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1.  Acute and Delayed Protective Effects of Pharmacologically Induced Hypothermia in an Intracerebral Hemorrhage Stroke Model of Mice 
Neuroscience  2013;252:489-500.
Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2 mg/kg bolus injection followed by 2 injections of 1 mg/kg, i.p.) were initiated at 1 or 24 hrs after ICH. HPI-201 induced mild hypothermia within 30 min and maintained body and brain temperatures at 32.7±0.4°C for at least 6 hrs without causing observable shivering. With the 1 hr delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24 hrs after the onset of ICH, it still significantly attenuated brain edema, cell death and blood brain barrier breakdown. HPI-201 significantly decreased the expression of MMP-9, reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-hr delayed HPI-201 treatment performed significantly better in the neurological behavior test 48 hrs after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.
doi:10.1016/j.neuroscience.2013.07.052
PMCID: PMC3961766  PMID: 23912033
Intracerebral hemorrhage; pharmacological hypothermia; PIH; neurotensin receptor; ABS-201; HPI-201
2.  Coordinated Development of Voltage-Gated Na+ and K+ Currents Regulates Functional Maturation of Forebrain Neurons Derived from Human Induced Pluripotent Stem Cells 
Stem Cells and Development  2012;22(10):1551-1563.
Like embryonic stem (ES) cells, human induced pluripotent stem (hiPS) cells can differentiate into neuronal cells. However, it is unclear how their exquisite neuronal function is electrophysiologically coordinated during differentiation and whether they are functionally identical to human ES cell-derived neurons. In this study, we differentiated hiPS and ES cells into pyramidal-like neurons and conducted electrophysiological characterization over the 4-week terminal differentiation period. The human neuron-like cells express forebrain pyramidal cell markers NeuN, neurofilament, the microtubule-associated protein 2 (MAP2), the paired box protein Pax-6 (PAX6), Tuj1, and the forkhead box protein G1 (FoxG1). The size of developing neurons increased continuously during the 4-week culture, and cell-resting membrane potentials (RMPs) underwent a negative shift from −40 to −70 mV. Expression of the muscarinic receptor-modulated K+ currents (IM) participated in the development of cell RMPs and controlled excitability. Immature neurons at week 1 could only fire abortive action potentials (APs) and the frequency of AP firing progressively increased with neuronal maturation. Interestingly, the developmental change of voltage-gated Na+ current (INa) did not correlate with the change in the AP firing frequency. On the other hand, the transient outward K+ current (IA), but not the delayed rectifier current (IK) contributed to the high frequency firing of APs. Synaptic activities were observed throughout the 4-week development. These morphological and electrophysiological features were almost identical between iPS and ES cell-derived neurons. This is the first systematic investigation showing functional evidence that hiPS cell-derived neurons possess similar neuronal activities as ES cell-derived neurons. These data support that iPS cell-derived neural progenitor cells have the potential for replacing lost neurons in cell-based therapy.
doi:10.1089/scd.2012.0556
PMCID: PMC3653388  PMID: 23259973
3.  Potential Role of KCNQ/M-Channels in Regulating Neuronal Differentiation in Mouse Hippocampal and Embryonic Stem Cell-Derived Neuronal Cultures 
Experimental neurology  2011;229(2):471-483.
Voltage-gated K+ channels are key regulators of neuronal excitability, playing major roles in setting resting membrane potential, repolarizing the cell membrane after action potentials and affecting transmitter release. The M-type channel or M-channel is a unique voltage- and ligand-regulated K+ channel. It is composed of the molecular counterparts KCNQ2 and KCNQ3 (also named Kv7.2 and Kv7.3) channels and expressed in the soma and dendrites of neurons. The present investigation examined the hypothesis that KCNQ2/3 channels played a regulatory role in neuronal differentiation and maturation. In cultured mouse embryonic stem (ES) cells undergoing neuronal differentiation and primary embryonic (E15-17) hippocampal cultures, KCNQ2 and KCNQ3 channels and underlying M-currents were identified. Blocking of KCNQ channels in these cells for 5 days using the specific channel blocker XE991 (10 μM) or linopirdine (30 μM) significantly decreased synaptophysin and syntaxin expression without affecting cell viability. Chronic KCNQ2/3 channel block reduced the expression of vesicular GABA transporter (v-GAT), but not vesicular glutamate transporter (v-GluT). Enhanced ERK1/2 phosphorylation was observed in XE991- and linopirdine-treated neural progenitor cells. In electrophysiological recordings, cells undergoing chronic block of KCNQ2/3 channels showed normal amplitude of mPSCs while the frequency of mPSCs was reduced. On the other hand, KCNQ channel opener N-Ethylmaleimide (NEM, 2 μM) increased mPSC frequency. Fluorescent imaging using fluorescent styryl-dye FM4-64 revealed that chronic blockade of KCNQ2/3 channels decreased endocytosis but facilitated exocytosis. These data indicate that KCNQ2/3 channels participate in regulation of neuronal differentiation and show a tonic regulation on pre-synaptic transmitter release and recycling in developing neuronal cells.
