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Chronic inflammation is associated with normal and pathological ageing. Here we show that chronic, progressive low-grade inflammation induced by knockout of the nfkb1 subunit of the transcription factor NF-κB induces premature ageing in mice. We also show that these mice have reduced regeneration in liver and gut. nfkb1 −/− fibroblasts exhibit aggravated cell senescence because of an enhanced autocrine and paracrine feedback through NF-κB, COX-2 and ROS, which stabilizes DNA damage. Preferential accumulation of telomere-dysfunctional senescent cells in nfkb1 −/− tissues is blocked by anti-inflammatory or antioxidant treatment of mice, and this rescues tissue regenerative potential. Frequencies of senescent cells in liver and intestinal crypts quantitatively predict mean and maximum lifespan in both short- and long-lived mice cohorts. These data indicate that systemic chronic inflammation can accelerate ageing via ROS-mediated exacerbation of telomere dysfunction and cell senescence in the absence of any other genetic or environmental factor. Many age-related diseases are associated with chronic inflammation. Here Jurk et al . use a mouse model of chronic, low-grade inflammation to support a model by which such inflammation promotes a vicious cycle of oxidative stress, telomere dysfunction and cell senescence that accelerates the ageing process.

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.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.

Some animals can regenerate large missing regions of their nervous system, requiring mechanisms to restore the pattern, numbers, and wiring of diverse neuron classes. Because injuries are unpredictable, regeneration must be accomplished from an unlimited number of starting points. Coordinated regeneration of neuron-glia architecture is thus a major challenge and remains poorly understood. In planarians, neurons and glia are regenerated from distinct progenitors. We found that planarians first regenerate neurons expressing a Delta-encoding gene, delta-2 , at key positions in the central and peripheral nervous systems. Planarian glia are specified later from dispersed Notch-1-expressing mesoderm-like phagocytic progenitors. Inhibition of delta-2 or notch-1 severely reduced glia in planarians, but did not affect the specification of other phagocytic cell types. Loss of several delta-2 -expressing neuron classes prevented differentiation of the glia associated with them, whereas transplantation of delta-2 -expressing photoreceptor neurons was sufficient for glia formation at an ectopic location. Our results suggest a model in which patterned delta-2- expressing neurons instruct phagocytic progenitors to locally differentiate into glia, presenting a mechanism for coordinated regeneration of numbers and pattern of cell types.

Tissue injury and the healing response lead to the release of endogenous danger signals including Toll-like receptor (TLR) and interleukin-1 receptor, type 1 (IL-1R1) ligands, which modulate the immune microenvironment. Because TLRs and IL-1R1 have been shown to influence the repair process of various tissues, we explored their role during bone regeneration, seeking to design regenerative strategies integrating a control of their signalling. Here we show that IL-1R1/MyD88 signalling negatively regulates bone regeneration, in the mouse. Furthermore, IL-1β which is released at the bone injury site, inhibits the regenerative capacities of mesenchymal stem cells (MSCs). Mechanistically, IL-1R1/MyD88 signalling impairs MSC proliferation, migration and differentiation by inhibiting the Akt/GSK-3β/β-catenin pathway. Lastly, as a proof of concept, we engineer a MSC delivery system integrating inhibitors of IL-1R1/MyD88 signalling. Using this strategy, we considerably improve MSC-based bone regeneration in the mouse, demonstrating that this approach may be useful in regenerative medicine applications. TLR and IL-1R1 ligands are danger signals released following tissue injury and during the healing response. Here, the authors show that IL-1β signalling via IL-1R1/MyD88 inhibits the Akt/GSK-3β/β-catenin pathway in mesenchymal stem cells, which suppresses their mobilization, proliferation, and differentiation into osteoblasts, processes necessary for bone regeneration.

