Mammalian CNS Axons Research Articles

Telomerase Reverse Transcriptase and p53 Regulate Mammalian Peripheral Nervous System and CNS Axon Regeneration Downstream of c-Myc.

Although several genes have been identified to promote axon regeneration in the CNS, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using female mouse sensory axon and optic nerve regeneration as model systems, we reveal an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provide evidence that TERT and p53 act downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells is sufficient to promote sensory axon and optic never regeneration, respectively. The study reveals a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration.SIGNIFICANCE STATEMENT Despite significant progress during the past decade, our understanding of the molecular mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented. By using sensory axon and optic nerve regeneration as model systems, the study revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. The results also delineated a c-Myc-TERT-p53 pathway in controlling axon growth. Last, our results demonstrated that p53 alone was sufficient to promote sensory axon and optic nerve regeneration in vivo Collectively, the study not only revealed a new mechanisms underlying mammalian axon regeneration, but also expanded the pool of potential targets that can be manipulated to enhance CNS axon regeneration.

Wnt signaling promotes axonal regeneration following optic nerve injury in the mouse

Adult mammalian CNS axons generally do not regenerate, creating an obstacle to effective repair and recovery after neuronal injury. The canonical Wnt/β-catenin signaling pathway is an essential signal transduction cascade that regulates axon growth and neurite extension in the developing mammalian embryo. In this study, we investigated whether a Wnt/β-catenin signaling activator could be repurposed to induce regeneration in the adult CNS after axonal injury. We used a retinal ganglion cell (RGC) axon crush injury model in a transgenic Wnt reporter mouse, and intravitreal injections were used to deliver Wnt3a or saline to the RGC cell bodies within the retina. Our findings demonstrated that Wnt3a induced Wnt signaling in RGCs and resulted in significant axonal regrowth past the lesion site when measured at two and four weeks post-injury. Furthermore, Wnt3a-injected eyes showed increased survival of RGCs and significantly higher pattern electroretinography (PERG) amplitudes compared to the control. Additionally, Wnt3a-induced axonal regeneration and RGC survival were associated with elevated activation of the transcription factor Stat3, and reducing expression of Stat3 using a conditional Stat3 knock-out mouse line led to diminished Wnt3a-dependent axonal regeneration and RGC survival. Therefore, these findings reveal a novel role for retinal Wnt signaling in axonal regrowth and RGC survival following axonal injury, which may lead to the development of novel therapies for axonal regeneration.

Dynamic Axonal Translation in Developing and Mature Visual Circuits

SummaryLocal mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.

Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f (Sac2) as an Endogenous Suppressor of Repair after Spinal Cord Injury.

Axonal growth and neuronal rewiring facilitate functional recovery after spinal cord injury. Known interventions that promote neural repair remain limited in their functional efficacy. To understand genetic determinants of mammalian CNS axon regeneration, we completed an unbiased RNAi gene-silencing screen across most phosphatases in the genome. We identified one known and 17 previously unknown phosphatase suppressors of injury-induced CNS axon growth. Silencing Inpp5f (Sac2) leads to robust enhancement of axon regeneration and growth cone reformation. Results from cultured Inpp5f(-/-) neurons confirm lentiviral shRNA results from the screen. Consistent with the nonoverlapping substrate specificity between Inpp5f and PTEN, rapamycin does not block enhanced regeneration in Inpp5f(-/-) neurons, implicating mechanisms independent of the PI3K/AKT/mTOR pathway. Inpp5f(-/-) mice develop normally, but show enhanced anatomical and functional recovery after mid-thoracic dorsal hemisection injury. More serotonergic axons sprout and/or regenerate caudal to the lesion level, and greater numbers of corticospinal tract axons sprout rostral to the lesion. Functionally, Inpp5f-null mice exhibit enhanced recovery of motor functions in both open-field and rotarod tests. This study demonstrates the potential of an unbiased high-throughput functional screen to identify endogenous suppressors of CNS axon growth after injury, and reveals Inpp5f (Sac2) as a novel suppressor of CNS axon repair after spinal cord injury. Significance statement: The extent of axon regeneration is a critical determinant of neurological recovery from injury, and is extremely limited in the adult mammalian CNS. We describe an unbiased gene-silencing screen that uncovered novel molecules suppressing axonal regeneration. Inpp5f (Sac2) gene deletion promoted recovery from spinal cord injury with no side effects. The mechanism of action is distinct from another lipid phosphatase implicated in regeneration, PTEN. This opens new pathways for investigation in spinal cord injury research. Furthermore the screening methodology can be applied on a genome wide scale to discovery the entire set of mammalian genes contributing to axonal regeneration.

