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Background: Fibroblast growth factor receptors (FGFRs) are key targets for nerve regeneration and repair. The therapeutic effect of exogenous recombinant FGFs in vivo is limited due to their high molecular weight. Small peptides with low molecular weight, easy diffusion, low immunogenicity, and nontoxic metabolite formation are potential candidates. The present study aimed to develop a novel low-molecular-weight peptide agonist of FGFR to promote nerve injury repair. Methods: Phage display technology was employed to screen peptide ligands targeting FGFR2. The peptide ligand affinity for FGFRs was detected by isothermal titration calorimetry. Structural biology-based computer virtual analysis was used to characterize the interaction between the peptide ligand and FGFR2. The peptide ligand effect on axon growth, regeneration, and behavioral recovery of sensory neurons was determined in the primary culture of sensory neurons and dorsal root ganglia (DRG) explants in vitro and a rat spinal dorsal root injury (DRI) model in vivo. The peptide ligand binding to other membrane receptors was characterized by surface plasmon resonance (SPR) and liquid chromatography-mass spectrometry (LC-MS)/MS. Intracellular signaling pathways primarily affected by the peptide ligand were characterized by phosphoproteomics, and related pathways were verified using specific inhibitors. Results: We identified a novel FGFR-targeting small peptide, CH02, with seven amino acid residues. CH02 activated FGFR signaling through high-affinity binding with the extracellular segment of FGFRs and also had an affinity for several receptor tyrosine kinase (RTK) family members, including VEGFR2. In sensory neurons cultured in vitro, CH02 maintained the survival of neurons and promoted axon growth. Simultaneously, CH02 robustly enhanced nerve regeneration and sensory-motor behavioral recovery after DRI in rats. CH02-induced activation of FGFR signaling promoted nerve regeneration primarily via AKT and ERK signaling downstream of FGFRs. Activation of mTOR downstream of AKT signaling augmented axon growth potential in response to CH02. Conclusion: Our study revealed the significant therapeutic effect of CH02 on strengthening nerve regeneration and suggested a strategy for treating peripheral and central nervous system injuries.

Beta amyloid (Aβ) is a key component of parenchymal Aβ plaques and vascular Aβ fibrils, which lead to cerebral amyloid angiopathy (CAA) in Alzheimer’s disease (AD). Recent studies have revealed that Aβ contained in the cerebrospinal fluid (CSF) can re-enter into brain through paravascular spaces. However, whether Aβ in CSF may act as a constant source of pathogenic Aβ in AD is still unclear. This study aimed to examine whether Aβ pathology could be worsened when CSF Aβ level was enhanced by intra-cisternal infusion of aged brain extract containing abundant Aβ in TgCRND8 host mice. TgCRND8 mouse is an AD animal model which develops predominant parenchymal Aβ plaques in the brain at as early as 3 months of age. Here, we showed that single intracisternal injection of Aβ seeds into TgCRND8 mice before the presence of Aβ pathology induced robust prion-like propagation of CAA within 90 days. The induced CAA is mainly distributed in the cerebral cortex, hippocampus and thalamus of TgCRND8 mice. Surprisingly, despite the robust increase in CAA levels, the TgCRND8 mice had a marked decrease in parenchymal Aβ plaques and the plaques related neuroinflammation in the brains compared with the control mice. These results amply indicate that Aβ in CSF may act as a source of Aβ contributing to the growth of vascular Aβ deposits in CAA. Our findings provide experimental evidence to unravel the mechanisms of CAA formation and the potential of targeting CSF Aβ for CAA.

The formation of a fluid-filled cystic cavity after spinal cord injury (SCI) is a major obstacle for neural regeneration. In this study, the post-SCI cavity was bridged by a functional self-assembling peptide (F-SAP) nanofiber hydrogel coupled with growth factor "cocktail". A sustained release of growth factors was achieved by carefully tailoring the physical hindrances and charge-induced interactions between the growth factors and the peptide nanofibers. Such an engineering microenvironment elicited axon regeneration, as determined by tracing of the descending pathway in the dorsal columns and immunochemical detection of regenerating axons beyond the lesion. Furthermore, the dynamic spatiotemporal activation line of endogenous NSCs (eNSCs) after severe SCI was thoroughly investigated. The results indicated that the growth factor-coupled F-SAP greatly facilitated eNSC proliferation, neuronal differentiation, maturation, myelination, and more importantly, the formation of interconnection with severed descending corticospinal tracts. The robust endogenous neurogenesis essentially led to the recovery of locomotion and electrophysiological properties. In conclusion, the growth factor-coupled F-SAP nanofiber hydrogel elucidated the therapeutic effect of eliciting endogenous neurogenesis by locally reassembling an extracellular matrix.

Axonopathy manifested by axon swellings might constitute one of the earliest pathological features of Alzheimer's disease. It has been proposed that axonopathy might be associated with the origin of Aβ plaques. However, how axonopathy leads to Aβ plaque pathogenesis remains elusive. Our previous studies have shown that Aβ neuropathology (mainly diffuse plaques) selectively occurred in the regions of corticospinal tract (CST) pathway and its innervated region in the spinal cord of TgCRND8 mice. In this study, we investigated the occurrence and progression of axonopathy and the possible implication in Aβ plaque pathogenesis in the spinal cord of TgCRND8 mice. By anterograde labeling of CST system with a neuroanatomical tracer, we found that dilated corticospinal axons started to appear at 7 months, then exhibited an age-dependent increase. These abnormal structures appear before any plaque deposits are visible in the spinal cord of the mice. Importantly, they colocalized with Aβ plaques in either the white matter or gray matter of the spinal cord at later stages, suggesting that these axonal swellings might represent the initial stages of Aβ plaque formation, and could play a role in Aβ plaque pathogenesis. Furthermore, using ultrastructural analysis we demonstrated that intracellular contents in the axonal dystrophies such as various dense vesicles leaked out into the extracellular matrix under a condition of axon swelling rupture in CST pathways of spinal cord. This provided precise structural evidence that how the Aβ leaks out from the axonal dystrophies into extracellular matrix and how an axonal swelling might serve as a nidus of amyloid plaque formation.

Summary N-methyl-D-aspartate (NMDA) receptor (NMDAR) is highly compartmentalized in neurons, and its dysfunction has been implicated in various neuropsychiatric and neurodegenerative disorders. Recent failure to exploit NMDAR antagonization as a potential therapeutic target has driven the need to identify molecular mechanisms that regulate NMDAR compartmentalization. Here, we report that the reduction of Kif5b, the heavy chain of kinesin-1, protected neurons against NMDA-induced excitotoxicity and ischemia-provoked neurodegeneration. Direct binding of kinesin-1 to the GluN2B cytoplasmic tails regulated the levels of NMDAR at extrasynaptic sites and the subsequent influx of calcium mediated by extrasynaptic NMDAR by regulating the insertion of NMDARs into neuronal surface. Transient increase of Kif5b restored the surface levels of NMDAR and the decreased neuronal susceptibility to NMDA-induced excitotoxicity. The expression of Kif5b was repressed in cerebral ischemia preconditioning. Our findings reveal that kinesin-1 regulates extrasynaptic NMDAR targeting and signaling, and the reduction of kinesin-1 could be exploited to defer neurodegeneration., Graphical Abstract, Highlights • Kif5b directly binds with GluN2B-containing NMDAR • Kinesin-1 mediates extrasynaptic NMDAR targeting and function • Reduction of kinesin-1 protects neurons against NMDAR-elicited excitotoxicity • Reduction of kinesin-1 protects brain against ischemia-elicited neurodegeneration, Neuroscience; Molecular Neuroscience; Cellular Neuroscience

The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy-lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.

Spinal cord injuries (SCIs) are followed by a complex series of events that contribute to the failure of regeneration. To date, there is no robust treatment that can restore the injury-induced loss of function. Since damaged spinal axons do not spontaneously regenerate in their native inhibitory microenvironment, a combined application of biomaterials and neurotrophic factors that induce nerve regeneration emerges as an attractive treatment for SCIs. In this study, we report the novel use of a three-dimensional (3D) hybrid scaffold to provide contact guidance for regrowth of axons in vivo. The scaffold comprises 3D aligned sparsely distributed poly(ε-caprolactone-co-ethyl ethylene phosphate) nanofibers that are supported and dispersed within a collagen hydrogel. Neurotrophin-3 was incorporated into the scaffold as an additional biochemical signal. To evaluate the efficacy of the scaffold in supporting nerve regeneration after SCIs, the construct was implanted into an incision injury, which was created at level C5 in the rat spinal cord. After 3 months of implantation, scaffolds with NT-3 incorporation showed the highest average neurite length (391.9 ± 12.9 μm, p ≤ 0.001) as compared to all the other experimental groups. In addition, these regenerated axons formed along the direction of the aligned nanofibers, regardless of their orientation. Moreover, the presence of the hybrid scaffolds did not affect tissue scarring and inflammatory reaction. Taken together, these findings demonstrate that our scaffold design can serve as a potential platform to support axonal regeneration following SCIs. NMRC (Natl Medical Research Council, S’pore) Accepted version

Both intracellular sigma peptide (ISP) and phosphatase and tensin homolog agonist protein (PAP4) promote nerve regeneration and motor functional recovery after spinal cord injury. However, the role of these two small peptides in peripheral nerve injury remains unclear. A rat model of brachial plexus injury was established by crush of the C6 ventral root. The rats were then treated with subcutaneous injection of PAP4 (497 µg/d, twice per day) or ISP (11 µg/d, once per day) near the injury site for 21 successive days. After ISP and PAP treatment, the survival of motoneurons was increased, the number of regenerated axons and neuromuscular junctions was increased, muscle atrophy was reduced, the electrical response of the motor units was enhanced and the motor function of the injured upper limbs was greatly improved in rats with brachial plexus injury. These findings suggest that ISP and PAP4 promote the recovery of motor function after peripheral nerve injury in rats. The animal care and experimental procedures were approved by the Laboratory Animal Ethics Committee of Jinan University of China (approval No. 20111008001) in 2011.

Glaucoma is the leading cause of irreversible blindness worldwide, and pathologically elevated intraocular pressure (IOP) is the major risk factor. Neuroprotection is one of the potential therapies for glaucomatous retinal damage. Intravitreal mesenchymal stem cell (MSC) transplantation provides a viable therapeutic option, and human umbilical cord- (hUC-) MSCs are attractive candidates for cell-based neuroprotection. Here, we investigated the ability of transplanted hUC-MSCs to survive and migrate within the vitreous cavity and their neuroprotective effects on chronic glaucomatous retina. For this, we developed a chronic ocular hypertension (COH) rat model through the intracameral injection of allogeneic Tenon’s fibroblasts. Green fluorescent protein-transduced hUC-MSCs were then injected into the vitreous cavity one week after COH induction. Results showed that a moderate IOP elevation lasted for two months. Transplanted hUC-MSCs migrated toward the area of damaged retina, but did not penetrate into the retina. The hUC-MSCs survived for at least eight weeks in the vitreous cavity. Moreover, the hUC-MSCs were efficient at decreasing the loss of retinal ganglion cells; retinal damage was attenuated through the inhibition of apoptosis. In this study, we have developed a novel COH rat model and demonstrated the prolonged neuroprotective potential of intravitreal hUC-MSCs in chronic glaucoma.

