after a prolonged treatment with bacteria endotoxin lipopolysaccharides (LPSs). LPSs activated Toll-like receptor 4 and its downstream Toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF) to regulate the mitochondrial translocation of NFAT in microglia, whereas genetic inhibition of NFAT1 compromised TRIF-mediated cytokine production and reduced ATP and reactive oxygen species generation. These findings reveal a previously undescribed mitochondrial translocation of NFAT in microglia responding to extended activation of Toll-like receptor-mediated signaling transduction pathways.
doi:10.1523/JNEUROSCI.2455-14.2015
PMCID: PMC4518054  PMID: 26224862
microglia; mitochondria; neuroinflammation; NFAT; Toll-like receptor
9.  A calcineurin- and NFAT-dependent pathway is involved in α-synuclein-induced degeneration of midbrain dopaminergic neurons 
Human Molecular Genetics  2014;23(24):6567-6574.
Parkinson's disease (PD), the most common degenerative movement disorder, is caused by a preferential loss of midbrain dopaminergic (mDA) neurons. Both α-synuclein (α-syn) missense and multiplication mutations have been linked to PD. However, the underlying intracellular signalling transduction pathways of α-syn-mediated mDA neurodegeneration remain elusive. Here, we show that transgenic expression of PD-related human α-syn A53T missense mutation promoted calcineurin (CN) activity and the subsequent nuclear translocation of nuclear factor of activated T cells (NFATs) in mDA neurons. α-syn enhanced the phosphatase activity of CN in both cell-free assays and cell lines transfected with either human wild-type or A53T α-syn. Furthermore, overexpression of α-syn A53T mutation significantly increased the CN-dependent nuclear import of NFATc3 in the mDA neurons of transgenic mice. More importantly, a pharmacological inhibition of CN by cyclosporine A (CsA) ameliorated the α-syn-induced loss of mDA neurons. These findings demonstrate an active involvement of CN- and NFAT-mediated signalling pathway in α-syn-mediated degeneration of mDA neurons in PD.
doi:10.1093/hmg/ddu377
PMCID: PMC4240205  PMID: 25051958
10.  No apparent transmission of transgenic α–synuclein into nigrostriatal dopaminergic neurons in multiple mouse models 
Background
α–synuclein (α–syn) is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson’s disease (PD) and certain other neurodegenerative disorders. Recent studies have explored the ability of α–syn to propagate between or across neighboring neurons and supposedly “infect” them with a prion–like mechanism. However, much of this research has used stereotaxic injections of heterologous α–syn fibrils to induce the spreading of inclusions in the rodent brains. Whether α–syn is able to transmit from the host cells to their neighboring cells in vivo is unclear.
Methods
Using immunestaining, we examined the potential propagation of α–syn into nigrostriatal dopaminergic (DA) neurons in three lines of transgenic mice that overexpress human wild–type α–syn (hα–syn) in different neuron populations.
Results
After testing for three different routes by which hα–syn propagation might occur, we were unable to find any evidence that hα–syn behaved like a prion and could be transmitted overtime into the DA neurons initially lack of hα–syn expression.
Conclusions
In transgenic mice hα–syn does not have the ability to propagate at pathologically significant levels between or across neurons. It must be noted that these observations do not disprove the studies that show its prion–like qualities, but rather that propagation is not detectable in transgenic models that do not use any injections of heterologous proteins or viral vectors to induce a spreading state.
doi:10.1186/s40035-015-0046-9
PMCID: PMC4668690  PMID: 26635953
Parkinson’s disease; α-synuclein; Propagation; Dopaminergic neurons; Transgenic mice
11.  HIV Protease Inhibitors Induce Endoplasmic Reticulum Stress and Disrupt Barrier Integrity in Intestinal Epithelial Cells 
Gastroenterology  2009;138(1):197-209.
BACKGROUND & AIMS
Human immunodeficiency virus (HIV) protease inhibitor (PI)-induced adverse effects have become a serious clinical problem. In addition to their metabolic and cardiovascular complications, these drugs also frequently cause severe gastrointestinal disorders, including mucosal erosions, epithelial barrier dysfunction, and diarrhea. However, the exact mechanisms underlying gastrointestinal adverse effects of HIV PIs remain unknown. This study investigated whether HIV PIs disrupt intestinal epithelial barrier integrity by activating endoplasmic reticulum (ER) stress.
