RNA sequence reveals mouse retinal transcriptome changes early after axonal injury
ABSTRACT: Purpose: The purpose of this study was to use RNA-seq to investigate the molecular mechanisms of damage in the early stages of the response to axonal injury, before the onset of RGC death. Methods: 12-week-old wild-type (WT) mice were used in this study. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Retinal mRNA profiles were generated by deep sequencing, in triplicate, using IlluminaHiseq2000. The sequence reads were analyzed by CLC genomics workbench and R software. qRT–PCR validation was performed using TaqMan assays. Results: Using an optimized data analysis workflow, we mapped about 66 million sequence reads per sample to the mouse genome (build mm9). Differential gene expression analysis showed that endoplasmic reticulum stress-related genes and antioxidative response-related genes have been shown to be significantly upregulated 2 days after ONC. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury. Retinal mRNA profiles of 12 week-old wild type (WT) after ONC or sham were generated by deep sequencing, in triplicate, using Illumina Hiseq2000.
Project description:Retinal ganglion cell (RGC) death is the final consequence of many blinding diseases, where there is considerable variation in the time course and severity of RGC loss. Indeed, this process appears to be influenced by a wide variety of genetic and environmental factors. In this study we explored the genetic basis for differences in ganglion cell death in two inbred strains of mice. We found that RGCs are more susceptible to death following optic nerve crush in C57BL/6J mice (54% survival) than in DBA2/J mice (62% survival). Using the Illumina Mouse-6 microarray, we identified 1,580 genes with significant change in expression following optic nerve crush in these two strains of mice. Our analysis of the changes occurring after optic nerve crush demonstrated that the greatest amount of change (44% of the variance) was due to the injury itself. This included changes associated with ganglion cell death, reactive gliosis, and abortive regeneration. The second pattern of gene changes (23% of the variance) was primarily related to differences in gene expressions observed between the C57BL/6J and DBA/2J mouse strains. The remaining changes in gene expression represent interactions between the effects of optic nerve crush and the genetic background of the mouse. We extracted one genetic network from this dataset that appears to be related to tissue remodeling. One of the most intriguing sets of changes included members of the crystallin family of genes, which may represent a signature of pathways modulating the susceptibility of cells to death. Differential responses to optic nerve crush between two widely used strains of mice were used to define molecular networks associated with ganglion cell death and reactive gliosis. These results form the basis for our continuing interest in the modifiers of retinal injury. 18 Samples: 9 per strain (C57BL/6J & DBA/2J); 3 conditions per strain
Project description:It is well-established that neurons in the adult mammalian central nervous system are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in ~21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after optic nerve crush can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. We used microarrays to detail the global gene expression patterns underlying a successful regeneration or the optic nerve following injury. Experiment Overall Design: Microarray analysis was performed on total RNA extracted from whole eye following optic nerve crush (ONX) or sham surgery at defined intervals (4, 12, & 21 days).
Project description:A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either Phosphatase and tensin homolog (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signaling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around two weeks after the crush injury. Remarkably, we now find that simultaneous deletion of both PTEN and SOCS3 enable robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only result in the induction of many growth-related genes, but also allow RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as a key for sustaining long-distance axon regeneration in adult CNS, a crucial step toward functional recovery. RNAs were extracted from FACS sorted YFP positive mouse retinal cells, and gene-profiled using affymetrix 1.0 ST expression arrays. Three hybridizations were performed for each group (Wild type after crush, PTEN Knockout+crush, SOCS3 Knockout+crush, and PTEN/SOCS3 double knockout+crush) with RNA samples collected from three independent FACS purifications. Data were analyzed using dChIP and SAM.
Project description:We used optic nerve injury as a model to study early signaling events in the neuronal soma following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury. We detected differences in phosphoproteins and gene expression within this time period that we used to interpret temporal events. Our studies revealed that the entire retina has been signaled by the RGC soma within 30 min after optic nerve injury and that pathways that modulate cell death are likely to be active in RGCs within 6 hrs following axonal injury Experiment Overall Design: In the treated animals, axons of the optic nerve were crushed with fine forceps for 10 sec, 1 mm posterior to the globe, under direct visualization, within an intact meningeal sheath. Controls were contralateral eyes from the same animals in each group that had not been injured. After 6 hr eyes were enucleated and processed for tissue sectionin
Project description:We used optic nerve injury as a model to study early signaling events in the neuronal soma following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury. We detected differences in phosphoproteins and gene expression within this time period that we used to interpret temporal events. Our studies revealed that the entire retina has been signaled by the RGC soma within 30 min after optic nerve injury and that pathways that modulate cell death are likely to be active in RGCs within 6 hrs following axonal injury Keywords: Stress Response Overall design: In the treated animals, axons of the optic nerve were crushed with fine forceps for 10 sec, 1 mm posterior to the globe, under direct visualization, within an intact meningeal sheath. Controls were contralateral eyes from the same animals in each group that had not been injured. After 6 hr eyes were enucleated and processed for tissue sectionin
Project description:Reactive gliosis is a complex process that involves profound changes in gene expression. We used microarray to indentify differentially expressed genes and to investigate the molecular mechanisms of reactive gliosis in optic nerve head in response to optic nerve crush injury. C57Bl/6 female mice were 6-8 weeks old at the time of optic nerve crush surgery. The optic nerve in the left eye was crush 1 mm behind the globe for 10 seconds and the right eye served as contralateral control. The animals were allowed to recover for 1 day, 3 day, 1 week, 3 weeks and 3 months before the optic nerve heads were collected. The naive control mice did not receive any surgery in either eye. Due to the small tissue size of the mouse optic nerve head, two optic nerve heads were pooled together for each microarray chip. The left eyes and the right eyes of two mice were combined respectively to form one pair of experiment and control samples. There were five biological replicates (10 mice) for each condition.
Project description:retinal ganglion cells die after optic nerve injury, either crush or transection. The molecular causesunderlying this degeneration are largely unkwon; the purpose of this job is to find which (if any) gene regulation triggers RGC death with the final goal of design neuroprotective protocols Experiment Overall Design: 3 groups: naive, IONT (intraorbital nerve transection) IONC (intraorbital nerve crush). IONT and IONC lesioned animals were kept the appropriate times postlesion (12h,. 24h, 48h, 3d, 7d, and 15d). For each time point 8-12 animals were used. RNA from 4 animals was pooled and extracted to hybridaze 1 array replica. All replicas were pooled biological replicas: 5 for naive RNA (each replica 4 retinas, therefore 5 independent RNA extractions were done with a total of 20 retinas), IONT: 12h 3 replicas, 24h 2 replicas, 48h 3replicas, 3d 2 replicas, 7d 3 replicas, 15d 2 replicas. IONC: 3 replicas per time point: 12h, 24h, 48h, 3d and 7d)
Project description:During brain wiring, mRNAs are trafficked into axons and growth cones where they are differentially translated in response to extrinsic signals. Differential control of local protein synthesis mediates neuronal compartment-specific behaviors that aid axon guidance. Yet little is understood about how specific mRNAs are selected for translation. Here we have investigated the local role of microRNAs (miRNAs) in mRNA-specific translation during axon pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Profiling experiments revealed a rich repertoire of axonal miRNAs in developing RGC axons and identified miR-182 as one of the most abundant. Loss of miR182 impairs Slit2-induced growth cone repulsion and causes RGC axon targeting defects in vivo. To aid miRNA target prediction, we also profiled mRNA expression in RGC axons. Our results show that miR-182 targets cofilin1 mRNA in RGC growth cones and modulates its local translation in response to Slit2. Overall design: Two biological replicates, one condition