MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

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.

Neurotrophin-3 promotes peripheral nerve regeneration by maintaining a repair state of Schwann cells after chronic denervation via the TrkC/ERK/c-Jun pathway.

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.

Unveiling adcyap1 as a protective factor linking pain and nerve regeneration through single-cell RNA sequencing of rat dorsal root ganglion neurons.

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.

PTBP1 regulates injury responses and sensory pathways in adult peripheral neurons.

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 ß1 (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.

Expression of thioredoxin-1 in the ASJ neuron corresponds with and enhances intrinsic regenerative capacity under lesion conditioning in C. elegans.

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.

SCG10 is required for peripheral axon maintenance and regeneration in mice.

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.

Spinal cord injury: molecular mechanisms and therapeutic interventions.

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.

Next-generation RNA sequencing elucidates transcriptomic signatures of pathophysiologic nerve regeneration.

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.

Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration.

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we used a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na+/K+-ATPase. In turn, both biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na+/K+-ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals.

Vision protection and robust axon regeneration in glaucoma models by membrane-associated Trk receptors.

Activation of neurotrophic factor signaling is a promising therapy for neurodegeneration. However, the transient nature of ligand-dependent activation limits its effectiveness. In this study, we solved this problem by inventing a system that forces membrane localization of the intracellular domain of tropomyosin receptor kinase B (iTrkB), which results in constitutive activation without ligands. Our system overcomes the small size limitation of the genome packaging in adeno-associated virus (AAV) and allows high expression of the transgene. Using AAV-mediated gene therapy in the eyes, we demonstrate that iTrkB expression enhances neuroprotection in mouse models of glaucoma and stimulates robust axon regeneration after optic nerve injury. In addition, iTrkB expression in the retina was also effective in an optic tract transection model, in which the injury site is near the superior colliculus. Regenerating axons successfully formed pathways to their brain targets, resulting in partial recovery of visual behavior. Our system may also be applicable to other trophic factor signaling pathways and lead to a significant advance in the field of gene therapy for neurotrauma and neurodegenerative disorders, including glaucoma.

Enhancing Motor and Sensory Axon Regeneration after Peripheral Nerve Injury Using Bioluminescent Optogenetics.

Introduction-Recovery from peripheral nerve injuries is poor even though injured peripheral axons can regenerate. Novel therapeutic approaches are needed. The most successful preclinical experimental treatments have relied on increasing the activity of the regenerating axons, but the approaches taken are not applicable to many nerve-injured patients. Bioluminescent optogenetics (BL-OG) is a novel method of increasing the excitation of neurons that might be similar to that found with activity-dependent experimental therapies. We investigated the use of BL-OG as an approach to promoting axon regeneration following peripheral nerve injury. Methods-BL-OG uses luminopsins, light-sensing ion channels (opsins) fused with a light-emitting luciferase. When exposed to a luciferase substrate, such as coelenterazine (CTZ), luminopsins expressed in neurons generate bioluminescence and produce excitation through their opsin component. Adeno-associated viral vectors encoding either an excitatory luminopsin (eLMO3) or a mutated form (R115A) that can generate bioluminescence but not excite neurons were injected into mouse sciatic nerves. After retrograde transport and viral transduction, nerves were cut and repaired by simple end-to-end anastomosis, and mice were treated with a single dose of CTZ. Results-Four weeks after nerve injury, compound muscle action potentials (M waves) recorded in response to sciatic nerve stimulation were more than fourfold larger in mice expressing the excitatory luminopsin than in controls expressing the mutant luminopsin. The number of motor and sensory neurons retrogradely labeled from reinnervated muscles in mice expressing eLMO3 was significantly greater than the number in mice expressing the R115A luminopsin and not significantly different from those in intact mice. When viral injection was delayed so that luminopsin expression was induced after nerve injury, a clinically relevant scenario, evoked M waves recorded from reinnervated muscles were significantly larger after injury in eLMO3-expressing mice. Conclusions-Treatment of peripheral nerve injuries using BL-OG has significant potential to enhance axon regeneration and promote functional recovery.

Transgenic Schwann cells overexpressing POU6F1 promote sciatic nerve regeneration within acellular nerve allografts.