Programmable RNA editing is harnessed for modifying mRNA. Besides mRNA, miRNA also regulates numerous biological activities, but current RNA editors have yet to be exploited for miRNA manipulation. To engineer primary miRNA (pri-miRNA), the miRNA precursor, we present a customizable editor REPRESS (RNA Editing of Pri-miRNA for Efficient Suppression of miRNA) and characterize critical parameters. The optimized REPRESS is distinct from other mRNA editing tools in design rationale, hence enabling editing of pri-miRNAs that are not editable by other RNA editing systems. We edit various pri-miRNAs in different cells including adipose-derived stem cells (ASCs), hence attenuating mature miRNA levels without disturbing host gene expression. We further develop an improved REPRESS (iREPRESS) that enhances and prolongs pri-miR-21 editing for at least 10 days, with minimal perturbation of transcriptome and miRNAome. iREPRESS reprograms ASCs differentiation, promotes in vitro cartilage formation and augments calvarial bone regeneration in rats, thus implicating its potentials for engineering miRNA and applications such as stem cell reprogramming and tissue regeneration. miRNAs play a crucial role in biological processes and their dysregulation is associated with many diseases and disorders. Here, the authors develop a method to manipulate miRNAs via RNA editing of their precursors and apply this method to program stem cell differentiation for bone tissue regeneration.

Gene therapy might represent a promising strategy for chondral and osteochondral defects repair by balancing the management of temporary joint mechanical incompetence with altered metabolic and inflammatory homeostasis. This review analysed preclinical and clinical studies on gene therapy for the repair of articular cartilage defects performed over the last 10 years, focussing on expression vectors (non-viral and viral), type of genes delivered and gene therapy procedures (direct or indirect). Plasmids (non-viral expression vectors) and adenovirus (viral vectors) were the most employed vectors in preclinical studies. Genes delivered encoded mainly for growth factors, followed by transcription factors, anti-inflammatory cytokines and, less frequently, by cell signalling proteins, matrix proteins and receptors. Direct injection of the expression vector was used less than indirect injection of cells, with or without scaffolds, transduced with genes of interest and then implanted into the lesion site. Clinical trials (phases I, II or III) on safety, biological activity, efficacy, toxicity or bio-distribution employed adenovirus viral vectors to deliver growth factors or anti-inflammatory cytokines, for the treatment of osteoarthritis or degenerative arthritis, and tumour necrosis factor receptor or interferon for the treatment of inflammatory arthritis.

Background Appendage regeneration in amphibians is regulated by the combinatorial actions of signaling molecules. The requirement of molecules secreted from specific tissues is reflected by the observation that the whole process of regeneration can be inhibited if a certain tissue is removed from the amputated stump. Interestingly, urodeles and anurans show different tissue dependencies during tail regeneration. The spinal cord is essential for tail regeneration in urodele but not in anuran larva, whereas the notochord but not the spinal cord is essential for tail regeneration in anuran tadpoles. Sonic hedgehog is one of the signaling molecules responsible for such phenomenon in axolotl, as hedgehog signaling is essential for overall tail regeneration and sonic hedgehog is exclusively expressed in the spinal cord. In order to know whether hedgehog signaling is involved in the molecular mechanism underlying the inconsistent tissue dependency for tail regeneration between anurans and urodeles, we investigated expression of hedgehog signal-related genes in the regenerating tail of Xenopus tadpole and examined the effect of the hedgehog signal inhibitor, cyclopamine, on the tail regeneration. Results In Xenopus , sonic hedgehog is expressed exclusively in the notochord but not in the spinal cord of the regenerate. Overall regeneration was severely impaired in cyclopamine-treated tadpoles. Notochord maturation in the regenerate, including cell alignment and vacuolation, and myofiber formation were inhibited. Proliferation of spinal cord cells in the neural ampulla and of mesenchymal cells was also impaired. Conclusion As in the axolotl, hedgehog signaling is required for multiple steps in tail regeneration in the Xenopus tadpole, although the location of the Shh source is quite different between the two species. This difference in Shh localization is the likely basis for the differing tissue requirement for tail regeneration between urodeles and anurans.

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 capacity 1 – 3 . Changes to DNA methylation patterns over time form the basis of ageing clocks 4 , 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 capacity 5 – 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.