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Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1–3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5–7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo. Expression of three Yamanaka transcription factors in mouse retinal ganglion cells restores youthful DNA methylation patterns, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice, suggesting that mammalian tissues retain a record of youthful epigenetic information that can be accessed to improve tissue function.

Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons1–4. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal—but not adult—microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system. In neonatal mice, scar-free healing after spinal cord injury is organized by microglia, and transplantation of neonatal microglia or peptidase-inhibitor-treated adult microglia into adult mice after injury improves healing and axon regrowth.

Central oscillators are primordial neural circuits that generate and control rhythmic movements (1,2). Mechanistic understanding of these circuits requires genetic identification of the oscillator neurons and their synaptic connections, to target electrophysiological recording and causal manipulation during behavior. Yet, such targeting remains a challenge with mammalian systems. Here, we delimit the oscillator circuit that drives rhythmic whisking, a motor action central to foraging and active sensing in rodents(3,4). We found that the whisking oscillator consists of parvalbumin-expressing inhibitory neurons located in the vibrissa intermediate reticular nucleus (vIRt(PV)) in the brainstem. vIRt(PV) neurons receive descending excitatory inputs and form recurrent inhibitory connections among themselves. Silencing vIRt(PV) neurons eliminated rhythmic whisking and resulted in sustained vibrissae protraction. In vivo recording of opto-tagged vIRt(PV) neurons in awake mice showed that these cells spike tonically when animals are at rest, and transition to rhythmic bursting at the onset of whisking, suggesting that rhythm generation is likely the result of network dynamics, as opposed to intrinsic cellular properties. Importantly, ablating inhibitory synaptic inputs to vIRt(PV) neurons quenched their rhythmic bursting, impaired the tonic-to-bursting transition and abolished regular whisking. Thus, the whisking oscillator is an all-inhibitory network and recurrent synaptic inhibition plays a key role in its rhythmogenesis.

Current models of somatosensory perception emphasize transmission from primary sensory neurons to the spinal cord and on to the brain1–4. Mental influence on perception is largely assumed to be acting locally within the brain. We have now examined if there is top-down control of sensory inflow through the spinal cord directly by the cortex. Although traditionally viewed as a primary motor pathway5, a subset of corticospinal neurons (CSNs) originating in the S1/S2 somatosensory cortex directly innervate the spinal dorsal horn via corticospinal tract (CST) axons. We show here that either reduction in somatosensory CSN activity or transection of the CST selectively impairs behavioral responses to light touch without altering responses to noxious stimuli. Moreover, such CSN manipulation greatly attenuates tactile allodynia in a peripheral neuropathic pain model. Tactile stimulation activates somatosensory CSNs and their corticospinal projections facilitate light touch-evoked activity of cholecystokinin (CCK+) interneurons in the deep dorsal horn. This represents a touch-driven feed forward spinal-cortical-spinal sensitization loop, which is important for the recruitment of spinal nociceptive neurons under tactile allodynia. These results reveal direct cortical modulation of normal and pathological tactile sensory processing in the spinal cord and open up opportunities for new treatments for neuropathic pain.

Transected axons fail to regrow across anatomically complete spinal cord injuries (SCI) in adults. Diverse molecules can partially facilitate or attenuate axon growth during development or after injury1-3, but efficient reversal of this regrowth failure remains elusive4. Here we show that three factorsthat are essential for axon growth during development but are attenuated or lacking in adults-(i) neuron intrinsic growth capacity2,5-9, (ii) growth-supportive substrate10,11 and (iii) chemoattraction12,13-are all individually required and, in combination, are sufficient to stimulate robust axon regrowth across anatomically complete SCI lesions in adult rodents. We reactivated the growth capacity of mature descending propriospinal neurons with osteopontin, insulin-like growth factor 1 and ciliary-derived neurotrophic factor before SCI14,15; induced growth-supportive substrates with fibroblast growth factor 2 and epidermal growth factor; and chemoattracted propriospinal axons with glial-derived neurotrophic factor16,17 delivered via spatially and temporally controlled release from biomaterial depots18,19, placed sequentially after SCI. We show in both mice and rats that providing these three mechanisms in combination, but not individually, stimulated robust propriospinal axon regrowth through astrocyte scar borders and across lesioncores of non-neuraltissue that was over 100-fold greater than controls. Stimulated, supported and chemoattracted propriospinal axons regrew a full spinal segment beyond lesion centres, passed well into spared neural tissue, formed terminal-like contacts exhibiting synaptic markers and conveyed a significant return of electrophysiological conduction capacity across lesions. Thus, overcoming the failure of axon regrowth across anatomically complete SCI lesions after maturity required the combined sequential reinstatement of several developmentally essential mechanisms that facilitate axon growth. These findings identify a mechanism-based biological repair strategy for complete SCI lesions that could be suitable to use with rehabilitation models designed to augment the functional recovery of remodelling circuits.

A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either Phosphatase and tensin homolog (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signaling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around two weeks after the crush injury1,2. Remarkably, we now find that simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as a key for sustaining long-distance axon regeneration in adult CNS, a crucial step toward functional recovery.

In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor has convergent axonal projections to two specific glomeruli in the olfactory bulb, thereby creating an odour map. However, it is unclear how this map is represented in the olfactory cortex. Here we combine rabies-virus-dependent retrograde mono-trans-synaptic labelling with genetics to control the location, number and type of 'starter' cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the olfactory bulb input differently. For example, the cortical amygdala preferentially receives dorsal olfactory bulb input, whereas the piriform cortex samples the whole olfactory bulb without obvious bias. These differences probably reflect different functions of these cortical areas in mediating innate odour preference or associative memory. The trans-synaptic labelling method described here should be widely applicable to mapping connections throughout the mouse nervous system.

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS)1,2. In addition to myelin-associated glycoprotein (MAG)3,4 and Nogo-A5,6,7, available evidence suggests the existence of additional inhibitors in CNS myelin8. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR)9 as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.

In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66)1, oligodendrocyte myelin glycoprotein (OMgp)2 and myelin-associated glycoprotein (MAG)3,4, exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors5,6, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75–NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR–p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.

HUMAN replication protein (RPA) functions in DNA replication1-4, homologous recombination5 and nucleotide excision repair6. This multisubunit single-stranded DNA-binding protein1,2 may be required to make unique protein-protein contacts because heterol-ogous single-stranded binding proteins cannot substitute for RPA in these diverse DNA transactions5-7. We report here that, by using affinity chromatography and immunoprecipitation, we found that human RPA bound specifically and directly to two excision repair proteins, the xeroderma pigmentosum damage-recognition protein XPA (refs 8, 9) and the endonuclease XPG (refs 10-13). Although it had been suggested that RPA might function before the DNA synthesis repair stage14,15, our finding that a complex of RPA and XPA showed a striking cooperativity in binding to DNA lesions indicates that RPA may function at the very earliest stage of excision repair. In addition, by binding XPG, RPA may target this endonuclease to damaged DNA.

New work on a rat model suggests that, after spinal-cord injury, restoration of sustained and robust respiratory function is possible using strategies that promote both neuronal plasticity and regeneration. See Article p.196 Patients with spinal cord injuries in the neck area often need mechanical ventilators to help them breathe, and impaired breathing is a leading cause of death in these patients. Two factors combine to make recovery difficult. First, an injury above the fourth cervical vertebra disrupts the passage of nerve impulses from the respiratory centre in the brainstem to the phrenic motor nuclei in the spinal cord, and second, in the event of injury, adult spinal cord axons tend not to regenerate. Working in a rat model of spinal cord injury, Jerry Silver and colleagues have identified an upregulation of extracellular matrix molecules that impairs axonal regeneration following injury. Using a strategy of specific extracellular component digestion with chondroitinase, combined with peripheral nerve autografting across the damaged section of the spinal cord, the authors demonstrate a significant recovery of respiratory activity after axon regeneration. This study suggests that regeneration and restoration of diaphragm function may be possible after some types of spinal cord trauma.