Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

Biological characterization of human amniotic epithelial cells in a serum-free system and their safety evaluation.

Human amniotic epithelial cells (hAECs), derived from the innermost layer of the term placenta closest to the fetus, have been shown to be potential seed cells for allogeneic cell therapy. Previous studies have shown a certain therapeutic effect of hAECs. However, no appropriate isolation and culture system for hAECs has been developed for clinical applications. In the present study, we established a serum-free protocol for hAEC isolation and cultivation, in which better cell growth was observed compared with that in a traditional culture system with serum. In addition to specific expression of cell surface markers (CD29, CD166 and CD90), characterization of the biological features of hAECs revealed expression of the pluripotent markers SSEA4, OCT4 and NANOG, which was greater than that in human mesenchymal stem cells, whereas very low levels of HLA-DR and HLA-DQ were detected, suggesting the weak immunogenicity of hAECs. Intriguingly, CD90+ hAECs were identified as a unique population with a powerful immunoregulatory capacity. In a systemic safety evaluation, intravenous administration of hAEC did not result in hemolytic, allergy, toxicity issues or, more importantly, tumorigenicity. Finally, the therapeutic effect of hAECs was demonstrated in mice with radiation-induced damage. The results revealed a novel function of hAECs in systemic injury recovery. Therefore, the current study provides an applicable and safe strategy for hAEC cell therapy administration in the clinical setting.

Cynomorium songaricum extracts functionally modulate transporters of gamma-aminobutyric acid and monoamine.

Cynomorium songaricum Rupr. (SY) is a central nervous system-oriented herb material that has actions of anti-dementia, anti-epilepsy, and anti-stress. It is unclear whether SY would be biologically active in functionally regulating neurotransmitter transporters. Here, we assessed these potential actions using Chinese hamster ovary cells expressing gamma-aminobutyric acid (GABA) transporter (GAT-1), dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. G1, D8, N1, or S6 cells, respectively). It was shown that SY extracts, such as SYw, SYa, SYp, SYc, SYe, and SYb (SY water, ethanol, petroleum ether, chloroform, ethyl acetate, and n-butyl alcohol extract, respectively) increased dopamine/norepinephrine (DA/NE) uptake by corresponding D8/N1 cells and decreased gamma-aminobutyric acid/serotoin (GABA/5HT) uptake by corresponding G1/S6 cells; wherein, the potency or efficacy of SYc for up-regulating DA/NE uptake and that of SYb for inhibiting GABA/5HT uptake were relatively stronger. Additionally, GABA/5H-uptake inhibition by SY extracts were also seen in cortical synaptosomes, and DA/NE-uptake enhancement by SYc was dependent on the activity of DAT and NET. Thus, SY extracts especially SYc and SYb are novel neurotransmitter-transporter modulators functioning as DAT/NET activators and/or GAT-1/SERT inhibitors.

Structural identification of a new tri-p-coumaroylspermidine with serotonin transporter inhibition from safflower.

We previously reported that safflower (Carthamus tinctorius L.) ethyl acetate extract (HE) possessed an inhibitory action on serotonin (5HT) uptake in Chinese hamster ovary (CHO) cells expressing 5HT transporter (SERT) (S6 cells). Here, HE was adopted to go through an activity-guided isolation, and then an ingredient with potent SERT inhibitory action was obtained, which was elucidated as N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (CX), a new coumaroylspermidine analog, by using spectroscopic methods including extensive 1D- and 2D-NMR analyses. Preliminary pharmacological study demonstrated that CX was a potent SERT inhibitor.

Caulis Sinomenii extracts activate DA/NE transporter and inhibit 5HT transporter.

