Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation,regeneration, and trauma

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up‐ or down‐regulated during two phases of the retinoic acid‐induced differentiation of the embryonal carcinoma line Ntera‐2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate‐resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented. J. Cell. Biochem. 80:171–180, 2000. © 2000 Wiley‐Liss, Inc.     

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.     

Inhibition of gecko GSK-3β promotes elongation of neurites and oligodendrocyte processes but decreases the proliferation of blastemal cells

GSK-3β signaling is involved in regulation of both neuronal and glial cell functions, and interference of the signaling affects central nervous system (CNS) development and regeneration. Thus, GSK-3β was proposed to be an important therapeutic target for promoting functional recovery of adult CNS injuries. To further clarify the regulatory function of the kinase on the CNS regeneration, we characterized gecko GSK-3β and determined the effects of GSK-3β inactivation on the neuronal and glial cell lines, as well as on the gecko tail (including spinal cord) regeneration. Gecko GSK-3β shares 91.7-96.7% identity with those of other vertebrates, and presented higher expression abundance in brain and spinal cord. The kinase strongly colocalized with the oligodendrocytes while less colocalized with neurons in the spinal cord. Phosphorylated GSK-3β (pGSK-3β) levels decreased gradually during the normally regenerating spinal cord ranging from L13 to the 6th caudal vertebra. Lithium injection increased the pGSK-3β levels of the corresponding spinal cord segments, and in vitro experiments on neurons and oligodendrocyte cell line revealed that the elevation of pGSK-3β promoted elongation of neurites and oligodendrocyte processes. In the normally regenerate tails, pGSK-3β kept stable in 2 weeks, whereas decreased at 4 weeks. Injection of lithium led to the elevation of pGSK-3β levels time-dependently, however destructed the regeneration of the tail including spinal cord. Bromodeoxyuridine (BrdU) staining demonstrated that inactivation of GSK-3β decreased the proliferation of blastemal cells. Our results suggested that species-specific regulation of GSK-3β was indispensable for the complete regeneration of CNS.     

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.     

Clemastine Enhances Myelination,Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.

     

Spearman Correlation Identifies Statistically Significant Gene Expression Clusters in Spinal Cord Development and Injury

An important problem in the analysis of large-scale gene expression data is the validation of gene expression clusters. By examining the temporal expression patterns of 74 genes expressed in rat spinal cord under three different experimental conditions, we have found evidence that some genes cluster together under multiple conditions. Using RT-PCR data from spinal cord development and two sets of microarray data from spinal injury, we applied Spearman correlation to identify clusters and to assign P values to pairs of genes with highly similar temporal expression patterns. We found that 15% of genes occurred in statistically significant pairs in all three experimental conditions, providing both statistical and experimental support for the idea that genes that cluster together are co-regulated. In addition, we demonstrated that DNA microarray and RT-PCR data are comparable, and can be combined to confirm gene expression relationships.     

Analysis of Gene Expression Profiles Associated with Functional Recovery After Spinal Cord Injury Caused by Sema4D Knockdown in Oligodendrocytes

We previously showed that sema4D Knockdown in oligodendrocytes promotes functional recovery after spinal cord injury. In this paper, we examined gene expression profiles associated with functional recovery by PCR array. For general observation during first 4 weeks, we found that sema4D knockdown could reduce edema and stimulate SCEP. Further, PCR array analysis indicated sema4D knockdown in OPCs inhibited wound tissue angiogenesis and inflammation genes expression and upregulated axon regeneration genes expression at early phase. Our findings provided the molecular mechanism for its potential application.     

Regulation of neurotransmitter synthesis: from neuron to gene.

We are interested in the molecular mechanisms of the regulation of neurotransmitter related gene expression by neurotrophic factors and neuronal activity. Previous work has shown that conditioned medium of muscle cells induces choline acetyltransferase (CAT) activity and represses tyrosine hydroxylase, dopamine-beta-hydroxylase and aromatic L-amino acid decarboxylase (AADC) activities in cultured sympathetic neurons. Here, we show that a new muscle-derived cell line secretes two factors which induce CAT activity in spinal cord cultures; one of those is related to LIF, a CAT inducing factor for sympathetic neurons. Preliminary data are presented on the structure of the human AADC and CAT genes. Putative promoter regions have been coupled to reporter genes; transient transfection experiments will allow us to determine the promoter elements responsible for the regulation by neurotrophic factors. We also summarize the distribution of AADC-immunoreactive cells in rat and cat brain, which will be used as a reference for the study of the specificity of expression of AADC promoter in transgenic mice.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.     

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.     

Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord

We have examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Here, we demonstrate that expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, we show that Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord.     

Adhesion to Carbon Nanotube Conductive Scaffolds Forces Action-Potential Appearance in Immature Rat Spinal Neurons

In the last decade, carbon nanotube growth substrates have been used to investigate neurons and neuronal networks formation in vitro when guided by artificial nano-scaled cues. Besides, nanotube-based interfaces are being developed, such as prosthesis for monitoring brain activity. We recently described how carbon nanotube substrates alter the electrophysiological and synaptic responses of hippocampal neurons in culture. This observation highlighted the exceptional ability of this material in interfering with nerve tissue growth. Here we test the hypothesis that carbon nanotube scaffolds promote the development of immature neurons isolated from the neonatal rat spinal cord, and maintained in vitro. To address this issue we performed electrophysiological studies associated to gene expression analysis. Our results indicate that spinal neurons plated on electro-conductive carbon nanotubes show a facilitated development. Spinal neurons anticipate the expression of functional markers of maturation, such as the generation of voltage dependent currents or action potentials. These changes are accompanied by a selective modulation of gene expression, involving neuronal and non-neuronal components. Our microarray experiments suggest that carbon nanotube platforms trigger reparative activities involving microglia, in the absence of reactive gliosis. Hence, future tissue scaffolds blended with conductive nanotubes may be exploited to promote cell differentiation and reparative pathways in neural regeneration strategies.     

Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 was initially expressed evenly along the dorsoventral axis and its expression became increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 was regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by their overexpressions in the spinal cord. When Zic1 was misexpressed on the ventral side of the chick spinal cord, neuronal differentiation was inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties were not grossly affected as revealed by molecular markers. Concordantly, when Zic1 was overexpressed in the dorsal spinal cord in transgenic mice, we observed hypercellularity in the dorsal spinal cord. The transgene-expressing cells were increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, we observed a significant cell number reduction without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differentiation is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick.