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Spinal cord injury (SCI) is a debilitating central nervous system (CNS) disorder that leads to significant motor and sensory impairments. Given the limited regenerative capacity of adult mammalian neurons, this study presents an innovative strategy to enhance axonal regeneration and functional recovery by identifying a novel factor that markedly promotes axonal regeneration. Employing a zebrafish model with targeted single axon injury in Mauthner cells (M-cells) and utilizing the Tg (Tol056: EGFP) transgenic line for in vivo monitoring, we investigate the intrinsic mechanisms underlying axonal regeneration. This research specifically examines the role of amino acid transport, emphasizing the role of the solute carrier 1A4 amino acid transporter in axonal regeneration. Our findings demonstrate that Slc1a4 overexpression significantly enhances axonal regeneration in M-cells, whereas Slc1a4 deficiency impedes this process, which is concomitant with the downregulation of the P53/Gap43 signaling pathway. By elucidating the fundamental role of Slc1a4 in axonal regeneration and uncovering its underlying mechanisms, this study thus provides novel insights into therapeutic strategies for SCI.

Facial nerve is an integral part of peripheral nerve. Schwann cells are important microglia involved in the repair and regulation of facial nerve injury. LncRNA growth arrest‑specific transcript 5 (GAS5) is involved in the behavioral regulation of Schwann cell and the regeneration of peripheral nervous system. However, there is little research about the effect of GAS5 on the repair of facial nerve injury (FNI) by regulating Schwann cells. This study aimed to investigate the role of GAS5 in Schwann cell function and FNI repair, focusing on the miR-138-5p/CXCL12 axis. Hematoxylin and eosin staining, Luxol fast blue staining, transmission electron microscope, and immunofluorescence (IF) experiments were used to verify the effect of GAS5 on FNI rats. Reverse transcription real-time polymerase chain reaction was performed to detect GAS5, miR-138-5p, and C-X-C motif chemokine ligand 12 (CXCL12) mRNA expression. IF staining was used to detect the inflorescence of S100 calcium binding protein B (S100β), SRY-box transcription factor 10 (SOX10), and tubulin beta 3 class III (β-Tubulin III). Glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100β, brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and CXCL12 proteins were detected using western blot. The 5-bromo-2'-deoxyuridine staining, Transwell, and flow cytometry assays were conducted to detect Schwann cell function. Dual-luciferase, RNA immunoprecipitation, and RNA pulldown assay were used to identify the interaction among GAS5, miR-138-5p, and CXCL12. Results found that GAS5 was downregulated in facial nerve tissues of FNI rats. Overexpressed GAS5 decreased facial grading, inhibited demyelination, and promoted proliferation, migration, and suppressed apoptosis of Schwann cells. Mechanistically, GAS5 was a sponge of miR-138-5p and positively regulated CXCL12 expression. GAS5 inhibition repressed CXCL12 expression and decreased cell proliferation and migration, increased apoptosis rate of Schwann cells by sponging miR-138-5p. In conclusion, overexpression of GAS5 accelerates facial nerve repair in FNI rats by regulating miR-138-5p/CXCL12 axis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This review explores the molecular and genetic underpinnings of axonal regeneration and functional recovery post-nerve injury, emphasizing its significance in reversing neurological deficits. It presents a systematic exploration of the roles of various genes in axonal regrowth across peripheral and central nerve injuries. Initially, it highlights genes and gene families critical for axonal growth and guidance, delving into their roles in regeneration. It then examines the regenerative microenvironment, focusing on the role of glial cells in neural repair through dedifferentiation, proliferation, and migration. The concept of "traumatic microenvironments" within the central nervous system (CNS) and peripheral nervous system (PNS) is discussed, noting their impact on regenerative capacities and their importance in therapeutic strategy development. Additionally, the review delves into axonal transport mechanisms essential for accurate growth and reinnervation, integrating insights from proteomics, genome-wide screenings, and gene editing advancements. Conclusively, it synthesizes these insights to offer a comprehensive understanding of axonal regeneration's molecular orchestration, aiming to inform effective nerve injury therapies and contribute to regenerative neuroscience., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microtubule stabilization is critical for axonal growth and regeneration, and many microtubule-associated proteins are involved in this process. In this study, we found that the knockdown of echinoderm microtubule-associated protein-like 1 (EML1) hindered axonal growth in cultured cortical and dorsal root ganglion neurons. We further revealed that EML1 facilitated the acetylation of microtubules and that the impairment of axonal growth due to EML1 inhibition could be restored by treatment with deacetylase inhibitors, suggesting that EML1 affected tubulin acetylation. Moreover, we verified an interaction between EML1 and the alpha-tubulin acetyltransferase 1, which is responsible for the acetylation of alpha-tubulin. We thus proposed that EML1 might regulate microtubule acetylation and stabilization via alpha-tubulin acetyltransferase 1 and then promote axon growth. Finally, we verified that the knockdown of EML1 in vivo also inhibited sciatic nerve regeneration. Our findings revealed a novel effect of EML1 on microtubule acetylation during axonal regeneration., Competing Interests: Declaration of competing interest The authors of the manuscript entitled “EML1 promotes neuronal axonal growth by enhancing ATAT1-mediated microtubule acetylation” to Biochimica et Biophysica Acta (BBA) - Molecular Cell Research declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Decellularized extracellular matrix (dECM) is an intriguing natural biomaterial that has garnered significant attention due to its remarkable biological properties. In our study, we employed a cell-matrixed nerve graft for the repair of sciatic nerve defects in rats. The efficacy of this approach was assessed, and concurrently, the underlying molecular regulatory mechanisms were explored to elucidate how such grafts facilitate nerve regeneration. Long noncoding RNAs (lncRNAs) regulate mRNA expression via multiple mechanisms, including post-transcriptional regulation, transcription factor effects, and competitive binding with miRNAs. These interactions between lncRNAs and mRNAs facilitate precise control of gene expression, allowing organisms to adapt to varying biological environments and physiological states. By investigating the expression profiles and interaction dynamics of mRNAs and lncRNAs, we can enhance our understanding of the molecular mechanisms through which cell-matrixed nerve grafts influence neural repair. Such studies are pivotal in uncovering the intricate networks of gene regulation that underpin this process., Results: Weighted gene co-expression network analysis (WGCNA) utilizes clustering algorithms, such as hierarchical clustering, to aggregate genes with similar expression profiles into modules. These modules, which potentially correspond to distinct biological functions or processes, can subsequently be analyzed for their association with external sample traits. By correlating gene modules with specific conditions, such as disease states or responses to treatments, WGCNA enables a deeper understanding of the genetic architecture underlying various phenotypic traits and their functional implications. We identified seven mRNA modules and five lncRNA modules that exhibited associations with treatment or time-related events by WGCNA. We found the blue (mRNAs) module which displayed a remarkable enrichment in "axonal guidance" and "metabolic pathways", exhibited strong co-expression with multiple lncRNA modules, including blue (related to "GnRH secretion" and "pyrimidine metabolism"), green (related to "arginine and proline metabolism"), black (related to "nitrogen metabolism"), grey60 (related to "PPAR signaling pathway"), and greenyellow (related to "steroid hormone biosynthesis"). All of the top 50 mRNAs and lncRNAs exhibiting the strongest correlation were derived from the blue module. Validation of key molecules were performed using immunohistochemistry and qRT-PCR., Conclusion: Revealing the principles and molecular regulatory mechanisms of the interaction between materials and biological entities, such as cells and tissues, is a direction for the development of biomimetic tissue engineering technologies and clinically effective products., (© 2024. The Author(s).)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

