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1.  mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis 
The Journal of Cell Biology  2015;210(7):1153-1164.
Neutrophil chemotaxis is regulated by opposing autocrine purinergic signaling mechanisms, which are stimulated by mitochondrial ATP formation that is up-regulated via mTOR and P2Y2 receptors at the front and down-regulated via A2a receptors and cAMP/PKA signaling at the back of cells.
Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca2+ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.
PMCID: PMC4586745  PMID: 26416965
2.  Novel method for real-time monitoring of ATP release reveals multiple phases of autocrine purinergic signaling during immune cell activation 
Acta physiologica (Oxford, England)  2014;213(2):334-345.
The activation of immune cells must be tightly regulated to allow an effective immune defense while limiting collateral damage to host tissues. Cellular ATP release and autocrine stimulation of purinergic receptors are recognized as critical regulators of immune cell activation. However, the study of purinergic signaling has been hampered by the short half-life of the released ATP and its breakdown products as well as the lack of real-time imaging methods to study spatiotemporal dynamics of ATP release.
To overcome these limitations, we optimized imaging methods that allow monitoring of ATP release with conventional microscopy using the recently developed small molecular ATP probes 1-2Zn(II) and 2-2Zn(II) for imaging of ATP in the extracellular space and release at the surface of living cells.
1-2Zn(II) allowed imaging of <1 µM ATP in the extracellular space, while 2-2Zn(II) provided unprecedented insights into the spatiotemporal dynamics of ATP release from neutrophils and T cells. Stimulation of these cells caused virtually instantaneous ATP release, which was followed by a second phase of ATP release that was localized to the immune synapse of T cells and the leading edge of polarized neutrophils. Imaging these ATP signaling processes along with mitochondrial probes provided evidence for a close spatial relationship between mitochondrial activation and localized ATP release in T cells and neutrophils.
We believe that these novel live cell imaging methods can be used to define the roles of purinergic signaling in immune cell activation and in the regulation of other complex physiological processes.
PMCID: PMC4293224  PMID: 25482154
ATP release; live cell imaging; fluorescence microscopy; autocrine purinergic signaling; neutrophils; T cells
3.  Uygur Medicine Xipayi Kui Jie’an Affects Gene Expression profiles in intestinal tissue Lesions in a Rat Model of Ulcerative Colitis 
The aim of this study was to investigate the mechanisms underlying the therapeutic effect of Uygur medicine KJA on UC in a rat model.
UC was induced in Wistar rats by application of 2, 4-dinitrochlorobenzene and acetic acid and were then treated with three different doses of KJA, and normal saline as control. After treatment for 20 days, the gene expression profile of colonic tissue was analyzed by microarray and verified by quantitative real-time RT-PCR.
Animals treated with the three different doses of KJA were compared with normal saline controls, wherein microarray analysis identified 1991, 2163, and 1677 differentially expressed genes respectively, of which 444 genes were raised and 670 genes were decrease spliced together in the three doses tested. The KEGG pathway analyses found commonly raised genes related to several different biological functions. Interesting genes included TRL2, IL-1β, TGF-β1, and NF-κB were confirmed by quantitative PCR.
The therapeutic effect of KJA on UC is likely explained by specific effects on the expression of genes, which are the effector molecules known to be involved in the development of UC. Further studies on differentially expressed genes will help explain the mechanism of action of Uygur medicine KJA.
PMCID: PMC4440515  PMID: 25997744
Uygur Medicine; Ulcerative colitis; Xipayi kuijie’an; Immunity and inflammation; Microarray
4.  Motor Impairments, Striatal Degeneration, and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95 
Journal of neurogenetics  2014;28(0):98-111.
Excessive activation of the N-Methyl-D-Aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.
PMCID: PMC4406490  PMID: 24702501
5.  Filamin A Regulates Neuronal Migration through Brefeldin A-Inhibited Guanine Exchange Factor 2-Dependent Arf1 Activation 
The Journal of Neuroscience  2013;33(40):15735-15746.