doi:10.1016/j.expneurol.2011.03.018
PMCID: PMC3100408  PMID: 21466805
M-current; synaptogenesis; Neuronal differentiation; Mouse ES cells; Hippocampal neurons; KCNQ channels; ERK1/2
4.  Novel Role of KCNQ2/3 channels in Neuronal Cell Viability 
Cell death and differentiation  2010;18(3):493-505.
Over-activation of certain K+ channels can mediate intracellular K+ depletion, which is an early ionic event in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected significantly greater M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K+ efflux, intracellular K+ depletion, and cell death in hippocampal cultures while little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular potassium. Supporting a mediating role in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in CHO cells initiated caspase-3 activation. Consistently, application of NEM (20 μM) in hippocampal cultures similarly caused caspase-3 activation. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondrial membrane depolarization, cytochrome c release, formation of apoptosome complex, and AIF translocation into the nucleus. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis.
doi:10.1038/cdd.2010.120
PMCID: PMC3017650  PMID: 20885443
Apoptosis; Hippocampal neurons; Cortical Neurons; Kv7 channel; M-current; KCNQ channel; ERK1/2
5.  Formation of Kv2.1-FAK Complex as a Mechanism of FAK Activation, Cell Polarization and Enhanced Motility 
Journal of cellular physiology  2008;217(2):544-557.
Focal adhesion kinase (FAK) plays key roles in cell adhesion and migration. We now report that the delayed rectifier Kv2.1 potassium channel, through its LD-like motif in N-terminus, may interact with FAK and enhance phosphorylation of FAK397 and FAK576/577. Overlapping distribution of Kv2.1 and FAK was observed on soma and proximal dendrites of cortical neurons. FAK expression promotes a polarized membrane distribution of the Kv2.1 channel. In Kv2.1-transfected CHO cells, formation of the Kv2.1-FAK complex was stimulated by fibronectin/integrin and inhibited by the K+ channel blocker tetraethylammonium (TEA). FAK phosphorylation was minimized by shRNA knockdown of the Kv2.1 channel, point mutations of the N-terminus, and TEA, respectively. Cell migration morphology was altered by Kv2.1 knockdown or TEA, hindering cell migration activity. In wound healing tests in vitro and a traumatic injury animal model, Kv2.1 expression and co-localization of Kv2.1 and FAK significantly enhanced directional cell migration and wound closure. It is suggested that the Kv2.1 channel may function as a promoting signal for FAK activation and cell motility.
doi:10.1002/jcp.21530
PMCID: PMC2562431  PMID: 18615577
Adhesion; Migration; FAK; Kv2.1 channel; Phosphorylation; Wound healing
6.  Purification of Cardiomyocytes from Differentiating Pluripotent Stem Cells using Molecular Beacons Targeting Cardiomyocyte-Specific mRNA 
Circulation  2013;128(17):10.1161/CIRCULATIONAHA.113.004228.
Background
While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs.
Method and Results
Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.
Conclusions
We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery.
doi:10.1161/CIRCULATIONAHA.113.004228
PMCID: PMC3878656  PMID: 23995537
pluripotent stem cell; cardiomyocyte; molecular beacons; cell selection; cardiac regeneration
7.  Inhibition of Na+/K+-ATPase induces hybrid cell death and enhanced sensitivity to chemotherapy in human glioblastoma cells 
BMC Cancer  2014;14(1):716.