The Krüppel-like factors (KLFs) have emerged as important transcriptional regulators of various cellular processes, including neural development. Some of them have been described as intrinsic factors involved in axon regeneration in the central nervous system (CNS) of vertebrates. Zebrafish are known for their ability to regenerate several tissues in adulthood, including the CNS, a capability lost during vertebrate evolution and absent in adult mammals. The role that KLFs could play in this differential ability remains unknown. Therefore, in this study, we analyzed the endogenous response of certain KLFs implicated in axon regeneration (KLFs 6, 7, 9, and 13) during retina development and after axon injury. The results showed that the expression of Klfs 6, 7, and 13 decreases in the developing retina of mice but not in zebrafish, while the mRNA levels of Klf9 strongly increase in both species. The response to injury was further analyzed using optic nerve crush (ONC) as a model of lesion. Our analysis during the acute phase (hours) demonstrated an induction of Klfs 6 and 7 expression exclusively in the zebrafish retina, while Klfs 9 and 13 mRNA levels increased in both species. Further analysis of the chronic response (days) showed that mRNA levels of Klf6 transiently increase in the retinas of both zebrafish and mice, whereas those of Klf7 decrease later after optic nerve injury. In addition, the analysis revealed that the expression of Klf9 decreases, while that of Klf13 increases in the retinas of zebrafish in response to optic nerve injury but remains unaltered in mice. Altogether, these findings support the hypothesis that KLFs may play a role in the differential axon regeneration abilities exhibited by fish and mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Application of polyethylene glycol (PEG) to a peripheral nerve injury at the time of primary neurorrhaphy is thought to prevent Wallerian degeneration via direct axolemma fusion. The molecular mechanisms of nerve fusion and recovery are unclear. Our study tested the hypothesis that PEG alters gene expression in neural and muscular environments as part of its restorative properties. Lewis rats underwent unilateral sciatic nerve transection with immediate primary repair. Subjects were randomly assigned to receive either PEG treatment or standard repair at the time of neurorrhaphy. Samples of sciatic nerve distal to the injury and tibialis muscle at the site of innervation were harvested at 24 hours and 4 weeks postoperatively. Total RNA sequencing and subsequent bioinformatics analyses were used to identify significant differences in differentially expressed genes (DEGs) and their related biological pathways (p<0.05) in PEG-treated subjects compared to non-PEG controls. No significant DEGs were identified in PEG-treated sciatic nerve compared to controls after 24 hours, but 1,480 DEGs were identified in PEG-treated tibialis compared to controls. At 4 weeks, 918 DEGs were identified in PEG-treated sciatic nerve, whereas only 3 DEGs remained in PEG-treated tibialis compared to controls. DEGs in sciatic were mostly upregulated (79%) and enriched in pathways present during nervous system development and growth, whereas DEGs in muscle were mostly downregulated (77%) and related to inflammation and tissue repair. Our findings indicate that PEG application during primary neurorrhaphy leads to significant differential gene regulation in the neural and muscular environment that is associated with improved functional recovery in animals treated with PEG compared to sham non-PEG controls. A detailed understanding of key molecules underlying PEG function in recovery after peripheral nerve repair may facilitate amplification of PEG effects through systemic or focal treatments at the time of neurotmesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Weiss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The regeneration of peripheral nerves after injury is often slow and impaired, which may be associated with weakened and denervated muscles subsequently leading to atrophy. Adipose-derived stem cells (ADSCs) are often regarded as cell-based therapeutic candidate due to their regenerative potential. The study aims to assess the therapeutic efficacy of gene-modified ADSCs on sciatic nerve injury. We lentivirally transduced ADSCs with shRNA-TWIST1 and transplanted modified cells to rats undergoing sciatic nerve transection and repair. Results showed that TWIST1 knockdown accelerated functional recovery of rats with sciatic nerve injury as faster nerve conduction velocity and higher wire hang scores obtained by rats transplanted with TWIST1-silenced ADSCs than scramble ADSCs. Although the rats experienced degenerated axons and decreased myelin sheath thickness after sciatic nerve injury 8 weeks after operation, those transplanted with TWIST1-silenced ADSCs exhibited more signs of regenerated nerve fibers surrounded by newly formed myelin sheaths than those with scramble ADSCs. The rats transplanted with TWIST1-silenced ADSCs presented increased expressions of neurotrophic factors including neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) in the sciatic nerves than those with scramble ADSCs. These results suggest that genetically modifying TWIST1 in ADSCs could facilitate peripheral nerve repair after injury in a more efficient way than that with ADSCs alone., (© 2024. The Author(s).)

Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration., (© 2024 Masin et al.)

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c -jun , a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hertzler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics., Competing Interests: Competing interests J.S.M. holds patents for the NTR inducible cell ablation system (US 7,514,595) and uses thereof (US 8,071,838 and US 8431768)., (© 2024. Published by The Company of Biologists Ltd.)

The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shimizu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The regenerative potential of injured axons displays considerable heterogeneity. However, the molecular mechanisms underlying the heterogeneity have not been fully elucidated. Here, we establish a method that can separate spinal motor neurons (spMNs) with low and high regenerative capacities and identify a set of transcripts revealing differential expression between two groups of neurons. Interestingly, oligodendrocyte transcription factor 1 (Olig1), which regulates the differentiation of various neuronal progenitors, exhibits recurrent expression in spMNs with enhanced regenerative capabilities. Furthermore, overexpression of Olig1 (Olig1 OE) facilitates axonal regeneration in various models, and down-regulation or deletion of Olig1 exhibits an opposite effect. By analyzing the overlapped differentially expressed genes after expressing individual Olig factor and functional validation, we find that the role of Olig1 is at least partially through the neurite extension factor 1 (Nrsn1). We therefore identify Olig1 as an intrinsic factor that promotes regenerative capacity of injured axons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The formation of vascular niche is pivotal during the early stage of peripheral nerve regeneration. Nevertheless, the mechanisms of vascular niche in the regulation of peripheral nerve repair remain unclear. Netrin-1 (NTN1) was found up-regulated in nerve stump after peripheral nerve injury (PNI). Herein, we demonstrated that NTN1-high endothelial cells (NTN1+ECs) were the critical component of vascular niche, fostering angiogenesis, axon regeneration, and repair-related phenotypes. We also found that NTN1+EC-derived exosomes (NTN1 EC-EXO) were involved in the formation of vascular niche as a critical role. Multi-omics analysis further verified that NTN1 EC-EXO carried a low-level expression of let7a-5p and activated key pathways associated with niche formation including focal adhesion, axon guidance, phosphatidylinositol 3-kinase-AKT, and mammalian target of rapamycin signaling pathway. Together, our study suggested that the construction of a pre-regenerative niche induced by NTN1 EC-EXO could establish a beneficial microenvironment for nerve repair and facilitate functional recovery after PNI.