Periventricular heterotopias is a malformation of cortical development, characterized by ectopic neuronal nodules around ventricle lining and caused by an initial migration defect during early brain development. Human mutations in the Filamin A (FLNA) and ADP-ribosylation factor guanine exchange factor 2 [ARFGEF2; encoding brefeldin-A-inhibited guanine exchange factor-2 (BIG2)] genes give rise to this disorder. Previously, we have reported that Big2 inhibition impairs neuronal migration and binds to FlnA, and its loss promotes FlnA phosphorylation. FlnA phosphorylation dictates FlnA–actin binding affinity and consequently alters focal adhesion size and number to effect neuronal migration. Here we show that FlnA loss similarly impairs migration, reciprocally enhances Big2 expression, but also alters Big2 subcellular localization in both null and conditional FlnA mice. FlnA phosphorylation promotes relocalization of Big2 from the Golgi toward the lipid ruffles, thereby activating Big2-dependent Arf1 at the cell membrane. Loss of FlnA phosphorylation or Big2 function impairs Arf1-dependent vesicle trafficking at the periphery, and Arf1 is required for maintenance of cell–cell junction connectivity and focal adhesion assembly. Loss of Arf1 activity disrupts neuronal migration and cell adhesion. Collectively, these studies demonstrate a potential mechanism whereby coordinated interactions between actin (through FlnA) and vesicle trafficking (through Big2-Arf) direct the assembly and disassembly of membrane protein complexes required for neuronal migration and neuroependymal integrity.
PMCID: PMC3787497  PMID: 24089482
6.  Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling 
PLoS ONE  2014;9(2):e89352.
Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins.
PMCID: PMC3925234  PMID: 24551245
7.  Brefeldin A-inhibited guanine exchange factor 2 regulates Filamin A phosphorylation and neuronal migration 
Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2 encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show that Arfgef2 null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at ser2152. Big2 physically interacts with FlnA and over-expression of phosphomimetic ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration.
PMCID: PMC3478955  PMID: 22956851
8.  Filamin A Regulates Neural Progenitor Proliferation and Cortical Size through Wee1-Dependent Cdk1 Phosphorylation 
The Journal of Neuroscience  2012;32(22):7672-7684.
Cytoskeleton-associated proteins play key roles not only in regulating cell morphology and migration but also in proliferation. Mutations in the cytoskeleton-associated gene filamin A (FlnA) cause the human disorder periventricular heterotopia (PH). PH is a disorder of neural stem cell development that is characterized by disruption of progenitors along the ventricular epithelium and subsequent formation of ectopic neuronal nodules. FlnA-dependent regulation of cytoskeletal dynamics is thought to direct neural progenitor migration and proliferation. Here we show that embryonic FlnA null mice exhibited a reduction in brain size, and decline in neural progenitor numbers over time. The drop in the progenitor population was not attributable to cell death or changes in premature differentiation, but to prolonged cell cycle duration. Suppression of FlnA led to prolongation of the entire cell cycle length, principally in M-phase. FlnA loss impaired degradation of cyclin b1-related proteins, thereby delaying the onset and progression through mitosis. We found that the cdk1 kinase Wee1 bound FlnA, demonstrated increased expression levels after loss of FlnA function, and was associated with increased phosphorylation of cdk1. Phosphorylation of cdk1 inhibited activation of the anaphase promoting complex degradation system, which was responsible for cyclin b1 degradation and progression through mitosis. Collectively, our results demonstrate a molecular mechanism whereby FlnA loss impaired G2 to M phase entry, leading to cell cycle prolongation, compromised neural progenitor proliferation, and reduced brain size.
PMCID: PMC3368379  PMID: 22649246
9.  Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses 
Journal of neurochemistry  2011;119(1):64-77.
Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance.