Background
Glioblastoma multiforme (GBM) is very difficult to treat with conventional anti-cancer/anti-apoptotic drugs. We tested the hypothesis that inhibition of Na+/K+-ATPase causes a mixed or hybrid form of concurrent apoptosis and necrosis and therefore should enhance anti-cancer effects of chemotherapy on glioblastoma cells.
Methods
In human LN229 and drug-resistant T98G glioblastoma cell cultures, cell death and signal pathways were measured using immunocytochemistry and Western blotting. Fluorescent dyes were applied to measure intracellular Ca2+, Na+ and K+ changes.
Results
The specific Na+/K+-ATPase blocker ouabain (0.1 - 10 μM) induced cell death and disruption of K+ homeostasis in a time- and concentration-dependent manner. Annexin-V translocation and caspase-3 activation indicated an apoptotic component in ouabain cytoxicity, which was accompanied with reduced Bcl-2 expression and mitochondrial membrane potential. Ouabain-induced cell death was partially attenuated by the caspase inhibitor Z-VAD (100 μM). Consistently, the K+ ionophore valinomycin initiated apoptosis in LN229 cells in a K+ efflux-dependent manner. Ouabain caused an initial cell swell, which was followed by a sustained cell volume decrease. Electron microscopy revealed ultrastructural features of both apoptotic and necrotic alterations in the same cells. Finally, human T98G glioblastoma cells that are resistant to the chemotherapy drug temozolomide (TMZ) showed a unique high expression of the Na+/K+-ATPase α2 and α3 subunits compared to the TMZ-sensitive cell line LN229 and normal human astrocytes. At low concentrations, ouabain selectively killed T98G cells. Knocking down the α3 subunit sensitized T98G cells to TMZ and caused more cell death.
Conclusion
This study suggests that inhibition of Na+/K+-ATPase triggers hybrid cell death and serves as an underlying mechanism for an enhanced chemotherapy effect on glioblastoma cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-716) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-716
PMCID: PMC4190379  PMID: 25255962
Na+ pump; Glioblastomas; Apoptosis; Hybrid cell death; K+ homeostasis; Intracellular Ca2+; Temozolomide
8.  Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells 
Background
Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines.
Methods
Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS.
Results and Conclusions
At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α –myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.
doi:10.1016/j.ijcard.2012.09.077
PMCID: PMC3556195  PMID: 23044428
Human embryonic stem cells; Human induced pluripotent stem cells; Cardiomyocytes; Directed differentiation
9.  Cell based therapies for ischemic stroke: From basic science to bedside 
Progress in neurobiology  2013;115:92-115.
Cell therapy is emerging as a viable therapy to restore neurological function after stroke. Many types of stem/progenitor cells from different sources have been explored for their feasibility and efficacy for the treatment of stroke. Transplanted cells not only have the potential to replace the lost circuitry, but also produce growth and trophic factors, or stimulate the release of such factors from host brain cells, thereby enhancing endogenous brain repair processes. Although stem/progenitor cells have shown a promising role in ischemic stroke in experimental studies as well as initial clinical pilot studies, cellular therapy is still at an early stage in humans. Many critical issues need to be addressed including the therapeutic time window, cell type selection, delivery route, and in vivo monitoring of their migration pattern. This review attempts to provide a comprehensive synopsis of preclinical evidence and clinical experience of various donor cell types, their restorative mechanisms, delivery routes, imaging strategies, future prospects and challenges for translating cell therapies as a neurorestorative regimen in clinical applications.
doi:10.1016/j.pneurobio.2013.11.007
PMCID: PMC4038267  PMID: 24333397
Stem cells; Cell-based therapies; Ischemic stroke; Neurorestoration
10.  Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms 
The Journal of Cell Biology  2013;202(7):1123-1138.
Polyglutamine-expanded huntingtin specifically targeted to synapses binds to synapsin-1, inhibits its phosphorylation, and causes defects in neurotransmitter release and age-dependent defects in neurological function.
Many genetic mouse models of Huntington’s disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.
doi:10.1083/jcb.201303146
PMCID: PMC3787372  PMID: 24081492
11.  Mobilization of Endogenous Bone Marrow Derived Endothelial Progenitor Cells and Therapeutic Potential of Parathyroid Hormone after Ischemic Stroke in Mice 
PLoS ONE  2014;9(2):e87284.