A significant public health burden is peripheral nerve damage (PNI), which is frequently brought on by trauma. Macrophages were essential to the effective regeneration of nerves and restoration of function. It is still not entirely understood how macrophages and Schwann cells interact after damage during remyelination. Here, we established an inflammatory model in bone marrow-derived macrophages (BMDMs) and a rat sciatic nerve damage model to investigate the possible relationship between lipopolysaccharides (LPS)-induced exosomes derived from Schwann cells (LPS SCs-Exos) and peripheral nerve repair. The pro-inflammatory macrophage was changed into a pro-regeneration macrophage by LPS SC-Exos. Notably, it was discovered that SC-Exos had a substantial enrichment of OTULIN. OTULIN was a key mediator in the regulatory effects of LPS SC-Exos by deubiquitinating ERBB2 and preventing its degradation. The local injection of SC-Exos into the nerve damage site led in a faster functional recovery, axon regeneration and remyelination, and an increased M2 macrophage polarization, whereas OTULIN knockdown reversed these effects in vivo. Our results indicate that LPS SC-Exos may offer a therapeutic avenue for peripheral nerve regeneration by promoting macrophage polarization toward an M2 phenotype through the shuttling of OTULIN and deubiquitination of ERBB2. SIGNIFICANCE STATEMENT: OTULIN protein from SC-Exos mediated the macrophages polarization and axonal growth in BMDMs through promoting ubiquitination of ERBB2 and triggering the degradation of ERBB2. The findings offered prospective therapeutic hints for PNI therapy approaches that target axonal regrowth., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Axonal regeneration is restricted in adults and causes irreversible motor dysfunction following spinal cord injury (SCI). In contrast, neonates have prominent regenerative potential and can restore their neural function. Although the distinct cellular responses in neonates have been studied, how they contribute to neural recovery remains unclear. To assess whether the secreted molecules in neonatal SCI can enhance neural regeneration, we re-analyzed the previously performed single-nucleus RNA-seq (snRNA-seq) and focused on Asporin and Cd109, the highly expressed genes in the injured neonatal spinal cord. In the present study, we showed that both these molecules were expressed in the injured spinal cords of adults and neonates. We treated the cortical neurons with recombinant Asporin or CD109 to observe their direct effects on neurons in vitro. We demonstrated that these molecules enhance neurite outgrowth in neurons. However, these molecules did not enhance re-growth of severed axons. Our results suggest that Asporin and CD109 influence neurites at the lesion site, rather than promoting axon regeneration, to restore neural function in neonates after SCI., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nerve injury often leads to severe dysfunction because of the lack of axon regeneration in adult mammal. Intriguingly a series of extracellular vesicles (EVs) have the obvious ability to accelerate the nerve repair. However, the detailed molecular mechanisms to describe that EVs switch neuron from a transmitter to a regenerative state have not been elucidated. This study elucidated the microRNA (miRNA) expression profiles of two types of EVs that promote nerve regeneration. The functions of these miRNAs were screened in vitro. Among the 12 overlapping miRNAs, miR-25-3p was selected for further analysis as it markedly promoted axon regeneration both in vivo and in vitro. Furthermore, knockdown experiments confirmed that PTEN and Klf4, which are the major inhibitors of axon regeneration, were the direct targets of miR-25-3p in dorsal root ganglion (DRG) neurons. The utilization of luciferase reporter assays and functional tests provided evidence that miR-25-3p enhances axon regeneration by targeting Tgif1. Additionally, miR-25-3p upregulated the phosphorylation of Erk. Furthermore, Rapamycin modulated the expression of miR-25-3p in DRG neurons. Finally, the pro-axon regeneration effects of EVs were confirmed by overexpressing miR-25-3p and Tgif1 knockdown in the optic nerve crush model. Thus, the enrichment of miR-25-3p in EVs suggests that it regulates axon regeneration, proving a potential cell-free treatment strategy for nerve injury., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The axons of retinal ganglion cells (RGCs) form the optic nerve, transmitting visual information from the eye to the brain. Damage or loss of RGCs and their axons is the leading cause of visual functional defects in traumatic injury and degenerative diseases such as glaucoma. However, there are no effective clinical treatments for nerve damage in these neurodegenerative diseases. Here, we report that LIM homeodomain transcription factor Lhx2 promotes RGC survival and axon regeneration in multiple animal models mimicking glaucoma disease. Furthermore, following N-methyl-D-aspartate (NMDA)-induced excitotoxicity damage of RGCs, Lhx2 mitigates the loss of visual signal transduction. Mechanistic analysis revealed that overexpression of Lhx2 supports axon regeneration by systematically regulating the transcription of regeneration-related genes and inhibiting transcription of Semaphorin 3C (Sema3C). Collectively, our studies identify a critical role of Lhx2 in promoting RGC survival and axon regeneration, providing a promising neural repair strategy for glaucomatous neurodegeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear., Methods: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs)., Results: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites., Conclusion: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Numerous micro-RNAs (miRNAs) affect neurodevelopment and neuroprotection, but potential roles of many miRNAs in regulating these processes are still unknown. Here, we used the retinal ganglion cell (RGC) central nervous system (CNS) projection neuron and optic nerve crush (ONC) injury model, to optimize a mature miRNA arm-specific quantification method for characterizing the developmental regulation of miR-1247-5p in RGCs, investigated whether injury affects its expression, and tested whether upregulating miR-1247-5p-mimic in RGCs promotes neuroprotection and axon regeneration. We found that, miR-1247-5p is developmentally-downregulated in RGCs, and is further downregulated after ONC. Importantly, RGC-specific upregulation of miR-1247-5p promoted neuroprotection and axon regeneration after injury in vivo. To gain insight into the underlying mechanisms, we analyzed by bulk-mRNA-seq embryonic and adult RGCs, along with adult RGCs transduced by miR-1247-5p-expressing viral vector, and identified developmentally-regulated cilial and mitochondrial biological processes, which were reinstated to their embryonic levels in adult RGCs by upregulation of miR-1247-5p. Since axon growth is also a developmentally-regulated process, in which mitochondrial dynamics play important roles, it is possible that miR-1247-5p promoted neuroprotection and axon regeneration through regulating mitochondrial functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sakai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Despite substantial progress in understanding the biology of axon regeneration in the CNS, our ability to promote regeneration of the clinically important corticospinal tract (CST) after spinal cord injury remains limited. To understand regenerative heterogeneity, we conducted patch-based single-cell RNA sequencing on rare regenerating CST neurons at high depth following PTEN and SOCS3 deletion. Supervised classification with Garnett gave rise to a Regeneration Classifier, which can be broadly applied to predict the regenerative potential of diverse neuronal types across developmental stages or after injury. Network analyses highlighted the importance of antioxidant response and mitochondrial biogenesis. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Our data demonstrate a universal transcriptomic signature underlying the regenerative potential of vastly different neuronal populations and illustrate that deep sequencing of only hundreds of phenotypically identified neurons has the power to advance regenerative biology., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

This study was to probe the protective effects and mechanisms of salvianolic acid A (SAA) on cerebral ischemia-reperfusion injury (CIRI). The middle cerebral artery occlusion model (MCAO) was established in rats. Rats' behavior, neurological deficits, brain injury, inflammation, and apoptosis in the brain tissue were evaluated. The inflammatory response and apoptosis of PC12 cells induced by oxygen glucose deprivation/reperfusion (OGD/R) were detected. SAA-mediated changes in miR-212-3p, SOX7, and Wnt/β-catenin pathway were determined, and the targeting relationship between miR-212-3p and SOX7 was clarified. SAA alleviated the neurological deficits and brain injury of MCAO rats and inhibited the inflammatory response and apoptosis of OGD/R-conditioned PC-12 cells. SAA upregulated miR-212-3p, Wnt3a, and β-catenin, whereas inhibited SOX7 levels. Silencing miR-212-3p counteracted the protective effect of SAA in the context of CIRI. SOX7 was a target protein of miR-212-3p. Silencing SOX7 based on SAA and miR-212-3p knockdown suppressed OGD/R-induced inflammation and apoptosis and increased Wnt3a and β-catenin levels in PC12 cells. SAA can improve the brain and nervous system injury caused by cerebral ischemia-reperfusion by upregulating miR-212-3p, thereby inhibiting SOX7 and activating the Wnt/βcatenin signaling pathway., (© 2023 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

In the lesioned zebrafish retina, Müller glia produce multipotent retinal progenitors that generate all retinal neurons, replacing lost cell types. To study the molecular mechanisms linking Müller glia reactivity to progenitor production and neuronal differentiation, we used single-cell RNA sequencing of Müller glia, progenitors and regenerated progeny from uninjured and light-lesioned retinae. We discover an injury-induced Müller glia differentiation trajectory that leads into a cell population with a hybrid identity expressing marker genes of Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. We further observe that neurogenic progenitors progressively differentiate to generate retinal ganglion cells first and bipolar cells last, similar to the events observed during retinal development. Our work provides a comprehensive description of Müller glia and progenitor transcriptional changes and fate decisions in the regenerating retina, which are key to tailor cell differentiation and replacement therapies for retinal dystrophies in humans., Competing Interests: LC, FR, AM, JB, AD, AW, SH, MB No competing interests declared, (© 2023, Celotto et al.)