METHODS
The most commonly used HIV PIs (lopinavir, ritonavir, and amprenavir) were used; their effects on ER stress activation and epithelial paracellular permeability were examined in vitro as well as in vivo using wild-type and CHOP−/− mice.
RESULTS
Treatment with lopinavir and ritonavir, but not amprenavir, induced ER stress, as indicated by a decrease in secreted alkaline phosphatase activities and an increase in the unfolded protein response. This activated ER stress partially impaired the epithelial barrier integrity by promoting intestinal epithelial cell apoptosis. CHOP silencing by specific small hairpin RNA prevented lopinavir-and ritonavir-induced barrier dysfunction in cultured intestinal epithelial cells, whereas CHOP−/− mice exhibited decreased mucosal injury after exposure to lopinavir and ritonavir.
CONCLUSIONS
HIV PIs induce ER stress and activate the unfolded protein response in intestinal epithelial cells, thus resulting in disruption of the epithelial barrier integrity.
doi:10.1053/j.gastro.2009.08.054
PMCID: PMC4644065  PMID: 19732776
12.  Longitudinal Metabolomics Profiling of Parkinson’s Disease-Related α-Synuclein A53T Transgenic Mice 
PLoS ONE  2015;10(8):e0136612.
Metabolic homeostasis is critical for all biological processes in the brain. The metabolites are considered the best indicators of cell states and their rapid fluxes are extremely sensitive to cellular changes. While there are a few studies on the metabolomics of Parkinson’s disease, it lacks longitudinal studies of the brain metabolic pathways affected by aging and the disease. Using ultra-high performance liquid chromatography and tandem mass spectroscopy (UPLC/MS), we generated the metabolomics profiling data from the brains of young and aged male PD-related α-synuclein A53T transgenic mice as well as the age- and gender-matched non-transgenic (nTg) controls. Principal component and unsupervised hierarchical clustering analyses identified distinctive metabolites influenced by aging and the A53T mutation. The following metabolite set enrichment classification revealed the alanine metabolism, redox and acetyl-CoA biosynthesis pathways were substantially disturbed in the aged mouse brains regardless of the genotypes, suggesting that aging plays a more prominent role in the alterations of brain metabolism. Further examination showed that the interaction effect of aging and genotype only disturbed the guanosine levels. The young A53T mice exhibited lower levels of guanosine compared to the age-matched nTg controls. The guanosine levels remained constant between the young and aged nTg mice, whereas the aged A53T mice showed substantially increased guanosine levels compared to the young mutant ones. In light of the neuroprotective function of guanosine, our findings suggest that the increase of guanosine metabolism in aged A53T mice likely represents a protective mechanism against neurodegeneration, while monitoring guanosine levels could be applicable to the early diagnosis of the disease.
doi:10.1371/journal.pone.0136612
PMCID: PMC4552665  PMID: 26317866
13.  A canine model of osteonecrosis of the femoral head induced by MRI guided argon helium cryotherapy system 
Objective: This study is to identify the reliability of osteonecrosis of the femoral head (ONFH) modeling established by MRI guided argon helium cryotherapy system in beagles. Methods: A total of 15 beagles were used to establish the ONFH model. The left femoral heads of the beagles received two cycles of argon helium freezing-thawing under MRI guidance and were considered as experimental group while the right femoral heads received only one cycle of argon helium freezing-thawing and were considered as the control group. X-ray, MRI, general shape and histological examinations were performed so as to identify the effect of modeling. Results: At 4 week after modeling, MRI showed obvious bilateral hip joint effusion and marked femoral head bone marrow high signal. At 8 week after surgery, abnormal signal appeared in bilateral femoral heads. T1WI showed irregular patchy low signal, T2WI showed irregular mixed signals and the joint capsule effusion showed long T1 and T2 changes. Twelve weeks after operation, T1WI showed a low signal strip with clear boundary and T2WI showed intermediate signal. The changes of the left femoral heads were significant while compared with those of the right sides. The lacunae rates of femoral heads in the experimental group at 4, 8, and 12 week after surgery (40.75 ± 3.77, 57.46 ± 4.01, 50.27 ± 2.98) were higher than those in control group (30.08 ± 3.61, 49.43 ± 2.82, 40.56 ± 2.73). Conclusion: Canine model of ONFH was successfully established using an argon helium cryotherapy system.