Stroke is a major neurovascular disorder threatening human life and health. Very limited clinical treatments are currently available for stroke patients. Stem cell transplantation has shown promising potential as a regenerative treatment after ischemic stroke. The present investigation explores a new concept of mobilizing endogenous stem cells/progenitor cells from the bone marrow using a parathyroid hormone (PTH) therapy after ischemic stroke in adult mice. PTH 1-34 (80 µg/kg, i.p.) was administered 1 hour after focal ischemia and then daily for 6 consecutive days. After 6 days of PTH treatment, there was a significant increase in bone marrow derived CD-34/Fetal liver kinase-1 (Flk-1) positive endothelial progenitor cells (EPCs) in the peripheral blood. PTH treatment significantly increased the expression of trophic/regenerative factors including VEGF, SDF-1, BDNF and Tie-1 in the brain peri-infarct region. Angiogenesis, assessed by co-labeled Glut-1 and BrdU vessels, was significantly increased in PTH-treated ischemic brain compared to vehicle controls. PTH treatment also promoted neuroblast migration from the subventricular zone (SVZ) and increased the number of newly formed neurons in the peri-infarct cortex. PTH-treated mice showed significantly better sensorimotor functional recovery compared to stroke controls. Our data suggests that PTH therapy improves endogenous repair mechanisms after ischemic stroke with functional benefits. Mobilizing endogenous bone marrow-derived stem cells/progenitor cells using PTH and other mobilizers appears an effective and feasible regenerative treatment after ischemic stroke.
doi:10.1371/journal.pone.0087284
PMCID: PMC3913619  PMID: 24503654
12.  Preconditioning Strategy in Stem Cell Transplantation Therapy 
Translational stroke research  2013;4(1):76-88.
Stem cell transplantation therapy has emerged as a promising regenerative medicine for ischemic stroke and other neurodegenerative disorders. However, many issues and problems remain to be resolved before successful clinical applications of the cell-based therapy. To this end, some recent investigations have sought to benefit from well-known mechanisms of ischemic/hypoxic preconditioning. Ischemic/hypoxic preconditioning activates endogenous defense mechanisms that show marked protective effects against multiple insults found in ischemic stroke and other acute attacks. As in many other cell types, a sub-lethal hypoxic exposure significantly increases the tolerance and regenerative properties of stem cells and progenitor cells. So far, a variety of preconditioning triggers have been tested on different stem cells and progenitor cells. Preconditioned stem cells and progenitors generally show much better cell survival, increased neuronal differentiation, enhanced paracrine effects leading to increased trophic support, and improved homing to the lesion site. Transplantation of preconditioned cells helps to suppress inflammatory factors and immune responses, and promote functional recovery. Although the preconditioning strategy in stem cell therapy is still an emerging research area, accumulating information from reports over the last few years already indicates it as an attractive, if not essential, prerequisite for transplanted cells. It is expected that stem cell preconditioning and its clinical applications will attract more attention in both the basic research field of preconditioning as well as in the field of stem cell translational research. This review summarizes the most important findings in this active research area, covering the preconditioning triggers, potential mechanisms, mediators, and functional benefits for stem cell transplant therapy.
doi:10.1007/s12975-012-0251-0
PMCID: PMC3728897  PMID: 23914259
Stem cell preconditioning; Stroke; Ischemia; Neurodegenerative disorder; Heart attack
13.  Neuro-Modulating Effects of Honokiol: A Review 
Honokiol is a poly-phenolic compound that exerts neuroprotective properties through a variety of mechanisms. It has therapeutic potential in anxiety, pain, cerebrovascular injury, epilepsy, and cognitive disorders including Alzheimer’s disease. It has been traditionally used in medical practices throughout much of Southeast Asia, but has now become more widely studied due to its pleiotropic effects. Most current research regarding this compound has focused on its chemotherapeutic properties. However, it has the potential to be an effective neuroprotective agent as well. This review summarizes what is currently known regarding the mechanisms involved in the neuroprotective and anesthetic effects of this compound and identifies potential areas for further research.
doi:10.3389/fneur.2013.00130
PMCID: PMC3769637  PMID: 24062717
honokiol; neuroprotection; GABA; stroke; inflammatory pain; amyloid; magnolol; analgesia
14.  Transplantation of Hypoxia Preconditioned Bone Marrow Mesenchymal Stem Cells Enhances Angiogenesis and Neurogenesis after Cerebral Ischemia in Rats 
Neurobiology of Disease  2012;46(3):635-645.