Objective.Acellular nerve allograft (ANA) is an effective surgical approach used to bridge the sciatic nerve gap. The molecular regulators of post-surgical recovery are not well-known. Here, we explored the effect of transgenic Schwann cells (SCs) overexpressing POU domain class 6, transcription factor 1 (POU6F1) on sciatic nerve regeneration within ANAs. We explored the functions of POU6F1 in nerve regeneration by using a cell model of H2O2-induced SCs injury and transplanting SCs overexpressing POU6F1 into ANA to repair sciatic nerve gaps.Approach.Using RNA-seq, Protein-Protein Interaction network analysis, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we identified a highly and differentially expressed transcription factor, POU6F1, following ANA treatment of sciatic nerve gap. Expressing a high degree of connectivity, POU6F1 was predicted to play a role in peripheral nervous system myelination.Main results.To test the role of POU6F1 in nerve regeneration after ANA, we infected SCs with adeno-associated virus-POU6F1, demonstrating that POU6F1 overexpression promotes proliferation, anti-apoptosis, and migration of SCsin vitro. We also found that POU6F1 significantly upregulated JNK1/2 and c-Jun phosphorylation and that selective JNK1/2 inhibition attenuated the effects of POU6F1 on proliferation, survival, migration, and JNK1/2 and c-Jun phosphorylation. The direct interaction of POU6F1 and activated JNK1/2 was subsequently confirmed by co-immunoprecipitation. In rat sciatic nerve injury model with a 10 mm gap, we confirmed the pattern of POU6F1 upregulation and co-localization with transplanted SCs. ANAs loaded with POU6F1-overexpressing SCs demonstrated the enhanced survival of transplanted SCs, axonal regeneration, myelination, and functional motor recovery compared to the ANA group loaded by SCs-only in line within vitrofindings.Significance.This study identifies POU6F1 as a novel regulator of post-injury sciatic nerve repair, acting through JNK/c-Jun signaling in SCs to optimize therapeutic outcomes in the ANA surgical approach.

Obtaining a New Gene-Cell Construct Based on Transduced Olfactory Ensheathing Cells for the Treatment of Spinal Cord Injuries.

We developed a viral vector Ad5/35-CAG-mBDNF expressing the mature form of BDNF (mBDNF). On the basis of olfactory ensheathing cells transduced with this adenovector, a new gene-cell construct was obtained. In experiments in vitro, high viability of the transduced olfactory ensheathing cells and enhanced secretion of BDNF by these cells were observed. It is possible that a new gene-cell construct will significantly increase the regenerative effects of transplanted olfactory ensheathing cells.

MiRNAs as Promising Translational Strategies for Neuronal Repair and Regeneration in Spinal Cord Injury.

Spinal cord injury (SCI) represents a devastating injury to the central nervous system (CNS) that is responsible for impaired mobility and sensory function in SCI patients. The hallmarks of SCI include neuroinflammation, axonal degeneration, neuronal loss, and reactive gliosis. Current strategies, including stem cell transplantation, have not led to successful clinical therapy. MiRNAs are crucial for the differentiation of neural cell types during CNS development, as well as for pathological processes after neural injury including SCI. This makes them ideal candidates for therapy in this condition. Indeed, several studies have demonstrated the involvement of miRNAs that are expressed differently in CNS injury. In this context, the purpose of the review is to provide an overview of the pre-clinical evidence evaluating the use of miRNA therapy in SCI. Specifically, we have focused our attention on miRNAs that are widely associated with neuronal and axon regeneration. "MiRNA replacement therapy" aims to transfer miRNAs to diseased cells and improve targeting efficacy in the cells, and this new therapeutic tool could provide a promising technique to promote SCI repair and reduce functional deficits.

Coordinated NADPH oxidase/hydrogen peroxide functions regulate cutaneous sensory axon de- and regeneration.

Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.

The Effect of Tension on Gene Expression in Primary Nerve Repair via the Epineural Suture Technique.

