Gene expression in mouse DRG neurons at 2 or 5 days post-sciatic lesion in WT and Nfil3-KO
ABSTRACT: Mice with a deletion of transcription factor Nfil3 show delayed functional regeneration after sciatic lesion compared to WT mice. To analyze differential gene expression, sciatic nerves were lesioned and nerve endings were coaptated. After two or 5 days, DRGs L4 and L5 were dissected out and gene expression was measured by microarray. Control tissue was taken from the non-injured side. We found that expression of classic regeneration associated genes was similar in WT and Nfil3 KO mice. We did identify a group of differentially expressed genes known for its role in olfactory signal transduction, but were not known as regeneration associated genes until now, which may explain the negative effects on functional recovery. Gene expression in DRGs L4 and L5 from WT and NFIL3 KO mice was analyzed at 2 and 5 days after receiving a sciatic nerve lesion. Control tissue was taken from the non-injured side. Four biological replicates were used per condition. Microarray chips were two color chips, but each channel was analyzed in single color fashion. Conditions were pseudo-randomized over chips in a balanced dye-swap setup.
Project description:Mice with a deletion of transcription factor Nfil3 show delayed functional regeneration after sciatic lesion compared to WT mice. To analyze differential gene expression, sciatic nerves were lesioned and nerve endings were coaptated. After two or 5 days, DRGs L4 and L5 were dissected out and gene expression was measured by microarray. Control tissue was taken from the non-injured side. We found that expression of classic regeneration associated genes was similar in WT and Nfil3 KO mice. We did identify a group of differentially expressed genes known for its role in olfactory signal transduction, but were not known as regeneration associated genes until now, which may explain the negative effects on functional recovery. Overall design: Gene expression in DRGs L4 and L5 from WT and NFIL3 KO mice was analyzed at 2 and 5 days after receiving a sciatic nerve lesion. Control tissue was taken from the non-injured side. Four biological replicates were used per condition. Microarray chips were two color chips, but each channel was analyzed in single color fashion. Conditions were pseudo-randomized over chips in a balanced dye-swap setup.
Project description:Parvalbumin (PV) is a calcium binding protein that identifies a subpopulation of proprioceptive dorsal root ganglion (DRG) neurons. Calcitonin gene-related peptide (CGRP) is also expressed in a high proportion of muscle afferents but its relationship to PV is unclear. Little is known of the phenotypic responses of muscle afferents to nerve injury. Sciatic nerve axotomy or L5 spinal nerve ligation and section (SNL) lesions were used to explore these issues in adult rats using immunocytochemistry.In naive animals, the mean PV expression was 25 % of L4 or L5 dorsal root ganglion (DRG) neurons, and this was unchanged 2 weeks after sciatic nerve axotomy. Colocalization studies with the injury marker activating transcription factor 3 (ATF3) showed that approximately 24 % of PV neurons expressed ATF3 after sciatic nerve axotomy suggesting that PV may show a phenotypic switch from injured to uninjured neurons. This possibility was further assessed using the spinal nerve ligation (SNL) injury model where injured and uninjured neurons are located in different DRGs. Two weeks after L5 SNL there was no change in total PV staining and essentially all L5 PV neurons expressed ATF3. Additionally, there was no increase in PV-ir in the adjacent uninjured L4 DRG cells. Co-labelling of DRG neurons revealed that less than 2 % of PV neurons normally expressed CGRP and no colocalization was seen after injury.These experiments clearly show that axotomy does not produce down regulation of PV protein in the DRG. Moreover, this lack of change is not due to a phenotypic switch in PV immunoreactive (ir) neurons, or de novo expression of PV-ir in uninjured neurons after nerve injury. These results further illustrate differences that occur when muscle afferents are injured as compared to cutaneous afferents.
Project description:Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest.
Project description:We generated whole-genome gene expression profiles of dorsal root ganglion (DRG) neurons following nerve damage. DRG neurons extend one peripheral axon into the spinal nerve and one central axon into the dorsal root. The peripheral axon regenerates vigorously, while in contrast the central axon has little regenerative capacity. For this study, two groups of animals were subjected either to sciatic nerve (SN) or dorsal root (DR) crush, and at 12, 24, 72 hours and 7 days after the crush, lumbar DRGs L4, L5 and L6 were dissected and total RNA was extracted. For each time point after lesion, three biological replicate RNA samples were hybridized together with the common reference sample consisting of labeld RNA pooled from three unlesioned animals.
Project description:Neuronal cells express considerable plasticity responding to environmental cues, in part, through subcellular mRNA regulation. Here we report on the extensive changes in distribution of mRNAs in the cell body and axon compartments of peripheral sensory neurons and the 3' untranslated region (3'UTR) landscapes after unilateral sciatic nerve entrapment (SNE) injury in rats. Neuronal cells dissociated from SNE-injured and contralateral L4 and L5 dorsal root ganglia were cultured in a compartmentalized system. Axonal and cell body RNA samples were separately subjected to high throughput RNA sequencing (RNA-Seq). The injured axons exhibited enrichment of mRNAs related to protein synthesis and nerve regeneration. Lengthening of 3'UTRs was more prevalent in the injured axons, including the newly discovered alternative cleavage and polyadenylation of NaV1.8 mRNA. Alternative polyadenylation was largely independent from the relative abundance of axonal mRNAs; but they were highly clustered in functional pathways related to RNA granule formation in the injured axons. These RNA-Seq data analyses indicate that peripheral nerve injury may result in highly selective mRNA enrichment in the affected axons with 3'UTR alterations potentially contributing to the mechanism of neuropathic pain.
