Neural control of tumor immunity.

Communication between the nervous system and the immune system has evolved to optimally respond to potentially dangerous stimuli both from within and outside the body. Tumors pose a severe threat to an organism and current therapies are insufficient for tumor regression in the majority of cases. Studies show that tumors are innervated by peripheral nerves from the sensory, parasympathetic and sympathetic nervous systems. Interactions between cancer cells, nerves and immune cells regulate overall tumor progression. Clinical studies have indicated the potential of targeting the peripheral nervous system for promoting anti-tumor immune responses. This view point provides an opinion on the current evidence and therapeutic potential of manipulating neuro-immune communications in cancer.

Three-dimensional chromatin mapping of sensory neurons reveals that promoter-enhancer looping is required for axonal regeneration.

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration.

Three-dimensional chromatin mapping of sensory neurons reveals that cohesin-dependent genomic domains are required for axonal regeneration.

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression programme at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C and RNA-seq. We find that cohesin is required for the full induction of the regenerative transcriptional program, by organising 3D genomic domains required for the activation of regenerative genes. Importantly, loss of cohesin results in disruption of chromatin architecture at regenerative genes and severely impaired nerve regeneration. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent chromatin interactions in neuronal regeneration.

The circadian clock time tunes axonal regeneration.

Nerve injuries cause permanent neurological disability due to limited axonal regeneration. Injury-dependent and -independent mechanisms have provided important insight into neuronal regeneration, however, common denominators underpinning regeneration remain elusive. A comparative analysis of transcriptomic datasets associated with neuronal regenerative ability revealed circadian rhythms as the most significantly enriched pathway. Subsequently, we demonstrated that sensory neurons possess an endogenous clock and that their regenerative ability displays diurnal oscillations in a murine model of sciatic nerve injury. Consistently, transcriptomic analysis showed a time-of-day-dependent enrichment for processes associated with axonal regeneration and the circadian clock. Conditional deletion experiments demonstrated that Bmal1 is required for neuronal intrinsic circadian regeneration and target re-innervation. Lastly, lithium enhanced nerve regeneration in wild-type but not in clock-deficient mice. Together, these findings demonstrate that the molecular clock fine-tunes the regenerative ability of sensory neurons and propose compounds affecting clock pathways as a novel approach to nerve repair.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Control of lymph node activity by direct local innervation.

The nervous system detects environmental and internal stimuli and relays this information to immune cells via neurotransmitters and neuropeptides. This is essential to respond appropriately to immunogenic threats and to support system homeostasis. Lymph nodes (LNs) act as sentinels where adaptive immune responses are generated. They are richly innervated by peripheral sympathetic and sensory nerves, which are responsible for the local secretion of neurotransmitters by sympathetic fibers, such as norepinephrine, and neuropeptides by sensory fibers, including calcitonin gene-related peptide (CGRP) and substance P. Additionally, time-of-day-dependent oscillations in nerve activity are associated with differential immune responses, suggesting a potential role for neuroimmune interactions in coordinating immunity in a circadian fashion. Here, we discuss how LN activity is controlled by local innervation.

The gut metabolite indole-3 propionate promotes nerve regeneration and repair.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.

Reversible CD8 T cell-neuron cross-talk causes aging-dependent neuronal regenerative decline.

Aging is associated with increased prevalence of axonal injuries characterized by poor regeneration and disability. However, the underlying mechanisms remain unclear. In our experiments, RNA sequencing of sciatic dorsal root ganglia (DRG) revealed significant aging-dependent enrichment in T cell signaling both before and after sciatic nerve injury (SNI) in mice. Lymphotoxin activated the transcription factor NF-κB, which induced expression of the chemokine CXCL13 by neurons. This in turn recruited CXCR5+CD8+ T cells to injured DRG neurons overexpressing major histocompatibility complex class I. CD8+ T cells repressed the axonal regeneration of DRG neurons via caspase 3 activation. CXCL13 neutralization prevented CXCR5+CD8+ T cell recruitment to the DRG and reversed aging-dependent regenerative decline, thereby promoting neurological recovery after SNI. Thus, axonal regeneration can be facilitated by antagonizing cross-talk between immune cells and neurons.

Enriched conditioning expands the regenerative ability of sensory neurons after spinal cord injury via neuronal intrinsic redox signaling.

Overcoming the restricted axonal regenerative ability that limits functional repair following a central nervous system injury remains a challenge. Here we report a regenerative paradigm that we call enriched conditioning, which combines environmental enrichment (EE) followed by a conditioning sciatic nerve axotomy that precedes a spinal cord injury (SCI). Enriched conditioning significantly increases the regenerative ability of dorsal root ganglia (DRG) sensory neurons compared to EE or a conditioning injury alone, propelling axon growth well beyond the spinal injury site. Mechanistically, we established that enriched conditioning relies on the unique neuronal intrinsic signaling axis PKC-STAT3-NADPH oxidase 2 (NOX2), enhancing redox signaling as shown by redox proteomics in DRG. Finally, NOX2 conditional deletion or overexpression respectively blocked or phenocopied enriched conditioning-dependent axon regeneration after SCI leading to improved functional recovery. These studies provide a paradigm that drives the regenerative ability of sensory neurons offering a potential redox-dependent regenerative model for mechanistic and therapeutic discoveries.

Lung innervation in the eye of a cytokine storm: neuroimmune interactions and COVID-19.

