The molecular microenvironment of the injured spinal cord does not support survival and differentiation of either grafted or endogenous NSCs, restricting the effectiveness of the NSC-based cell replacement strategy. Studying the biology of NSCs in in vivo usually requires a considerable amount of time and cost, and the complexity of the in vivo system makes it difficult to identify individual environmental factors. The present study sought to establish the organotypic spinal cord slice culture that closely mimics the in vivo environment. The cultured spinal cord slices preserved the cytoarchitecture consisting of neurons in the gray matter and interspersed glial cells. The majority of focally applied exogenous NSCs survived up to 4 weeks. Pre-exposure of the cultured slices to a hypoxic chamber markedly reduced the survival of seeded NSCs on the slices. Differentiation into mature neurons was severely limited in this co-culture system. Endogenous neural progenitor cells were marked by BrdU incorporation, and applying an inflammatory cytokine IL-1beta significantly increased the extent of endogenous neural progenitors with the oligodendrocytic lineage. The present study shows that the organotypic spinal cord slice culture can be properly utilized to study molecular factors from the post-injury microenvironment affecting NSCs in the injured spinal cord.
Anoxia ; Biology ; Bromodeoxyuridine ; Cellular Microenvironment ; Coculture Techniques ; Neural Stem Cells ; Neuroglia ; Neurons ; Seeds ; Spinal Cord ; Spinal Cord Injuries ; Stem Cells ; Transplants

Neurite outgrowth and its maintenance are essential aspects of neuronal cells for their connectivity and communication with other neurons. Recent studies showed that over-expression of either leucine-rich repeat kinase 2 (LRRK2), whose mutations are associated with familial Parkinson's disease (PD), or Rab5b, an early endosomal marker protein, induces reduction in neurite length. Based on our recent findings that LRRK2 co-localizes and interacts with Rab5, we tested the hypothesis that LRRK2 and Rab5 may functionally interplay while controlling neurite outgrowth. Firstly, we confirmed previous reports that over-expression of either the LRRK2 PD-specific G2019S mutant or the Rab5 constitutively active Q79L mutant, but not of dominant negative N133I mutant, significantly reduces neurite outgrowth. Secondly, when over-expression of either LRRK2 wild type (WT) or G2019S was accompanied with over-expression of one of the Rab5 variants (WT, Q79L and N133I), or with down-regulation of Rab5, the reduction extent of its neurite length was similar to that of cells over-expressing LRRK2 alone, regardless of Rab5's status. Finally, we observed similar patterns of neurite length regulation in embryonic rat hippocampal neuron cultures. Taken together, our results suggest that LRRK2 and Rab5 functionally coordinate their regulation of neurite outgrowth and that LRRK2 is a more critical factor than Rab5.
Animals ; Down-Regulation ; Neurites ; Neurons ; Parkinson Disease ; PC12 Cells ; Phosphotransferases ; Rats

Previously, we reported that glucose-deprived astrocytes are more vulnerable to the cytotoxicity of peroxynitrite, the reaction product of nitric oxide and superoxide anion. The augmented vulnerability of glucose-deprived astrocytes to peroxynitrite cytotoxicity was dependent on their proliferation rate. Inhibition of cell cycle progression has been shown to inhibit the apoptotic cell death occurring in cerebral ischemia-reperfusion. In the present study, we demonstrate that the increased death of glucose-deprived astrocytes by peroxynitrte was largely blocked by the cell cycle phase G2/M transition blocker etoposide. However, the cytoprotective effect of etoposide was not associated with its inhibition of cell cycle progression. Instead, etoposide effectively scavenged peroxynitrite. However, etoposide did not scavenge individual nitric oxide and superoxide anion and it did not prevent the hydrogen peroxide-induced cytotoxicity. The present results indicate that etoposide prevents the toxicity of peroxynitrite in astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression.
Astrocytes ; Cell Cycle ; Cell Death ; Etoposide ; Hydrogen ; Nitric Oxide ; Peroxynitrous Acid ; Superoxides

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.
Animals ; Butyrates ; Cyclic N-Oxides ; Embryonic Structures ; Hand ; Imidazoles ; Neurites ; Neurons ; Nitric Oxide ; Nitric Oxide Synthase ; PC12 Cells ; Rats ; RNA, Messenger

Foxg1 (previously named BF1) is a winged-helix transcription factor with restricted expression pattern in the telencephalic neuroepithelium of the neural tube and in the anterior half of the developing optic vesicle. Previous studies have shown that the targeted disruption of the Foxg1 gene leads to hypoplasia of the cerebral hemispheres with severe defect in the structures of the ventral telencephalon. To further investigate the molecular mechanisms by which Foxg1 plays essential roles during brain development, we have adopted a strategy to isolate genes whose expression changes immediately after introduction of Foxg1 in cultured neural precursor cell line, HiB5. Here, we report that seventeen genes were isolated by ordered differential displays that are up-regulated by over-expression of Foxg1, in cultured neuronal precursor cells. By nucleotide sequence comparison to known genes in the GeneBank database, we find that nine of these clones represent novel genes whose DNA sequences have not been reported. The results suggest that these genes are closely related to developmental regulation of Foxg1.
Animals ; Base Sequence ; Brain ; Cell Line ; Cerebrum ; Clone Cells ; Neural Tube ; Neurons ; Rats ; Stem Cells ; Telencephalon ; Transcription Factors

