Shijie Song

Expression of neural markers in human umbilical cord blood.

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain.

Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III β-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.

Oxidative DNA damage in the aging mouse brain

The brain exhibits regional vulnerabilities to many insults, and age itself has differential effects on neuronal populations as exemplified by the age‐dependent loss of dopaminergic neurons in the nigrostriatal system. We hypothesized that oxidative damage to DNA was more likely to occur in the nigrostriatal system which undergoes significant neurochemical and functional changes with age. To test this hypothesis, oxidative damage to DNA, indicated by levels of 8‐hydroxy‐2′‐deoxyguanosine (oxo8dG), was measured in pons‐medulla (PM), midbrain (MB), caudate‐putamen (CP), hippocampus (HP), cerebellum (CB), and cerebral cortex (CX) at 3, 18, and 34 months of age in C57/bl mice. Steady‐state levels of oxo8dG increased significantly with age in MB, CP, and CB, but not in PM, HP, or CX. Manganese superoxide dismutase (MnSOD) activity decreased with age in MB, CP, and HP, but not in PM, CB, or CX. Regional activities of Cu/Zn superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (Glut Px) did not change significantly with age. Concomitant with the regional alterations in DNA damage, there was a significant age‐dependent decline in locomotor activity, motor coordination, and striatal dopamine content especially during the interval between 18 and 34 months. In conclusion, oxyradical‐associated damage to DNA did not accumulate uniformly across brain regions with age and was highest in brain regions that subserve spontaneous locomotor activity and motor coordination.

Improving solubility and pharmacokinetics of meloxicam via multiple-component crystal formation.

Meloxicam is a nonsteroidal anti-inflammatory drug prescribed for rheumatoid arthritis, osteoarthritis, postoperative pain and fever. Meloxicam exhibits low solubility in acidic aqueous media and a slow onset of action in biological subjects. An oral dosage form of meloxicam with enhanced aqueous solubility is desired to enable a faster onset of action and its use for mild-to-medium-level acute pain relief. With this in mind, we examine the solubility and pharmacokinetics of 12 meloxicam cocrystals with carboxylic acids. Dissolution studies of meloxicam and its cocrystals were performed in pH 6.5 phosphate buffer solutions at 37 °C. In addition, pharmacokinetic profiles over four hours were acquired after oral administration of a 10 mg/kg (meloxicam equivalent) solid suspension in rats. The majority of meloxicam cocrystals were found to achieve higher meloxicam concentrations in dissolution media and enhanced oral absorption compared to that of pure meloxicam. All meloxicam cocrystals were converted to meloxicam form I when the slurry reached equilibrium. To better understand how cocrystallization impacts the absorption of meloxicam after oral administration, correlations between the in vitro and in vivo data were explored. The results suggest that the meloxicam cocrystals with a faster dissolution rate would exhibit increased oral absorption and an earlier onset of action.

Combination Therapy of Human Umbilical Cord Blood Cells and Granulocyte Colony Stimulating Factor Reduces Histopathological and Motor Impairments in an Experimental Model of Chronic Traumatic Brain Injury

Traumatic brain injury (TBI) is associated with neuro-inflammation, debilitating sensory-motor deficits, and learning and memory impairments. Cell-based therapies are currently being investigated in treating neurotrauma due to their ability to secrete neurotrophic factors and anti-inflammatory cytokines that can regulate the hostile milieu associated with chronic neuroinflammation found in TBI. In tandem, the stimulation and mobilization of endogenous stem/progenitor cells from the bone marrow through granulocyte colony stimulating factor (G-CSF) poses as an attractive therapeutic intervention for chronic TBI. Here, we tested the potential of a combined therapy of human umbilical cord blood cells (hUCB) and G-CSF at the acute stage of TBI to counteract the progressive secondary effects of chronic TBI using the controlled cortical impact model. Four different groups of adult Sprague Dawley rats were treated with saline alone, G-CSF+saline, hUCB+saline or hUCB+G-CSF, 7-days post CCI moderate TBI. Eight weeks after TBI, brains were harvested to analyze hippocampal cell loss, neuroinflammatory response, and neurogenesis by using immunohistochemical techniques. Results revealed that the rats exposed to TBI treated with saline exhibited widespread neuroinflammation, impaired endogenous neurogenesis in DG and SVZ, and severe hippocampal cell loss. hUCB monotherapy suppressed neuroinflammation, nearly normalized the neurogenesis, and reduced hippocampal cell loss compared to saline alone. G-CSF monotherapy produced partial and short-lived benefits characterized by low levels of neuroinflammation in striatum, DG, SVZ, and corpus callosum and fornix, a modest neurogenesis, and a moderate reduction of hippocampal cells loss. On the other hand, combined therapy of hUCB+G-CSF displayed synergistic effects that robustly dampened neuroinflammation, while enhancing endogenous neurogenesis and reducing hippocampal cell loss. Vigorous and long-lasting recovery of motor function accompanied the combined therapy, which was either moderately or short-lived in the monotherapy conditions. These results suggest that combined treatment rather than monotherapy appears optimal for abrogating histophalogical and motor impairments in chronic TBI.