COVID-19 is an infectious disease caused by the coronavirus SARS-CoV-2, which was first reported in Wuhan, China, in December 2019 and has caused a global pandemic. Acute respiratory distress syndrome (ARDS) is a common feature of severe forms of COVID-19 and can lead to respiratory failure, especially in older individuals. The increasing recognition of the neurotropic potential of SARS-CoV-2 has sparked interest in the role of the nervous system in respiratory failure in people with COVID-19. However, the neuroimmune interactions in the lung in the context of ARDS are poorly understood. In this Perspectives article, we propose the concept of the neuroimmune unit as a critical determinant of lung function in the context of COVID-19, inflammatory conditions and ageing, focusing particularly on the involvement of the vagus nerve. We discuss approaches such as neurostimulation and pharmacological neuromodulation to reduce tissue inflammation with the aim of preventing respiratory failure.

AMPK controls the axonal regenerative ability of dorsal root ganglia sensory neurons after spinal cord injury.

Regeneration after injury occurs in axons that lie in the peripheral nervous system but fails in the central nervous system, thereby limiting functional recovery. Differences in axonal signalling in response to injury that might underpin this differential regenerative ability are poorly characterized. Combining axoplasmic proteomics from peripheral sciatic or central projecting dorsal root ganglion (DRG) axons with cell body RNA-seq, we uncover injury-dependent signalling pathways that are uniquely represented in peripheral versus central projecting sciatic DRG axons. We identify AMPK as a crucial regulator of axonal regenerative signalling that is specifically downregulated in injured peripheral, but not central, axons. We find that AMPK in DRG interacts with the 26S proteasome and its CaMKIIα-dependent regulatory subunit PSMC5 to promote AMPKα proteasomal degradation following sciatic axotomy. Conditional deletion of AMPKα1 promotes multiple regenerative signalling pathways after central axonal injury and stimulates robust axonal growth across the spinal cord injury site, suggesting inhibition of AMPK as a therapeutic strategy to enhance regeneration following spinal cord injury.

Cyclin-dependent-like kinase 5 is required for pain signaling in human sensory neurons and mouse models.

Cyclin-dependent-like kinase 5 (CDKL5) gene mutations lead to an X-linked disorder that is characterized by infantile epileptic encephalopathy, developmental delay, and hypotonia. However, we found that a substantial percentage of these patients also report a previously unrecognized anamnestic deficiency in pain perception. Consistent with a role in nociception, we found that CDKL5 is expressed selectively in nociceptive dorsal root ganglia (DRG) neurons in mice and in induced pluripotent stem cell (iPS)-derived human nociceptors. CDKL5-deficient mice display defective epidermal innervation, and conditional deletion of CDKL5 in DRG sensory neurons impairs nociception, phenocopying CDKL5 deficiency disorder in patients. Mechanistically, CDKL5 interacts with calcium/calmodulin-dependent protein kinase II α (CaMKIIα) to control outgrowth and transient receptor potential cation channel subfamily V member 1 (TRPV1)-dependent signaling, which are disrupted in both CDKL5 mutant murine DRG and human iPS-derived nociceptors. Together, these findings unveil a previously unrecognized role for CDKL5 in nociception, proposing an original regulatory mechanism for pain perception with implications for future therapeutics in CDKL5 deficiency disorder.

PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

Cbp-dependent histone acetylation mediates axon regeneration induced by environmental enrichment in rodent spinal cord injury models.

After a spinal cord injury, axons fail to regenerate in the adult mammalian central nervous system, leading to permanent deficits in sensory and motor functions. Increasing neuronal activity after an injury using electrical stimulation or rehabilitation can enhance neuronal plasticity and result in some degree of recovery; however, the underlying mechanisms remain poorly understood. We found that placing mice in an enriched environment before an injury enhanced the activity of proprioceptive dorsal root ganglion neurons, leading to a lasting increase in their regenerative potential. This effect was dependent on Creb-binding protein (Cbp)-mediated histone acetylation, which increased the expression of genes associated with the regenerative program. Intraperitoneal delivery of a small-molecule activator of Cbp at clinically relevant times promoted regeneration and sprouting of sensory and motor axons, as well as recovery of sensory and motor functions in both the mouse and rat model of spinal cord injury. Our findings showed that the increased regenerative capacity induced by enhancing neuronal activity is mediated by epigenetic reprogramming in rodent models of spinal cord injury. Understanding the mechanisms underlying activity-dependent neuronal plasticity led to the identification of potential molecular targets for improving recovery after spinal cord injury.

Paracrine Mechanisms of Redox Signalling for Postmitotic Cell and Tissue Regeneration.

Adult postmitotic mammalian cells, including neurons and cardiomyocytes, have a limited capacity to regenerate after injury. Therefore, an understanding of the molecular mechanisms underlying their regenerative ability is critical to advance tissue repair therapies. Recent studies highlight how redox signalling via paracrine cell-to-cell communication may act as a central mechanism coupling tissue injury with regeneration. Post-injury redox paracrine signalling can act by diffusion to nearby cells, through mitochondria or within extracellular vesicles, affecting specific intracellular targets such as kinases, phosphatases, and transcription factors, which in turn trigger a regenerative response. Here, we review redox paracrine signalling mechanisms in postmitotic tissue regeneration and discuss current challenges and future directions.

Publisher Correction: Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

In the version of this Article originally published, the affiliations for Roland A. Fleck and José Antonio Del Río were incorrect due to a technical error that resulted in affiliations 8 and 9 being switched. The correct affiliations are: Roland A. Fleck: 8Centre for Ultrastructural Imaging, Kings College London, London, UK. José Antonio Del Río: 2Cellular and Molecular Neurobiotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain; 9Department of Cell Biology, Physiology and Immunology, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 10Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain. This has now been amended in all online versions of the Article.

Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

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-ß1-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.