Hypoxic preconditioning of stem cells and neural progenitor cells has been tested for promoting cell survival after transplantation. The present investigation examined the hypothesis that hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) could not only enhance their survival but also reinforce regenerative properties of these cells. BMSCs from eGFP engineered rats or pre-labeled with BrdU were pre-treated with normoxia (20% O2, N-BMSCs) or sublethal hypoxia (0.5% O2. H-BMSCs). The hypoxia exposure up-regulated HIF-1α and trophic/growth factors in BMSCs, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) and its receptor FIK-1, erythropoietin (EPO) and its receptor EPOR, stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). Meanwhile, many pro-inflammatory cytokines/chemokines were downregulated in H-BMSCs. N-BMSCs or H-BMSCs were intravenously injected into adult rats 24 hrs after 90-min middle cerebral artery occlusion. Comparing to N-BMSCs, transplantation of H-BMSCs showed greater effect of suppressing microglia activity in the brain. Significantly more NeuN-positive and Glut1-positive cells were seen in the ischemic core and peri-infarct regions of the animals received H-BMSC transplantation than that received N-BMSCs. Some NeuN-positive and Glut-1-positive cells showed eGFP or BrdU immunoflourescent reactivity, suggesting differentiation from exogenous BMSCs into neuronal and vascular endothelial cells. In Rota-rod test performed 15 days after stroke, animals received H-BMSCs showed better locomotion recovery compared with stroke control and N-BMSC groups. We suggest that hypoxic preconditioning of transplanted cells is an effective means of promoting their regenerative capability and therapeutic potential for the treatment of ischemic stroke.
doi:10.1016/j.nbd.2012.03.002
PMCID: PMC3353023  PMID: 22426403
hypoxic preconditioning; bone marrow mesenchymal stem cell; transplantation; angiogenesis; neurogenesis
15.  SRPK2 Phosphorylates Tau and Mediates the Cognitive Defects in Alzheimer’s Disease 
Serine-arginine protein kinases 2 (SRPK2) is a cell cycle-regulated kinase that phosphorylates serine/arginine domain-containing proteins and mediates pre-mRNA splicing with unclear function in neurons. Here, we show that SRPK2 phosphorylates tau on S214, suppresses tau-dependent microtubule polymerization and inhibits axonal elongation in neurons. Depletion of SRPK2 in dentate gyrus inhibits tau phosphorylation in APP/PS1 mouse and alleviates the impaired cognitive behaviors. The defective LTP in APP/PS1 mice is also improved after SRPK2 depletion. Moreover, active SRPK2 is increased in the cortex of APP/PS1 mice and the pathological structures of human Alzheimer’s disease (AD) brain. Therefore, our study suggests SRPK2 may contribute to the formation of hyperphosphorylated tau and the pathogenesis of AD.
doi:10.1523/JNEUROSCI.3300-12.2012
PMCID: PMC3518045  PMID: 23197718
16.  Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice 
PLoS ONE  2013;8(5):e64160.
Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.
doi:10.1371/journal.pone.0064160
PMCID: PMC3662762  PMID: 23717557
17.  Stem Cells and Ion Channels 
Stem Cells International  2013;2013:238635.
doi:10.1155/2013/238635
PMCID: PMC3671535  PMID: 23766766
18.  Protective Effect of Apelin on Cultured Rat Bone Marrow Mesenchymal Stem Cells against Apoptosis 
Stem Cell Research  2011;8(3):357-367.
