Rattus norvegicus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA106033 | ENA |
REPOSITORIES: ENA
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Project description:Efficient growth cone regeneration requires protein synthesis in the adult mammalian brain and spinal cord. Recent evidence suggests that the local availability of protein synthesis machinery in adult mammalian axons may be an indicator of their regenerative capacity. Here we investigated the local protein synthesis capacity in matured cortical axons, which have poor regenerative capacity, yet are critical for recovery following injury due to traumatic brain injury and stroke. This work is the first to biochemically isolate and identify mRNA from mammalian cortical axons, making use of a unique microfluidic platform to isolate axons free of other cellular debris. We first sought to identify mRNA in naïve axons that makes up the pool of mRNA available for translation initiated following axotomy. Next, we investigated changes in the mRNA population localized to axons 2 days following axotomy and growth cone regeneration. Experiment Overall Design: Cortical axons were harvested using the compartmentalized microfluidic platform after 13 days in culture at a time when they express mature synaptic proteins. A total of 7 Genechips were used for the uninjured cortical axons from 3 different culture batches. 3 Genechips were used for neurons isolated from the neuronal compartment of the microfluidic platfrom. The neuronal Genechip were used to compare mRNA populations with axonal Genechips and for quality control purposes. 3 Genechips were used for regenerating axons; for these, axons were axotomized at 11 days in culture, then allowed to regrow for 2 days before harvesting.
2009-01-14 | E-GEOD-11730 | biostudies-arrayexpress
Project description:Using an experimental TBI rat model of mild/moderate Controlled Cortical Impact (CCI) injury, we combined large-scale proteomics identification and relative quantification using Spatially-Resolved Microproteomics with MALDI MS Imaging of Lipids. Spatially by studying different regions in the brain post injury in a coronal view, with main focus on the injury site itself. Temporally by studying the acute and subacute phase post injury, including injured rat brains at 1 day, 3 days, 7 days, and 10 days post injury. Direct on-tissue micro-digestion followed by micoextraction from 1 mm2 surface area within the injured cortical tissue were subjected to LC-MS & MS/MS analysis using HR MS. In addition, several identified potential biomarkers within our study were used to stimulate dorsal root ganglion (DRG), astrocyte, and macrophage cell lines to obtain a better understanding of their role and contribution in the injury.
2019-06-19 | PXD011262 | Pride
Project description:The identification of axonal mRNAs in model organisms has led to the discovery of many axonally translated proteins required for axon guidance and injury response. The extent to which these axonal mRNAs are conserved in humans is unknown. Here we report on the axonal transcriptome of glutamatergic neurons derived from human embryonic stem cells (hESC-neurons) grown in axon isolating microfluidic chambers. We identified mRNAs enriched in axons, representing a functionally unique local transcriptome as compared to the whole neuron transcriptome. Further, we found that the enriched functional categories within high confidence axonal transcripts resemble those in the axonal transcriptome of rat cortical neurons. Comparing our list of human axonal transcripts to similar datasets generated from embryonic and adult rat dorsal root ganglia and rat cortical neurons we found 60 mRNAs common to all four neuron types. We found that over half of these genes are associated with neurological phenotypes or diseases in model organisms and human. This data provides an important resource for studying local mRNA translation in human axons and has the potential to reveal both conserved and unique axonal mechanisms across species and neuronal types. We analyzed the axonal and whole hESC-neuron transcriptome in triplicate using the Affymetrix Human Gene 2.0 ST Array platform.
2016-07-29 | E-GEOD-84975 | biostudies-arrayexpress
Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.
2010-01-30 | E-GEOD-13788 | biostudies-arrayexpress
Project description:The goal of this study is to compare the mRNA interactome of different RBPs in regenerating axons utilizing RNA-immunoprecipitation (RIP). Interacting mRNAs to each target RBPs were co-immunoprecipitated from axoplasm of sciatic nerve, injured 7 days ago. Interactome of each RBPs were identified by Next-generation sequencing (NGS).