Current treatment approaches toward spinal cord injuries (SCI) have mainly focused on overcoming the inhibitory microenvironment that surrounds lesion sites. Unfortunately, the mere modulation of the cell/tissue microenvironment is often insufficient to achieve desired functional recovery. Therefore, stimulating the intrinsic growth ability of injured neurons becomes crucial. MicroRNAs (miRs) play significant roles during axon regeneration by regulating local protein synthesis at growth cones. However, one challenge of using miRs to treat SCI is the lack of efficient delivery approaches. Here, a 3D fiber‐hydrogel scaffold is introduced which can be directly implanted into a spinal cord transected rat. This 3D scaffold consists of aligned electrospun fibers which provide topographical cues to direct axon regeneration, and collagen matrix which enables a sustained delivery of miRs. Correspondingly, treatment with Axon miRs (i.e., a cocktail of miR‐132/miR‐222/miR‐431) significantly enhances axon regeneration. Moreover, administration of Axon miRs along with anti‐inflammatory drug, methylprednisolone, synergistically enhances functional recovery. Additionally, this combined treatment also decreases the expression of pro‐inflammatory genes and enhance gene expressions related to extracellular matrix deposition. Finally, increased Axon miRs dosage with methylprednisolone, significantly promotes functional recovery and remyelination. Altogether, scaffold‐mediated Axon miR treatment with methylprednisolone is a promising therapeutic approach for SCI., A novel approach of targeting axon local protein synthesis to promote the intrinsic growth ability of neurons by scaffold‐mediated microRNA delivery is introduced. The biofunctional 3D fiber‐hydrogel scaffold provides topographical cues that direct axon regrowth and a novel microRNA cocktail (miR‐132/miR‐222/miR‐431), which in the presence of methylprednisolone, enhances both nerve regeneration and functional recovery after spinal cord injury.

Intracerebral hemorrhage (ICH) is associated with high morbidity and mortality rates. Currently, there is no promising treatment that improves prognosis significantly. While a thorough investigation of the pathological process within the primary site of injury in the brain has been conducted by the research field, the focus was mainly on gray matter injury, which partly accounted for the failure of discovery of clinically efficacious treatments. It is not until recent years that white matter (WM) injury in the brain after subcortical ICH was examined. As WM tracts form networks between different regions, damage to fibers should impair brain connectivity, resulting in functional impairment. Although WM changes have been demonstrated in the brain after ICH, alterations distant from the initial injury site down in the spinal cord are unclear. This longitudinal study, for the first time, revealed prolonged morphological changes of the contralesional dorsal corticospinal tract (CST) in the spinal cord 5 weeks after experimental ICH in mice by confocal microscopy and transmission electron microscopy, implying that the structural integrity of the CST was compromised extensively after ICH. Given the important role of CST in motor function, future translational studies targeting motor recovery should delineate the treatment effects on CST integrity.

Brachial plexus root avulsion (BPRA) is a type of injury that leads to motor function loss as a result of motoneurons (MNs) degeneration. Here we identified that the reduced expression of rat miR-137-3p in the ventral horn of spinal cord was associated with MNs death. However, the pathophysiological role of miR-137-3p in root avulsion remains poorly understood. We demonstrated that the calcium-activated neutral protease-2 (calpain-2) was a direct target gene of miR-137-3p with miR-137-3p binding to the 3'-untranslated region of calpain-2. Silencing of calpain-2 suppressed the expression of neuronal nitric oxide synthase (nNOS), a primary source of nitric oxide (NO). After avulsion 2 weeks, up-regulation of miR-137-3p in the spinal cord reduced calpain-2 levels and nNOS expression inside spinal MNs, resulting in an amelioration of the MNs death. These events provide new insight into the mechanism by which upregulation of miR-137-3p can impair MN survival in the BPRA.

When spinal roots are torn off from the spinal cord, both the peripheral and central nervous system get damaged. As the motoneurons lose their axons, they start to die rapidly, whereas target muscles atrophy due to the denervation. In this kind of complicated injury, different processes need to be targeted in the search for the best treatment strategy. In this study, we tested glial cell-derived neurotrophic factor (GDNF) treatment and fetal lumbar cell transplantation for their effectiveness to prevent motoneuron death and muscle atrophy after the spinal root avulsion and delayed reimplantation. Application of exogenous GDNF to injured spinal cord greatly prevented the motoneuron death and enhanced the regeneration and axonal sprouting, whereas no effect was seen on the functional recovery. In contrast, cell transplantation into the distal nerve did not affect the host motoneurons but instead mitigated the muscle atrophy. The combination of GDNF and cell graft reunited the positive effects resulting in better functional recovery and could therefore be considered as a promising strategy for nerve and spinal cord injuries that involve the avulsion of spinal roots.