Effects of psilocybin on hippocampal neurogenesis and extinction of trace fear conditioning.

Drugs that modulate serotonin (5-HT) synaptic concentrations impact neurogenesis and hippocampal (HPC)-dependent learning. The primary objective is to determine the extent to which psilocybin (PSOP) modulates neurogenesis and thereby affects acquisition and extinction of HPC-dependent trace fear conditioning. PSOP, the 5-HT2A agonist 25I-NBMeO and the 5-HT2A/C antagonist ketanserin were administered via an acute intraperitoneal injection to mice. Trace fear conditioning was measured as the amount of time spent immobile in the presence of the conditioned stimulus (CS, auditory tone), trace (silent interval) and post-trace interval over 10 trials. Extinction was determined by the number of trials required to resume mobility during CS, trace and post-trace when the shock was not delivered. Neurogenesis was determined by unbiased counts of cells in the dentate gyrus of the HPC birth-dated with BrdU co-expressing a neuronal marker. Mice treated with a range of doses of PSOP acquired a robust conditioned fear response. Mice injected with low doses of PSOP extinguished cued fear conditioning significantly more rapidly than high-dose PSOP or saline-treated mice. Injection of PSOP, 25I-NBMeO or ketanserin resulted in significant dose-dependent decreases in number of newborn neurons in hippocampus. At the low doses of PSOP that enhanced extinction, neurogenesis was not decreased, but rather tended toward an increase. Extinction of “fear conditioning” may be mediated by actions of the drugs at sites other than hippocampus such as the amygdala, which is known to mediate the perception of fear. Another caveat is that PSOP is not purely selective for 5-HT2A receptors. PSOP facilitates extinction of the classically conditioned fear response, and this, and similar agents, should be explored as potential treatments for post-traumatic stress disorder and related conditions.

Insulin Promotes Neuronal Survival via the Alternatively Spliced Protein Kinase CδII Isoform

Background: Alternatively spliced PKCδII is a pro-survival protein. Results: Insulin regulates alternative splicing of PKCδII pre-mRNA, which promotes Bcl2 and bcl-xL expression. Conclusion: PKCδII is a key regulator of insulin-mediated neuronal survival. Significance: Elucidation of the molecular mechanisms by which insulin promotes survival in neuronal cells is necessary to understand how intranasal insulin improves cognition. Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood

The bone marrow is clearly much more than a reservoir of stem cells that repopulates blood cell lineages throughout life. The marrow also contains nonhematopoietic stem cells, which are much more versatile than previously appreciated. These nonhematopoietic stem/progenitor cells are found in the bone marrow stromal cell (BMSC) population. BMSCs also are known as colony-forming unit fibroblasts and mesenchymal stem cells (MSCs). MSCs also can be generated from umbilical cord blood and other tissues. MSCs have been shown to express properties of neuroectodermal cells in vitro by many researchers and in vivo after transplantation into the brain and spinal cord. Many investigators have developed variations on the original method described 6 years ago for the preparation of neural progenitors from BMSCs. We bring up to date the materials and procedures used to prepare BMSCs from bone marrow and from human umbilical cord blood for the induction of neural progenitor cells and subsequent differentiation into neurons and glia.

Effects of MDMA (“ecstasy”) during adolescence on place conditioning and hippocampal neurogenesis

The use of 3,4,methylenedioxymethamphetamine (MDMA), the active agent in ecstasy, during adolescence is widespread yet the effects on adolescent behavior and brain development are unknown. The aim of the present study was 1) to evaluate effects of MDMA in adolescent rats using the conditioned place preference (CPP) paradigm to measure MDMA-induced reward and 2) assess effects of MDMA administration on cellular proliferation, survival and neurogenesis in the dentate gyrus of the hippocampus. During the adolescent period, MDMA CPP was measured in adolescents [postnatal day (PND) 28-39] by training rats to associate 1.25, 2.5, 5.0mg/kg MDMA or saline administration with environmental cues. After CPP ended, bromodeoxyuridine (BrdU) was injected and rats were euthanized either 24h (to evaluate cell proliferation) or 2 weeks (to assess neurogenesis) after the last MDMA injection. Adolescents expressed a CPP for 2.5mg/kg MDMA. Repeated exposure to 5.0mg/kg MDMA during adolescence increased cell proliferation, yet diminished neurogenesis, an effect that was replicated using flow cytometry. These findings suggest differential dose effects of adolescent MDMA exposure on reward related behaviors and hippocampal neurogenesis.

Vitamin A Metabolite, All-trans-retinoic Acid, Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cδ (PKCδ), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCδ, PKCδVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCδVIII via utilization of a downstream 5′ splice site of exon 10 on PKCδ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCδVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCδVIII alternative splicing. To identify the cis-elements involved in 5′ splice site selection we cloned a minigene, which included PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5′ splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCδ minigene promoted selection of 5′ splice site II. Mutation of the SC35 binding site in the PKCδ minigene abolished RA-mediated utilization of 5′ splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCδ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCδ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCδVIII.