The regenerative capacity of central nervous system (CNS) axons after injury is severely impaired compared to axons of the peripheral nervous system (PNS). We hypothesized that mechanisms both intrinsic and extrinsic to the neuron influence the ability of CNS axons to regenerate. To investigate this hypothesis we explored two model systems. In the first model system, we identified a regeneration transcriptome in injured corticospinal motor neurons that is associated with enhanced central axon regeneration after spinal cord injury. The genetic mechanisms identified in this model include cAMP-Erk-CREB, Huntingtin, NFE2L2, ephrin and semaphorin signaling, and provide a dataset for potential therapeutic intervention to improve axonal regeneration in vivo after spinal cord injury. In the second model, we tested the hypothesis that glial cells of the peripheral nerve, Schwann cells, are an essential mechanism contributing to central axonal regeneration after "conditioning" lesions, wherein injury to the peripheral branch of a dorsal root ganglion sensory neuron enhances regeneration of the central branch of the sensory neuron. The gene encoding Low-density lipoprotein Receptor-related Protein-1 (LRP1) was conditionally deleted in Schwann cells, impairing the survival and function of Schwann cells after injury; animals with Schwann cell-specific deletion of LRP1 exhibited a significant reduction in axon regeneration in vitro and a trend towards central sensory axon regeneration after conditioning lesions, confirming that glial cells exhibit an essential but partial role in supporting axonal regeneration. Overall, these studies identify novel molecular and cellular mechanisms that influence central axon regeneration, and suggest therapeutic approaches to improve neural repair after CNS injury

Abstract Background To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems. Results Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology. Conclusions NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity^1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

Neural progenitor cell (NPC) transplantation has high therapeutic potential in neurological disorders. Functional restoration may depend on the formation of reciprocal connections between host and graft. While it has been reported that axons extending out of neural grafts in the brain form contacts onto phenotypically appropriate host target regions, it is not known whether adult, injured host axons regenerating into NPC grafts also form appropriate connections. We report that spinal cord NPCs grafted into the injured adult rat spinal cord self-assemble organotypic, dorsal horn-like domains. These clusters are extensively innervated by regenerating adult host sensory axons and are avoided by corticospinal axons. Moreover, host axon regeneration into grafts increases significantly after enrichment with appropriate neuronal targets. Together, these findings demonstrate that injured adult axons retain the ability to recognize appropriate targets and avoid inappropriate targets within neural progenitor grafts, suggesting that restoration of complex circuitry after SCI may be achievable.

Sensory neurons in the PNS demonstrate substantial capacity for regeneration following injury. Recent studies have identified changes in the transcriptome of sensory neurons, which are instrumental for axon regeneration. The role of Schwann cells (SCs) in mediating these changes remains undefined. We tested the hypothesis that SCs regulate expression of genes in sensory neurons before and after PNS injury by comparing mice in which LDL Receptor-related Protein-1 (LRP1) is deleted in SCs (scLRP1-/- mice) with wild-type (scLRP1+/+ ) littermates. LRP1 is an endocytic and cell-signaling receptor that is necessary for normal SC function and the SC response to nerve injury. scLRP1-/- mice represent a characterized model in which the SC response to nerve injury is abnormal. Adult DRG neurons, isolated from scLRP1-/- mice, with or without a conditioning nerve lesion, demonstrated increased neurite outgrowth when cultured ex vivo, compared with neurons from wild-type mice. Following sciatic nerve crush injury, nerve regeneration was accelerated in vivo in scLRP1-/- mice. These results were explained by transcriptional activation of RAGs in DRG neurons in scLRP1-/- mice prior to nerve injury. Although the presence of abnormal SCs in scLRP1-/- mice primed DRG neurons for repair, nerve regeneration in scLRP1-/- mice resulted in abnormalities in ultrastructure, principally in Remak bundles, and with the onset of neuropathic pain. These results demonstrate the importance of SCs in controlling RAG expression by neurons and the potential for this process to cause chronic pain when abnormal. The SC may represent an important target for preventing pain following PNS injury.

Sensory neurons in the PNS demonstrate substantial capacity for regeneration following injury. Recent studies have identified changes in the transcriptome of sensory neurons, which are instrumental for axon regeneration. The role of Schwann cells (SCs) in mediating these changes remains undefined. We tested the hypothesis that SCs regulate expression of genes in sensory neurons before and after PNS injury by comparing mice in which LDL Receptor-related Protein-1 (LRP1) is deleted in SCs (scLRP1-/- mice) with wild-type (scLRP1+/+ ) littermates. LRP1 is an endocytic and cell-signaling receptor that is necessary for normal SC function and the SC response to nerve injury. scLRP1-/- mice represent a characterized model in which the SC response to nerve injury is abnormal. Adult DRG neurons, isolated from scLRP1-/- mice, with or without a conditioning nerve lesion, demonstrated increased neurite outgrowth when cultured ex vivo, compared with neurons from wild-type mice. Following sciatic nerve crush injury, nerve regeneration was accelerated in vivo in scLRP1-/- mice. These results were explained by transcriptional activation of RAGs in DRG neurons in scLRP1-/- mice prior to nerve injury. Although the presence of abnormal SCs in scLRP1-/- mice primed DRG neurons for repair, nerve regeneration in scLRP1-/- mice resulted in abnormalities in ultrastructure, principally in Remak bundles, and with the onset of neuropathic pain. These results demonstrate the importance of SCs in controlling RAG expression by neurons and the potential for this process to cause chronic pain when abnormal. The SC may represent an important target for preventing pain following PNS injury.

Human induced pluripotent stem cells (iPSCs) from a healthy 86-year-old male were differentiated into neural stem cells and grafted into adult immunodeficient rats after spinal cord injury. Three months after C5 lateral hemisections, iPSCs survived and differentiated into neurons and glia and extended tens of thousands of axons from the lesion site over virtually the entire length of the rat CNS. These iPSC-derived axons extended through adult white matter of the injured spinal cord, frequently penetrating gray matter and forming synapses with rat neurons. In turn, host supraspinal motor axons penetrated human iPSC grafts and formed synapses. These findings indicate that intrinsic neuronal mechanisms readily overcome the inhibitory milieu of the adult injured spinal cord to extend many axons over very long distances; these capabilities persist even in neurons reprogrammed from very aged human cells.

Video abstract

The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In murine models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of SCI. Corticospinal regeneration requires grafts to be driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, a process that is potentially related to the attenuation of the glial scar. Rat corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord 'replacement' with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of SCI research and offering new possibilities for clinical translation.

Axon regeneration after spinal cord injury (SCI) is attenuated by growth inhibitory molecules associated with myelin. We report that rat myelin stimulated the growth of axons emerging from rat neural progenitor cells (NPCs) transplanted into sites of SCI in adult rat recipients. When plated on a myelin substrate, neurite outgrowth from rat NPCs and from human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) was enhanced threefold. In vivo, rat NPCs and human iPSC-derived NSCs extended greater numbers of axons through adult central nervous system white matter than through gray matter and preferentially associated with rat host myelin. Mechanistic investigations excluded Nogo receptor signaling as a mediator of stem cell-derived axon growth in response to myelin. Transcriptomic screens of rodent NPCs identified the cell adhesion molecule neuronal growth regulator 1 (Negr1) as one mediator of permissive axon-myelin interactions. The stimulatory effect of myelin-associated proteins on rodent NPCs was developmentally regulated and involved direct activation of the extracellular signal-regulated kinase (ERK). The stimulatory effects of myelin on NPC/NSC axon outgrowth should be investigated further and could potentially be exploited for neural repair after SCI.