Project description:Unlike adult mammals, adult frogs regrow and regenerate their optic nerve following a crush injury. Using Translational Ribosome Affinity Purification (TRAP), a method to isolate mRNAs actively undergoing translation in a target cell population, we have generated a transcriptional profile by RNA-Seq for retinal ganglion cells (RGC) during the period of recovery following an optic nerve injury. Based on bioinformatics analysis using the JGI 9.1 Xenopus laevis gene models, our results reveal a profound shift in the composition of actively translating mRNAs during the early stages of RGC regeneration: as factors involved in cell signaling are rapidly downregulated, and those involved in core metabolism are upregulated. We identified one highly upregulated gene in response to injury, uchl1, which coupled to downregulation of the synucleins (snca, scng), was previously implicated in neurodegenerative diseases. Our injury-screen in Xenopus identified a previously unknown gene, gng8, as being associated with the regenerative process. Our generated online database provides the Xenopus community a valuable resource for the identification of genes involved in the regeneration process to target for future functional studies.
Project description:CNS injuries of the anuran amphibian, Xenopus laevis, are uniquely befitted for studying the molecular compositions of neuronal regeneration of retinal ganglion cells (RGC) due to a functional recovery of optic axons disparate to adult mammalian analogues. RGCs and their optic nerve axons undergo irreversible neurodegeneration in glaucoma and associated optic neuropathies, resulting in blindness in mammals. Conversely, Xenopus demonstrates RGC lifetime-spanning regenerative capabilities after optic nerve crush, inciting opportunities to compare de novo regeneration and develop efficient pharmaceutical approaches for vision restoration. Studies revealing lipidome alterations during optic nerve regeneration are sparse and could serve as a solid foundation for these underlying molecular changes. We profile the lipid changes in a transgenic line of 1 year old Xenopus laevis Tg(islet2b:gfp) frogs that were either left untreated (naïve) or had a monocular surgery of either a left optic crush injury (crush) or sham surgery (sham). Matching controls of uninjured right optic nerves were also collected (control). Tg(islet2b:gfp) frogs were allowed to recover for 7,12,18, and 27 days post optic nerve crush. Following euthanasia, the optic nerves were collected for lipidomic analysis. A modified Bligh and Dyer method [PMID: 13671378] was used for lipid extraction, followed by untargeted mass spectrometry lipid profiling with a Q-Exactive Orbitrap Liquid Chromatography-Mass Spectrometer (LC MS-MS) coupled with Vanquish Horizon Binary UHPLC LC-MS system.
Project description:Dnmt3a deficiency in retinal ganglion cells promotes axon regeneration in an optic nerve crush mouse model. To investigate the role of Dnmt3a in axon regeneration, retinal ganglion cell nuclei were sorted from control and retinal ganglion cell-specific Dnmt3a knockout mice at 2 days post optic nerve crush and applied for single nuclei RNA-seq analysis by 10X Genomics technology.
Project description:Dnmt3a deficiency in retinal ganglion cells promotes axon regeneration in an optic nerve crush mouse model. To investigate the role of Dnmt3a in axon regeneration, retinal ganglion cells isolated from control and retinal ganglion cell-specific Dnmt3a knockout mice at 2 days post-crush were applied for Bulk RNA-seq analysis by NovaSeq platform.
Project description:Dnmt3a deficiency in retinal ganglion cells promotes axon regeneration in an optic nerve crush mouse model. To investigate the contribution of Dnmt3a to DNA methylation and axon regeneration, retinal ganglion cells isolated from control and retinal ganglion cell-specific Dnmt3a knockout mice at 2 days post-crush were applied for whole genome bisulfite-seq analysis by Illumina NovaSeq 6000.
Project description:Expressing HDAC5 mutant whose serine 259 and 488 have been replaced by alanine (HDAC5AA) promotes optic nerve regeneration in retinal ganglion cells. However, expressing GFP, HDAC5WT and HDAC5DAD, whose serine 259 and 498 have been replaced by aspartic acid and serine 280 by alanine, do not promote optic nerve regeneration. The goal of this experiment was to determine the underlying mechanisms leading to the phenotypical differences in optic nerve regeneration between control GFP, HDAC5DAD, and HDAC5AA by analyzing the retinal transcriptome of the different treatments.
Project description:To investigate the role of aldose reductase (AR) inhibition using Sorbinil on retinal microglia (RMG) activation, retinal ganglion cell (RGC) survival, and axon regeneration after optic nerve trauma. We observed that AR inhibition using Sorbinil attenuates RMG activation and subsequently promotes RGC survival and delays axon degeneration one week after optic nerve crush.