Caulis Sinomenii (QFT) has analgesic, sedative, and anxiolytic-like actions, and is proven effective for improving drug dependence that is known to be associated with abnormal monoaminergic transmission. We assessed whether QFT would be biologically active in functionally regulating monoamine transporters using CHO cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. D8, N1, or S6 cells, respectively). Here, we showed that its primary extracts, such as QA, QC, QE, QD, and QB (QFT ethanol, chloroform, ethyl acetate, alkaloid-free chloroform, and alkaloid-containing chloroform extract, respectively), and secondary extracts, such as QE-2, - 3, - 5, - 7, QD-1, - 2, - 3, - 4, - 5, and QB-1, - 2, - 3, - 4, - 5 (fractioned from QE, QD, and QB, respectively), in differing degrees, either increased DA/ NE uptake by corresponding D8/N1 cells or decreased 5HT uptake by S6 cells; wherein, QE-2, QD-3, and QE-7 were potent DA/NE uptake activators while both QE-7 and QB-5 were potent 5HT uptake inhibitors. Furthermore, the enhancement of DA/NE uptake was dependent of DAT/NET activity, and the inhibition of 5HT uptake was typical of competition. Thus, QFT extracts, especially QE-2 and QE-7 (both with stronger potencies), are novel monoamine transporter modulators functioning as DAT/ NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine transporters.

Dopamine transporter inhibitory and antiparkinsonian effect of common flowering quince extract.

Common flowering quince (FQ) is the fruit of Chaenomeles speciosa (Sweet) Nakai. FQ-containing cocktails have been applied to the treatment of neuralgia, migraine, and depression in traditional Chinese medicine. The present study assessed whether FQ is effective in dopamine transporter (DAT) regulation and antiparkinsonism by utilizing in vitro and in vivo assays, respectively. FQ at concentrations of 1-1000 microg/ml concentration-dependently inhibited dopamine uptake by Chinese hamster ovary (CHO) cells stably expressing DAT (D8 cells) and by synaptosomes. FQ had a slight inhibitory action on norepinephrine uptake by CHO cells expressing the norepinephrine transporter and no inhibitory effect on gamma-aminobutyric acid (GABA) uptake by CHO cells expressing GABA transporter-1 or serotonin uptake by the serotonin transporter. A viability assay showed that FQ mitigated 1-methyl-4-phenylpyridinium-induced toxicity in D8 cells. Furthermore, in behavioral studies, FQ alleviated rotational behavior in 6-hydroxydopamine-treated rats and improved deficits in endurance performance in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Furthermore, immunohistochemistry revealed that FQ markedly reduced the loss of tyrosine hydroxylase-positive neurons in the substantia nigra in MPTP-treated mice. In summary, FQ is a selective, potent DAT inhibitor and has antiparkinsonian-like effects that are mediated possibly by DAT suppression. FQ has the potential to be further developed for Parkinson's disease treatment.

Δ3,2-Hydroxybakuchiol Attenuates Depression in Multiple Rodent Models Possibly by Inhibition of Monoamine Transporters in Brain.

Δ3,2-Hydroxybakuchiol is isolated from Psoralea corylifolia (L.), which has therapeutic applications in traditional Chinese medicine. Our previous studies have showed that Δ3,2-hydroxybakuchiol inhibited the decreased activity of reserpinized mice, suggestive of its antidepressive potential. In this study, we explored the antidepressant profile of Δ3,2-hydroxybakuchiol in various rodent models and its possible monoamine-modulating mechanism. Δ3,2-Hydroxybakuchiol significantly reduced immobility time of mice in forced swim test and tail suspension test. Δ3,2-Hydroxybakuchiol also significantly increased sucrose consumption in chronic unpredictable mild stress (CUMS) rat model. Furthermore, isotope uptake study showed that Δ3,2-hydroxybakuchiol inhibited the activity of human dopamine transporter (DAT) and norepinephrine transporter (NET) in transporter-overexpressing pheochromocytoma (PC12) cells with IC50 values similar to the potency of bupropion. Microdialysis showed that Δ3,2-hydroxybakuchiol increased dopamine and norepinephrine concentration in rat striatum. In summary, Δ3,2-hydroxybakuchiol exerts antidepressant effects on various types of depression models through a possible mechanism of monoamine transporter inhibition.

Inhibitive effects of Fructus Psoraleae extract on dopamine transporter and noradrenaline transporter.