Background: Severe peripheral nerve injury (PNI) often leads to significant movement disorders and intractable pain. Therefore, promoting nerve regeneration while avoiding neuropathic pain is crucial for the clinical treatment of PNI patients. However, established animal models for peripheral neuropathy fail to accurately recapitulate the clinical features of PNI. Additionally, researchers usually investigate neuropathic pain and axonal regeneration separately, leaving the intrinsic relationship between the development of neuropathic pain and nerve regeneration after PNI unclear. To explore the underlying connections between pain and regeneration after PNI and provide potential molecular targets, we performed single-cell RNA sequencing and functional verification in an established rat model, allowing simultaneous study of the neuropathic pain and axonal regeneration after PNI., Results: First, a novel rat model named spared nerve crush (SNC) was created. In this model, two branches of the sciatic nerve were crushed, but the epineurium remained unsevered. This model successfully recapitulated both neuropathic pain and axonal regeneration after PNI, allowing for the study of the intrinsic link between these two crucial biological processes. Dorsal root ganglions (DRGs) from SNC and naïve rats at various time points after SNC were collected for single-cell RNA sequencing (scRNA-seq). After matching all scRNA-seq data to the 7 known DRG types, we discovered that the PEP1 and PEP3 DRG neuron subtypes increased in crushed and uncrushed DRG separately after SNC. Using experimental design scRNA-seq processing (EDSSP), we identified Adcyap1 as a potential gene contributing to both pain and nerve regeneration. Indeed, repeated intrathecal administration of PACAP38 mitigated pain and facilitated axonal regeneration, while Adcyap1 siRNA or PACAP6-38, an antagonist of PAC1R (a receptor of PACAP38) led to both mechanical hyperalgesia and delayed DRG axon regeneration in SNC rats. Moreover, these effects can be reversed by repeated intrathecal administration of PACAP38 in the acute phase but not the late phase after PNI, resulting in alleviated pain and promoted axonal regeneration., Conclusions: Our study reveals that Adcyap1 is an intrinsic protective factor linking neuropathic pain and axonal regeneration following PNI. This finding provides new potential targets and strategies for early therapeutic intervention of PNI., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Neuritin plays a key role in neural development and regeneration by promoting neurite outgrowth and synapse maturation. Our previous research revealed the mechanism by which neuritin inhibits Notch signaling through interaction with neuralized-like 1 (Neurl1) to promote neurite growth. However, how neuritin regulates Notch signaling through Neurl1 has not been elucidated. Here, we first confirm that neuritin is an upstream regulator of Neurl1 and inhibits Notch signaling through Neurl1. Neurl1 is an E3 ubiquitin ligase that can promote ubiquitination and endocytosis of the Notch1 ligand Jagged1. Therefore, we observe the effect of neuritin on the ligase activity of Neurl1. The results indicate that neuritin inhibits Neurl1 activity by reducing the ubiquitination level and endocytosis of the target protein Jagged1. Moreover, we find that decreased activity of Neurl1 results in reduced expression of Notch receptor Notch intracellular domain (NICD) and downstream target gene hairy and enhancer of split-1 ( HES1 ). Furthermore, we investigate how neuritin affects Neurl1 enzyme activity. The results show that neuritin not only weakens the affinity between Neurl1 and Jagged1 but also promotes the degradation of Neurl1 by the 26S proteasome pathway. Taken together, our results suggest that neuritin negatively regulates Notch signaling by inhibiting the activity of Neurl1, promoting the degradation of Neurl1 and weakening the affinity of Neurl1 for Jagged1. Our study clarifies the molecular mechanisms of neuritin in regulating the Notch signaling pathway and provides new clues about how neuritin mediates neural regeneration and plasticity.

Background: Maintaining the repair phenotype of denervated Schwann cells in the injured distal nerve is crucial for promoting peripheral nerve regeneration. However, when chronically denervated, the capacity of Schwann cells to support repair and regeneration deteriorates, leading to peripheral nerve regeneration and poor functional recovery. Herein, we investigated whether neurotrophin-3 (NT-3) could sustain the reparative phenotype of Schwann cells and promote peripheral nerve regeneration after chronic denervation and aimed to uncover its potential molecular mechanisms., Methods: Western blot was employed to investigate the relationship between the expression of c-Jun and the reparative phenotype of Schwann cells. The inducible expression of c-Jun by NT-3 was examined both in vitro and in vivo with western blot and immunofluorescence staining. A chronic denervation model was established to study the role of NT-3 in peripheral nerve regeneration. The number of regenerated distal axons, myelination of regenerated axons, reinnervation of neuromuscular junctions, and muscle fiber diameters of target muscles were used to evaluate peripheral nerve regeneration by immunofluorescence staining, transmission electron microscopy (TEM), and hematoxylin and eosin (H&E) staining. Adeno-associated virus (AAV) 2/9 carrying shRNA, small molecule inhibitors, and siRNA were employed to investigate whether NT-3 could signal through the TrkC/ERK pathway to maintain c-Jun expression and promote peripheral nerve regeneration after chronic denervation., Results: After peripheral nerve injury, c-Jun expression progressively increased until week 5 and then began to decrease in the distal nerve following denervation. NT-3 upregulated the expression of c-Jun in denervated Schwann cells, both in vitro and in vivo. NT-3 promoted peripheral nerve regeneration after chronic denervation, mainly by upregulating or maintaining a high level of c-Jun rather than NT-3 itself. The TrkC receptor was consistently presented on denervated Schwann cells and served as NT-3 receptors following chronic denervation. NT-3 mainly upregulated c-Jun through the TrkC/ERK pathway., Conclusion: NT-3 promotes peripheral nerve regeneration by maintaining the repair phenotype of Schwann cells after chronic denervation via the TrkC/ERK/c-Jun pathway. It provides a potential target for the clinical treatment of peripheral nerve injury after chronic denervation., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increases in ischemia and expression of genes linked to neuronal regeneration. Post NPC injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, the proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved the mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/β-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/β-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Ribosomal proteins are involved in neurodevelopment and central nervous system (CNS) disease and injury. However, the roles of specific ribosomal protein subunits in developmental axon growth, and their potential as therapeutic targets for treating CNS injuries, are still poorly understood. Here, we show that ribosomal protein large (Rpl) and small (Rps) subunit genes are substantially (56-fold) enriched amongst the genes, which are downregulated during maturation of retinal ganglion cell (RGC) CNS projection neurons. We also show that Rpl and Rps subunits are highly co-regulated in RGCs, and partially re-upregulated after optic nerve crush (ONC). Because developmental downregulation of ribosomal proteins coincides with developmental decline in neuronal intrinsic axon growth capacity, we hypothesized that Rpl/Rps incomplete re-upregulation after injury may be a part of the cellular response which attempts to reactivate intrinsic axon growth mechanisms. We found that experimentally upregulating Rpl7 and Rpl7A promoted axon regeneration after ONC in vivo. Finally, we characterized gene networks associated with Rpl/Rps, and showed that Rpl7 and Rpl7A belong to the cluster of genes, which are shared between translational and neurodevelopmental biological processes (based on gene-ontology) that are co-downregulated in maturing RGCs during the decline in intrinsic axon growth capacity., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI., Methods: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 μL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1 st , 7 th , 14 th and 21 st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining., Results: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased ( P <0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group ( P <0.05, P <0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased ( P <0.05, P <0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group ( P <0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200( P <0.01, P <0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group., Conclusion: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.