Accumulating evidences suggest that peripheral nerve injury (PNI) may initiate astrocytic responses in the central nervous system (CNS). However, the response of astrocytes in the spinal ventral horn and its potential role in nerve regeneration after PNI remain unclear. Herein, we firstly illustrated that astrocytes in the spinal ventral horn were dramatically activated in the early stage following sciatic nerve injury, and these profiles were eliminated in the chronic stage. Additionally, we found that the expression of neurotrophins, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), also accompanied with astrocyte activation. In comparison with the irreversible transected subjects, astrocyte activation and the neurotrophic upregulation in the early stage were more drastic in case the transected nerve was rebridged immediately after injury. Furthermore, administering fluorocitrate to inhibit astrocyte activation resulted in decreased neurotrophin expression in the spinal ventral horn and delayed axonal regeneration in the nerve as well as motor function recovery. Overall, the present study indicates that peripheral nerve injury can initiate astrocyte activation accompanied with neurotrophin upregulation in the spinal ventral horn. The above responses mainly occur in the early stage of PNI and may contribute to nerve regeneration and motor function recovery.

// Qiuju Yuan 1, 2 , Jian Yang 3 , Wutian Wu 3, 4, 5, 6 and Zhi-Xiu Lin 1, 2 1 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Brain Research Centre, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 3 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 4 State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 5 Research Center of Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 6 GHM Institute of CNS regeneration, Jinan University, Guangzhou, China Correspondence to: Zhi-Xiu Lin, email: linzx@cuhk.edu.hk Wutian Wu, email: wtwu@hkucc.hku.hk Keywords: axonal transport, dystrophic axon swellings, corticospinal tract, motor deficits Received: February 03, 2017 Accepted: May 08, 2017 Published: June 09, 2017 ABSTRACT There have been an increasing number of reports of non-cognitive symptoms in Alzheimer’s disease (AD). Some symptoms are associated with the loss of motor functions, e.g. gait disturbances, disturbed activity level and balance. Consistent with clinical findings, several AD mouse models harboring amyloid pathology develop motor impairment. Although the factors that contribute to the motor deficits have not yet been determined, it has been suggested that axonopathy is one of the key factors that may contribute to this particular feature of the disease. Our previous study found that TgCRND8 mice exhibited profound motor deficits as early as 3 months old. In this study, we explored the possible factors that may be related to motor deficits in TgCRND8 mice. Results from silver, neurofilament and amyloid precursor protein (APP) staining revealed no axonopathy occurred in the brain and spinal cord of TgCRND8 mice at the age of 3 months. Anterograde labeling of corticospinal tract of spinal cord and electronic microscopy (EM) analysis showed that no axonopathy occurred in TgCRND8 mice at the age of 3 months. According to these results, it could be concluded that no axonal alterations were evident in the TgCRND8 mice when motor deficits was overt. Thus, axonopathy may play a less prominent role in motor deficits in AD. These results suggest that mechanisms by which motor function undergo impairment in AD need to be further studied.

A spinal root avulsion injury disconnects spinal roots with the spinal cord. The rampant motoneuron death, inhibitory CNS/PNS transitional zone (TZ) for axonal regrowth and limited regeneration speed together lead to motor dysfunction. Microtubules rearrange to assemble a new growth cone and disorganized microtubules underline regeneration failure. It has been shown that microtubule-stabilizing drug, Epothilone B, enhanced axonal regeneration and attenuated fibrotic scaring after spinal cord injury. Here, we are reporting that after spinal root avulsion+ re-implantation in adult rats, EpoB treatment improved motor functional recovery and potentiated electrical responses of motor units. It facilitated axons to cross the TZ and promoted more and bigger axons in the peripheral nerve. Neuromuscular junctions were reformed with better preserved postsynaptic structure, and muscle atrophy was prevented by EpoB administration. Our study showed that EpoB was a promising therapy for promoting axonal regeneration after peripheral nerve injury.

Peripheral nerve injury (PNI) usually results in poor functional recovery. Nerve repair is the common clinical treatment for PNI but is always obstructed by the chronic degeneration of the distal stump and muscle. Cell transplantation can alleviate the muscle atrophy after PNI, but the subsequent recovery of the locomotive function is seldom described. In this study, we combined cell transplantation and nerve repair to investigate whether the transplantation of embryonic spinal cord cells could benefit the delayed nerve repair. The experiment consisted of 3 stages: transection of the tibial nerve to induce 'pre-degeneration', a second surgery performed 2 weeks later for transplantation of E14 embryonic spinal cord cells or vehicle (culture medium) at the distal end of the injured nerve, and, 3 months later, the removal of the grafted cells and the cross-suturing of the residual distal end to the proximal end of a freshly cut ipsilateral common peroneal (CP) nerve. Cell survival and fate after the transplantation were investigated, and the functional recovery after the cross-suturing was compared between the groups. The grafted cells could survive and generate motor neurons, extending axons that were subsequently myelinated and forming synapses with the muscle. After the cross-suturing, the axonal regeneration from the proximal stump of the injured CP nerve and the functional recovery of the denervated gastrocnemius muscle were significantly promoted in the group receiving the cells. Our study presents a new perspective indicating that the transplantation of embryonic spinal cord neurons may be a valuable therapeutic strategy for PNI.

Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications.

Cerebral amyloid angiopathy (CAA) refers to pathological changes occurring in cerebral blood vessels caused by deposition of beta amyloid (Aβ) protein. However, the mechanisms involved in the origin of Aβ for the formation of CAA and its link to parenchymal amyloid depositions remained to be unraveled. Here, we found CAA and parenchymal plaques distributed separately instead of mingling with each other in the spinal cord of TgCRND8 mice. Parenchymal plaques predominantly located in the dorsal horn whereas CAA distributed in the ventral horn. We further found that the ratio of Aβ40/Aβ42 was significantly higher in the ventral than that in the dorsal by ELISA assay, suggesting that origin of Aβ forming parenchymal plaques may be different from that of CAA in the spinal cord. This hypothesis was further demonstrated by the surgical methods which indicated eliminating parenchymal plaques did not alter CAA in the affected spinal cord. We also examined the ratio of Aβ40/Aβ42 in the cerebral spinal fluid (CSF) in order to identify the origin of the CAA formation, and found the Aβ40/Aβ42 ratio was similar to that of CAA formation in the ventral horn. We further demonstrated that CSF tracer distributed along ventral horn vessels, in exactly the same pattern as Aβ deposition in CAA in ventral part of spinal cord. These findings verified the concept that CSF influx may act as a constant source for delivering Aβ, and contribute to the growth of paraarterial deposits in CAA. Taken together, the results of the present study highlight the important role of the Aβ40/Aβ42 ratio in determining vascular versus parenchymal amyloid deposition. Unlike parenchymal plaques, Aβ of CAA comes from CSF; thus, manipulation of CSF Aβ could represent a novel strategy to treat CAA.

