Genome wide chromatin accessibility analysis reveals a role for CREB in retinal ganglion cells axon growth decline in development and regeneration after optic nerve injury [ATAC-seq]
Ontology highlight
ABSTRACT: CNS neurons lose their ability to grow and regenerate axons during development. This is the case for Retinal Ganglion Cells (RGCs) in the retina, which transmit visual information to the brain via axons projecting into the optic nerve. RGCs are unable to regenerate their axon after injury, and start a degeneration process that leads to cell death and loss of vision. To identifying molecular mechanisms that increase regeneration of RGC and may offer new treatment strategies for patients with glaucoma or other types of optic neuropathies, we focused on the identification of transcription factors and chromatin accessible sites that are enriched in RGC during developmental stages, in which axon growth capacity is robust. We find that stage-specific gene expression changes are correlated with temporal changes in promoter chromatin accessibility. We also find that Creb binding motifs are enriched in the differentially opened regions of the chromatin at embryonic developmental stage. Overexpression of active Creb promotes axon regeneration after optic nerve injury. Our results provide a map of the chromatin accessibility during RGC development and highlights that manipulating TF associated with developmental stages can stimulate axon growth in adulthood.
Project description:CNS neurons lose their ability to grow and regenerate axons during development. This is the case for Retinal Ganglion Cells (RGCs) in the retina, which transmit visual information to the brain via axons projecting into the optic nerve. RGCs are unable to regenerate their axon after injury, and start a degeneration process that leads to cell death and loss of vision. To identifying molecular mechanisms that increase regeneration of RGC and may offer new treatment strategies for patients with glaucoma or other types of optic neuropathies, we focused on the identification of transcription factors and chromatin accessible sites that are enriched in RGC during developmental stages, in which axon growth capacity is robust. We find that stage-specific gene expression changes are correlated with temporal changes in promoter chromatin accessibility. We also find that Creb binding motifs are enriched in the differentially opened regions of the chromatin at embryonic developmental stage. Overexpression of active Creb promotes axon regeneration after optic nerve injury. Our results provide a map of the chromatin accessibility during RGC development and highlights that manipulating TF associated with developmental stages can stimulate axon growth in adulthood.
Project description:Irreversible blindness from glaucoma and optic neuropathies is attributed to retinal ganglion cells (RGCs) losing the ability to regenerate axons. While several transcription factors and proteins have demonstrated enhancement of axon regeneration after optic nerve injury, mechanisms contributing to the age-related decline in axon regenerative capacity remains elusive. Here, we show that microRNAs are differentially expressed during RGC development, and identify microRNA-19a (miR-19a) as a heterochronic marker; developmental decline of miR-19a relieves suppression of PTEN, a key regulator of axon regeneration, and serves as a temporal indicator of decreasing axon regenerative capacity. Intravitreal injection of miR-19a promotes axon regeneration after optic nerve crush in adult mice, and increases axon extension in RGCs isolated from aged human donors. This uncovers a previously unrecognized involvement of the miR-19a-PTEN axis in RGC axon regeneration, and demonstrates therapeutic potential of microRNA-mediated restoration of axon regenerative capacity via intravitreal injection in patients with optic neuropathies.
Project description:Collapsin response mediator proteins (Crmps) play roles in neuronal development and axon growth. However, neuronal-specific roles of Crmp1, Crmp4, and Crmp5 in regeneration of injured central nervous system (CNS) axons in vivo are unclear. Here, we analyzed developmental and subtype-specific expression of Crmp genes in retinal ganglion cells (RGCs), tested whether overexpressing Crmp1, Crmp4, or Crmp5 in RGCs through localized intralocular AAV2 delivery promotes axon regeneration after optic nerve injury in vivo, and characterized developmental co-regulation of gene-concept networks associated with Crmps. We found that all Crmp genes are developmentally downregulated in RGCs during maturation. However, while Crmp1, Crmp2, and Crmp4 were expressed to a varying degree in most RGC subtypes, Crmp3 and Crmp5 were expressed only in a small subset of RGC subtypes. We then found that after optic nerve injury, Crmp1, Crmp4, and Crmp5 promote RGC axon regeneration to varying extents, with Crmp4 promoting the most axon regeneration and also localizing to axons. We also found that Crmp1 and Crmp4, but not Crmp5, promote RGC survival. Finally, we found that Crmp1, Crmp2, Crmp4, and Crmp5's ability to promote axon regeneration is associated with neurodevelopmental mechanisms, which control RGC’s intrinsic axon growth capacity.
Project description:Collapsin response mediator proteins (Crmps) play roles in neuronal development and axon growth. However, neuronal-specific roles of Crmp1, Crmp4, and Crmp5 in regeneration of injured central nervous system (CNS) axons in vivo are unclear. Here, we analyzed developmental and subtype-specific expression of Crmp genes in retinal ganglion cells (RGCs), tested whether overexpressing Crmp1, Crmp4, or Crmp5 in RGCs through localized intralocular AAV2 delivery promotes axon regeneration after optic nerve injury in vivo, and characterized developmental co-regulation of gene-concept networks associated with Crmps. We found that all Crmp genes are developmentally downregulated in RGCs during maturation. However, while Crmp1, Crmp2, and Crmp4 were expressed to a varying degree in most RGC subtypes, Crmp3 and Crmp5 were expressed only in a small subset of RGC subtypes. We then found that after optic nerve injury, Crmp1, Crmp4, and Crmp5 promote RGC axon regeneration to varying extents, with Crmp4 promoting the most axon regeneration and also localizing to axons. We also found that Crmp1 and Crmp4, but not Crmp5, promote RGC survival. Finally, we found that Crmp1, Crmp2, Crmp4, and Crmp5's ability to promote axon regeneration is associated with neurodevelopmental mechanisms, which control RGC’s intrinsic axon growth capacity.