Birthdate and Outgrowth Timing Predict Cellular Mechanisms of Axon Target Matching in the Developing Visual Pathway

How axons select their appropriate targets in the brain remains poorly understood. Here, we explore the cellular mechanisms of axon target matching in the developing visual system by comparing four transgenic mouse lines, each with a different population of genetically labeled retinal ganglion cells (RGCs) that connect to unique combinations of brain targets. We find that the time when an RGC axon arrives in the brain is correlated with its target selection strategy. Early-born, early-arriving RGC axons initially innervate multiple targets. Subsequently, most of those connections are removed. By contrast, later-born, later-arriving RGC axons are highly accurate in their initial target choices. These data reveal the diversity of cellular mechanisms that mammalian CNS axons use to pick their targets and highlight the key role of birthdate and outgrowth timing in influencing this precision. Timing-based mechanisms may underlie the assembly of the other sensory pathways and complex neural circuitry in the brain.

Building Bridges for Spinal Cord Repair

For nearly a century, neuroscientists have sought to restore neurological function across spinal cord lesions. Lu etal. now present significant progress toward this goal, showing in rats that transplanted neural stem cells establish a functional bridge across completely transected spinal cords.

Keratan Sulfate Proteoglycans in Plasticity and Recovery after Spinal Cord Injury: Figure 1.

The limited potential for regeneration and plasticity of adult mammalian CNS axons is a major impediment to functional recovery following spinal cord injury (SCI). Inhibitory extracellular matrix, particularly chondroitin sulfate proteoglycans (CSPGs), associated with the lesion penumbra and

Semi-automated Sholl analysis for quantifying changes in growth and differentiation of neurons and glia

There is a need to develop therapies that promote growth or remyelination of mammalian CNS axons. Although the feasibility of pre-clinical treatment strategies should be tested in animal models, in vitro assays are usually faster and less expensive. As a result, in vitro models are ideal for screening large numbers of potential therapeutics prior to use in more complex in vivo systems. In 1953, Sholl introduced a technique that is a reliable and sensitive method for quantifying indices of neurite outgrowth. However, application of the technique is limited because it is labor-intensive. Several methods have been developed to reduce the analysis time for the Sholl technique; but these methods require extensive pre-processing of digital images, they introduce user bias or they have not been compared to manual analysis to ensure accuracy. Here we describe a new, semi-automated Sholl technique for quantifying neuronal and glial process morphology. Using MetaMorph®, we developed an unbiased analysis protocol that can be performed ∼3× faster than manual quantification with a comparable level of accuracy regardless of cell morphology. The laborious image processing typical of most computer-aided analysis is avoided by embedding image correction functions into the automated portion of the analysis. The sensitivity and validity of the technique was confirmed by quantifying neuron growth treated with growth factors or oligodendroglial maturation in the presence or absence of thyroid hormone. Thus, this technique provides a rapid and sensitive method for quantifying changes in cell morphology and screening for treatment effects in multiple cell types in vitro.

Axonal Injury and Regeneration in the Adult Brain of Drosophila

Drosophila melanogaster is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the CNS. We developed a versatile and physiologically robust preparation for the long-term culture of the whole Drosophila brain. We use this method to develop a novel Drosophila model for CNS axonal injury and regeneration. We first show that, similar to mammalian CNS axons, injured adult wild-type fly CNS axons fail to regenerate, whereas adult-specific enhancement of protein kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms after injury. We next exploit this model to explore pathways that induce robust regeneration and find that adult-specific activation of c-Jun N-terminal protein kinase signaling is sufficient for de novo CNS axonal regeneration injury, including the growth of new axons past the lesion site and into the normal target area.