Bone marrow-derived mesenchymal stem cells (BMSCs) have shown great promise for ischemic tissue repair. However, poor viability of transplanted BMSCs within ischemic tissues has limited their therapeutic potential. Apelin, an endogenous peptide, whose level is elevated following ischemia, has been shown to enhance survival of cardiomyocytes and neuronal cells during ischemia. We hypothesized that apelin-13 protects BMSCs from apoptotic death. In this paper we determined the potential mechanism of apelin-13 effects using cultured BMSCs from adult rats. Apoptosis was induced by the specific apoptotic insult serum deprivation (SD) for up to 36 hrs. Apoptotic cell death was measured using immunostaining and Western blotting in the presence and absence of apelin-13 (0.1 to 5.0 nM) co-applied during SD exposure. SD-induced apoptosis was significantly reduced by apelin-13 in a concentration-dependent manner. SD-induced mitochondrial depolarization, cytochrome c release, and caspase-3 activation were largely prevented by apelin-13. The apelin-13 anti-apoptotic effects were blocked by inhibiting the MAPK/ERK1/2 and PI3K/Akt signaling pathways. Taken together, our findings indicate that apelin-13 is a survival factor for BMSCs and its anti-apoptotic property may prove to be of therapeutic significance in terms of exploiting BMSC-based transplantation therapy.
doi:10.1016/j.scr.2011.12.004
PMCID: PMC3319486  PMID: 22289418
Apelin-13; Bone marrow mesenchymal stem cells; Serum deprivation; Apoptosis
19.  Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells 
Neurons in the adult mammalian CNS do not spontaneously regenerate axons after injury due to CNS myelin and other inhibitory factors. Previous studies have showed that inhibition of the Rho-ROCK pathway promotes axonal outgrowth in primary neurons or in spinal cord injury models. Furthermore, RhoA inhibitor C3 transferase has a potential effect to induce neural differentiation in primary cultured neurons and cell lines. As stem cells and stem cell-derived neural progenitor cells have emerged as a regenerative medicine for stroke, Parkinson’s disease and other neurological disorders, strategies that can promote axonal outgrowth and neuronal differentiation appear to have promising benefits in the cell-based therapy. Currently, how changes in the Rho-ROCK pathway may affect the neurite outgrowth and neuronal differentiation of stem cells has been poorly understood. The present investigation examined the effects of RhoA inhibition on neurite outgrowth and neuronal differentiation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of the mouse. Our results show that inhibition of RhoA leads to neurite outgrowth of NSCs not only on normal culture substrate, poly-D-lysine (PDL), but also on myelin substrate. Moreover, inhibition of RhoA improves neuronal differentiation of NSCs and up-regulates biomarkers of neuronal gene expression. These results support that the Rho signaling pathway plays an important role in neurite development and neuronal differentiation of NSCs.
PMCID: PMC3601458  PMID: 23525456
Neural stem cells; Rho signaling pathway; neurite outgrowth; neuronal differentiation; myelin
20.  Expression of Heparanase in Vasculature Cells and Astrocytes of the Mouse Brain after Focal Cerebral Ischemia 
Brain Research  2011;1433:137-144.
Heparanase is a heparan sulfate degrading endoglycosidase. Previous work has demonstrated that heparanase plays important roles in various biological processes including angiogenesis, wound healing and metastasis. However, the role of heparanase in the post-ischemic brain is not well defined. Transient focal cerebral ischemia in adult mice was induced by ligations of the right middle cerebral artery (MCA) and both common carotid arteries (CCAs). All mice were subjected to bromodeoxyuridine (BrdU) injection and sacrificed at different time points after stroke for immunohistochemical and Western blot analyses. Heparanase expression increased after ischemia in both cell-specific and time-dependent manners. Three to 7 days after stroke, levels of the 50-kD heparanase, basic fibroblast growth factor (FGF-2), and angiopoietin-2 (Ang-2) increased in the peri-infarct region. At early time points, heparanase expression was largely confined to proliferating vascular endothelial cells. At 14 days after ischemia, this expression had shifted to astrocytes in the same region. These data show that cerebral ischemia markedly increases heparanase levels in endothelial cells and then in astrocytes. The unique features of the heparanase upregulation imply that heparanase may play specific roles in the pathological and regenerative processes during the acute and sub-acute/chronic phase in the post-stroke brain.
doi:10.1016/j.brainres.2011.11.032
PMCID: PMC3286644  PMID: 22169133
Heparanase; Cerebral ischemia; Endothelial cells; Astrocytes; Angiogenesis; FGF-2; Angiopoietin-2
21.  dl-3-n-Butylphthalide Prevents Neuronal Cell Death after Focal Cerebral Ischemia in Mice via the JNK Pathway 
Brain research  2010;1359:216-226.
