Wei-Bin Shen

Environmental neurotoxin-induced progressive model of parkinsonism in rats

Exposure to a number of drugs, chemicals, or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant Cycas micronesica by the Chamorro population of Guam and the development of amyotrophic lateral sclerosis/parkinsonism dementia complex.

Sex differences in GABA turnover and glutamic acid decarboxylase (GAD65 and GAD67) mRNA in the rat hypothalamus

GABAergic neurons are estimated to make up more than half of the neuronal population of the hypothalamus and they likely account for some of the structural and functional sexual dimorphisms observed in the mammalian brain. We previously reported sex differences in the rate of GABA turnover in discrete hypothalamic structures of adult rats. In the present study, we extended our search for sex differences in GABA turnover to additional structures, and further determined whether these differences were associated with differences in GAD(65) and or GAD(67) mRNA levels. Utilizing the GABA transaminase inhibition method, we determined GABA turnover in 14 microdissected brain regions. The rate of GABA turnover was about 2-fold greater in male than in diestrous day one (D(1)) female rats in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], anteroventral periventricular nucleus (AVPv), median eminence (ME), and dorsomedial portion of the ventromedial nucleus (VMNdm). A sex difference also was noted in the DBB(ovlt) for GAD(65) mRNA determined by microlysate RNase protection assay. Here, GAD(65) levels were almost 2-fold greater in male rats, which suggests that differences in the activity of this GAD enzyme isoform contributes to the difference in turnover in this area. Additionally, in the dorsomedial nucleus (DMN), the GAD(65) mRNA level was significantly higher in female rats, and in the medial amygdaloid nucleus (Am), GAD(67) mRNA was higher in male rats. These data reveal striking sexual dimorphisms in the rate of GABA turnover and in GAD mRNA levels in specific populations of hypothalamic GABAergic neurons. The functional relationships between these GABAergic neurons and sexually dimorphic phenotypes associated with these structures, such as gonadotropin secretion, reproductive behaviors, seizure threshold and others, warrant further investigation.

Castration rapidly decreases hypothalamic γ-aminobutyric acidergic neuronal activity in both male and female rats

The postcastration LH response is greater and somewhat more rapid in male than female rats. We have previously demonstrated that hypothalamic gamma-aminobutyric acid (GABA)ergic neuronal activity decreases following gonadectomy in male rats. To investigate whether these same hypothalamic GABA neurons decrease their activity postcastration in female rats, and whether more rapid and or greater postcastration decreases occur in male rats, we determined the timing and magnitude of the postcastration decreases in GABA turnover which are associated with the sexually dimorphic postcastration LH response. Adult male and 4-day cycling female rats were castrated between 0800 and 1000 h (females ovariectomized on diestrus day 1). Serum LH levels increased significantly by 12 h postcastration in both males and females with the magnitude of the increases being 6.2-fold in males and 2.8-fold in females. GABA turnover was determined in 16 microdissected brain structures by the GABA transaminase inhibition method at 0 h (sham-operated controls), 6 h, 12 h and 1, 2, 4 and 6 days postcastration. In male rats, in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], the rate of GABA turnover decreased significantly already by 6 h postcastration compared with the 0 h controls, and remained suppressed through 6 days. This rapid down regulation of DBB(ovlt) GABAergic neurons also occurred in female rats, however, the duration of the decrease was not as prolonged as in male rats. Similar changes occurred in the tuberoinfundibular GABAergic (TIGA) neurons projecting to the median eminence in both males and females. Down regulation of these GABAergic neurons precedes or is coincident with increased postcastration LH secretion in both sexes, and the duration of the decreases is consistent with the less robust postcastration LH response in female rats. In addition, the rate of GABA turnover decreased after castration in the interstitial (bed) nucleus of the stria terminalis, ventral aspect (INSTv), the medial preoptic nucleus, dorsomedial aspect (MPNdm) and the ventromedial nucleus, ventrolateral aspect (VMNvl) in male rats, and in the INSTv and VMNvl of female rats, while there was no effect of castration in other hypothalamic regions or control structures. The result in the female VMNvl is consistent with reports that GABA facilitates lordosis behavior in this hypothalamic structure. These findings are consistent with the hypothesis that discrete hypothalamic populations of sex steroid-sensitive GABAergic neurons mediate the postcastration LH responses in both male and female rats, and may underlie other sexually dimorphic adult phenotypes such as sex behavior.

Human neural progenitor cells retain viability, phenotype, proliferation, and lineage differentiation when labeled with a novel iron oxide nanoparticle, Molday ION Rhodamine B

Ultrasmall superparamagnetic iron-oxide particles (USPIOs) loaded into stem cells have been suggested as a way to track stem cell transplantation with magnetic resonance imaging, but the labeling, and post-labeling proliferation, viability, differentiation, and retention of USPIOs within the stem cells have yet to be determined for each type of stem cell and for each type of USPIO. Molday ION Rhodamine B™ (BioPAL, Worcester, MA, USA) (MIRB) has been shown to be a USPIO labeling agent for mesenchymal stem cells, glial progenitor cells, and stem cell lines. In this study, we have evaluated MIRB labeling in human neuroprogenitor cells and found that human neuroprogenitor cells are effectively labeled with MIRB without use of transfection reagents. Viability, proliferation, and differentiation properties are unchanged between MIRB-labeled neuroprogenitors cells and unlabeled cells. Moreover, MIRB-labeled human neuroprogenitor cells can be frozen, thawed, and replated without loss of MIRB or even without loss of their intrinsic biology. Overall, those results show that MIRB has advantageous properties that can be used for cell-based therapy.

