Promotes Neurite Outgrowth Research Articles

Design of IKVAV peptide/gold nanoparticle decorated, micro/nano-channeled PCL/PLGA film scaffolds for neuronal differentiation and neurite outgrowth

In the field of neural tissue engineering, intensive efforts are being made to develop tissue scaffolds that can support an effective functional recovery and neural development by guiding damaged axons and neurites. Micro/nano-channeled conductive biomaterials are considered a promising approach for repairing the injured neural tissues. Many studies have demonstrated that the micro/nano-channels and aligned nanofibers could guide the neurites to extend along the direction of alignment. However, an ideal biocompatible scaffold containing conductive arrays that could promote effective neural stem cell differentiation and development, and also stimulate high neurite guidance has not been fully developed. In the current study, we aimed to fabricate micro/nano-channeled polycaprolactone (PCL)/Poly-d,l-lactic-co-glycolic acid (PLGA) hybrid film scaffolds, decorate their surfaces with IKVAV pentapeptide/gold nanoparticles (AuNPs), and investigate the behavior of PC12 cells and neural stem cells (NSCs) on the developed biomaterial under static/bioreactor conditions. Here we show that channeled groups decorated with AuNPs highly promote neurite outgrowth and neuronal differentiation along linear lines in the presence of electrical stimulation, compared with the polypyrrole (PPy) coating, which has been used traditionally for many years. Hopefully, this newly developed channeled scaffold structure (PCL/PLGA-AuNPs-IKVAV) could help to support long-distance axonal regeneration and neuronal development after different neural damages.

Synthesis and biological evaluation of C-4 substituted phenoxazine-bearing hydroxamic acids with potent class II histone deacetylase inhibitory activities

Class II histone deacetylases (HDACs) are considered as potential targets to treat Alzheimer’s disease (AD). Previously, C-3 substituted phenothiazine-containing compounds with class II HDAC-inhibiting activities was found to promote neurite outgrowth. This study replaced phenothiazine moiety with phenoxazine that contains many C-3 and C-4 substituents. Some resulting compounds bearing the C-4 substituent on a phenoxazine ring displayed potent class II HDAC inhibitory activities. Structure-activity relationship (SAR) of these compounds that inhibited HDAC isoenzymes was disclosed. Molecular modelling analysis demonstrates that the potent activities of C-4 substituted compounds probably arise from π-π stacked interactions between these compounds and class IIa HDAC enzymes. One of these, compound 7d exhibited the most potent class II HDAC inhibition (IC50= 3–870 nM). Notably, it protected neuron cells from H2O2-induced neuron damage at sub-μM concentrations, but with no significant cytotoxicity. These findings show that compound 7d is a lead compound for further development of anti-neurodegenerative agents.

R-spondin 3/LGR4 (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4) Axis Is a Novel Inflammatory and Neurite Outgrowth Signaling System in the Ischemic Brain in Mice

Although stimulation of Wnt/β-catenin signaling is an important strategy to treat ischemic stroke, its signaling pathway has not been fully clarified yet. Recently, RSPO3 (R-spondin 3)/LGR4 (leucine-rich repeat-containing G protein-coupled receptor 4) signaling has resolved TLR4 (toll-like receptor 4)-induced inflammation in lung injury; however, whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of RSPO3/LGR4 signaling in the ischemic brain. BALB/c mice were exposed to permanent distal middle cerebral artery and common carotid artery occlusion. Temporal RSPO3 and LGR4 expressions were examined, and the mice were randomly assigned to receive vehicle or recombinant RSPO3. The underlying mechanisms were investigated using microglial cell lines and primary mixed glia-endothelia-neuron and primary neuronal cultures. In the ischemic brain, RSPO3 and LGR4 were expressed in endothelial cells and microglia/macrophages and neurons, respectively. Stimulation of RSPO3/LGR4 signaling by recombinant RSPO3 recovered neurological deficits with decreased Il1β and iNOS mRNA on day 3 and increased Gap43 on day 9. In cultured cells, LGR4 was expressed in neuron and microglia, whereas RSPO3 promoted nuclear translocation of β-catenin. Neuroprotective effects with reduced expression of inflammatory cytokines were observed in lipopolysaccharide-stimulated glia-endothelium-neuron cultures but not in glutamate-, CoCl2-, H2O2-, or oxygen glucose deprivation-stimulated neuronal cultures, indicating that RSPO3/LGR4 can protect neurons by regulating inflammatory cytokines. LGR4-Fc chimera, which was used to block endogenous RSPO3/LGR4 signaling, increased LPS-induced production of inflammatory cytokines, suggesting that endogenous RSPO3 suppresses inflammation. RSPO3 decreased TLR4-related inflammatory cytokine expression by decreasing TLR4 expression without affecting the M1/M2 phenotype. RSPO3 also inhibited TLR2- and TLR9-induced inflammation but not TLR7-induced inflammation, and promoted neurite outgrowth. RSPO3/LGR4 signaling plays a critical role in regulating TLR-induced inflammation and neurite outgrowth in the ischemic brain. Enhancing this signal will be a promising approach for treating ischemic stroke.

Neuronal Differentiation and Outgrowth Effect of Thymol in Trachyspermum ammi Seed Extract via BDNF/TrkB Signaling Pathway in Prenatal Maternal Supplementation and Primary Hippocampal Culture.

Reviving the neuronal functions in neurodegenerative disorders requires the promotion of neurite outgrowth. Thymol, which is a principal component of Trachyspermum ammi seed extract (TASE), is reported to have neuroprotective effects. However, the effects of thymol and TASE on neuronal differentiation and outgrowth are yet to be studied. This study is the first report investigating the neuronal growth and maturation effects of TASE and thymol. Pregnant mice were orally supplemented with TASE (250 and 500 mg/kg), thymol (50 and 100 mg/kg), vehicle, and positive controls. The supplementation significantly upregulated the expression of brain-derived neurotrophic factor (BDNF) and early neuritogenesis markers in the pups' brains at post-natal day 1 (P1). Similarly, the BDNF level was significantly upregulated in the P12 pups' brains. Furthermore, TASE (75 and 100 µg/mL) and thymol (10 and 20 µM) enhanced the neuronal polarity, early neurite arborization, and maturation of hippocampal neurons in a dose-dependent manner in primary hippocampal cultures. The stimulatory activities of TASE and thymol on neurite extension involved TrkB signaling, as evidenced by attenuation via ANA-12 (5 µM), which is a specific TrkB inhibitor. Moreover, TASE and thymol rescued the nocodazole-induced blunted neurite extension in primary hippocampal cultures, suggesting their role as a potent microtubule stabilizing agent. These findings demonstrate the potent capacities of TASE and thymol in promoting neuronal development and reconstruction of neuronal circuitry, which are often compromised in neurodegenerative diseases and acute brain injuries.

Aligned core–shell fibrous nerve wrap containing Bletilla striata polysaccharide improves functional outcomes of peripheral nerve repair

Peripheral nerve injuries are commonly encountered in extremity traumas. Their motor and sensory recovery following microsurgical repair is limited by slow regeneration speed (<1 mm/d) and subsequent muscle atrophy, which are consequently correlated with the activity of local Schwann cells and efficacy of axon outgrowth. To promote post-surgical nerve regeneration, we synthesized a nerve wrap consisting of an aligned polycaprolactone (PCL) fiber shell with a Bletilla striata polysaccharide (BSP) core (APB). Cell experiments demonstrated that the APB nerve wrap markedly promoted neurite outgrowth and Schwann cell migration and proliferation. Animal experiments applying a rat sciatic nerve repair model indicated that the APB nerve wrap restored conduction efficacy of the repaired nerve and the compound action potential as well as contraction force of the related leg muscles. Histology of the downstream nerves disclosed significantly higher fascicle diameter and myelin thickness with the APB nerve wrap compared to those without BSP. Thus, the BSP-loaded nerve wrap is potentially beneficial for the functional recovery after peripheral nerve repair and offers sustained targeted release of a natural polysaccharide with good bioactivity.

Neuron-Derived Exosomes Promote the Recovery of Spinal Cord Injury by Modulating Nerve Cells in the Cellular Microenvironment of the Lesion Area.

During spinal cord injury (SCI), the homeostasis of the cellular microenvironment in the injured area is seriously disrupted, which makes it extremely difficult for injured neurons with regenerative ability to repair, emphasizing the importance of restoring the cellular microenvironment at the injury site. Neurons interact closely with other nerve cells in the central nervous system (CNS) and regulate these cells. However, the specific mechanisms by which neurons modulate the cellular microenvironment remain unclear. Exosomes were isolated from the primary neurons, and their effects on astrocytes, microglia, oligodendrocyte progenitor cells (OPCs), neurons, and neural stem cells were investigated by quantifying the expression of related proteins and mRNA. A mouse SCI model was established, and neuron-derived exosomes were injected into the mice by the caudal vein to observe the recovery of motor function in mice and the changes in the nerve cells in the lesion area. Neuron-derived exosomes could reverse the activation of microglia and astrocytes and promote the maturation of OPCs in vivo and in vitro. In addition, neuron-derived exosomes promoted neurite outgrowth of neurons and the differentiation of neural stem cells into neurons. Moreover, our experiments showed that neuron-derived exosomes enhanced motor function recovery and nerve regeneration in mice with SCI. Our findings highlight that neuron-derived exosomes could promote the repair of the injured spinal cord by regulating the cellular microenvironment of neurons and could be a promising treatment for spinal cord injury.

The neurotrophic activities of brain-derived neurotrophic factor are potentiated by binding with apigenin, a common flavone in vegetables, in stimulating the receptor signaling.

We aimed to identify the neurotrophic activities of apigenin (4',5,7-trihydroxyflavone) via its coordination with brain-derived neurotrophic factor (BNDF) and an elevated signaling of tyrosine kinase receptor B (Trk B receptor). The direct binding of apigenin to BDNF was validated by ultrafiltration and biacore assay. Neurogenesis, triggered by apigenin and/or BDNF, was determined in cultured SH-SY5Y cells and rat cortical neurons. The amyloid-beta (Aβ)25-35 -induced cellular stress was revealed by propidium iodide staining, mitochondrial membrane potential, bioenergetic analysis, and formation of reactive oxygen species levels. Activation of Trk B signaling was tested by western blotting. Apigenin and BDNF synergistically maintained the cell viability and promoted neurite outgrowth of cultured neurons. In addition, the BDNF-induced neurogenesis of cultured neurons was markedly potentiated by applied apigenin, including the induced expressions of neurofilaments, PSD-95 and synaptotagmin. Moreover, the synergy of apigenin and BDNF alleviated the (Aβ)25-35 -induced cytotoxicity and mitochondrial dysfunction. The synergy could be accounted by phosphorylation of Trk B receptor, and which was fully blocked by a Trk inhibitor K252a. Apigenin potentiates the neurotrophic activities of BDNF through direct binding, which may serve as a possible treatment for its curative efficiency in neurodegenerative diseases and depression.

Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress.

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3′-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.

Abstract 3042: BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy

Abstract Background: Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating side effect associated with many antineoplastic chemotherapeutic agents including cytotoxic and targeted agents. It significantly impacts cancer patients’ quality of life during treatment, and may cause lasting neuropathy and shorten treatment regimen, potentially impacting clinical benefit. The pathology of CIPN is complex and still not completely understood. Damages to nerve cells are believed to be directly resulting from the antineoplastic agents’ cytotoxicity, and inflammation and the immune system are believed to be involved as well. Evidence suggests that intervention of certain GPCRs (e.g., cannabinoid receptors and others) may be useful as potential treatments. Altered neuronal sphingolipid metabolism has been linked to neuropathic pain and elevated plasma levels of sphingosine-1-phosphate (S1P) have also been associated with patients receiving chemotherapy and developing CIPN; therefore, modulating S1P levels may also be a potential treatment. Method: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that lowers systemic S1P; Saposin C has been reported to activate certain GPCRs, including GPR37. BXQ-350 was investigated in an adult Phase 1 dose-escalation safety study in heavily pretreated all-comer cancer patients with advanced solid malignancies (NCT02859857). The cytoprotective properties of BXQ-350 against multiple agents known to induce CIPN were investigated in vitro in neuronal PC12 cells and BXQ-350’s CIPN mitigation properties were investigated in vivo in a CIPN preclinical model. Results: A pancreatic cancer patient with chronic CIPN at time of enrollment in the Phase 1 study spontaneously reported a significant improvement of her neuropathic symptoms shortly after receiving BXQ-350; several patients had similar improvements. Results from preclinical experiments in PC12 cells revealed that BXQ-350 at concentration as low as 50 nM protected cells from oxaliplatin, paclitaxel, bortezomib or MMAE and promoted neurite growth. BXQ-350 was subsequently investigated in a murine oxaliplatin-induced CIPN preclinical model, showing a dose-dependent reduction of thermal and mechanical allodynia correlating with decreasing systemic S1P levels. Conclusions: Preclinical results demonstrated that BXQ-350 was highly effective at protecting neuronal cells from antineoplastic agents known to induce CIPN and prevent CIPN in a preclinical model. These results appeared to support the clinical observation that BXQ-350 alleviated CIPN symptoms in several patients soon after receiving BXQ-350. Additional studies are on-going. Citation Format: Nikhil Wilkins, Tim Stephens, Robin Furnish, Charlie Cruze, Darren Wolfe, Gilles H. Tapolsky, Ray Takigiku. BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3042.

Fucosylated heparan sulfate from the midgut gland of Patinopecten yessoensis

The structural and functional relationships of glycosaminoglycans (GAGs) derived from marine organisms have been investigated, suggesting that marine invertebrates, particularly Bivalvia, are abundant sources of highly sulfated or branched GAGs. In this study, we identified a novel fucosylated heparan sulfate (Fuc-HS) from the midgut gland of the Japanese scallop, Patinopecten yessoensis. Scallop HS showed resistance to GAG-degrading enzymes, including chondroitinases and heparinases, and susceptibility to heparinases increased when scallop HS was treated with mild acid hydrolysis, which removes the fucosyl group. Moreover, 1H NMR detected significant signals near 1.2–1.3 ppm corresponding to the H-6 methyl proton of fucose residues and small H-3 (3.59 ppm) or H-2 (3.39 ppm) signals of glucuronate (GlcA) were detected, suggesting that the fucose moiety is attached to the C-3 position of GlcA in scallop HS. GC–MS detected peaks corresponding to 1, 3, 5-tri-O-acetyl-2, 4-di-O-methyl-L-fucitol and 1, 4, 5-tri-O-acetyl-2, 3-di-O-methyl-L-fucitol, suggesting that the fucose moiety is 3-O- or 4-O-sulfated. Furthermore, scallop HS showed anti-coagulant and neurite outgrowth-promoting (NOP) activities. These results suggest that the midgut gland of scallops is a valuable source of Fuc-HS with biological activities.

Deletion of Transglutaminase 2 from Mouse Astrocytes Significantly Improves Their Ability to Promote Neurite Outgrowth on an Inhibitory Matrix.

Astrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with an altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on are poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. Selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2-/- and wild-type (WT) mouse astrocytes on neurite outgrowth and synapse formation. Neurons were grown on a control substrate or an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2-/- astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating gene expression through interactions with transcription factors and transcription complexes. Based on the results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Co-immunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Overexpression or knockdown of Zbtb7a levels in WT and TG2-/- astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.

β3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, β-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the β-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, β3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the β3AR-agonist, BRL-37344, increased myocyte BDNF content, while β3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the β1AR blocker, metoprolol, via upregulated β3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac β3AR stimulation, or β-blockers (via upregulated β3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Chitosan Nerve Grafts Incorporated with SKP-SC-EVs Induce Peripheral Nerve Regeneration.