dl-3-n-Butylphthalide (NBP) has shown cytoprotective effects in animal models of stroke and has passed clinical trails as a therapeutic drug for stroke in China. Hence, as a potential clinical treatment for stroke, understanding the mechanism(s) of action of NBP is essential. This investigation aimed to delineate the cellular and molecular mechanism of NBP protection in neuronal cultures and in the ischemic brain. NBP (10 M) attenuated serum deprivation-induced neuronal apoptosis and the production of reactive oxygen species (ROS) in cortical neuronal cultures. Adult male 129 S2/sv mice were subjected to permanent occlusion of the middle cerebral artery (MCA). NBP (100 mg/kg, i.p.) administrated 2 hrs before or 1 hr after ischemia reduced ischemia-induced infarct formation, attenuated caspase-3 and caspase-9 activation in the ischemic brain. TUNEL-positive cells and mitochondrial release of cytochrome c and apoptosis-inducing-factor (AIF) in the penumbra region were reduced by NBP. The pro-apoptotic signaling mediated by phospho-JNK and p38 expression was down-regulated by NBP treatment in vitro and in vivo. It is suggested that NBP protects against ischemic damage via multiple mechanisms including mitochondria associated caspase-dependent and -independent apoptotic pathways. Previous and current studies and recent clinical trials encourage exploration of NBP as a neuroprotective drug for the treatment of ischemic stroke.
doi:10.1016/j.brainres.2010.08.061
PMCID: PMC3099257  PMID: 20800583
dl-3-n-butylphthalide; Ischemic stroke; Apoptosis; Caspase; AIF; Cytochrome C; Mitochondria; MAP kinase
22.  Neuroprotective Effect of the Endogenous Neural Peptide Apelin in Cultured Mouse Cortical Neurons 
Experimental cell research  2010;316(11):1773-1783.
The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 hrs induced nuclear fragmentation and apoptotic death; apelin-13 (1.0 – 5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca2+ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.
doi:10.1016/j.yexcr.2010.02.005
PMCID: PMC3155990  PMID: 20152832
Cortical neurons; Apelin; Serum deprivation; Apoptosis
23.  Sublethal Transient Global Ischemia Stimulates Migration of Neuroblasts and Neurogenesis in Mice 
Translational stroke research  2010;1(3):184-196.
Increasing evidence has shown the potential of neuronal plasticity in adult brain after injury. Neural proliferation can be triggered by a focal sublethal ischemic preconditioning event; whether mild global ischemia could cause neurogenesis has been not clear. The present study investigated stimulating effects of sublethal transient global ischemia (TGI) on endogenous neurogenesis and neuroblast migration in the subventricular zone (SVZ), dentate gyrus, and peri-infarct areas of the adult cortex. Adult mice of 129S2/Sv strain were subjected to 8-min bilateral common carotid artery ligation followed by 5-bromo-2′-deoxyuridine (BrdU; 50 mg/kg, intraperitoneal) administration every day until being sacrificed at 1–21 days after reperfusion. The mild TGI did not induce neuronal cell death for up to 7 days after TGI, as evidenced by negative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining among NeuN-positive cells in the hippocampus and neocortex. In TGI animals, BrdU staining revealed enhanced proliferation of neuroblasts and their migration track from the SVZ into the striatum and neocortex. In the corpus callosum, there were more BrdU-positive cells in the TGI group in the first 2 days. Increasing numbers of BrdU-positive cells were seen 7–21 days later in the striatum and cortex of TGI mice. The cortex of TGI animals showed increased expression of erythropoietin, erythropoietin receptor, fibroblast growth factor 2, vascular endothelial growth factor, and phosphorylated Jun N-terminal kinase; the expression was peaked 2 to 3 days after reperfusion. BrdU and NeuN double staining in the dentate gyrus, striatum, and cortex implied increased neurogenesis induced by the TGI preconditioning. Doublecortin (DCX)-positive cells increased in the cortex of TGI mice, localized to cortical layers II, III, and V, and many stained positive for the mature neuronal markers NeuN, neurofilament, N-methyl-d-aspartic acid receptor subunit gene NR1, or the gamma-aminobutyric-acid-synthesizing enzyme glutamic acid decarboxylase (GAD67). The atypical localization of DCX-positive cells and the colabeling with mature neuronal markers suggested that, in addition to indentifying migrating neuroblasts, DCX might also be a stress marker in the cortex. It is suggested that the sublethal TGI-induced regenerative responses may contribute to the beneficial effects of ischemic preconditioning.