Project description:The failure of adult CNS neurons to survive and regenerate their axons after injury or in neurodegenerative disease remains a major target for basic and clinical neuroscience. Recent data demonstrated in the adult mouse that exogenous expression of Sry-related high-mobility-box 11 (Sox11) promotes optic nerve regeneration after optic nerve injury, but exacerbates the death of a subset of retinal ganglion cells, alpha-RGCs. During development, Sox11 is required for RGC differentiation from retinal progenitor cells (RPCs), and we found that mutation of a single residue to prevent sumoylation at K91 increased nuclear localization and RGC differentiation in vitro. Here we explored whether this Sox11 manipulation similarly has stronger effects on RGC survival and optic nerve regeneration. In vitro, we found that non-SUMOylatable Sox11 K91A leads to RGC death and suppresses axon outgrowth in primary neurons. We furthermore found that Sox11 K91A more strongly promotes axon regeneration but also increases RGC death after optic nerve injury in vivo in adult mouse. RNA-seq data showed that Sox11 and Sox11 K91A increase the expression of key signaling pathway genes associated with axon growth and regeneration but downregulated Spp1 and Opn4 expression in RGC cultures, consistent with negatively regulating the survival of α-RGCs and ipRGCs. Thus Sox11 and its sumoylation site at K91 regulate gene expression, survival and axon growth in RGCs and may be explored further as potential regenerative therapies for optic neuropathy.
Project description:Adult mammalian CNS neurons undergo a developmental switch in intrinsic axon growth ability associated with their failure to regenerate axons after injury. Krüppel-like transcription factors (KLF) regulate intrinsic axon growth ability, but signaling regulation upstream and downstream is poorly understood. Here we find that suppressing expression of KLF9, an axon growth suppressor normally upregulated 250-fold in retinal ganglion cell (RGC) development, promotes long-distance optic nerve regeneration in vivo. We identify a novel binding partner, MAPK10/JNK3, critical for KLF9’s axon growth suppressive activity. Additionally, by screening genes regulated by KLFs in RGCs, we identify dual-specificity phosphatase 14 (Dusp14) as key to limiting axon growth and regenerative ability downstream of KLF9, associated with its dephosphorylation of MAPKs critical to neurotrophic signaling of RGC axon elongation. These results now link intrinsic and extrinsic regulation of axon growth and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.
Project description:At least 30 types of retinal ganglion cell (RGC) send distinct messages through the optic nerve to the brain. Strategies for promoting regeneration of RGC axons following injury act on only some of these types. Here we tested the hypothesis that over-expressing developmentally important transcription factors in adult RGCs could reprogram them to a “youthful” growth-competent state and promote regeneration of other types. From a screen of transcription factors expressed by developing RGCs, we found one, Sox11, that induced substantial axon regeneration. Transcriptome profiling confirmed that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, alpha-RGCs, which preferentially regenerate following treatments such as PTEN deletion, were killed by Sox 11. Thus, Sox 11 promotes regeneration of non-alpha RGCs, which are refractory to PTEN. We conclude that Sox11 can reprogram adult RGCs to a growth-competent state and that different growth-promoting interventions act on distinct neuronal types.
Project description:The retina constitutes a segment of the central nervous system (CNS). Both retinal and CNS neurons lack the inherent capacity to spontaneously regenerate axons following injury. Retinal ganglion cells (RGCs) serve as the neurons connecting the eyes to the brain. Diseases such as glaucoma initiate damage to RGC axons at the optic nerve, ultimately leading to cell death and irreversible vision loss. While enhancing the survival of RGCs is a crucial initial step, especially for those with pre-existing axonal injuries in the optic nerve due to prolonged insults, effective therapies should also promote axon regeneration. Neuro-regeneration and reintegration into the brain remain to be enormous challenges in neurology and ophthalmology. Despite decades of effort and resources invested in the research of neuro-regeneration, scientists are still rather far from comprehensively identifying all intrinsic axonal growth regulators and their collaborative roles. Data-independent acquisition mass spectrometry (DIA-MS) is a next-generation proteomic methodology that generates permanent digital proteome maps offering highly reproducible retrospective analysis of cellular and tissue specimens. Compared to conventional mass-spectrometry, DIA-MS provides better reproducibility and sensitivity. In this study, we employed DIA-MS to conduct a comprehensive protein expression mapping in retinas from mouse models of degeneration and regeneration, and to gain insights into the complex molecular mechanisms associated with degeneration and regeneration processes in the retina.
Project description:Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cell (RGC) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here we developed a strategy to specifically label and purify regRGCs and surRGCs respectively from the same Pten deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserves visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.
Project description:Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cell (RGC) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here we developed a strategy to specifically label and purify regRGCs and surRGCs respectively from the same Pten deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserves visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.