PMCID: PMC4612890  PMID: 26550205
Osteonecrosis; femoral head; magnetic resonance imaging; cryotherapy; canine model
15.  Release Behaviour of Single Pellets and Internal Fine 3D Structural Features Co-define the In Vitro Drug Release Profile 
The AAPS Journal  2014;16(4):860-871.
Multi-pellet formulations are advantageous for the controlled release of drugs over single-unit dosage forms. To understand the diffusion controlled drug release mechanism, the pellet structure and drug release from a single pellet (not at dose level) were studied using synchrotron radiation X-ray computed microtomography (SR-μCT) and a sensitive LC/MS/MS method. The purpose of this article is to introduce a powerful, non-invasive and quantitative technique for studying individual pellet microstructures and to investigate the relationship between the microstructure and drug release from single pellets. The data from the single pellet dissolution measurements demonstrated that the release profile of capsules containing approximately 1,000 pellets per unit dose was the summation of the release profiles of the individual pellets. The release profiles of single tamsulosin hydrochloride (TSH) pellets formed three groups when a cluster analysis was performed, and the dissolution rate of the individual pellets correlated well with the combined effects of the drug loading, volume and surface area of the pellets (R2 = 0.9429). In addition, the void microstructures within the pellet were critical during drug release. Therefore, SR-μCT is a powerful tool for quantitatively elucidating the three-dimensional microstructure of the individual pellets; because the microstructure controls drug release, it is an important parameter in the quality control of multi-pellet formulations.
doi:10.1208/s12248-014-9611-x
PMCID: PMC4070264  PMID: 24875050
microstructure; release kinetics; single pellet; synchrotron radiation X-ray computed microtomography
16.  Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells: an exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics 
Acta Pharmaceutica Sinica. B  2015;5(3):223-230.
Nucleotide pools in mammalian cells change due to the influence of antitumor drugs, which may help in evaluating the drug effect and understanding the mechanism of drug action. In this study, an ion-pair RP-HPLC method was used for a simple, sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs (daunorubicin, epirubicin and dactinomycin D). Through the use of this targeted metabolomics approach to find potential biomarkers, UTP and ATP were verified to be the most appropriate biomarkers. Moreover, a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action. This model can be further validated by evaluating drugs with different mechanisms of action. This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs.
Graphical abstract
A RP-HPLC method was used for determination 12 nucleotides in cells treated with antibiotic antitumor drugs. Four categories of drugs with different mechanisms of action were investigated by principal component analysis to develop a recognition model to predict a possible mechanism of an antitumor-compound action.
doi:10.1016/j.apsb.2015.03.010
PMCID: PMC4629260  PMID: 26579450
ADP, adenosine diphosphate; AMP, adenosine monophosphate; ANOVA, analysis of variance; ATP, adenosine triphosphate; AUC, area under the curve; CDP, cytidine diphosphate; CTP, cytidine triphosphate; dATP, deoxyadenosine triphosphate; dCDP, deoxycytidine diphosphate; dCTP, deoxycytidine triphosphate; dGMP, deoxyribonucleic monophosphate; dGTP, deoxyguanosine triphosphate; DMEM, Dulbecco׳s modified eagle׳s cell culture media; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; dUDP, deoxyuridine diphpsphate; dUTP, deoxyuridine triphosphate; EC, energy charge; EDTA, ethylene diamine tetra-acetic acid; FCS, fetal calf serum; GDP, guanosine diphosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PCA, principal component analysis; RNA, ribonucleic acid; ROC, receiver operating characteristic; RPMI-1640, Roswell Park Memorial Institute-1640; TBAHS, tetrabutylammonium hydrogen sulfate; TCA, trichloroacetic acid; UDP, uridine diphosphate; UTP, uridine triphosphate; Nucleotides; Targeted metabolomics analysis; Tumor cells; Potential biomarkers; Mechanisms of antitumor drug action; Antibiotic anticancer drugs; Principal component analysis; Ion-pair HPLC
17.  Aldehyde Dehydrogenase 1 making molecular inroads into the differential vulnerability of nigrostriatal dopaminergic neuron subtypes in Parkinson’s disease 
A preferential dysfunction/loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) accounts for the main motor symptoms of Parkinson’s disease (PD), the most common degenerative movement disorder. However, the neuronal loss is not stochastic, but rather displays regionally selectivity, indicating the existence of different DA subpopulations in the SNpc. To identify the underlying molecular determinants is thereby instrumental in understanding the pathophysiological mechanisms of PD-related neuron dysfunction/loss and offering new therapeutic targets. Recently, we have demonstrated that aldehyde dehydrogenase 1 (ALDH1A1) is one such molecular determinant that defines and protects an SNpc DA neuron subpopulation preferentially affected in PD. In this review, we provide further analysis and discussion on the roles of ALDH1A1 in the function and survival of SNpc DA neurons in both rodent and human brains. We also explore the feasibility of ALDH1A1 as a potential biomarker and therapeutic target for PD.