PMCID: PMC3184177  PMID: 21797867
membrane rafts; DRM; postsynaptic density; PSD
10.  catena-Poly[[(5-phenyl-2,2′-bipyridine-κ2 N,N′)copper(I)]-μ-thio­cyanido-κ2 N:S] 
The title compound, [Cu(NCS)(C16H12N2)]n, was synthesised under hydro­thermal conditions. The CuI ion shows distorted tetra­hedral geometry being coordinated by two N atoms from a 5-phenyl-2,2′-bipyridine ligand and by the N and S atoms from two different thio­cyanate anions. The CuI ions are bridged by thio­cyanide groups, forming a one-dimensional coordination polymer along the b axis. The crystal packing is through van der Waals contacts and C—H⋯π inter­actions.
PMCID: PMC3274895  PMID: 22346842
11.  Poly[penta­kis­(μ-cyanido-κ2 N:C)tris­(5-phenyl-2,2′-bipyridine-κ2 N,N′)penta­copper(I)] 
The hydro­thermal reaction of Cu(acetate)2 and K3[Fe(CN)6] with 5-phenyl-2,2′-bipyridine (5-ph-2,2′-bpy) in water yields the polymeric title complex, [Cu5(CN)5(C16H12N2)3]n, which consists of ribbons along the a axis, constructed from 26-membered {Cu10(CN)8} rings. In these rings, the metal atoms are bridged by cyanide groups, except for one close Cu⋯Cu contact [2.7535 (12) Å], which can be considered as ligand-unsupported. Within the rings, one Cu atom has a distorted tetra­hedral geometry through the coordination to two N atoms from 5-ph-2,2′-bpy and two N/C atoms from two cyanide groups. Two Cu atoms have a trigonal planar environment being coordinated by three cyanide groups and two other Cu atoms have a distorted square planar geometry through coordination to two N atoms from 5-ph-2,2′-bpy and two N/C atoms from two cyanide groups.
PMCID: PMC3238629  PMID: 22199520
12.  Repeated carbon nanotube administrations in male mice cause reversible testis damage without affecting fertility 
Nature nanotechnology  2010;5(9):683-689.
Soluble carbon nanotubes are promising materials for in vivo delivery and imaging applications. Several reports have described the in vivo toxicity of carbon nanotubes, however, their effects on male reproduction have not been examined. Here we show that repeated intravenous injections of water-soluble multi-walled carbon nanotubes into male mice can cause reversible testis damage without affecting fertility. Nanotubes accumulated in the testes, generated oxidative stress, and decreased the thickness of the seminiferous epithelium in the testis at day 15, but the damage was repaired after 60 and 90 days. The quantity, quality, and integrity of the sperm and the levels of three major sex hormones were not significantly affected throughout the 90-day period. The fertility of treated male mice was unaffected; the pregnancy rate and delivery success of female mice that mated with the treated male mice did not differ from those that mated with untreated male mice.
PMCID: PMC2934866  PMID: 20693989
carbon nanotube; nano-reproductive toxicity; nanotoxicity; nanotechnology; male fertility; mice
13.  Hyperdopaminergic Tone Erodes Prefrontal LTP via a D2 Receptor-operated Protein Phosphatase Gate 
Dopamine (DA) plays crucial roles in the cognitive functioning of the prefrontal cortex (PFC), which, to a large degree, depends on lasting neural traces formed in prefrontal networks. The establishment of these permanent traces requires changes in cortical synaptic efficacy. DA, via the D1-class receptors, is thought to gate or facilitate synaptic plasticity in the PFC, with little role recognized for the D2-class receptors. Here we show that, when significantly elevated, DA erodes, rather than facilitates, the induction of long-term potentiation (LTP) in the PFC by acting at the far less abundant cortical D2-class receptors through a dominant coupling to the protein phosphatase 1 (PP1) activity in postsynaptic neurons. In mice with persistently elevated extracellular DA, resulting from inactivation of the DA transporter (DAT) gene, LTP in layer V PFC pyramidal neurons can not be established, regardless of induction protocols. Acute increase of dopaminergic transmission by DAT blockers or overstimulation of D2 receptors in normal mice have similar LTP shut-off effects. LTP in mutant mice can be rescued by a single in vivo administration of D2-class antagonists. Suppression of postsynaptic PP1 mimics and occludes the D2-mediated rescue of LTP in mutant mice, and prevents the acute erosion of LTP by D2 agonists in normal mice. Our studies reveal a mechanistically unique heterosynaptic PP1 gate that is constitutively driven by background DA to influence LTP induction. By blocking prefrontal synaptic plasticity, excessive DA may prevent storage of lasting memory traces in PFC networks and impair executive functions.
