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A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord 1 – 3 applied during neurorehabilitation 4 , 5 (EES REHAB ) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking. We hypothesized that this unexpected reduction reflects activity-dependent selection of specific neuronal subpopulations that become essential for a patient to walk after spinal cord injury. To identify these putative neurons, we modelled the technological and therapeutic features underlying EES REHAB in mice. We applied single-nucleus RNA sequencing 6 – 9 and spatial transcriptomics 10 , 11 to the spinal cords of these mice to chart a spatially resolved molecular atlas of recovery from paralysis. We then employed cell type 12 , 13 and spatial prioritization to identify the neurons involved in the recovery of walking. A single population of excitatory interneurons nested within intermediate laminae emerged. Although these neurons are not required for walking before spinal cord injury, we demonstrate that they are essential for the recovery of walking with EES following spinal cord injury. Augmenting the activity of these neurons phenocopied the recovery of walking enabled by EES REHAB , whereas ablating them prevented the recovery of walking that occurs spontaneously after moderate spinal cord injury. We thus identified a recovery-organizing neuronal subpopulation that is necessary and sufficient to regain walking after paralysis. Moreover, our methodology establishes a framework for using molecular cartography to identify the neurons that produce complex behaviours. Transcriptomic analysis following epidural electrical stimulation of the lumbar spinal cord during neurorehabilitation in mice identifies a population of neurons that orchestrates the restoration of walking following paralysis.

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3–IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3–IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SOD G93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases. Madaro et al. show that denervation induces accumulation of IL-6–STAT3-activated fibro-adipogenic progenitors without inflammation or muscle regeneration, leading to muscle atrophy and fibrosis.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1–dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K–phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2–PI3K–p-Akt signalling pathway. Hervera et al. show that extracellular vesicles containing NOX2 complexes are released from macrophages and incorporated into injured axons, leading to axonal regeneration through PI3K–p-Akt signalling.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo . Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants ( tgfb1a , tgfb3 , tnfa , sparc ) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

Spinal cord injury (SCI) remains a severe condition with an extremely high disability rate. The challenges of SCI repair include its complex pathological mechanisms and the difficulties of neural regeneration in the central nervous system. In the past few decades, researchers have attempted to completely elucidate the pathological mechanism of SCI and identify effective strategies to promote axon regeneration and neural circuit remodeling, but the results have not been ideal. Recently, new pathological mechanisms of SCI, especially the interactions between immune and neural cell responses, have been revealed by single-cell sequencing and spatial transcriptome analysis. With the development of bioactive materials and stem cells, more attention has been focused on forming intermediate neural networks to promote neural regeneration and neural circuit reconstruction than on promoting axonal regeneration in the corticospinal tract. Furthermore, technologies to control physical parameters such as electricity, magnetism and ultrasound have been constantly innovated and applied in neural cell fate regulation. Among these advanced novel strategies and technologies, stem cell therapy, biomaterial transplantation, and electromagnetic stimulation have entered into the stage of clinical trials, and some of them have already been applied in clinical treatment. In this review, we outline the overall epidemiology and pathophysiology of SCI, expound on the latest research progress related to neural regeneration and circuit reconstruction in detail, and propose future directions for SCI repair and clinical applications.

Over the past decade, we have witnessed a flourishing of novel strategies to enhance neuroplasticity and promote axon regeneration following spinal cord injury, and results from preclinical studies suggest that some of these strategies have the potential for clinical translation. Spinal cord injury leads to the disruption of neural circuitry and connectivity, resulting in permanent neurological disability. Recovery of function relies on augmenting neuroplasticity to potentiate sprouting and regeneration of spared and injured axons, to increase the strength of residual connections and to promote the formation of new connections and circuits. Neuroplasticity can be fostered by exploiting four main biological properties: neuronal intrinsic signalling, the neuronal extrinsic environment, the capacity to reconnect the severed spinal cord via neural stem cell grafts, and modulation of neuronal activity. In this Review, we discuss experimental evidence from rodents, nonhuman primates and patients regarding interventions that target each of these four properties. We then highlight the strengths and challenges of individual and combinatorial approaches with respect to clinical translation. We conclude by considering future developments and providing views on how to bridge the gap between preclinical studies and clinical translation.