Project description:BACKGROUND AND PURPOSE: Substance P (SP), a representative member of the tachykinin family, is involved in nociception under physiological and pathological conditions. Recently, hemokinin-1 (HK-1) was identified as a new member of this family. Although HK-1 acts on NK(1) tachykinin receptors that are thought to be innate for SP, the roles of HK-1 in neuropathic pain are still unknown. EXPERIMENTAL APPROACH: Using rats that had been subjected to chronic constrictive injury (CCI) of the sciatic nerve as a neuropathic pain model, we examined the changes in expression of SP- and HK-1-encoding genes (TAC1 and TAC4, respectively) in the L4/L5 spinal cord and L4/L5 dorsal root ganglia (DRGs) in association with changes in pain-related behaviours in this neuropathic pain state. KEY RESULTS: The TAC4 mRNA level was increased on the ipsilateral side of the dorsal spinal cord, but not in DRGs, at day 3 after CCI. In contrast, the TAC1 mRNA level was significantly increased in the DRGs at day 3 after CCI without any changes in the dorsal spinal cord. Analysis of a cultured microglial cell line revealed the presence of TAC4 mRNA in microglial cells. Minocycline, an inhibitor of microglial activation, blocked the increased expression of TAC4 mRNA after CCI and inhibited the associated pain-related behaviours and microglial activation in the spinal cord. CONCLUSIONS AND IMPLICATIONS: The present results suggest that HK-1 expression is increased at least partly in activated microglial cells after nerve injury and is clearly involved in the early phase of neuropathic pain.
Project description:Purpose:Neuropathic pain is a complex chronic condition occurring post-nervous system damage. The transcriptional reprogramming of injured dorsal root ganglia (DRGs) drives neuropathic pain. However, few comparative analyses using high-throughput platforms have investigated uninjured DRG in neuropathic pain, and potential interactions among differentially expressed genes (DEGs) and pathways were not taken into consideration. The aim of this study was to identify changes in genes and pathways associated with neuropathic pain in uninjured L4 DRG after L5 spinal nerve ligation (SNL) by using bioinformatic analysis. Materials and methods:The microarray profile GSE24982 was downloaded from the Gene Expression Omnibus database to identify DEGs between DRGs in SNL and sham rats. The prioritization for these DEGs was performed using the Toppgene database followed by gene ontology and pathway enrichment analyses. The relationships among DEGs from the protein interactive perspective were analyzed using protein-protein interaction (PPI) network and module analysis. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of DEGs in the rodent neuropathic pain model. Results:A total of 206 DEGs that might play a role in neuropathic pain were identified in L4 DRG, of which 75 were upregulated and 131 were downregulated. The upregulated DEGs were enriched in biological processes related to transcription regulation and molecular functions such as DNA binding, cell cycle, and the FoxO signaling pathway. Ctnnb1 protein had the highest connectivity degrees in the PPI network. The in vivo studies also validated that mRNA and protein levels of Ctnnb1 were upregulated in both L4 and L5 DRGs. Conclusion:This study provides insight into the functional gene sets and pathways associated with neuropathic pain in L4 uninjured DRG after L5 SNL, which might promote our understanding of the molecular mechanisms underlying the development of neuropathic pain.
Project description:Changes in microRNA (miRNA) expression in the mouse L4 and L5 dorsal root ganglion following unilateral sciatic nerve transection. The timepoint of 7 days post-axotomy was chosen to capture miRNA expression profiles at a time when the injured neurons were beginning to regenerate. Two condition experiment, paired control DRG vs axotomised DRG following unilateral sciatic nerve transection. 3 biological replicates, one replicate per array. Dye swap in Replicate 2.
Project description:Dorsal root ganglia (DRG) neurons regenerate spontaneously after traumatic or surgical injury. Long noncoding RNAs (lncRNAs) are involved in various biological regulation processes. Conditions of lncRNAs in DRG neuron injury deserve to be further investigated. Transcriptomic analysis was performed by high-throughput Illumina HiSeq2500 sequencing to profile the differential genes in L4-L6 DRGs following rat sciatic nerve tying. A total of 1,228 genes were up-regulated and 1,415 down-regulated. By comparing to rat lncRNA database, 86 known and 26 novel lncRNA genes were found to be differential. The 86 known lncRNA genes modulated 866 target genes subject to gene ontology (GO) and KEGG enrichment analysis. The genes involved in the neurotransmitter status of neurons were downregulated and those involved in a neuronal regeneration were upregulated. Known lncRNA gene rno-Cntnap2 was downregulated. There were 13 credible GO terms for the rno-Cntnap2 gene, which had a putative function in cell component of voltage-gated potassium channel complex on the cell surface for neurites. In 26 novel lncRNA genes, 4 were related to 21 mRNA genes. A novel lncRNA gene AC111653.1 improved rno-Hypm synthesizing huntingtin during sciatic nerve regeneration. Real time qPCR results attested the down-regulation of rno-Cntnap lncRNA gene and the upregulation of AC111653.1 lncRNA gene. A total of 26 novel lncRNAs were found. Known lncRNA gene rno-Cntnap2 and novel lncRNA AC111653.1 were involved in neuropathic pain of DRGs after spared sciatic nerve injury. They contributed to peripheral nerve regeneration via the putative mechanisms.
Project description:Changes in microRNA (miRNA) expression in the mouse L4 and L5 dorsal root ganglion following unilateral sciatic nerve transection. The timepoint of 7 days post-axotomy was chosen to capture miRNA expression profiles at a time when the injured neurons were beginning to regenerate. Overall design: Two condition experiment, paired control DRG vs axotomised DRG following unilateral sciatic nerve transection. 3 biological replicates, one replicate per array. Dye swap in Replicate 2.