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Comparative Transcriptomics of Rat and Axolotl After Spinal Cord Injury Dissects Differences and Similarities in Inflammatory and Matrix Remodeling Gene Expression Patterns.

ABSTRACT: Following spinal cord injury in mammals, maladaptive inflammation, and matrix deposition drive tissue scarring and permanent loss of function. In contrast, axolotls regenerate their spinal cord after severe injury fully and without scarring. To explore previously unappreciated molecules and pathways that drive tissue responses after spinal cord injury, we performed a 4-way intersection of rat and axolotl transcriptomics datasets and isolated shared genes with similar or differential expression at days 1, 3, and 7 after spinal cord injury in both species. Systems-wide differences and similarities between the two species are described in detail using public-domain computational tools and key differentially regulated genes are highlighted. Amongst persistent differential expression in matching neuronal genes (upregulated in axolotls but downregulated in rats) and nucleic acid metabolism genes (downregulated in axolotls but upregulated in rats), we found multiple extracellular matrix genes that were upregulated in both species after spinal cord injury and all time-points (days 1, 3, and 7), indicating the importance of extracellular matrix remodeling in wound healing. Moreover, the archetypal transcription factor SP1, which was consistently upregulated in rats but was unchanged in axolotls, was predicted as a potential transcriptional regulator of classic inflammatory response genes in rats most of which were not regulated in regenerating axolotls. This analysis offers an extensive comparative platform between a non-regenerating mammal and a regenerating urodele after spinal cord injury. To better understand regeneration vs. scarring mechanisms it is important to understand consistent molecular differences as well as similarities after experimental spinal cord injury.

SUBMITTER: Tica J

PROVIDER: S-EPMC6262295 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparative Transcriptomics of Rat and Axolotl After Spinal Cord Injury Dissects Differences and Similarities in Inflammatory and Matrix Remodeling Gene Expression Patterns.

Tica Jure J Didangelos Athanasios A

Frontiers in neuroscience 20181113

Following spinal cord injury in mammals, maladaptive inflammation, and matrix deposition drive tissue scarring and permanent loss of function. In contrast, axolotls regenerate their spinal cord after severe injury fully and without scarring. To explore previously unappreciated molecules and pathways that drive tissue responses after spinal cord injury, we performed a 4-way intersection of rat and axolotl transcriptomics datasets and isolated shared genes with similar or differential expression a ...[more]

PMID: 30519154

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Project description:The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50-53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole cord and injury-responsive adult Axolotl cord ependymal cells showed an identical growth factor response. Epidermal growth factor (EGF) maintains mesenchymal outgrowth in vitro, the cells are proliferative and maintain msi-1 expression. Non-regeneration competent Xenopus ependymal cells, NF 62+, failed to attach or grow well in EGF+ medium. Ependymal Msi-1 expression in vivo and in vitro is a strong indicator of regeneration competence in the amphibian spinal cord.