The prosthetics that are currently available for use in neural injuries are limited in their medical applications because of poor biointegration in native neural tissues. Using nanotechnology to address this issue is highly appealing because nanoscale control over the synthetic-native interface could allow a much more precise physical matching between the host tissues and the implant. The inherent complexity of the nervous system requires tailored and highly specialized structures that can interact with their surroundings in well-defined and controllable ways for each application in question, be it within the central or peripheral nervous system. Nanoengineered materials have a fundamentally flexible nature that makes them highly appealing in designing regenerative strategies for the nervous system. This chapter focuses on the use of nanomaterials in bridging nerve gaps and for serving as a scaffold for stem cells in neuroregeneration, highlighting the different types of injuries and the subsequent requirements for recovery.

Motoneurons undergo retrograde degeneration following axonal injuries. The degree of degeneration varies with types of injuries, proximity of injury site to the cell body, species and age of the animal. Unlike other injuries, spinal root avulsion induces a rapid and massive cell loss of the motoneurons. It not only provides a mean to study the degenerative changes of spinal motoneurons but also resembles the brachial plexus injury in human. Despite they situate in the central nervous system, motoneurons have a strong ability to regenerate under a suitable environment. We herein present step-by-step procedures for performing extravertebral and intravertebral avulsion at seventh cervical segment in rat spinal cord and peripheral nerve implantation to allow regeneration of the motoneurons.

Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury. Spastin plays an important role in the regulation of microtubule severing. Both spastin and collapsin response mediator proteins can regulate neurite growth and branching; however, whether spastin interacts with collapsin response mediator protein 3 (CRMP3) during this process remains unclear, as is the mechanism by which CRMP3 participates in the repair of spinal cord injury. In this study, we used a proteomics approach to identify key proteins associated with spinal cord injury repair. We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin. Then, co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3. Finally, we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth. Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process. Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin. CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo. Lastly, CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching. Therefore, our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching. These proteins may therefore be novel targets for spinal cord injury repair. The Institutional Animal Care and Use Committee of Jinan University, China approved this study (approval No. IACUS-20181008-03) on October 8, 2018.

Although a large number of trials in the SCI field have been conducted, few proven gains have been realized for patients. In the present study, we determined the efficacy of a novel combination treatment involving surgical intervention and long-term weight-bearing walking training in spinal cord injury (SCI) subjects clinically diagnosed as complete or American Spinal Injury Association Impairment Scale (AIS) Class A (AIS-A). A total of 320 clinically complete SCI subjects (271 male and 49 female), aged 16-60 years, received early (≤ 7 days, n = 201) or delayed (8-30 days, n = 119) surgical interventions to reduce intraspinal or intramedullary pressure. Fifteen days post-surgery, all subjects received a weight-bearing walking training with the "Kunming Locomotion Training Program (KLTP)" for a duration of 6 months. The neurological deficit and recovery were assessed using the AIS scale and a 10-point Kunming Locomotor Scale (KLS). We found that surgical intervention significantly improved AIS scores measured at 15 days post-surgery as compared to the pre-surgery baseline scores. Significant improvement of AIS scores was detected at 3 and 6 months and the KLS further showed significant improvements between all pair-wise comparisons of time points of 15 days, 3 or 6 months indicating continued improvement in walking scores during the 6-month period. In conclusion, combining surgical intervention within 1 month post-injury and weight-bearing locomotor training promoted continued and statistically significant neurological recoveries in subjects with clinically complete SCI, which generally shows little clinical recovery within the first year after injury and most are permanently disabled. This study was approved by the Science and Research Committee of Kunming General Hospital of PLA and Kunming Tongren Hospital, China and registered at ClinicalTrials.gov (Identifier: NCT04034108) on July 26, 2019.

Peripheral nerves are fragile and easily damaged, usually resulting in nervous tissue loss, motor and sensory function loss. Advances in neuroscience and engineering have been significantly contributing to bridge the damage nerve and create permissive environment for axonal regrowth across lesions. We have successfully designed two self-assembling peptides by modifying RADA 16-I with two functional motifs IKVAV and RGD. Nanofiber hydrogel formed when combing the two neutral solutions together, defined as RADA 16-Mix that overcomes the main drawback of RADA16-I associated with low pH. In the present study, we transplanted the RADA 16-Mix hydrogel into the transected rat sciatic nerve gap and effect on axonal regeneration was examined and compared with the traditional RADA16-I hydrogel. The regenerated nerves were found to grow along the walls of the large cavities formed in the graft of RADA16-I hydrogel, while the nerves grew into the RADA 16-Mix hydrogel toward distal position. RADA 16-Mix hydrogel induced more axons regeneration and Schwann cells immigration than RADA16-I hydrogel, resulting in better functional recovery as determined by the gait-stance duration percentage and the formation of new neuromuscular junction structures. Therefore, our results indicated that the functional SAP RADA16-Mix nanofibrous hydrogel provided a better environment for peripheral nerve regeneration than RADA16-I hydrogel and could be potentially used in peripheral nerve injury repair.