doi:10.1007/s12975-010-0016-6
PMCID: PMC3142584  PMID: 21792374
Transient global ischemia; Neuroblasts; Neurogenesis; Cell migration; Doublecortin (DCX); Subventricular zone (SVZ)
24.  Erythropoietin reduces neuronal cell death and hyperalgesia induced by peripheral inflammatory pain in neonatal rats 
Molecular Pain  2011;7:51.
Painful stimuli during neonatal stage may affect brain development and contribute to abnormal behaviors in adulthood. Very few specific therapies are available for this developmental disorder. A better understanding of the mechanisms and consequences of painful stimuli during the neonatal period is essential for the development of effective therapies. In this study, we examined brain reactions in a neonatal rat model of peripheral inflammatory pain. We focused on the inflammatory insult-induced brain responses and delayed changes in behavior and pain sensation. Postnatal day 3 pups received formalin injections into the paws once a day for 3 days. The insult induced dysregulation of several inflammatory factors in the brain and caused selective neuronal cell death in the cortex, hippocampus and hypothalamus. On postnatal day 21, rats that received the inflammatory nociceptive insult exhibited increased local cerebral blood flow in the somatosensory cortex, hyperalgesia, and decreased exploratory behaviors. Based on these observations, we tested recombinant human erythropoietin (rhEPO) as a potential treatment to prevent the inflammatory pain-induced changes. rhEPO treatment (5,000 U/kg/day, i.p.), coupled to formalin injections, ameliorated neuronal cell death and normalized the inflammatory response. Rats that received formalin plus rhEPO exhibited normal levels of cerebral blood flow, pain sensitivity and exploratory behavior. Treatment with rhEPO also restored normal brain and body weights that were reduced in the formalin group. These data suggest that severe inflammatory pain has adverse effects on brain development and rhEPO may be a possible therapy for the prevention and treatment of this developmental disorder.
doi:10.1186/1744-8069-7-51
PMCID: PMC3161880  PMID: 21777449
pain; erythropoietin; neonates; inflammatory; cerebral blood flow; cell death
25.  Dual roles of tumor necrosis factor-α receptor-1 in a mouse model of hindlimb ischemia 
Signals in the tumor necrosis factor α (TNF-α) pathway are upregulated after ischemia, yet its role in peripheral ischemia remains unclear. We investigated the effect of TNF-α receptor 1 (TNFR-1) in acute limb ischemia of TNFR-1 knockout (TNFR-1−/−) and wild type (WT, TNFR-1+/+) mice. Laser Doppler scanning showed that although pre-ischemia blood flow levels were similar in these mice, the limb reperfusion after ischemia was significantly higher in TNFR-1−/− mice 1–7 days after injury. Consistently, fewer TUNEL-positive cells, less DNA fragmentation, and a lower ischemic score were detected in the TNFR-1−/− group when compared to WT controls. Western blot analysis revealed less expression of pro-apoptotic markers Bax and cleaved caspase-3 in TNFR-1−/− mice 1 day after ischemia, supporting the hypothesis that the absence of TNFR-1 results in a reduction of apoptosis. The rate of post-ischemia amputation was 50% in WT mice versus 0% in TNFR-1−/− mice. However, immunohistochemical co-staining of microvessel marker CD31 and cellular proliferation marker BrdU 21 days after ischemia showed an impaired angiogenic activity in the TNFR-1−/− mice. These data were supported by Western blot analysis, which indicated a decreased expression of angiopoietin-1 (Ang-1) and its receptor Tie-2 in TNFR-1−/− mice. Our results suggest that a deficiency in TNFR-1 prevents the activation of death-related proteins downstream to TNF-α and attenuates apoptosis in acute limb ischemia, but the lack of TNFR-1 signaling hinders the belated angiogenesis mediated by the Ang-1/Tie-2 pathway.
doi:10.1177/1358863X08098143
PMCID: PMC3107068  PMID: 19144778
angiogenesis; apoptosis; peripheral ischemia; TNFR-1

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