doi:10.1186/2047-9158-3-27
PMCID: PMC4334846  PMID: 25705376
Parkinson’s disease; Substantia nigra pars compacta; Dopaminergic neuron; Aldehyde dehydrogenase 1; α-synuclein; Neurodegeneration; Aging
18.  Amyotrophic lateral sclerosis-related VAPB P56S mutation differentially affects the function and survival of corticospinal and spinal motor neurons 
Human Molecular Genetics  2013;22(21):4293-4305.
The substitution of Proline with Serine at residue 56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) has been linked to an atypical autosomal dominant form of familial amyotrophic lateral sclerosis 8 (ALS8). To investigate the pathogenic mechanism of P56S VAPB in ALS, we generated transgenic (Tg) mice that heterologously express human wild-type (WT) and P56S VAPB under the control of a pan-neuronal promoter Thy1.2. While WT VAPB Tg mice did not exhibit any overt motor behavioral phenotypes, P56S VAPB Tg mice developed progressive hyperactivities and other motor abnormalities. VAPB protein was accumulated as large punctate in the soma and proximal dendrites of both corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs) in P56S VAPB Tg mice. Concomitantly, a significant increase of endoplasmic reticulum stress and unfolded protein response and the resulting up-regulation of pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein expression were observed in the CSMNs and SMNs of P56S VAPB Tg mice. However, only a progressive loss of CSMNs but not SMNs was found in P56S VAPB Tg mice. In SMNs, P56S VAPB promoted a rather selective translocation of VAPB protein onto the postsynaptic site of C-boutons that altered the morphology of C-boutons and impaired the spontaneous rhythmic discharges of SMNs. Therefore, these findings provide new pathophysiological mechanisms of P56S VAPB that differentially affect the function and survival of CSMNs and SMNs in ALS8.
doi:10.1093/hmg/ddt279
PMCID: PMC3792689  PMID: 23771029
19.  Optogenetic Measurement of Presynaptic Calcium Transients Using Conditional Genetically Encoded Calcium Indicator Expression in Dopaminergic Neurons 
PLoS ONE  2014;9(10):e111749.
Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.
doi:10.1371/journal.pone.0111749
PMCID: PMC4216119  PMID: 25360513
20.  LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity 
Nature neuroscience  2014;17(3):367-376.
Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). Here we show that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIβ (PKARIIβ). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged LRRK2-null mice after treatment with a Drd1 agonist. Notably, a Parkinson’s disease-related LRRK2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIβ also induced excessive PKA activity in the SPNs. Our findings reveal a new regulatory role of LRRK2 in PKA signaling, and provide a new pathogenic mechanism of SPN dysfunction in Parkinson’s disease.
doi:10.1038/nn.3636
PMCID: PMC3989289  PMID: 24464040
21.  Aldehyde dehydrogenase 1 defines and protects a nigrostriatal dopaminergic neuron subpopulation 
The Journal of Clinical Investigation  2014;124(7):3032-3046.
