Samuel Sam Wah Tay

Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After Hypoglossal Nerve Injury

Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal‐cell‐derived factor 1 (SDF‐1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF‐1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF‐1 alpha (rhSDF‐1α) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G‐protein‐dependent manner. Intracerebral injection of rhSDF‐1α has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine‐CX3CR1 and SDF‐1–CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.

Retinoic acid inhibits expression of TNF-α and iNOS in activated rat microglia

The release of proinflammatory mediators such as tumor necrosis factor‐α (TNF‐α) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimers disease. As all‐trans‐retinoic acid (RA) has been reported to exert anti‐inflammatory actions in various cell types, we have examined its effects on the expression of TNF‐α and inducible nitric oxide synthase (iNOS) in microglia activated by β‐amyloid peptide (Aβ) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Aβ or LPS stimulated the mRNA expression level of TNF‐α (6–116‐fold) and iNOS (8–500‐fold) significantly. RA acted in a dose‐dependent manner (0.1–10 μM) by attenuating both TNF‐α (29–97%) and iNOS (61–96%) mRNA expression in microglia exposed to Aβ or LPS. RA‐induced inhibition of TNF‐α and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF‐α proteins as revealed by nitrite assay and ELISA, respectively. The anti‐inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor‐β, and transforming growth factor‐β1 as well as the inhibition of NF‐κB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.

Expression of chemokine receptors CXCR4, CCR2, ccr5 and CX3CR1 in neural progenitor cells isolated from the subventricular zone of the adult rat brain

We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.

Mesenchymal stem cell transplantation attenuates blood brain barrier damage and neuroinflammation and protects dopaminergic neurons against MPTP toxicity in the substantia nigra in a model of Parkinson's disease

Immunomodulatory effects of transplanted mesenchymal stem cells (MSCs) in the treatment of Parkinsons disease were studied in the MPTP-induced mouse model. MPTP treatment induced a significant loss of dopaminergic neurons, decreased expressions of claudin 1, claudin 5 and occludin in the substantia nigra compacta (SNc), and functional damage of the blood brain barrier (BBB). Our study further discovered that infiltration of MBLs into the brain to bind with microglia was detected in the SNc of MPTP-treated mice, suggesting that the BBB compromise and MBL infiltration might be involved in the pathogenesis of MPTP-induced PD. In addition, MPTP treatment also increased the expression of mannose-binding lectins (MBLs) in the liver tissue. Intravenous transplantation of MSCs into MPTP-treated mice led to recovery of BBB integrity, suppression of MBL infiltration at SNc and MBL expression in the liver, suppression of microglial activation and prevention of dopaminergic neuron death. No transplanted MSCs were observed to differentiate into dopaminergic neurons, while the MSCs migrated into the SNc and released TGF-beta1 there. Therefore, intravenous transplantation of MSCs which protect dopaminergic neurons from MPTP toxicity may be engaged in anyone or a combination of these mechanisms: repair of the BBB, reduction of MBL in the brain, inhibition of microglial cytotoxicity, and direct protection of dopaminergic neurons.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BackgroundCongenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice.Methods and resultsWe analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos.ConclusionIt is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells

Aims/hypothesisMaternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs.Materials and methodsCell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2′-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT–PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically.ResultsHigh glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells.Conclusions/interpretationThe changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

Recent Studies on Neural Tube Defects in Embryos of Diabetic Pregnancy: An Overview

Maternal diabetes develops in 2-6% of total pregnancies, depending on geographical and ethnic background. About 10% of fetuses from diabetic pregnancy display congenital malformations in various organ systems including cardiovascular, gastrointestinal, genitourinary and neurological systems, among which the neural tube defects (NTDs) such as anencephaly, holoprosencephaly and syntelencephaly were more frequently demonstrated. Recent studies by the Diabetes Control and Complications Trial Research Group show that tight glycemic control early in pregnancy decreases the progression of a number of diabetic complications. However, it appears that the pre-existing tissue damage cannot be reversed even after normoglycemic levels are achieved during pregnancy. In recent years, considerable efforts have been made to investigate the etiology of birth defects among infants of diabetic mothers. It has been shown that diabetes-induced fetal abnormalities are accompanied by some metabolic disturbances including elevated superoxide dismutase (SOD) activity, reduced levels of myoinositol and arachidonic acid and inhibition of the pentose phosphate shunt pathway. Moreover, the frequency of fetal malformations in diabetic pregnancy has been reported to be markedly reduced by dietary supplements of antioxidants such as vitamin E, vitamin C and butylated hy- droxytoluene, suggesting that oxidative stress is involved in the etiology of fetal dysmorphogenesis. Furthermore, several experimental studies have shown that NTDs in embryos of diabetic mice are associated with altered expression of genes, which control development of the neural tube. In this review, recent findings of possible molecular mechanisms which cause morphological changes during neural tube development in embryos of diabetic pregnancy are discussed.