A petroleum ether extract (FP) from Fructus Psoraleae, seeds of Psoralea corylifolia L. (Leguminosae), was found to strongly inhibit dopamine (DA) uptake by dopamine transporter (DAT) heterogeneously expressed cells (D8 cells) and noradrenaline (NE) uptake by noradrenaline transporter (NET) heterogeneously expressed cells, which, however, had no effect on gamma-aminobutyric acid transporter heterogeneously expressed cells and serotonin transporter heterogeneously expressed cells at the concentration up to 100 microg/ml. These inhibitory effects were also confirmed by experiments on SK-N-SH cell line and synaptosomes from rats' brains. In addition, FP showed a significantly mitigating effect on 1-methyl-4-pyridinium induced injury of D8 cells. Meanwhile, FP dose-dependently reduced the binding of tritium-labeled cocaine analog (-)-2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane to DAT of D8 cells, which suggests that FP may inhibit DAT activity in the same way as cocaine does. Behavioral study showed FP had a long-lasting stimulant effects on the activity of intact mice and reserpinized mice. So FP is proposed as a kind of DAT and NET inhibitor and may be involved in the process of regulating the DA and NE system, and FP or its unknown bioactive compounds may be developed into new medicines for disorders such as Parkinson's disease, depression, Attention Deficit Hyperactivity Disorder (ADHD) or cocaine addiction.

[Construction of bicistronic lentiviral vectors carrying HSV-tk and EGFP gene].

OBJECTIVE: To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease. METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were co-transfected with the lentiviral vector (three plasmids) by calcium phosphate DNA precipitation. HLEC, HXO-Rb(44), SH-SY-5Y and Hela cells were transfected with viral production and the expression of EGFP was examined under fluorescent microscope after transfection. The expression of HSV-tk and EGFP was examined by RT-PCR. RESULTS: Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfection efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months. CONCLUSION: The bicistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.

[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells].

OBJECTIVE: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. METHODS: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated. RESULT: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect). CONCLUSION: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

Protopine inhibits serotonin transporter and noradrenaline transporter and has the antidepressant-like effect in mice models.

The protopine isolated from a Chinese herb Dactylicapnos scandens Hutch was identified as an inhibitor of both serotonin transporter and noradrenaline transporter in vitro assays. 5-hydroxy-DL-tryptophan(5-HTP)-induced head twitch response (HTR) and tail suspension test were adopted to study whether protopine has anti-depression effect in mice using reference antidepressant fluoxetine and desipramine as positive controls. In HTR test, protopine at doses of 5, 10, 20 mg/kg dose dependently increase the number of 5-HTP-induced HTR. Protopine at doses of 3.75 mg/kg, 7.5 mg/kg and 30 mg/kg also produces a dose-dependent reduction in immobility in the tail suspension test. The present results open up new possibilities for the use of protopine in the treatment of mood disorders, such as mild and moderate states of depression.

Transactivated minimal E1b promoter is capable of driving the expression of short hairpin RNA.

The strategy that transcribes short hairpin RNAs (shRNAs) by RNA polymerase II promoters is expected to present flexible approaches for regulating the patterns of shRNA expression. The capacity of generating shRNA by a modified adenovirus RNA polymerase II E1b promoter was studied. This 49bp promoter consists of a TATA-box and an initiation element. It is demonstrated that this modified E1b promoter is capable of driving shRNA transcription and causing either long-term suppression against the target gene in response to the transactivation of constitutively expressed Gal4-VP16 fusion protein or inducible suppression given that the expression of Gal4-VP16 is subject to a dexamethasone inducer.

Transplantation of human amniotic epithelial cells improves hindlimb function in rats with spinal cord injury.

