Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost following spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. A week later BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immuno-electron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked cFos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.

Nuclear exclusion of the transcriptional regulators and potent oncoproteins, YAP/TAZ, is considered necessary for adult tissue homeostasis. Here we show that nuclear YAP/TAZ are essential regulators of peripheral nerve development and myelin maintenance. To proliferate, developing Schwann cells (SCs) require YAP/TAZ to enter S-phase and, without them, fail to generate sufficient SCs for timely axon sorting. To differentiate, SCs require YAP/TAZ to upregulate Krox20 and, without them, completely fail to myelinate, resulting in severe peripheral neuropathy. Remarkably, in adulthood, nuclear YAP/TAZ are selectively expressed by myelinating SCs, and conditional ablation results in severe peripheral demyelination and mouse death. YAP/TAZ regulate both developmental and adult myelination by driving TEAD1 to activate Krox20. Therefore, YAP/TAZ are crucial for SCs to myelinate developing nerve and to maintain myelinated nerve in adulthood. Our study also provides a new insight into the role of nuclear YAP/TAZ in homeostatic maintenance of an adult tissue.DOI: http://dx.doi.org/10.7554/eLife.20982.001

Activity-based therapies such as passive bicycling and step-training on a treadmill contribute to motor recovery after spinal cord injury (SCI), leading to a greater number of steps performed, improved gait kinematics, recovery of phase-dependent modulation of spinal reflexes, and prevention of decrease in muscle mass. Both tasks consist of alternating movements that rhythmically stretch and shorten hindlimb muscles. However, the paralyzed hindlimbs are passively moved by a motorized apparatus during bike-training, whereas locomotor movements during step-training are generated by spinal networks triggered by afferent feedback. Our objective was to compare the task-dependent effect of bike-and step-training after SCI on physiological measures of spinal cord plasticity in relation to changes in levels of neurotrophic factors. Thirty adult female Sprague-Dawley rats underwent complete spinal transection at a low thoracic level (T12). The rats were assigned to one of three groups: bike-training, step-training, or no training. The exercise regimen consisted of 15 min/d, 5 days/week, for 4 weeks, beginning 5 days after SCI. During a terminal experiment, H-reflexes were recorded from interosseus foot muscles following stimulation of the tibial nerve at 0.3, 5, or 10 Hz. The animals were sacrificed and the spinal cords were harvested for Western blot analysis of the expression of neurotrophic factors in the lumbar spinal cord. We provide evidence that bike-and step-training significantly increase the levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 in the lumbar enlargement of SCI rats, whereas only step-training increased glial cell-derived neurotrophic factor (GDNF) levels. An increase in neurotrophic factor protein levels that positively correlated with the recovery of H-reflex frequency-dependent depression suggests a role for neurotrophic factors in reflex normalization.

In spinal cats, locomotor recovery without rehabilitation is limited, but weight-bearing stepping returns with treadmill training. We studied whether neurotrophins administered to the injury site also restores locomotion in untrained spinal cats and whether combining both neurotrophins and training further improves recovery. Ordinary rat fibroblasts or a mixture of fibroblasts secreting brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (Fb-NTF) were grafted into T12 spinal transection sites. Cats with each type of transplant were divided into two groups: one receiving daily training and the other receiving no training. As expected, trained cats with/without neurotrophin-producing transplants could step on the treadmill. Untrained cats without neurotrophin-producing transplants could not locomote. However, untrained cats with neurotrophin-secreting transplants performed plantar weight-bearing stepping at speeds up to 0.8 m/s as early as 2 wk after transection. Locomotor capability and stance lengths in these animals were similar to those in animals receiving training alone, suggesting that administration of BDNF/NT-3 was equivalent to treadmill training in restoring locomotion in chronically spinalized cats. Cats receiving both interventions showed the greatest improvement in step length. Anatomical evaluation indicated that all transections were complete and that axons did not enter the cord caudal to the graft. Thus BDNF/NT-3 secreting fibroblasts were equivalent to training in their ability to engage the locomotor circuitry in chronic spinal cats. Furthermore, the rapid time-course of recovery and the absence of axonal growth through the transplants indicate that the restorative mechanisms were not related to supraspinal axonal growth. Finally, the results show that transplants beneficial in rodents are applicable to larger mammals.

Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft-host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF-PBS treatment, GDNF-ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.

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A simple neuromechanical model has been developed that describes a spinal central pattern generator (CPG) controlling the locomotor movement of a single-joint limb via activation of two antagonist (flexor and extensor) muscles. The limb performs rhythmic movements under control of the muscular, gravitational and ground reaction forces. Muscle afferents provide length-dependent (types Ia and II) and force-dependent (type Ib from the extensor) feedback to the CPG. We show that afferent feedback adjusts CPG operation to the kinematics and dynamics of the limb providing stable “locomotion.” Increasing the supraspinal drive to the CPG increases locomotion speed by reducing the duration of stance phase. We show that such asymmetric, extensor-dominated control of locomotor speed (with relatively constant swing duration) is provided by afferent feedback independent of the asymmetric rhythmic pattern generated by the CPG alone (in “fictive locomotion” conditions). Finally, we demonstrate the possibility of reestablishing stable locomotion after removal of the supraspinal drive (associated with spinal cord injury) by increasing the weights of afferent inputs to the CPG, which is thought to occur following locomotor training.