One of the most complex systems in the human body is the nervous system, which is divided into the central and peripheral nervous systems. The regeneration of the CNS is a complex and challenging biological phenomenon hindered by the low regenerative capacity of neurons and the prohibition factors in response to nerve injuries. To date, no effective approach can achieve complete recovery and fully restore the functions of the nervous system once it has been damaged. Developments in neuroscience have identified properties of the local environment with a critical role in nerve regeneration. Advances in biomaterials and biomedical engineering have explored new approaches of constructing permissive environments for nerve regeneration, thereby enabling optimism with regard to nerve-injury treatment. This article reviews recent progress in nanoengineered environments for aiding nerveinjury repair and regeneration, including nanofibrous scaffolds, functional molecules, and stem cells.

Multiple sclerosis (MS) is a progressive autoimmune disease of the CNS with its underlying mechanisms not fully understood. In the present study, we tested the hypothesis that caveolin-1, a major membrane scaffolding protein, plays a critical role in the pathogenesis of experimental autoimmune encephalomyelitis, a laboratory murine model of MS. We found increased expression of caveolin-1 in serum and spinal cord tissues in association with disease incidence and severity in wild-type mice with active encephalomyelitis. After immunization, Cav-1 knock-out mice showed remarkable disease resistance with decreased incidence and clinical symptoms. Furthermore, Cav-1 knock-out mice had alleviated encephalitogenic T cells trafficking into the CNS with decreased expressions of adhesion molecules ICAM-1 and VCAM-1 within the lesions. In agreement within vivostudies,in vitroknockdown of caveolin-1 compromised the upregulation of ICAM-1 in endothelial cells, leading to the amelioration of the transendothelial migration of pathogenic TH1 and TH17 cells. Together, those results indicate that caveolin-1 serves as an active modulator of CNS-directed lymphocyte trafficking and could be a therapeutic target for neuroinflammatory diseases, such as multiple sclerosis.SIGNIFICANCE STATEMENTThe hallmark feature of neuroinflammatory diseases is the massive infiltrations of encephalitogenic leukocytes into the CNS parenchyma, a process that remains largely unclear. Our study demonstrates the critical contribution of caveolin-1 to encephalomyelitis pathogenesis and CNS-directed lymphocyte trafficking by modulation of adhesion molecules ICAM-1 and VCAM-1, highlighting the pathological involvement of caveolin-1 in neuroinflammatory diseases.

RhoA, a member of Rho GTPases family, is known to play an important role in remodeling actin cytoskeleton. During the development of the peripheral nervous system (PNS), Schwann cells undergo proliferation, migration, and radial sorting and finally wrap the related axons compactly to form myelin sheath. All these processes involve actin cytoskeletal remodeling. However, the role of RhoA on Schwann cell during development is still unclear. To address this question, we first used a lentiviral vector-mediated short hairpin (sh) RNA targeting RhoA to knock down the expression of RhoA in the cultured Schwann cells in vitro. Effects of RhoA on Schwann cell proliferation and migration were examined by BrdU assay and transwell assay, respectively. Results of the present study indicated that downregulated RhoA expression in cultured Schwann cells significantly slacked the cells' capabilities of migration and proliferation. Then, we investigated the role of RhoA in the developing rat sciatic nerves. Immunohistology and Western blotting showed that RhoA was mainly expressed in Schwann cells in the sciatic nerves and was peaked at 2 weeks postnatal then kept in low level up to 8 weeks. In the subjected rats whose sciatic nerves were microinjected with lentiviral vectors at postnatal 3 days, we found that the lentiviruses mainly transfected Schwann cells, and the RhoA expression in the transfected Schwann cells was significantly knocked down. Four weeks after lentivirus microinjection, immunohistology and transmission electron microscopy illustrated that RhoA knockdown resulted in hypomyelination and significant decrease of the thickness of myelin in the transfected area. Overall data of current study suggested that RhoA plays a critical role in Schwann cell biology and is essential for myelination in developing peripheral nerve.

Increasing evidence suggests that activation of satellite glia cells (SGCs) in sensory ganglia play important roles in the development of neuropathic pain. The present study aimed to investigate the involvement of SGC activation in a novel model of motor nerve injury induced pain hypersensitivity. The neuropathic pain model was established by cervical 8 ventral root avulsion (C8VA). Glial fibrillary acidic protein (GFAP) was used as a marker of SGC activation. Unilateral C8VA resulted in mechanical allodynia, but not thermal hyperalgesia in bilateral paws. Expectedly, SGCs were robustly activated on as early as 1 day and persisted for at least 7 days in the ipsilateral and contralateral dorsal root ganglia (DRG) of C6, C7 and C8 after C8VA. Double immunofluorescence showed that almost all the activated SGCs enveloped neurofilament 200 (NF200) positive myelinated neurons in DRG. Local application of fluorocitrate (FC), a glial metabolism inhibitor, significantly decreased the number of activated SGCs and alleviated bilateral mechanical allodynia. These results suggest that SGC activation contributed to ipsilateral and mirror-image pain hypersensitivity after C8VA. Inhibition of SGC activation represented a promising therapeutic strategy for the management of neuropathic pain following brachial plexus root avulsion.