BACKGROUND: Human amniotic epithelial cells (HAECs), which have several characteristics similar to stem cells, therefore could possibly be used in cell therapy without creating legal or ethical problems. In this study, we transplanted HEACs into the injured spinal cord of rats to investigate if the cells can improve the rats' hindlimb motor function. METHODS: HAECs were obtained from a piece of fresh amnion, labeled with Hoechst33342, and transplanted into the site of complete midthoracic spinal transections in adult rats. The rats (n = 21) were randomly divided into three groups: Sham-operation group (n = 7), cells-graft group (n = 7), and PBS group (n = 7). One rat of each group was killed for histological analysis at the second week after the transplantation. The other six rats of each group were killed for histological analysis after an 8-week behavioral testing. Hindlimb motor function was assessed by using the open-field BBB scoring system. Survival rate of the graft cells was observed at second and eighth weeks after the transplantation. We also detected the myelin sheath fibers around the lesions and the size of the axotomized red nucleus. A one-way ANOVA was used to compare the means among the groups. The significance level was set at P < 0.05. RESULTS: The graft HAECs survived for a long time (8 weeks) and integrated into the host spinal cord without immune rejection. Compared with the control group, HAECs can promote the regeneration and sprouting of the axons, improve the hindlimb motor function of the rats (BBB score: cells-graft group 9.0 +/- 0.89 vs PBS group 3.7 +/- 1.03, P < 0.01), and inhibit the atrophy of axotomized red nucleus [cells-graft group (526.47 +/- 148.42) microm(2) vs PBS group (473.69 +/- 164.73) microm(2), P < 0.01]. CONCLUSION: Transplantation of HAECs can improve the hindlimb motor function of rats with spinal cord injury.

CMV-hFasL transgenic mice prevent from experimental autoimmune thyroiditis.

BACKGROUND: Previous studies showed that the role of Fas ligand (FasL) is not consistent in the pathogenesis of autoimmune thyroiditis. This study was designed to investigate the effects of FasL on the pathogenesis of experimental autoimmune thyroiditis (EAT) using CMV-human FasL (hFasL) transgenic mice. METHODS: Transgenic mice ubiquitously expressing hFasL were used as an animal model of EAT by injection of porcine thyroglobulin (pTg). Expression of hFasL was detected by RT-PCR and Western blot. The activity of hFasL transgenic thyrocytes killing Jurket cells was determined. CMV-hFasL transgenic mice and wild type (WT) mice were immunized with pTg and killed 28 days later to evaluate the lymphocytic infiltration of their thyroids. The number of CD4+ and CD8+ lymphocytes from the spleen was detected using FACS. The serum interferon-gamma (IFN-gamma) concentration was measured by ELISA. RESULTS: hFasL expression in the thyroid of CMV-hFasL transgenic mice was confirmed. After co-incubation of Jurket thymocytes with thyroid tissues of CMV-hFasL transgenic mice, the percentage of apoptotic cells in the CMV-hFasL transgenic thyroid group was significantly higher than that of the control WT thyroid group [(23.4 +/- 4.3)% vs (6.6 +/- 2.5)%, P < 0.01]. On day 28 after immunization with pTg, the infiltration index of lymphocytes in thyroids of the CMV-hFasL transgenic mice was significantly lower than that of the WT mice [(1.0 +/- 0.5) vs (2.1 +/- 0.7), P < 0.001]. Moreover, the number of CD4+ and CD8+ lymphocytes of the spleen and serum IFN-gamma concentration were significantly decreased in the CMV-hFasL transgenic mice. CONCLUSIONS: FasL plays an important role in the pathogenesis of autoimmune thyroiditis. Transgenic mice ubiquitously expressing hFasL may strongly inhibit lymphocytic infiltration of the thyroid of EAT and ameliorate the course of this disease.

[Intraventricular transplantation of human neural stem cells into embryonic rats].

OBJECTIVE: To explore the migration and distribution of human neural stem cells (NSCs) after they were transplanted into lateral cerebroventricles of embryonic rats. METHODS: This study was conducted with the proxy consent and the permission from the Hospitals Ethical Review Committee. The cells from the embryonic human cortices were mechanically dissociated. N2 medium was used to culture and expand the cells. And the cells were identified by immunocytochemistry; then after being labelled with BrdU or adenovirus carrying LacZ gene, they were implanted into the lateral cerebroventricles of embryonic rats. The rats were sacrificed after they were born and the brain sections were examined by immunocytochemistry at different time points. RESULTS: NSCs from embryonic humans were successfully cultured; they formed typical neurospheres in suspension, and the majorities of the cells expressed nestin, which was the marker for neural progenitor cells, and the cells could differentiate into neurons, astrocytes and oligodendrocytes. NSCs could steadily expressed exogenous gene in vivo and in vitro; after transplantation into embryonic rat brains, the fetal human NSCs incorporated individually into all major compartments of the brains, generating widespread CNS chimerism and having a wide distribution in the host fore-, mid-, and hindbrain. CONCLUSION: NSCs could express foreign gene steadily and were the ideal cell sources for cell and gene therapy. These cells could incorporate into developing brains and generate widespread chimerism. These chimeras provide a unique model for studying human neural cell migration and differentiation in a functional nervous system.