Axon regeneration can be induced across anatomically complete spinal cord injury (SCI), but robust functional restoration has been elusive. Whether restoring neurological functions requires directed regeneration of axons from specific neuronal subpopulations to their natural target regions remains unclear. To address this question, we applied projection-specific and comparative single-nucleus RNA sequencing to identify neuronal subpopulations that restore walking after incomplete SCI. We show that chemoattracting and guiding the transected axons of these neurons to their natural target region led to substantial recovery of walking after complete SCI in mice, whereas regeneration of axons simply across the lesion had no effect. Thus, reestablishing the natural projections of characterized neurons forms an essential part of axon regeneration strategies aimed at restoring lost neurological functions.

In the adult mammalian central nervous system (CNS), axons fail to regenerate spontaneously after injury because of a combination of extrinsic and intrinsic factors. Despite recent advances targeting the intrinsic regenerative properties of adult neurons, the molecular mechanisms underlying axon regeneration are not fully understood. Here, we uncover a regulatory mechanism that controls the expression of key proteins involved in regeneration at the translational level. Our results show that mRNA-specific translation is critical for promoting axon regeneration. Indeed, we demonstrate that specific ribosome-interacting proteins, such as the protein Huntingtin (HTT), selectively control the translation of a specific subset of mRNAs. Moreover, modulating the expression of these translationally regulated mRNAs is crucial for promoting axon regeneration. Altogether, our findings highlight that selective translation through the customization of the translational complex is a key mechanism of axon regeneration with major implications in the development of therapeutic strategies for CNS repair., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration., (© 2023. Springer Nature Limited.)

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals., (© 2023. Springer Nature Limited.)

A key limiting factor of successful axon regeneration is the intrinsic regenerative ability in both the peripheral nervous system (PNS) and central nervous system (CNS). Previous studies have identified intrinsic regenerative ability regulators that act on gene expression in injured neurons. However, it is less known whether RNA modifications play a role in this process. Here, we systematically screened the functions of all common m 6 A modification-related enzymes in axon regeneration and report ALKBH5, an evolutionarily conserved RNA m 6 A demethylase, as a regulator of axonal regeneration in rodents. In PNS, knockdown of ALKBH5 enhanced sensory axonal regeneration, whereas overexpressing ALKBH5 impaired axonal regeneration in an m 6 A-dependent manner. Mechanistically, ALKBH5 increased the stability of Lpin2 mRNA and thus limited regenerative growth associated lipid metabolism in dorsal root ganglion neurons. Moreover, in CNS, knockdown of ALKBH5 enhanced the survival and axonal regeneration of retinal ganglion cells after optic nerve injury. Together, our results suggest a novel mechanism regulating axon regeneration and point ALKBH5 as a potential target for promoting axon regeneration in both PNS and CNS., Competing Interests: DW, TZ, SZ, ML, YL, XG, SM, BY No competing interests declared, (© 2023, Wang, Zheng et al.)

Schwann cells are functional cells in nerve regeneration and are commonly used as seed cells in tissue engineering. Enhanced Schwann cell migration capacity improves recovery effects, and thus, the identification of Schwann cells with greater migration ability is of great importance. In the present study, we examined the biological activities of Schwann cells collected from rat sciatic nerves (SN) and dorsal root ganglia (DRG). Observations from transwell migration assay and wound healing assay demonstrate that DRG Schwann cells migrate at a faster speed as compared with SN Schwann cells. Sequencing and bioinformatics suggest that differentially expressed genes between SN and DRG Schwann cells are associated with cell motility and migration. miR-140 and miR-200, two microRNAs (miRNAs) that are highly expressed in SN Schwann cells negatively influence Schwann cell migration and thus may be key regulators of Schwann cell phenotype. Igsf10, Plxna2, and Lcp1 are screened as candidate downstream targets of miR-140 and miR-200 based on bioinformatic analysis and their expression correlation with miRNAs. Our comparative analysis displays the unique characteristics of Schwann cells in different anatomical localizations and demonstrates that DRG Schwann cells are suitable seed cells for tissue engineering and regenerative medicine., (© 2023 International Society for Neurochemistry.)