Here we describe characterization of chicken neuronal Per-Arnt-Sim domain 3 (NPAS3) gene during embryogenesis including examinations of expression pattern and function of the gene. RTPCR assay showed that the primary tissue of expression for this gene is the central nervous system (CNS) while RNA in situ hybridization assay confirmed that NPAS3 was expressed in the ventricular zone of developing neural tube as early as Hamburger-Hamilton (HH) stage 20. Ectopic over-expression of the gene in ovo in the developing chicken neural tube by electroporation had little effect on stem cell population, overall neurogenesis, and motor neuron differentiation. We discuss the implications of our observation.
Central Nervous System ; Chickens ; Electroporation ; Embryonic Development ; Female ; In Situ Hybridization ; Motor Neurons ; Neural Tube ; Neurogenesis ; Neurons ; Pregnancy ; RNA ; Stem Cells

Tetrahydropapaveroline (THP), a neurotoxic tetrahydroisoquinoline alkaloid formed by condensation between dopamine and dopaldehyde, has been speculated to cause Parkinson's disease and also to contribute to alcohol dependence. Having two catechol moieties, THP may readily undergo oxidation to form an o-quinone intermediate with concomitant production of reactive oxygen species, which can cause neuronal cell death and DNA damage. This review will deal with the current knowledge of neurotoxic effects of this endogenous alkaloid and underlying biochemical mechanisms.
Alcoholism ; Catechols ; Cell Death ; DNA Damage ; Dopamine ; Neurons ; Parkinson Disease ; Reactive Oxygen Species ; Tetrahydroisoquinolines ; Tetrahydropapaveroline

Transient receptor potential cation channel, subfamily V, member 1 (TRPV1, also known as vanilloid receptor 1) is a receptor that detects capsaicin, a pungent component of chili peppers, and noxious heat. Although its function in the primary nociceptor as a pain receptor is well established, whether TRPV1 is expressed in the brain is still under debate. In this study, the responses of primary cortical neurons were investigated. Here, we report that 1) capsaicin induces caspase-3-dependent programmed cell death, which coincides with increased production of nitric oxide and peroxynitrite ; that 2) the prolonged capsaicin treatment induces a steady increase in the degree of capase-3 activation, which is prevented by the removal of capsaicin; 3) and that blocking calcium entry and calcium-mediated signaling prevents capsaicin-induced cell death. These results indicate that cortical neurons express TRPV1 whose prolonged activation causes cell death.
Apoptosis ; Brain ; Calcium ; Capsaicin ; Capsicum ; Caspase 3 ; Cell Death ; Hot Temperature ; Neurons ; Nitric Oxide ; Nociceptors ; Peroxynitrous Acid

Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter, regulates neurological functions such as mood, sleep, and appetite. Erythropoietin (EPO) is well known for erythropoiesis but has recently emerged as a therapeutic agent in brain diseases. However, the mechanisms that induce EPO in the brain remain unclear. The present study was undertaken to investigate whether the effects of 5-HT involve EPO in murine hippocampal neurons. 5-HT produced a significant increase in neuronal differentiation of hippocampal neural progenitor cells. Expression of erythropoietin was increased in 5-HT-treated cells as well. The actions of 5-HT and EPO appeared to be similar in neurite outgrowth and spine formation. In addition, we show that hippocampal expression of EPO was decreased by chronic unpredictable stress (CUS) and that antidepressant treatment to maintain 5-HT concentration in synaptic cleft reversed this effect. In conclusion, actions of antidepressants might involve EPO induction in the brain.
Animals ; Antidepressive Agents ; Appetite ; Brain ; Brain Diseases ; Depression ; Erythropoiesis ; Erythropoietin ; Hippocampus ; Mice ; Neurites ; Neurons ; Neurotransmitter Agents ; Serotonin ; Spine ; Stem Cells

Parkinson's disease (PD) is the second most common neurodegenerative motor disease caused by degeneration of dopaminergic neurons in the substantia nigra. Because brain inflammation has been considered a risk factor for PD, we analyzed whether PTEN induced putative kinase 1 (PINK1), an autosomal recessive familial PD gene, regulates brain inflammation during injury states. Using acutely prepared cortical slices to mimic injury, we analyzed expression of the pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 at the mRNA and protein levels. Both mRNA and protein expression of these cytokines was higher at 6-24 h after slicing in PINK1 knockout (KO) slices compared to that in wild-type (WT) slices. In serial experiments to understand the signaling pathways that increase inflammatory responses in KO slices, we found that IkappaB degradation was enhanced but Akt phosphorylation decreased in KO slices compared to those in WT slices. In further experiments, an inhibitor of PI3K (LY294002) upstream of Akt increased expression of pro-inflammatory cytokines. Taken together, these results suggest that PINK1 deficiency enhance brain inflammation through reduced Akt activation and enhanced IkappaB degradation in response to brain injury.
Brain ; Brain Injuries ; Cytokines ; Dopaminergic Neurons ; Encephalitis ; Hydrazines ; Inflammation ; Interleukin-6 ; Interleukins ; Parkinson Disease ; Phosphorylation ; Phosphotransferases ; Risk Factors ; RNA, Messenger ; Substantia Nigra ; Tumor Necrosis Factor-alpha