2018-12-21 | GSE103444 | GEO
Project description:The identification of axonal mRNAs in model organisms has led to the discovery of many axonally translated proteins required for axon guidance and injury response. The extent to which these axonal mRNAs are conserved in humans is unknown. Here we report on the axonal transcriptome of glutamatergic neurons derived from human embryonic stem cells (hESC-neurons) grown in axon isolating microfluidic chambers. We identified mRNAs enriched in axons, representing a functionally unique local transcriptome as compared to the whole neuron transcriptome. Further, we found that the enriched functional categories within high confidence axonal transcripts resemble those in the axonal transcriptome of rat cortical neurons. Comparing our list of human axonal transcripts to similar datasets generated from embryonic and adult rat dorsal root ganglia and rat cortical neurons we found 60 mRNAs common to all four neuron types. We found that over half of these genes are associated with neurological phenotypes or diseases in model organisms and human. This data provides an important resource for studying local mRNA translation in human axons and has the potential to reveal both conserved and unique axonal mechanisms across species and neuronal types.
2016-07-29 | GSE84975 | GEO
Project description:We have conducted quantitative proteomic analyses of the axons of cultured rat cortical neurons. Axons are isolated by using glass chips that enable the axons and their cell bodies of neurons to grow in separated regions on the chip surface. Proteins extracted from the isolated axons, as well as those extracted from whole cortical neurons are subjected to two-dimensional liquid chromatography (2D-LC)-mass spectrometry (MS)-MS analyses. The abundances of proteins in the axon are found to be strongly correlated with their average abundances in whole neurons. Based upon these data, a quantitative description of the protein distribution among various subcellular structures in the axon has been generated. The proteins extracted from the axons and whole neurons are also subjected to stable isotope dimethyl labeling reaction and then to 2D-LC-MS/MS analysis. Proteins enriched in the axon compartment of rat cortical neurons are thus identified.
2018-04-17 | PXD005527 | Pride
Project description:Cortical development is a complex process involving the generation of neuronal progenitors, which proliferate and migrate to form the stratified layers of the maturing cortex. To identify microRNAs (miRNAs) and genes that may be important during early cortical development, we analyzed the expression profiles of rat neuronal progenitors obtained at embryonic day 11 (E11), E12 and E13 using microarrays. Neuronal progenitors were purified from telencephalic dissociates with a positive-selection strategy using surface labeling tetanus-toxin and cholera-toxin and fluorescence-activated cell sorting. We identified classes of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors as cortical development progressed from E11 to E13. We present data that supports a regulatory role for miRNAs during the transition from neuronal progenitors into differentiating cortical neurons. Experiment Overall Design: Flow cytometry was used to isolate tetanus toxin+ and cholera toxin+ neuronal progenitors from embryonic days 11, 12 and 13 rat telencephalon. 4 biological replicates were obtained for each group using a pooled litters for each biological replicate on different prepararation days
2009-01-13 | E-GEOD-11334 | biostudies-arrayexpress
Project description:Local protein synthesis in sensory neuron axons is necessary for axonal regeneration with the efficiency of regeneration decreasing with age. Because the full repertoire of transcripts in embryonic and adult rat sensory axons is unknown we asked how the pool of mRNAs dynamically changes during ageing. We isolated mRNA from pure axons and growth cones devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localised in sensory axons and that this repertoire changes during development toward adulthood. Embryonic sensory axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are highly enriched in mRNAs encoding immune molecules with a role in nociception. To validate our experimental approach we show that Tubulin-beta3 mRNA is present only in embryonic axons where it is locally synthesised. In summary, we show that the population of axonal mRNAs dynamically changes during development, which may partly contribute to the intrinsic capacity of axons at different ages to regenerate after injury and to modulate pain. Pure axonal RNA were extracted from the axons of embryonic and adult dorsal root ganglion neurons, each with 5 biological replicates. The axonal transcriptomes were analysed using Affymetrix Rat Genome 230 2.0 Arrays.
2010-11-23 | E-GEOD-22638 | biostudies-arrayexpress