Type 2 diabetes mellitus induces congenital heart defects in murine embryos by increasing oxidative stress, endoplasmic reticulum stress, and apoptosis

BACKGROUNDnMaternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects remain largely unknown.nnnOBJECTIVEnWe aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus-induced congenital heart defects.nnnSTUDY DESIGNnA mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination.nnnRESULTSnMaternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin protein; endoplasmic reticulum chaperone gene expression; and XBP1 messenger RNA splicing, as well as increased cleaved caspase 3 and 8 in embryonic hearts. Furthermore, maternal type 2 diabetes mellitus triggered excessive apoptosis in ventricular myocardium, endocardial cushion, and outflow tract of the embryonic heart.nnnCONCLUSIONnSimilar to those observations in type 1 diabetic embryopathy, maternal type 2 diabetes mellitus causes heart defects in the developing embryo manifested with oxidative stress, endoplasmic reticulum stress, and excessive apoptosis in heart cells.

Development of the perforating pathway: An ipsilaterally projecting pathway between the medial septum/diagonal band of Broca and the cingulate cortex that intersects the corpus callosum

The perforating pathway (PFP) intersects the corpus callosum perpendicularly at the midline in the dorsoventral axis. Therefore axons in either the PFP or the corpus callosum make different axonal guidance decisions in the same anatomical region of the developing cortical midline. The mechanisms underlying these axonal choices are not known. To begin to identify these guidance mechanisms, we characterized the development of these two pathways in detail. The development of the corpus callosum and its pioneering projections has been described elsewhere (Shu and Richards [2001] J. Neurosci. 21:2749–2758; Rash and Richards [2001] J. Comp. Neurol. 434:147–157). Here we examine the development, origins, and projections of axons that make up the PFP. The majority of axons within the PFP originate from neurons in the medial septum and diagonal band of Broca complex. These neurons project in a topographic manner to the cingulate cortex. In contrast to previous reports, we find that a much smaller projection originating from the cingulate cortex also contributes to this pathway. The pioneering projections of the PFP and the corpus callosum arrive at the corticoseptal boundary at around the same developmental stage. These findings show that ipsilaterally projecting PFP axons and contralaterally projecting callosal axons make distinct guidance decisions at the same developmental stage when they reach the corticoseptal boundary. J. Comp. Neurol. 436:411–422, 2001.

Induction of neural differentiation by the transcription factor neuroD2.

Pro‐neural basic helix loop helix (bHLH) transcription factors are involved in many aspects of normal neuronal development, and over‐expression of genes for several of these factors has been shown to induce aspects of neuronal differentiation in cell lines and stem cells. Here we show that over‐expression of NeuroD2 (ND2), Neurogenin1 and 2 leads to morphological differentiation of N18‐RE‐105 neuroblastoma cells and increased expression of synaptic proteins. Particularly ND2 induced neurite formation and increases in the expression of synaptic proteins such as synaptotagmin, that is not expressed normally in this cell type, as well as the redistribution of another synaptic protein, SNAP25, to a cell membrane location. Infection of human neural progenitor cells using adeno associated viral (AAV) vectors also promoted neuronal differentiation. Over‐expressing cells demonstrated a significant increase in the neuron specific form of tubulin as well as increased expression of synaptotagmin. Genetic modification of neural progenitor cell with bHLH factors such as ND2 may be a viable strategy to enhance differentiation of these cells into replacement neurons for human disease.

Cell-Based Therapy in TBI: Magnetic Retention of Neural Stem Cells In Vivo.

Stem cell therapy is under active investigation for traumatic brain injury (TBI). Noninvasive stem cell delivery is the preferred method, but retention of stem cells at the site of injury in TBI has proven challenging and impacts effectiveness. To investigate the effects of applying a magnetic field on cell homing and retention, we delivered human neuroprogenitor cells (hNPCs) labeled with a superparamagnetic nanoparticle into post-TBI animals in the presence of a static magnetic field. We have previously devised a method of loading hNPCs with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles Molday ION Rhodamine B (MIRB™). Labeling of hNPCs (MIRB-hNPCs) does not affect hNPC viability, proliferation, or differentiation. The 0.6 tesla (T) permanent magnet was placed ~4 mm above the injured parietal cortex prior to intracarotid injection of 4 × 104 MIRB-hNPCs. Fluorescence imaging, Perls Prussian blue histochemistry, immunocytochemistry with SC121, a human-specific antibody, and T2-weighted magnetic resonance imaging ex vivo revealed there was increased homing and retention of MIRB-hNPCs in the injured cortex as compared to the control group in which MIRB-hNPCs were injected in the absence of a static magnetic field. Fluoro-Jade C staining and immunolabeling with specific markers confirmed the viability status of MIRB-hNPCs posttransplantation. These results show that increased homing and retention of MIRB-hNPCs post-TBI by applying a static magnetic field is a promising technique to deliver cells into the CNS for treatment of neurological injuries and neurodegenerative diseases.

High glucose suppresses embryonic stem cell differentiation into neural lineage cells

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25xa0mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5xa0mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14xa0cells. Under low glucose conditions, the GR-E14xa0cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14xa0cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14xa0cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14xa0cell line is a useful inxa0vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright