Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.
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ABSTRACT:
SUBMITTER: Yang LQ
PROVIDER: S-EPMC7719141 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Axon regeneration, fundamental to nerve repair, and functional recovery, relies on rapid changes in gene expression attributable to microRNA (miRNA) regulation. MiR-133b has been proved to play an important role in different organ regeneration in zebrafish, but its role in regulating axon regeneration in vivo is still controversial. Here, combining single-cell electroporation with a vector-based miRNA-expression system, we have modulated the expression of miR-133b in Mauthner-cells (M-cells) at the single-cell level in zebrafish. Through in vivo imaging, we show that overexpression of miR-133b inhibits axon regeneration, whereas down-regulation of miR-133b, promotes axon outgrowth. We further show that miR-133b regulates axon regeneration by directly targeting a novel regeneration-associated gene, tppp3, which belongs to Tubulin polymerization-promoting protein family. Gain or loss-of-function of tppp3 experiments indicated that tppp3 was a novel gene that could promote axon regeneration. In addition, we observed a reduction of mitochondrial motility, which have been identified to have a positive correlation with axon regeneration, in miR-133b overexpressed M-cells. Taken together, our work provides a novel way to study the role of miRNAs in individual cell and establishes a critical cell autonomous role of miR-133b in zebrafish M-cell axon regeneration. We propose that up-regulation of the newly founded regeneration-associated gene tppp3 may enhance axonal regeneration.
| S-EPMC5702462 | biostudies-literature
Project description:MicroRNA (miRNA), functioning as a post-transcriptional regulatory element, plays a significant role in numerous regulatory mechanisms and serves as a crucial intrinsic factor influencing axon regeneration. Prior investigations have elucidated the involvement of miRNA-9 in various processes, however, its specific contribution to axon regeneration in the central nervous system (CNS) remains uncertain. Hence, the zebrafish Mauthner axon regeneration model was employed to manipulate the expression of miRNA-9 in single cells, revealing that upregulation of miRNA-9 facilitated axon regeneration. Additionally, her6, a downstream target gene of miRNA-9, was identified as a novel gene associated with axon regeneration. Suppression of her6 resulted in enhanced Mauthner axon regeneration, as evidenced by the significantly improved regenerative capacity observed in her6 knockout zebrafish. In addition, modulation of her6 expression affects intracellular calcium levels in neurons and promoting her6 expression leads to a decrease in calcium levels in vivo using the new NEMOf calcium indicator. Moreover, the administration of the neural activity activator, pentylenetetrazol (PTZ) partially compensated for the inhibitory effect of her6 overexpression on the calcium level and promoted axon regeneration. Taken together, our study revealed a role for miRNA-9 in the process of axon regeneration in the CNS, which improved intracellular calcium activity and promoted axon regeneration by inhibiting the expression of downstream target gene her6. In our study, miRNA-9 emerged as a novel and intriguing target in the intricate regulation of axon regeneration and offered compelling evidence for the intricate relationship between calcium activity and the facilitation of axon regeneration.
| S-EPMC10899279 | biostudies-literature
Project description:Mitochondrial transport is essential for neuronal function, but the evidence of connections between mitochondrial transport and axon regeneration in the central nervous system (CNS) of living vertebrates remains limited. Here, we developed a novel model to explore mitochondrial transport in a single Mauthner axon (M axon) of zebrafish with non-invasive in vivo imaging. To confirm the feasibility of using this model, we treated labeled zebrafish with nocodazole and demonstrated that it could disrupt mitochondrial transport. We also used two-photon laser axotomy to precisely axotomize M axons and simultaneously recorded their regeneration and the process of mitochondrial transport in living zebrafish larvae. The findings showed that the injured axons with stronger regenerative capability maintain greater mitochondrial motility. Furthermore, to stimulate axon regeneration, treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP) could also augment mitochondrial motility. Taken together, our results provide new evidence that mitochondrial motility is positively correlated with axon regeneration in the living vertebrate CNS. This promising model will be useful for further studies on the interaction between axon regeneration and mitochondrial dynamics, using various genetic and pharmacological techniques.
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Project description:Wolfram Syndrome (WS) is a fatal human inherited disease with symptoms of diabetes, vision decreasing, and neurodegeneration caused by mutations in the endoplasmic reticulum (ER)-resident protein WFS1. WFS1 has been reported to play an important role in glucose metabolism. However, the role of WFS1 in axonal regeneration in the central nervous system has so far remained elusive. Herein, we established a model of the wfs1b globally deficient zebrafish line. wfs1b deficiency severely impeded the Mauthner-cell (M-cell) axon regeneration, which was partly dependent on the ER stress response. The administration of ER stress inhibitor 4-Phenylbutyric acid (4-PBA) promoted M-cell axon regeneration in wfs1b-/- zebrafish larvae, while the ER stress activator Tunicamycin (TM) inhibited M-cell axon regeneration in wfs1b+/+ zebrafish larvae. Moreover, complementation of wfs1b at the single-cell level stimulated M-cell axon regeneration in the wfs1b-/- zebrafish larvae. Altogether, our results revealed that wfs1b promotes M-cell axon regeneration through the ER stress signal pathway and provide new evidence for a therapeutic target for WS and axon degeneration.
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Project description:Despite robust effects on immature neurons, growth factors minimally promote axon regeneration in the adult central nervous system (CNS). Attempting to improve growth-factor responsiveness in mature neurons by dedifferentiation, we overexpressed Lin28 in the retina. Lin28-treated retinas responded to insulin-like growth factor-1 (IGF1) by initiating retinal ganglion cell (RGC) axon regeneration after axotomy. Surprisingly, this effect was cell non-autonomous. Lin28 expression was required only in amacrine cells, inhibitory neurons that innervate RGCs. Ultimately, we found that optic-nerve crush pathologically upregulated activity in amacrine cells, which reduced RGC electrical activity and suppressed growth-factor signaling. Silencing amacrine cells or pharmacologically blocking inhibitory neurotransmission also induced IGF1 competence. Remarkably, RGCs regenerating across these manipulations localized IGF1 receptor to their primary cilia, which maintained their signaling competence and regenerative ability. Thus, our results reveal a circuit-based mechanism that regulates CNS axon regeneration and implicate primary cilia as a regenerative signaling hub.
| S-EPMC7350660 | biostudies-literature
Project description:BackgroundPeripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells.ResultsUsing pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored.ConclusionThe accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.
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Project description:Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce 'effector' RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types.
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Project description:Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.
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Project description:Regeneration after peripheral nerve damage requires that axons re-grow to the correct target tissues in a process called target-specific regeneration. Although much is known about the mechanisms that promote axon re-growth, re-growing axons often fail to reach the correct targets, resulting in impaired nerve function. We know very little about how axons achieve target-specific regeneration, particularly in branched nerves that require distinct targeting decisions at branch points. The zebrafish vagus motor nerve is a branched nerve with a well-defined topographic organization. Here, we track regeneration of individual vagus axons after whole-nerve laser severing and find a robust capacity for target-specific, functional re-growth. We then develop a new single-cell chimera injury model for precise manipulation of axon-environment interactions and find that (1) the guidance mechanism used during regeneration is distinct from the nerve's developmental guidance mechanism, (2) target selection is specified by neurons' intrinsic memory of their position within the brain, and (3) targeting to a branch requires its pre-existing innervation. This work establishes the zebrafish vagus nerve as a tractable regeneration model and reveals the mechanistic basis of target-specific regeneration.
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Project description:Many neurons have limited capacity to regenerate their axons after injury. Neurons in the mammalian central nervous system do not regenerate, and even neurons in the peripheral nervous system often fail to regenerate to their former targets. This failure is likely due in part to pathways that actively restrict regeneration; however, only a few factors that limit regeneration are known. Here, using single-neuron analysis of regeneration in vivo, we show that Notch/lin-12 signaling inhibits the regeneration of mature C. elegans neurons. Notch signaling suppresses regeneration by acting autonomously in the injured cell to prevent growth cone formation. The metalloprotease and gamma-secretase cleavage events that lead to Notch activation during development are also required for its activity in regeneration. Furthermore, blocking Notch activation immediately after injury improves regeneration. Our results define a postdevelopmental role for the Notch pathway as a repressor of axon regeneration in vivo.
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