N-methyl-D-aspartate receptor (NMDAR) is highly compartmentalized in neurons and the dysfunction has been implicated in various neuropsychiatric and neurodegenerative disorders. Recent failure to exploit NMDAR antagonization as a potential therapeutic target has driven the need to identify molecular mechanisms that regulate NMDAR compartmentalization. Here, we report that neural activity-dependent reduction of Kif5b, the heavy chain of kinesin-1, protected neurons against NMDA-induced excitotoxicity and ischemia-provoked neurodegeneration. Direct binding of Kinesin-1 to the GluN2B cytoplasmic tails regulated levels of NMDAR at extrasynaptic sites and the subsequent influx of calcium mediated by extrasynaptic NMDAR via regulating the insertion of NMDARs into neuronal surface. Transient increase of Kif5b restored the surface levels of NMDAR and the decreased neuronal susceptibility to NMDA-induced excitotoxicity. Our findings reveal that kinesin-1 regulates extrasynaptic NMDAR targeting and signaling, and the reduction of kinesin-1 could be regulated by neural activity and could be exploited to postpone or halt neurodegeneration.

Brachial plexus root avulsion (BPRA) results in the complete loss of motor function in the upper limb, mainly due to the death of spinal motoneurons (MNs). The survival of spinal MNs is the key to the recovery of motor function. Neuregulin-1 (Nrg1) plays fundamental roles in nervous system development and nerve repair. However, its functional role in BPRA remains unclear. On the basis of our findings that Nrg1 is down-regulated in the ventral horn in a mouse model of BPRA, Nrg1 may be associated with BPRA. Here, we investigated whether recombinant Nrg1β (rNrg1β) can enhance the survival of spinal MNs and improve functional recovery in mice following BPRA. In vitro studies on primary cultured mouse MNs showed that rNrg1β increased the survival rate in a dose-dependent manner, reaching a peak at 5 nM, which increased the survival rate and enhanced the pERK levels in MNs under H2O2-induced oxidative stress. In vivo studies revealed that rNrg1β improved the functional recovery of elbow flexion, promoted the survival of MNs, enhanced the re-innervation of biceps brachii, and decreased the muscle atrophy. These results suggest that Nrg1 may provide a potential therapeutic strategy for root avulsion.

Dorsal root injury commonly results in irreversible loss of sensory functions because of the limited intrinsic regenerative capacity of adult sensory axons and the growth-inhibitory environment at the dorsal root entry zone (DREZ) between the dorsal root and the spinal cord. Chondroitin sulfate proteoglycans (CSPGs) are the dominant suppressors of axonal regeneration, acting via neuronal receptors including protein tyrosine phosphatase-σ (PTPσ). ISP (Intracellular Sigma Peptide) is a small peptide mimetic of the PTPσ wedge region that has been developed to target PTPσ and relieve CSPG inhibition. Extracellular regulated kinases (Erks) and cAMP response element binding protein (CREB) are signaling molecules downstream of CSPGs and PTPσ; they are expressed in neurons and essential for axon growth. In this study, we observed that ISP administration could promote sensory function restoration in adult rats after dorsal spinal root crush injury. Our results show that systemic ISP administration would not only significantly increase sensory axon regeneration and functional recovery, but also activate Erk and CREB signaling pathway. Furthermore, ISP has also been verified to increase dorsal root ganglion axonal remyelination in vitro. These results suggest that modulation of PTPσ by ISP represents a promising therapeutic strategy for sensory neuronal injuries.

RADA16-I is a synthetic type I self-assembling peptide nanofiber scaffold (SAPNS) which may serve as a novel biocompatible hemostatic agent. Its application in neurosurgical hemostasis, however, has not been explored. Although RADA16-I is nontoxic and nonimmunogenic, its intrinsic acidity may potentially provoke inflammation in the surgically injured brain. We conducted an animal study to compare RADA16-I with fibrin sealant, a commonly used agent, with the hypothesis that the former would be a comparable alternative. Using a standardized surgical brain injury model, 30 Sprague-Dawley rats were randomized into three treatment groups: RADA16-I, fibrin sealant or gelatin sponge (control). Animals were sacrificed on day 3 and 42. Astrocytic and microglial infiltrations within the cerebral parenchyma adjacent to the operative site were significantly lower in the RADA16-I and fibrin sealant groups than control. RADA16-I did not cause more cellular inflammatory response despite its acidity when compared with fibrin sealant. Immunohistochemical studies showed infiltration by astrocytes and microglia into the fibrin sealant and RADA16-I grafts, suggesting their potential uses as tissue scaffolds. RADA16-I is a promising candidate for further translational and clinical studies that focus on its applications as a safe and effective hemostat, proregenerative nanofiber scaffold as well as drug and cell carrier.

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.

After complete transection of the thoracic spinal segment, neonatal rats exhibit spontaneous locomotor recovery of hindlimbs, but this recovery is not found in adult rats after similar injury. The potential mechanism related to the difference in recovery of neonatal and adult rats remains unknown. In this study, 342 animals were analyzed. The vascular endothelial growth factor (VEGF) level in spinal segments below injury sites was significantly higher in postnatal day 1 rats (P1) compared with 28-day-old adult rats (P28) following a complete T9 transection. VEGF administration in P28 rats with T9 transection significantly improved the functional recovery; by contrast, treatment with VEGF receptor inhibitors in P1 rats with T9 transection slowed down the spontaneous functional recovery. Results showed more neurons reduced in the lumbar spinal cord and worse local neural network reorganization below injury sites in P28 rats than those in P1 rats. Transynaptic tracing with pseudorabies virus and double immunofluorescence analysis indicated that VEGF treatment in P28 rats alleviated the reduced number of neurons and improved their network reorganization. VEGF inhibition in neonates resulted in high neuronal death rate and deteriorated network reorganization. In in vivo studies, T9 transection induced less increase in the number of microglia in the spinal cord in P1 animals than P28 animals. VEGF treatment reduced the increase in microglial cells in P28 animals. VEGF administration in cultured spinal motoneurons prevented lipopolysaccharide (LPS)-induced neuronal death and facilitated neurite growth. Western blots of the samples of lumbar spinal cord after spinal transection and cultured spinal motoneurons showed a lower level of Erk1/2 phosphorylation after the injury or LPS induction compared with that in the control. The phosphorylation level increased after VEGF treatment. In conclusion, VEGF is a critical mediator involved in functional recovery after spinal transection and can be considered a potential target for clinical therapy.

Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain.

Injuries to peripheral nerves are frequent in serious traumas and spinal cord injuries. In addition to surgical approaches, other interventions, such as cell transplantation, should be considered to keep the muscles in good condition until the axons regenerate. In this study, E14.5 rat embryonic spinal cord fetal cells and cultured neural progenitor cells from different spinal cord segments were injected into transected musculocutaneous nerve of 200–300 g female Sprague Dawley (SD) rats, and atrophy in biceps brachii was assessed. Both kinds of cells were able to survive, extend their axons towards the muscle and form neuromuscular junctions that were functional in electromyographic studies. As a result, muscle endplates were preserved and atrophy was reduced. Furthermore, we observed that the fetal cells had a better effect in reducing the muscle atrophy compared to the pure neural progenitor cells, whereas lumbar cells were more beneficial compared to thoracic and cervical cells. In addition, fetal lumbar cells were used to supplement six weeks delayed surgical repair after the nerve transection. Cell transplantation helped to preserve the muscle endplates, which in turn lead to earlier functional recovery seen in behavioral test and electromyography. In conclusion, we were able to show that embryonic spinal cord derived cells, especially the lumbar fetal cells, are beneficial in the treatment of peripheral nerve injuries due to their ability to prevent the muscle atrophy.

Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Aim Apurinic/apyrimidinic endonuclease 1 (APE1) is an intermediate enzyme in base excision repair which is important for removing damaged nucleotides under normal and pathological conditions. Accumulation of damaged bases causes genome instability and jeopardizes cell survival. Our study is to examine APE1 regulation under oxidative stress in spinal motor neurones which are vulnerable to oxidative insult. Methods We challenged the motor neurone-like cell line NSC-34 with hydrogen peroxide and delineated APE1 function by applying various inhibitors. We also examined the expression of APE1 in spinal motor neurones after spinal root avulsion in adult rats. Results We showed that hydrogen peroxide induced APE1 down-regulation and cell death in a differentiated motor neurone-like cell line. Inhibiting the two functional domains of APE1, namely, DNA repair and redox domains potentiated hydrogen peroxide induced cell death. We further showed that p53 phosphorylation early after hydrogen peroxide treatment might contribute to the down-regulation of APE1. Our in vivo results similarly showed that APE1 was down-regulated after root avulsion injury in spinal motor neurones. Delay of motor neurone death suggested that APE1 might not cause immediate cell death but render motor neurones vulnerable to further oxidative insults. Conclusion We conclude that spinal motor neurones down-regulate APE1 upon oxidative stress. This property renders motor neurones susceptible to continuous challenge of oxidative stress in pathological conditions.

Object The purpose of this study was to examine whether choline acetyltransferase (ChAT) staining can be used for assessing the rate of motor neuron regeneration at an early phase of axon outgrowth. Methods The authors developed a new sciatic nerve crush model in adult mice. In this model, in addition to performing a sciatic nerve crush injury, the authors excised the ipsilateral lumbar L3–6 dorsal root ganglion (DRG), which resulted in degeneration of the sensory fibers entering into the sciatic nerve. Crushed nerve sections obtained at Day 3 or Day 7 postinjury were analyzed by means of immunostaining. Results The immunostaining showed that ChAT, a motor axon–specific antigen, was totally co-localized with growth-associated protein 43 (GAP-43), which is expressed in regenerating nerves and transported into growth cones. Conclusions Our results suggest that measuring the length of motor axon outgrowth by ChAT immunostaining is reliable. ChAT staining provides a more convenient method for evaluating the rate of motor axon outgrowth in a mixed nerve.

The spinal cord plays a key role in motor behavior. It relays major sensory information, receives afferents from supraspinal centers and integrates movement in the central pattern generators. Spinal motor output is controlled via corticofugal pathways including corticospinal and cortico-subcortical projections. Spinal cord injury damages descending supraspinal as well as ascending sensory pathways. In adult rodent models, plasticity of the spinal cord is thought to contribute to functional recovery. How much spinal cord function depends on cortical input is not well known. Here, we address this question using Celsr3/Foxg1 mice, in which cortico-subcortical connections (including corticospinal tract (CST) and the terminal sensory pathway, the thalamocortical tract) are genetically ablated during early development. Although Celsr3/Foxg1 mice are able to eat, walk, climb on grids and swim, open-field tests showed them to be hyperactive. When compared with normal littermates, mutant animals had reduced number of spinal motor neurons, with atrophic dendritic trees. Furthermore, motor axon terminals were decreased in number, and this was confirmed by electromyography. The number of cholinergic, calbindin, and calretinin-positive interneurons was moderately increased in the mutant spinal cord, whereas that of reelin and parvalbumin-positive interneurons was unchanged. As far as we know, our study provides the first genetic evidence that the spinal motor network does not mature fully in the absence of corticofugal connections, and that some motor function is preserved despite congenital absence of the CST.