Subpopulations of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) display a differential vulnerability to loss in Parkinson’s disease (PD); however, it is not clear why these subsets are preferentially selected in PD-associated neurodegeneration. In rodent SNpc, DA neurons can be divided into two subpopulations based on the expression of aldehyde dehydrogenase 1 (ALDH1A1). Here, we have shown that, in α-synuclein transgenic mice, a murine model of PD-related disease, DA neurodegeneration occurs mainly in a dorsomedial ALDH1A1-negative subpopulation that is also prone to cytotoxic aggregation of α-synuclein. Notably, the topographic ALDH1A1 pattern observed in α-synuclein transgenic mice was conserved in human SNpc. Postmortem evaluation of brains of patients with PD revealed a severe reduction of ALDH1A1 expression and neurodegeneration in the ventral ALDH1A1-positive DA subpopulations. ALDH1A1 expression was also suppressed in α-synuclein transgenic mice. Deletion of Aldh1a1 exacerbated α-synuclein–mediated DA neurodegeneration and α-synuclein aggregation, whereas Aldh1a1-null and control DA neurons were comparably susceptible to 1-methyl-4-phenylpyridinium–, glutamate-, or camptothecin-induced cell death. ALDH1A1 overexpression appeared to preferentially protect against α-synuclein–mediated DA neurodegeneration but did not rescue α-synuclein–induced loss of cortical neurons. Together, our findings suggest that ALDH1A1 protects subpopulations of SNpc DA neurons by preventing the accumulation of dopamine aldehyde intermediates and formation of cytotoxic α-synuclein oligomers.
doi:10.1172/JCI72176
PMCID: PMC4071380  PMID: 24865427
22.  A novel animal model of osteonecrosis of the femoral head induced using a magnetic resonance imaging-guided argon-helium cryotherapy system 
The aim of the present study was to establish a novel animal model of osteonecrosis of the femoral head (ONFH) using a magnetic resonance imaging (MRI)-guided argon-helium cryotherapy system. A total of 48 rabbits were used to generate the ONFH models. In group I, the left femoral head of the rabbits received two cycles of argon-helium freezing-thawing under MRI guidance, while in group II, the right femoral head of each rabbit received only one cycle of argon-helium freezing-thawing. X-ray and histological examinations were performed. The percentages of lacunae in the femoral heads of group I at weeks 4, 8 and 12 following surgery (49.75±3.17, 62.06±4.12 and 48.25±2.76%, respectively) were higher than those in group II (39.13±4.48, 50.69±3.84 and 37.50±3.86%, respectively). In addition, the percentage of empty lacunae in group I was 62.06% at week 8 following surgery. Therefore, an animal model of ONFH was successfully established using an argon-helium cryotherapy system. The percentage of empty lacunae in group I was higher than that in group II at weeks 4, 8 and 12 after surgery.
doi:10.3892/etm.2014.1625
PMCID: PMC4043595  PMID: 24926337
osteonecrosis; femoral head; magnetic resonance imaging; cryotherapy; animal model
23.  MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein 
Human Molecular Genetics  2012;22(3):608-620.
Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3′-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.
doi:10.1093/hmg/dds470
PMCID: PMC3542867  PMID: 23125283
24.  Association between the g.296596G > A genetic variant of RELN gene and susceptibility to autism in a Chinese Han population 
Genetics and Molecular Biology  2013;36(4):486-489.
Autism is a childhood neuro-developmental disorder, and Reelin (RELN) is an important candidate gene for influencing autism. This study aimed at investigating the influence of genetic variants of the RELN gene on autism susceptibility. In this study, 205 autism patients and 210 healthy controls were recruited and the genetic variants of the RELN gene were genotyped by the created restriction site-polymerase chain reaction (CRS-PCR) method. The influence of genetic variants on autism susceptibility was analyzed by association analysis, and the g.296596G > A genetic variant in exon10 of the RELN gene was detected. The frequencies of allele/genotype in autistic patients were significantly different from those in healthy controls, and a statistically significant association was detected between this genetic variant and autism susceptibility. Our data lead to the inference that the g.296596G > A genetic variant in the RELN gene has a potential influence on autism susceptibility in the Chinese Han population.
doi:10.1590/S1415-47572013005000037
PMCID: PMC3873176  PMID: 24385848
autism; susceptibility; association analysis; RELN gene; genetic variants
25.  Conditional Expression of Parkinson disease-related Mutant α-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1 
α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
doi:10.1523/JNEUROSCI.1731-12.2012
PMCID: PMC3417246  PMID: 22764233

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