Project description:A previously unreported population of foam cells (foamy macrophages) accumulates in the invasive fibrotic meninges during gap regeneration of transected adult Axolotl spinal cord (salamander Ambystoma mexicanum) and may act beneficially. Multinucleated giant cells (MNGCs) also occurred in the fibrotic meninges. Actin-label localization and transmission electron microscopy showed characteristic foam cell and MNGC podosome and ruffled border-containing sealing ring structures involved in substratum attachment, with characteristic intermediate filament accumulations surrounding nuclei. These cells co-localized with regenerating cord ependymal cell (ependymoglial) outgrowth. Phase contrast-bright droplets labeled with Oil Red O, DiI, and DyRect polar lipid live cell label showed accumulated foamy macrophages to be heavily lipid-laden, while reactive ependymoglia contained smaller lipid droplets. Both cell types contained both neutral and polar lipids in lipid droplets. Foamy macrophages and ependymoglia expressed the lipid scavenger receptor CD36 (fatty acid translocase) and the co-transporter toll-like receptor-4 (TLR4). Competitive inhibitor treatment using the modified fatty acid Sulfo-N-succinimidyl Oleate verified the role of the lipid scavenger receptor CD36 in lipid uptake studies in vitro. Fluoromyelin staining showed both cell types took up myelin fragments in situ during the regeneration process. Foam cells took up DiI-Ox-LDL and DiI-myelin fragments in vitro while ependymoglia took up only DiI-myelin in vitro. Both cell types expressed the cysteine proteinase cathepsin K, with foam cells sequestering cathepsin K within the sealing ring adjacent to the culture substratum. The two cell types act as sinks for Ox-LDL and myelin fragments within the lesion site, with foamy macrophages showing more Ox-LDL uptake activity. Cathepsin K activity and cellular localization suggested that foamy macrophages digest ECM within reactive meninges, while ependymal cells act from within the spinal cord tissue during outgrowth into the lesion site, acting in complementary fashion. Small MNGCs also expressed lipid transporters and showed cathepsin K activity. Comparison of 3H-glucosamine uptake in ependymal cells and foam cells showed that only ependymal cells produce glycosaminoglycan and proteoglycan-containing ECM, while the cathepsin studies showed both cell types remove ECM. Interaction of foam cells and ependymoglia in vitro supported the dispersion of ependymal outgrowth associated with tissue reconstruction in Axolotl spinal cord regeneration.

Project description:BackgroundAstrocytomas are the most common primary brain tumors distinguished into four histological grades. Molecular analyses of individual astrocytoma grades have revealed detailed insights into genetic, transcriptomic and epigenetic alterations. This provides an excellent basis to identify similarities and differences between astrocytoma grades.MethodsWe utilized public omics data of all four astrocytoma grades focusing on pilocytic astrocytomas (PA I), diffuse astrocytomas (AS II), anaplastic astrocytomas (AS III) and glioblastomas (GBM IV) to identify similarities and differences using well-established bioinformatics and systems biology approaches. We further validated the expression and localization of Ang2 involved in angiogenesis using immunohistochemistry.ResultsOur analyses show similarities and differences between astrocytoma grades at the level of individual genes, signaling pathways and regulatory networks. We identified many differentially expressed genes that were either exclusively observed in a specific astrocytoma grade or commonly affected in specific subsets of astrocytoma grades in comparison to normal brain. Further, the number of differentially expressed genes generally increased with the astrocytoma grade with one major exception. The cytokine receptor pathway showed nearly the same number of differentially expressed genes in PA I and GBM IV and was further characterized by a significant overlap of commonly altered genes and an exclusive enrichment of overexpressed cancer genes in GBM IV. Additional analyses revealed a strong exclusive overexpression of CX3CL1 (fractalkine) and its receptor CX3CR1 in PA I possibly contributing to the absence of invasive growth. We further found that PA I was significantly associated with the mesenchymal subtype typically observed for very aggressive GBM IV. Expression of endothelial and mesenchymal markers (ANGPT2, CHI3L1) indicated a stronger contribution of the micro-environment to the manifestation of the mesenchymal subtype than the tumor biology itself. We further inferred a transcriptional regulatory network associated with specific expression differences distinguishing PA I from AS II, AS III and GBM IV. Major central transcriptional regulators were involved in brain development, cell cycle control, proliferation, apoptosis, chromatin remodeling or DNA methylation. Many of these regulators showed directly underlying DNA methylation changes in PA I or gene copy number mutations in AS II, AS III and GBM IV.ConclusionsThis computational study characterizes similarities and differences between all four astrocytoma grades confirming known and revealing novel insights into astrocytoma biology. Our findings represent a valuable resource for future computational and experimental studies.

Dataset Information

Comparative Transcriptomics of Rat and Axolotl After Spinal Cord Injury Dissects Differences and Similarities in Inflammatory and Matrix Remodeling Gene Expression Patterns.

Publications

Comparative Transcriptomics of Rat and Axolotl After Spinal Cord Injury Dissects Differences and Similarities in Inflammatory and Matrix Remodeling Gene Expression Patterns.

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