Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development

Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.     

The inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis

Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.     

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.     

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain

Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.     

The Neuroprotective Potential of Phase II Enzyme Inducer on Motor Neuron Survival in Traumatic Spinal Cord Injury In vitro

(1) Phase II enzyme inducer is a kind of compound which can promote the expression of antioxidative enzymes through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Recently, it has been reported that these compounds show neuroprotective effect via combating oxidative stress. The purpose of this study is to determine whether phase II enzyme inducers have neuroprotective effects on traumatic spinal cord injury. (2) An organotypic spinal cord culture system was used, Phase II enzyme inducers were added to culture medium for 1 week, motor neurons were counted by SMI-32 staining, glutamate, Nrf2, and Heme oxygenase-1(HO-1) mRNA were tested. (3) This study showed motor neuron loss within 1 week in culture. After 1 week in culture, the system was stable. Moreover, Glutamate was increased when in culture 48 h and decreased after 1 week in culture. There was no significant change between 1 and 4 weeks in culture. Necrotic motor neuron and damaged mitochondrial were observed in culture 48 h. Furthermore, phase II enzyme inducers: tert-butyhydroquinone (t-BHQ), 3H-1,2-dithiole-3-thione (D3T), and 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) were shown to promote motor neuron survival after dissection, it was due to increasing Nrf2 and HO-1 mRNA expression and protecting mitochondrial not due to decreasing glutamate level. (4) The loss of motor neuron due to dissection can mimic severe traumatic spinal cord injury. These results demonstrate that glutamate excitotoxicity and the damage of mitochondrial is possibly involve in motor neuron death after traumatic spinal cord injury and phase II enzyme inducers show neuroprotective potential on motor neuron survival in traumatic spinal cord injury in vitro.     

The expression and function of MTG/ETO family proteins during neurogenesis

The proneural basic helix-loop-helix (bHLH) proteins promote neurogenesis by inducing changes in gene expression required for neuronal differentiation. Here we characterize one aspect of this differentiation program by analyzing a small family of putative corepressors encoded by MTG genes. We show that MTG genes are expressed sequentially during neurogenesis as cells undergo neuronal differentiation in both the chick spinal cord and in the Xenopus primary nervous system. Using in ovo electroporation, we show that misexpressing wild-type forms of MTG proteins in the developing chick spinal cord does not detectably alter neuronal differentiation. By contrast, the number of differentiated neurons is markedly reduced when a putative dominant-negative mutant of the MTG proteins is expressed in neural precursors in a manner that can be rescued by wild-type MTGR1. Together, these results suggest that MTG family members act downstream of proneural proteins, presumably as corepressors, to promote neuronal differentiation.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.     

Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. We report that Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent, as ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity.     

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.     

The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.

Mice homozygous for null alleles of the putative signaling molecule Wnt-1 have a reproducible phenotype: loss of the midbrain and adjacent cerebellar component of the metencephalon. By examining embryonic expression of the mouse engrailed (En) genes, from 8.0 to 9.5 days postcoitum, we demonstrate that Wnt-1 primarily regulates midbrain development. The midbrain itself is required for normal development of the metencephalon. Thus, the observed neonatal phenotype is explained by a series of early events, within 48 hr of neural plate induction, that leads to a complete loss of En domains in the anterior central nervous system. Wnt-1 and a related gene, Wnt-3a, are coexpressed from early somite stages in dorsal aspects of the myelencephalon and spinal cord. We suggest that functional redundancy between these two genes accounts for the lack of a caudal central nervous system phenotype.