ABSTRACT
Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.
Subject(s)
Cell Proliferation/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Nerve Regeneration/genetics , Neural Stem Cells/cytology , Neurons/metabolism , Neurons/pathology , Transcription, Genetic , Animals , Axons/pathology , Axons/physiology , Disease Models, Animal , Female , Gene Expression Profiling , Huntingtin Protein/genetics , Mice , Neural Stem Cells/transplantation , Neuronal Plasticity , Neurons/cytology , Neurons/transplantation , Protein Biosynthesis , Pyramidal Tracts/cytology , Pyramidal Tracts/metabolism , Pyramidal Tracts/pathology , RNA-Seq , Spinal Cord Injuries/genetics , Spinal Cord Injuries/pathology , TranscriptomeABSTRACT
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.
Subject(s)
Aging/metabolism , Axons/metabolism , Myelin Sheath/metabolism , Neural Stem Cells/cytology , Neuronal Outgrowth , Animals , Axons/ultrastructure , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Cyclic AMP/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gray Matter/cytology , Humans , Mice, Inbred C57BL , Myelin Sheath/ultrastructure , Neural Stem Cells/ultrastructure , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Inbred F344 , Rats, Nude , Spinal Cord/cytology , White Matter/cytologyABSTRACT
BACKGROUND: Quantification of axon regeneration in spinal cord tissue sections is a fundamental step to adequately determine if an applied treatment leads to an anatomical benefit following spinal cord injury. Recent advances have led to the development of therapies that can promote regeneration of thousands of injured axons in vivo. Axon labeling methods and in the application of regeneration-enabling stem cell grafts have increased the number of detectable regenerating axons by orders of magnitudes. Manual axon tracing in such cases is challenging and laborious, and as such there is a great need for automated algorithms that can perform accurate tracing and quantification in axon-dense tissue sections. RESULTS: We developed "AxonTracer", a fully automated software algorithm that traces and quantifies regenerating axons in spinal cord tissue sections. AxonTracer is an open source plugin for the freely available image-processing program ImageJ. The plugin identifies transplanted cells grafts or other regions of interest (ROIs) based on immunohistological staining and quantifies regenerating axons within the ROIs. Individual images or groups of images (batch mode) can be analyzed sequentially. In batch mode, a unique algorithm identifies a reference image for normalization, as well as a suitable image for defining detection parameters. An interactive user interface allows for adjustment of parameters defining ROI size, axon detection sensitivity and debris cleanup. Automated quantification of regenerating axons by AxonTracer correlates strongly with semi-manual quantification by the widely-used ImageJ plugin NeuronJ. However, quantification with AxonTracer is automated and reduces the need for user input compared to alternative methods. CONCLUSIONS: AxonTracer is a freely available open-source tool for automated analysis of regenerating axons in the injured nervous system. An interactive user interface provides detection-parameter adjustment, and usage does not require prior image analysis experience. Raw data as well as normalized results are stored in spreadsheet format and axon tracings are superimposed on raw images allowing for subjective visual verification. This software allows for automated, unbiased analysis of hundreds of axon-dense images, thus providing a useful tool in enabling in vivo screens of axon regeneration following spinal cord injury.