INTRODUCTION: The precise mechanism through which excessive tension confers poor outcomes in nerve gap repair is yet to be elucidated. Furthermore, the effect of tension on gene expression in regenerating nerves has not been characterized. This study investigated differential gene expression in transected nerves repaired under high and minimal tension. METHODS: Male Lewis rats underwent right sciatic nerve transection with either minimal-tension or high-tension repair. Fourteen weeks postoperatively, segments of the right sciatic nerves were harvested along with equal-length segments from the contralateral, healthy nerve to serve as internal controls (naïve nerve). Differentially expressed genes (DEGs) and differentially regulated biochemical pathways between the samples were identified. RESULTS: Seventeen animals were studied. The gene expression profiles of naïve nerve and minimal-tension repair demonstrated minimal within-group variation, whereas that of high-tension repair demonstrated heterogeneity. Relative to naïve nerve, high-tension repair samples had 4276 DEGs (1941 upregulated and 2335 downregulated) and minimal-tension repair samples had 3305 DEGs (1479 upregulated and 1826 downregulated). High-tension repair samples had 360 DEGs relative to minimal-tension repair samples (68 upregulated and 292 downregulated). Upregulated biological pathways in all repaired nerves included steroid biosynthesis, extracellular matrix-receptor interaction, and ferroptosis. Finally, upregulated pathways in high-tension repair samples relative to minimal-tension repair samples included tumor necrosis factor signaling, interleukin-17 signaling, cytokine-cytokine receptor interaction, and mitogen-activated protein kinase signaling. CONCLUSIONS: The improved outcomes achieved with minimal-tension nerve repair may take root in a favorable gene expression profile. Future elucidation of biochemical pathways in nerve regeneration may identify potential therapeutic targets to optimize primary nerve repair outcomes.

Dendrite regeneration in C. elegans is controlled by the RAC GTPase CED-10 and the RhoGEF TIAM-1.

Neurons are vulnerable to physical insults, which compromise the integrity of both dendrites and axons. Although several molecular pathways of axon regeneration are identified, our knowledge of dendrite regeneration is limited. To understand the mechanisms of dendrite regeneration, we used the PVD neurons in C. elegans with stereotyped branched dendrites. Using femtosecond laser, we severed the primary dendrites and axon of this neuron. After severing the primary dendrites near the cell body, we observed sprouting of new branches from the proximal site within 6 hours, which regrew further with time in an unstereotyped manner. This was accompanied by reconnection between the proximal and distal dendrites, and fusion among the higher-order branches as reported before. We quantified the regeneration pattern into three aspects-territory length, number of branches, and fusion phenomena. Axonal injury causes a retraction of the severed end followed by a Dual leucine zipper kinase-1 (DLK-1) dependent regrowth from the severed end. We tested the roles of the major axon regeneration signalling hubs such as DLK-1-RPM-1, cAMP elevation, let-7 miRNA, AKT-1, Phosphatidylserine (PS) exposure/PS in dendrite regeneration. We found that neither dendrite regrowth nor fusion was affected by the axon injury pathway molecules. Surprisingly, we found that the RAC GTPase, CED-10 and its upstream GEF, TIAM-1 play a cell-autonomous role in dendrite regeneration. Additionally, the function of CED-10 in epidermal cell is critical for post-dendrotomy fusion phenomena. This work describes a novel regulatory mechanism of dendrite regeneration and provides a framework for understanding the cellular mechanism of dendrite regeneration using PVD neuron as a model system.

Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration.

Purpose: Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods: Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results: In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions: Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

Complement C5aR/LPS-induced BDNF and NGF modulation in human dental pulp stem cells.

Stem cells with the ability to differentiate into a variety of cells and secrete nerve regeneration factors have become an emerging option in nerve regeneration. Dental pulp stem cells (DPSCs) appear to be a good candidate for nerve regeneration given their accessibility, neural crest origin, and neural repair qualities. We have recently demonstrated that the complement C5a system, which is an important mediator of inflammation and tissue regeneration, is activated by lipoteichoic acid-treated pulp fibroblasts, and governs the production of brain-derived nerve growth factor (BDNF). This BDNF secretion promotes neurite outgrowth towards the injury site. Here, we extend our observation to DPSCs and compare their neurogenic ability to bone marrow-derived mesenchymal stem cells (BM-MSCs) under inflammatory stimulation. Our ELISA and immunostaining data demonstrate that blocking the C5a receptor (C5aR) reduced BDNF production in DPSCs, while treatment with C5aR agonist increased the BDNF expression, which suggests that C5aR has a positive regulatory role in the BDNF modulation of DPSCs. Inflammation induced by lipopolysaccharide (LPS) treatment potentiated this effect and is C5aR dependent. Most important, DPSCs produced significantly higher levels of C5aR-mediated BDNF compared to BM-MSCs. Taken together, our data reveal novel roles for C5aR and inflammation in modulation of BDNF and NGF in DPSCs.