Transcriptional regulation of gene expression clusters in motor neurons following spinal cord injury.

BACKGROUND: Spinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity. RESULTS: Here we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability. CONCLUSIONS: This analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper-excitability, the manipulation of which potentially could be used to alter the transcriptional response to prevent the motor neurons from entering a state of hyper-excitability.

Gene expression profiling of two distinct neuronal populations in the rodent spinal cord.

BACKGROUND: In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. METHODOLOGY/PRINCIPAL FINDINGS: We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. CONCLUSIONS/SIGNIFICANCE: We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord.

Effects of sphingosine 1-phosphate on calcium signaling, proliferation and S1P2 receptor expression in PC Cl3 rat thyroid cells.

Sphingosine 1-phosphate (S1P) regulates diverse biological processes, including mitosis, by binding to the S1P family of G-protein coupled receptors. The aim of the study was to determine the pattern of S1P receptor expression and to investigate the effects of S1P on intracellular calcium levels and proliferation in the rat thyroid cell line PC Cl(3). S1P(2) and S1P(3) mRNA and proteins were detected in PC Cl(3) cells, as well as in FRTL-5 rat thyroid cells. In addition, S1P(5) mRNA was present at low levels, but not S1P(1) or S1P(4). In PC Cl(3) cells, S1P invoked calcium release from intracellular stores, but not calcium entry. The Ca(2+) release was mediated by phospholipase C and inositol 1,4,5-trisphosphate. S1P attenuated the TSH-evoked cAMP increase in a pertussis toxin-sensitive manner. S1P per se did not affect the proliferation of the cells, but attenuated the proliferation evoked by a combination of insulin and TSH. Furthermore, S1P attenuated the PMA-evoked proliferation. S1P(2) expression was positively regulated by insulin and PMA. S1P itself transiently upregulated S1P(2) receptor mRNA, while TSH had a net downregulating effect on S1P(2) expression. In summary, S1P modulates central intracellular signaling cascades and is antiproliferative in PC Cl(3) cells. S1P(2) receptor expression is modulated by insulin and TSH, two central growth factors in thyroid cell regulation.