Lipidomics dataset of PTEN deletion-induced optic nerve regeneration mouse model.
Ontology highlight
ABSTRACT:
SUBMITTER: Arcuri J
PROVIDER: S-EPMC7797512 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We present the lipidome of adult PTENloxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of regeneration. At 4-week-old, PTENloxP/loxP mice were intravitreally-injected with 2-3 μl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. The Bligh and Dyer method was used for lipid extraction, followed by mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS). The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0
2020-08-31 | ST001477 | MetabolomicsWorkbench
Project description:The right optic nerve of adult, 6 month to 1 year old, female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured left optic nerves were also collected. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) n = 24 to obtain sufficient tissue for analysis. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [1] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, Study ID ST001725.
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Project description:We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718).
| S-EPMC6723204 | biostudies-literature
Project description:In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography-mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53.
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Project description:A quantitative proteomic profiling was performed on PTEN deletion-induced optic nerve regeneration mouse models and controls. At postnatal day 28 (week 4), PTENloxP/loxP mice were subjected to intravitreal injection (2-3ul) of adeno-associated viruses expressing Cre (AAV2-Cre) or control placenta alkaline phosphatase (AAV2-PLAP). Optic Nerve crush was performed 2 weeks post AAV injections at three times points (0, 7, 14 days post injury) and collected for proteomics analysis.
A protein extraction was carried out by homogenization of the tissue in extraction buffer (TEAB, NaCl and SDS). During extraction, three internal peptide standards, named as Regen III, were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 1 sets of 18 tags from a 18plex TMT kit for quantification. After combination and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled human peptides to be used as an ionization control and cross-sample quantification standard (Regen II). Overall, Regen V standards (refers to a combination of Regen III and Regen II, for extraction and ionization normalization) were used to compare and normalize against any future axon regeneration sample cohort. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10.
After animals were euthanized, optic nerves were collected by dissection. There were 6 experimental conditions and three biological replicates for each condition for a total of 18 nerves. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic bacterial peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 18uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin.
All samples were labelled using 1 set of 18 tags from a 18plex TMT (Tandem Mass Tag) kit for quantification. After combination and drying of all peptide samples, the samples were fractionated via Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher). Each combined TMT sample was spiked with two additional human peptide standards containing isobaric labels (Regen II). The final concentration of Regen II was 54uM. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards, known as Regen V (Regen III + Regen II), serve as a normalization method that may be used to compare protein abundance data across multiple datasets.
Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One contains the Regen V internal standard peptide sequences, and the other contains BSA peptide sequences. The identification of the Regen V and BSA standards was run in a separate Proteome Discoverer workflow targeted to identify the internal standards separately from experimental proteins. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. Post translational modifications of the Regen II, (biotin, carbon-13 and nitrogen-15) were added during this step to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.
2023-11-27 | MSV000093512 | MassIVE
Project description:The right optic nerve of 1 year old female and male Danio rerio were crushed and collected three days after. Matching controls of uninjured eyes were also collected. The tissue was dissected from euthanized fish and “flash” frozen on dry ice in Eppendorf tubes. Due to the small size of the nerves, for each category (female crush, female control, male crush, male control) n=24 the samples were pooled. The brain from one male fish was also collected for control/calibration. Lipid extraction was done with the Bligh and Dyer [2] method, followed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. The lipids were identified and quantified with LipidSearch 4.2.21 and the statistical analysis was conducted through Metaboanalyst 5.0.
2021-03-12 | ST001725 | MetabolomicsWorkbench
Project description:Although mammalian retinal ganglion cells (RGCs) normally cannot regenerate axons nor survive after optic nerve injury, this failure is partially reversed by inducing sterile inflammation in the eye. Infiltrative myeloid cells express the axogenic protein oncomodulin (Ocm) but additional, as-yet-unidentified, factors are also required. We show here that infiltrative macrophages express stromal cell–derived factor 1 (SDF1, CXCL12), which plays a central role in this regard. Among many growth factors tested in culture, only SDF1 enhances Ocm activity, an effect mediated through intracellular cyclic AMP (cAMP) elevation and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation. SDF1 deficiency in myeloid cells (CXCL12flx/flxLysM-Cre−/+ mice) or deletion of the SDF1 receptor CXCR4 in RGCs (intraocular AAV2-Cre in CXCR4flx/flx mice) or SDF1 antagonist AMD3100 greatly suppresses inflammation-induced regeneration and decreases RGC survival to baseline levels. Conversely, SDF1 induces optic nerve regeneration and RGC survival, and, when combined with Ocm/cAMP, SDF1 increases axon regeneration to levels similar to those induced by intraocular inflammation. In contrast to deletion of phosphatase and tensin homolog (Pten), which promotes regeneration selectively from αRGCs, SDF1 promotes regeneration from non-αRGCs and enables the latter cells to respond robustly to Pten deletion; however, SDF1 surprisingly diminishes the response of αRGCs to Pten deletion. When combined with inflammation and Pten deletion, SDF1 enables many RGCs to regenerate axons the entire length of the optic nerve. Thus, SDF1 complements the effects of Ocm in mediating inflammation-induced regeneration and enables different RGC subtypes to respond to Pten deletion.
| S-EPMC9169637 | biostudies-literature
Project description:Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.
| S-EPMC2796263 | biostudies-literature
Project description:The inability of retinal ganglion cells (RGCs) to regenerate damaged axons through the optic nerve has dire consequences for victims of traumatic nerve injury and certain neurodegenerative diseases. Several strategies have been shown to induce appreciable regeneration in vivo, but the regrowth of axons through the entire optic nerve and on into the brain remains a major challenge. We show here that the induction of a controlled inflammatory response in the eye, when combined with elevation of intracellular cAMP and deletion of the gene encoding pten (phosphatase and tensin homolog), enables RGCs to regenerate axons the full length of the optic nerve in mature mice; approximately half of these axons cross the chiasm, and a rare subset (?1%) manages to enter the thalamus. Consistent with our previous findings, the axon-promoting effects of inflammation were shown to require the macrophage-derived growth factor Oncomodulin (Ocm). Elevation of cAMP increased the ability of Ocm to bind to its receptors in the inner retina and augmented inflammation-induced regeneration twofold. Inflammation combined with elevated cAMP and PTEN deletion increased activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways and augmented regeneration ?10-fold over the level induced by either pten deletion or Zymosan alone. Thus, treatments that synergistically alter the intrinsic growth state of RGCs produce unprecedented levels of axon regeneration in the optic nerve, a CNS pathway long believed to be incapable of supporting such growth.
| S-EPMC3001271 | biostudies-literature
Project description:Traumatic optic neuropathy (TON) is the loss of vision secondary to trauma. Approximately two weeks after traumatic damage, diffuse retinal ganglion cell loss and axon degeneration of the optic nerve are exhibited [1]. Here we present the changes that occur in the optic nerve lipidome of two-month-old C57BL/6J mice following sonication-induced TON (SI-TON), which closely models the indirect clinical mechanism in TON. Optic nerves were harvested at three time points following injury: 1-day, 7-days, and 14-days for comparison with the control group (uninjured optic nerves from 2-month-old mice). The optic nerves were subjected to mass spectrometry and bioinformatic analysis using LipidSearch 4.1.3 and Metaboanalyst 4.0. This data pertains to the lipidome at each time point following indirect trauma to the optic nerve. The data presented here will augment investigation into the neurodegenerative process. The data is available at Metabolomics Workbench [http://www.metabolomicsworkbench.org (Project ID: PR000859)].
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