PMCID: PMC2818669  PMID: 19906957
Dopamine; prefrontal cortex; LTP; D2-class receptor; PP1; psychiatric disorder
14.  The HECT-Type E3 Ubiquitin Ligase AIP2 Inhibits Activation-Induced T-Cell Death by Catalyzing EGR2 Ubiquitination▿  
Molecular and Cellular Biology  2009;29(19):5348-5356.
E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here, we report that the HECT-type E3 ligase AIP2 positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and interleukin-2 production by suppressing the apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T-cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2, inhibits EGR2 ubiquitination and FasL expression, and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T-cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.
PMCID: PMC2747983  PMID: 19651900
15.  PSD-95 Uncouples Dopamine-Glutamate Interaction in the D1/PSD-95/NMDA Receptor Complex 
Classical dopaminergic signaling paradigms and emerging studies on direct physical interactions between the D1 dopamine (DA) receptor and the N-Methyl-D-Aspartate (NMDA) glutamate receptor predict a reciprocally facilitating, positive feedback loop. This loop, if not controlled, may cause concomitant overactivation of both D1 and NMDA receptors, triggering neurotoxicity. Endogenous protective mechanisms must exist. Here we show that PSD-95, a prototypical structural and signaling scaffold in the postsynaptic density, inhibits D1-NMDA receptor association and uncouples NMDA receptor-dependent enhancement of D1 signaling. This uncoupling is achieved, at least in part, via a disinhibition mechanism by which PSD-95 abolishes NMDA receptor-dependent inhibition of D1 internalization. Knockdown of PSD-95 immobilizes D1 receptors on the cell surface and escalates NMDA receptor-dependent D1 cAMP signaling in neurons. Thus, in addition to its role in receptor stabilization and synaptic plasticity, PSD-95 acts as a brake on the D1-NMDA receptor complex and dampens the interaction between them.
PMCID: PMC2693913  PMID: 19261890
Dopamine D1 receptor; NMDA receptor; synaptic scaffold; cAMP; trafficking; dendritic spine
16.  Dopaminergic Signaling in Dendritic Spines 
Biochemical pharmacology  2008;75(11):2055-2069.
Dopamine regulates movement, motivation, reward, and learning and is implicated in numerous neuropsychiatric and neurological disorders. The action of dopamine is mediated by a family of seven-transmembrane G protein-coupled receptors encoded by at least five dopamine receptor genes (D1, D2, D3, D4, and D5), some of which are major molecular targets for diverse neuropsychiatric medications. Dopamine receptors are present throughout the soma and dendrites of the neuron, but accumulating ultrastructural and biochemical evidence indicates that they are concentrated in dendritic spines, where most of the glutamatergic synapses are established. By modulating local channels, receptors, and signaling modules in spines, this unique population of postsynaptic receptors is strategically positioned to control the excitability and synaptic properties of spines and mediate both the tonic and phasic aspects of dopaminergic signaling with remarkable precision and versatility. The molecular mechanisms that underlie the trafficking, targeting, anchorage, and signaling of dopamine receptors in spines are, however, largely unknown. The present commentary focuses on this important subpopulation of postsynaptic dopamine receptors with emphases on recent molecular, biochemical, pharmacological, ultrastructural, and physiological studies that provide new insights about their regulatory mechanisms and unique roles in dopamine signaling.
PMCID: PMC2443745  PMID: 18353279
17.  Inhibition of the Dopamine D1 Receptor Signaling by PSD-95*◆ 
The Journal of biological chemistry  2007;282(21):15778-15789.
Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing gluta-mate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH2terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.
PMCID: PMC2649122  PMID: 17369255

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