The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/β-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/β-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/β-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/β-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/β-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species. Following spinal injury in zebrafish, non-neural cells establish an extracellular matrix to promote axon re-growth but how this is regulated is unclear. Here, the authors show that Wnt/β-catenin signaling in fibroblast-like cells at a lesion activates axon re-growth via deposition of Collagen XII.

Remodeling of cytoskeleton structures, such as microtubule assembly, is believed to be crucial for growth cone initiation and regrowth of injured axons. Autophagy plays important roles in maintaining cellular homoeostasis, and its dysfunction causes neuronal degeneration. The role of autophagy in axon regeneration after injury remains speculative. Here we demonstrate a role of autophagy in regulating microtubule dynamics and axon regeneration. We found that autophagy induction promoted neurite outgrowth, attenuated the inhibitory effects of nonpermissive substrate myelin, and decreased the formation of retraction bulbs following axonal injury in cultured cortical neurons. Interestingly, autophagy induction stabilized microtubules by degrading SCG10, a microtubule disassembly protein in neurons. In mice with spinal cord injury, local administration of a specific autophagy-inducing peptide, Tat-beclin1, to lesion sites markedly attenuated axonal retraction of spinal dorsal column axons and cortical spinal tract and promoted regeneration of descending axons following long-term observation. Finally, administration of Tat-beclin1 improved the recovery of motor behaviors of injured mice. These results show a promising effect of an autophagy-inducing reagent on injured axons, providing direct evidence supporting a beneficial role of autophagy in axon regeneration.

The interaction of coagulation factors with the perivascular environment affects the development of disease in ways that extend beyond their traditional roles in the acute hemostatic cascade. Key molecular players of the coagulation cascade like tissue factor, thrombin, and fibrinogen are epidemiologically and mechanistically linked with diseases with an inflammatory component. Moreover, the identification of novel molecular mechanisms linking coagulation and inflammation has highlighted factors of the coagulation cascade as new targets for therapeutic intervention in a wide range of inflammatory human diseases. In particular, a proinflammatory role for fibrinogen has been reported in vascular wall disease, stroke, spinal cord injury, brain trauma, multiple sclerosis, Alzheimer’s disease, rheumatoid arthritis, bacterial infection, colitis, lung and kidney fibrosis, Duchenne muscular dystrophy, and several types of cancer. Genetic and pharmacologic studies have unraveled pivotal roles for fibrinogen in determining the extent of local or systemic inflammation. As cellular and molecular mechanisms for fibrinogen functions in tissues are identified, the role of fibrinogen is evolving from a marker of vascular rapture to a multi-faceted signaling molecule with a wide spectrum of functions that can tip the balance between hemostasis and thrombosis, coagulation and fibrosis, protection from infection and extensive inflammation, and eventually life and death. This review will discuss some of the main molecular links between coagulation and inflammation and will focus on the role of fibrinogen in inflammatory disease highlighting its unique structural properties, cellular targets, and signal transduction pathways that make it a potent proinflammatory mediator and a potential therapeutic target.

Spinal cord injury (SCI) involves diverse injury responses in different cell types in a temporally and spatially specific manner. Here, using single-cell transcriptomic analyses combined with classic anatomical, behavioral, electrophysiological analyses, we report, with single-cell resolution, temporal molecular and cellular changes in crush-injured adult mouse spinal cord. Data revealed pathological changes of 12 different major cell types, three of which infiltrated into the spinal cord at distinct times post-injury. We discovered novel microglia and astrocyte subtypes in the uninjured spinal cord, and their dynamic conversions into additional stage-specific subtypes/states. Most dynamic changes occur at 3-days post-injury and by day-14 the second wave of microglial activation emerged, accompanied with changes in various cell types including neurons, indicative of the second round of attacks. By day-38, major cell types are still substantially deviated from uninjured states, demonstrating prolonged alterations. This study provides a comprehensive mapping of cellular/molecular pathological changes along the temporal axis after SCI, which may facilitate the development of novel therapeutic strategies, including those targeting microglia.