Background: Preinjury of peripheral nerves triggers dorsal root ganglia (DRG) axon regeneration, a biological change that is more pronounced in young mice than in old mice, but the complex mechanism has not been clearly explained. Here, we aim to gain insight into the mechanisms of axon regeneration after conditioning lesion in different age groups of mice, thereby providing effective therapeutic targets for central nervous system (CNS) injury., Methods: The microarray GSE58982 and GSE96051 were downloaded and analyzed to identify differentially expressed genes (DEGs). The protein-protein interaction (PPI) network, the miRNA-TF-target gene network, and the drug-hub gene network of conditioning lesion were constructed. The L4 and L5 DRGs, which were previously axotomized by the sciatic nerve conditioning lesions, were harvested for qRT-PCR. Furthermore, histological and behavioral tests were performed to assess the therapeutic effects of the candidate drug telmisartan in spinal cord injury (SCI)., Results: A total of 693 and 885 DEGs were screened in the old and young mice, respectively. Functional enrichment indicates that shared DEGs are involved in the inflammatory response, innate immune response, and ion transport. QRT-PCR results showed that in DRGs with preinjury of peripheral nerve, Timp1, P2ry6, Nckap1l, Csf1, Ccl9, Anxa1, and C3 were upregulated, while Agtr1a was downregulated. Based on the bioinformatics analysis of DRG after conditioning lesion, Agtr1a was selected as a potential therapeutic target for the SCI treatment. In vivo experiments showed that telmisartan promoted axonal regeneration after SCI by downregulating AGTR1 expression., Conclusion: This study provides a comprehensive map of transcriptional changes that discriminate between young and old DRGs in response to injury. The hub genes and their related drugs that may affect the axonal regeneration program after conditioning lesion were identified. These findings revealed the speculative pathogenic mechanism involved in conditioning-dependent regenerative growth and may have translational significance for the development of CNS injury treatment in the future., (© 2023. The Author(s).)

Polypyrimidine tract binding protein 1 (PTBP1) is thought to be expressed only at embryonic stages in central neurons. Its down-regulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently tested for generation of neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Unexpectedly, we found that both proteins are coexpressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 also shows axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and it associates in axons with mRNAs involved in injury responses and nerve regeneration, including importin β1 (KPNB1) and RHOA. Perturbation of PTBP1 affects local translation in axons, nociceptor neuron regeneration and both thermal and mechanical sensation. Thus, PTBP1 has functional roles in adult axons. Hence, caution is required before considering targeting of PTBP1 for therapeutic purposes.

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. We trigger conditioned regeneration in the Caenorhabditis elegans ASJ neuron by laser surgery or genetic disruption of sensory pathways. Conditioning upregulates thioredoxin-1 (trx-1) expression, as indicated by trx-1 promoter-driven expression of green fluorescent protein and fluorescence in situ hybridization (FISH), suggesting trx-1 levels and associated fluorescence indicate regenerative capacity. The redox activity of trx-1 functionally enhances conditioned regeneration, but both redox-dependent and -independent activity inhibit non-conditioned regeneration. Six strains isolated in a forward genetic screen for reduced fluorescence, which suggests diminished regenerative potential, also show reduced axon outgrowth. We demonstrate an association between trx-1 expression and the conditioned state that we leverage to rapidly assess regenerative capacity., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2023. The Author(s).)

Proper microtubule dynamics are critical for neuronal morphogenesis and functions, and their dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as stathmin-2 or STMN2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Additionally, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport were observed in Scg10 knockout dorsal root ganglion (DRG) neurons. Furthermore, SCG10 depletion impaired axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The cellular and molecular underpinnings of Wallerian degeneration have been robustly explored in laboratory models of successful nerve regeneration. In contrast, there is limited interrogation of failed regeneration, which is the challenge facing clinical practice. Specifically, we lack insight on the pathophysiologic mechanisms that lead to the formation of neuromas-in-continuity (NIC). To address this knowledge gap, we have developed and validated a novel basic science model of rapid-stretch nerve injury, which provides a biofidelic injury with NIC development and incomplete neurologic recovery. In this study, we applied next-generation RNA sequencing to elucidate the temporal transcriptional landscape of pathophysiologic nerve regeneration. To corroborate genetic analysis, nerves were subject to immunofluorescent staining for transcripts representative of the prominent biological pathways identified. Pathophysiologic nerve regeneration produces substantially altered genetic profiles both temporally and in the mature neuroma microenvironment, in contrast to the coordinated genetic signatures of Wallerian degeneration and successful regeneration. To our knowledge, this study presents as the first transcriptional study of NIC pathophysiology and has identified cellular death, fibrosis, neurodegeneration, metabolism, and unresolved inflammatory signatures that diverge from pathways elaborated by traditional models of successful nerve regeneration., (© 2023. The Author(s).)

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show that the cells directing expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1 kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)