A mRNA molecule encoding truncated excitatory amino acid carrier 1 (EAAC1) protein (EAAC2) is transcribed from an independent promoter but not an alternative splicing event.

Glutamate transporter EAAC1 removes excitatory neurotransmitter in central nervous system, and also absorbs glutamate in epithelia of intestine, kidney, liver and heart for normal cell growth. When a mouse cDNA was screened using EAAC1 cDNA fragment as probe in our lab, a transcript (GenBank U75214) encoding an EAAC1 protein with 148 residues truncated at N-terminal was cloned and named as EAAC2. Sequence analysis shows that EAAC2 has it's own start code and unique 5'UTR that is different from that of EAAC1. A mouse genomic library was screened and a positive clone including EAAC1 CDS was sequenced (GenBank AF 322393) and indicates that normal EAAC1 transcript (GenBank U73521) is transcribed from 10 exons in terms of exon I, II, III, IV, V, VI, VII, VIII, IX, X, and EAAC2 transcript is consisted by exons from IV to IX as same as that of EAAC1 and with its unique exon beta upstream to exon IV and exon delta downstream to IX. EAAC2 transcript has a cluster of transcriptional start sites not overlapping with the transcriptional start sites of EAAC1. These results indicate that EAAC2 is transcribed from an independent promoter but not an alternative splicing event.

Impaired reproduction in transgenic mice overexpressing Gamma-aminobutyric acid transporter I (GAT1).

It is well documented that g-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis, and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand.

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITC-conjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.

Cognitive impairment in mice over-expressing gamma-aminobutyric acid transporter 1 (GAT1).

There is increasing evidence that GABAergic system plays an important role in the neural control of learning and memory processes. GAT1 over-expressing mice (NA) were generated, in which GAT1 is under the control of a neuron-specific enolase (NSE) promoter, to investigate effects of GABA transporter on cognitive function. Our results revealed that NA mice displayed cognitive deterioration in associative learning ability and new object recognition retention, compared with the wild-type littermates (WT2). However, the impaired cognitive function of transgenic mice could be rescued after chronic administration of GAT1 selective inhibitor for 6 days. In addition, there was no change of the expression of NMDA receptors in NA mice. Taken together, we show a potentially important role for GAT1 in the neural control of cognitive processes, and indicate great potential for GAT1 as a clinical target of cognitive disorders.

Identification of glutamate receptors and transporters in mouse and human sperm.

gamma-Aminobutyric acid (GABA) and glutamate (Glu) are considered as the predominant inhibitory and excitatory neurotransmitters in mammalian central nervous systems (CNS), respectively. The presence of the GABA system and metabotropic glutamate receptors in sperm prompted us to explore the existence of ionotropic glutamate receptors and glutamate transporters in sperm. Immunofluorescent analysis was used to investigate the existence and location of glutamate, glutamate receptor (NR2B), and glutamate transporter (GLT1) in mouse and human sperm. Our present results showed that NR2B was located in the midpiece of sperm, whereas GLT1 mainly existed in the head. Moreover, glutamate uptake activity was detected in mouse sperm and it could be blocked by dihydrokainic acid (DHK, GLT1-selective inhibitor) and DL-threo-beta-hydroxyaspartic acid (THA, nonselective inhibitor). In addition, reverse transcription-polymerase chain reaction technique and sequencing analysis revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6, and KA2) existed in mouse sperm as well as in human sperm. The present findings are the first direct evidence for the existence of ionotropic glutamate receptors and glutamate transporters in sperm. It also indicates that, in sperm, glutamate receptors and transporters might have functions other than neurotransmission.