Effects of an embryonic repair graft on recovery from spinal cord injury

It is widely believed that mammalian CNS axons have little regenerative capacity because their environment is non-permissive to regrowth. This viewpoint is based, in large part, on the fact that in virtually all previous studies on regeneration following spinal cord injury, regenerated axonal projections have been few in number, quite short, and considered to be mostly aberrant. As a result, motor recovery has been very limited in both experimental preparations and the human. In this chapter, we describe use of a neonatal, spinally transected animal model in which selected spinal cord segments were carefully replaced with equivalent tissue from embryonic tissue of the same species. We demonstrate that the new spinal environment is indeed permissive, and reconstruction is possible of neural connections, which are similar to the pre-injury, normal projections. Moreover, the distribution and number of regenerated axons are closely related to the extent of functional motor recovery. Our results suggest that contrary to doctrinaire thought, the mammalian CNS possesses a remarkable capacity for regrowth. For this to be efficacious, however, regenerating axons must contact the inherent, pre-injury guidance system, whose cues were used for establishing appropriate neural connections in the developing animal, and are retained in the adult. It is argued that by use of these guidance cues, regenerating axons that traverse the site of a spinal cord injury, can project on to locate their pre-injury pathways and targets, and thereby restore function.

Chapter 17 Cellular and molecular correlates of the regeneration of adult mammalian CNS axons into peripheral nerve grafts

Studies of the regeneration of CNS axons into peripheral nerve grafts have provided information crucial to our understanding of the regenerative potential of CNS neurons. Injured axons in the thalamus and corpus striatum produce regenerative sprouts within a few days of graft implantation, apparently in response to living cells in the grafts. The regenerating axons often grow directly towards the grafts, and enter Schwann cell columns where they elongate surrounded by Schwann cell processes. The regenerating CNS axons, and the Schwann cell processes along which they grow, initially express the cell adhesion molecules NCAM, and L1. The axons also express polysialic acid and, unlike regenerating peripheral axons, bind tenascin-C derived from Schwann cells. Wherever peripheral nerve grafts are implanted into the CNS they appear to promote the differential regeneration of CNS axons. Most of the axons which grow into grafts in the thalamus are derived from the thalamic reticular nucleus (TRN), whereas grafts in the striatum promote regeneration of axons from the substantia nigra pars compacta (SNpc) and grafts in the cerebellum promote regeneration from deep cerebellar nuclei (DCN) and brainstem precerebellar neurons. In contrast most thalamocortical projection neurons, striatal projection neurons and Purkinje cells in the cerebellar cortex are poor at regenerating. There are patterns to the expression of regeneration-related molecules by axons injured by nerve grafts in the CNS. Most neurons which regenerate well (e.g. TRN and DCN neurons) upregulate GAP-43, L1 and the transcription factor c-jun in response to a graft, whereas those neurons which do not regenerate well (e.g. Purkinje cells, thalamocortical and striatal projection neurons) do not upregulate these molecules. These observations suggest that some classes of CNS neurons may be intrinsically unable to regenerate axons and the repair of injuries in the brain and spinal cord may consequently require some form of gene therapy for axotomised neurons.

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified β-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.

Samuel William Johnson

Although several genes have been identified to promote axon regeneration in the CNS, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using female mouse sensory axon and optic nerve regeneration as model systems, we reveal an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provide evidence that TERT and p53 act downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells is sufficient to promote sensory axon and optic never regeneration, respectively. The study reveals a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration. <b>SIGNIFICANCE STATEMENT</b> Despite significant progress during the past decade, our understanding of the molecular mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented. By using sensory axon and optic nerve regeneration as model systems, the study revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. The results also delineated a c-Myc-TERT-p53 pathway in controlling axon growth. Last, our results demonstrated that p53 alone was sufficient to promote sensory axon and optic nerve regeneration <i>in vivo</i>. Collectively, the study not only revealed a new mechanisms underlying mammalian axon regeneration, but also expanded the pool of potential targets that can be manipulated to enhance CNS axon regeneration.