Downregulation of miR-124 in MPTP-treated mouse model of Parkinson's disease and MPP iodide-treated MN9D cells modulates the expression of the calpain/cdk5 pathway proteins.

Parkinsons disease (PD) is a debilitating neurodegenerative disorder causing severe motor disabilities resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) region of the midbrain. MicroRNAs (miRNAs) are small, non-coding RNAs which play a major role in several cellular processes in health and disease by regulating gene expression post-transcriptionally. Aberrant miRNA expression has been detected in post-mortem human PD brain samples, in vitro and in vivo PD models. However, none of the studies have focused on the role of the brain-abundant miR-124 in PD. In this study, we have evaluated the expression changes of miR-124 in the SN of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. MiRNA expression analysis by qPCR revealed a decrease in the expression of brain-enriched miR-124 in the SN of MPTP-treated mice as compared to controls. Further, in vitro study revealed a decrease in the expression of miR-124 in MN9D dopaminergic neurons treated with methyl phenyl pyridinium (MPP) iodide. The expression of calpains 1 and 2 which is modulated by miR-124 was increased in the SNc of MPTP-treated mice as observed at different time points after treatment and in the MN9D dopaminergic neurons treated with MPP iodide leading to increased expression of the p35 cleavage product, p25 and cyclin-dependent kinase 5 (cdk5). Calpain-p25-mediated increase in cdk5 expression leading to dopaminergic neuronal death has been demonstrated in human PD and MPTP-PD models. Increased expression of calpain 1/cdk5 pathway proteins was observed in anti-miR-124-transfected MN9D cells in our studies. Knockdown of miR-124 led to increased production of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) both known to increase oxidative stress. Further, experiments with miR-124 target protector sequences specific to calpain 1 revealed an interaction of miR-124 with calpain 1. Overexpression of miR-124 after MPP iodide treatment on MN9D cells was found to attenuate the expression of the calpain 1/p25/cdk5 proteins while improving cell survival. These results suggest that miR-124 acts to modulate the expression of calpain/cdk5 pathway proteins in the dopaminergic neurons. A better understanding of the mechanisms controlling the expression of miR-124 will aid in targeting miR-124 for better treatment strategies for PD.

Global gene expression analysis of cranial neural tubes in embryos of diabetic mice.

Maternal diabetes causes congenital malformations in various organs including the neural tube in fetuses. In this study, we have analyzed the differential gene expression profiling in the cranial neural tube of embryos from diabetic and control mice by using the oligonucleotide microarray. Expression patterns of genes and proteins that are differentially expressed in the cranial neural tube were further examined by the real‐time reverse transcriptase–polymerase chain reaction, in situ hybridization, and immunohistochemistry. Proliferation index and apoptosis were examined by BrdU (5‐bromo‐2‐deoxyuridine) labeling and TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labeling) assay, respectively. Embryos (E11.5) of diabetic pregnancies displayed distortion in neuroepithelia of the cranial neural tube. Microarray analysis revealed that a total of 390 genes exhibited more than twofold changes in expression level in the cranial neural tube of embryos from diabetic mice. Several genes involving apoptosis, proliferation, migration, and differentiation of neurons in the cranial neural tube were differentially expressed in embryos of diabetic pregnancy. In addition, maternal diabetes perturbed the development of choroid plexus and ventricular systems and reduced the production of proteins such as Ttr and Igf2 in the developing brain, indicating that these changes could impair the survival and proliferation of neuroepithelial cells and neurogenesis in embryos of diabetic mice. It is concluded that altered expression of a variety of genes involved in brain development is associated with cranial neural tube dysmorphogenesis that may subsequently contribute to intellectual impairment of the offspring of a diabetic mother.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.