Repair of long-distance peripheral nerve defects remains an important clinical problem. Nerve grafts incorporated with extracellular vesicles (EVs) from various cell types have been developed to bridge peripheral nerve defects. In our previous research, EVs obtained from skin-derived precursor Schwann cells (SKP-SC-EVs) were demonstrated to promote neurite outgrowth in cultured cells and facilitate nerve regeneration in animal studies. To further assess the functions of SKP-SC-EVs in nerve repair, we incorporated SKP-SC-EVs and Matrigel into chitosan nerve conduits (EV-NG) for repairing a 15-mm long-distance sciatic nerve defect in a rat model. Behavioral analysis, electrophysiological recording, histological investigation, molecular analysis, and morphometric assessment were carried out. The results revealed EV-NG significantly improved motor and sensory function recovery compared with nerve conduits (NG) without EVs incorporation. The outgrowth and myelination of regenerated axons were improved, while the atrophy of target muscles induced by denervation was alleviated after EVs addition. Our data indicated SKP-SC-EVs incorporation into nerve grafts represents a promising method for extended peripheral nerve damage repair.

Peripheral nerve-derived fibroblasts promote neurite outgrowth in adult dorsal root ganglion neurons more effectively than skin-derived fibroblasts.

What is the central question of this study? Although fibroblasts are involved in the regenerative process associated with peripheral nerve injury, detailed information regarding their characteristics is largely lacking. What is the main finding and its importance? Nerve-derived fibroblasts have a greater neurite-promoting effect than skin-derived fibroblasts, and epineurium-derived fibroblasts can promote neurite outgrowth more effectively than parenchyma-derived fibroblasts. The epineurium-derived fibroblasts and parenchyma-derived fibroblasts have distinctly different molecular profiles, including genes of soluble factors to promote axonal growth. Fibroblasts are molecularly and functionally different depending on their localization in nerve tissue, and epineurium-derived fibroblasts might be involved in axon regeneration after peripheral nerve injury more than previously thought. Although fibroblasts (Fb) are components of a peripheral nerve involved in the regenerative process associated with peripheral nerve injury, detailed information regarding their characteristics is largely lacking. The objective of the present study was to investigate the capacity of Fb derived from peripheral nerves to stimulate the outgrowth of neurites from adult dorsal root ganglion neurons and to clarify their molecular characteristics. Fibroblasts were prepared from the epineurium and parenchyma of rat sciatic nerves and skin. The Fb derived from epineurium showed the greatest effect on neurite outgrowth, followed by the Fb derived from parenchyma, indicating that Fb derived from nerves promote neurite outgrowth more effectively than skin-derived Fb. Although both soluble and cell-surface factors contributed evenly to the neurite-promoting effect of nerve-derived Fb, in crush and transection injury models, Fb were not closely associated with regenerating axons, indicating that only soluble factors from Fb are available to regenerating axons. A transcriptome analysis revealed that the molecular profiles of these Fb were distinctly different and that the gene expression profiles of soluble factors that promote axonal growth are unique to each Fb. These findings indicate that Fb are molecularly and functionally different depending on their localization in nerve tissue and that Fb derived from epineurium might be involved more than was previously thought in axon regeneration after peripheral nerve injury.

Cell-free matrix derived from adipose mesenchymal stromal cells enhances corneal rehabilitation via delivery of nerve regenerative PGRN

Promotion of corneal dense network of nerve is a promising method to treat severe corneal injury, yet with limited effective drugs. In our previous work, decellularized matrix of adipose derived mesenchymal stromal cells (ADMSCs), named as DMA, presented excellent conjunctival repair capacity with proper suture resistance and immune modulatory characteristics. Thus, we intended to investigate their potential application in facilitating corneal repair after alkali burn. Owing to the transparent characteristic of DMA, DMA could be applied as novel ocular bandages with nutritious function. In this study we found that cornea treated with DMA showed accelerated recovery compared with those received amniotic membrane (AM) treatment by fluorescein staining, slip-lamp examination, OCT and corneal nerve immunostaining. Further, in order to find key components responsible for the faster recovery, high-throughput proteomic analyses were conducted and more than 2000 proteins, including associated signaling pathways, were identified in DMA. Intriguingly, progranulin (PGRN), one of enriched proteins identified in DMA, was firstly recognized for its neurotrophic role in alleviation of corneal damage. To sum up, DMA exhibited a potent clinical application with a cocktail of key therapeutic factors, in which PGRN was a crucial factor in promoting neurite outgrowth and re-establishing homeostasis of the cornea.

Increased level of exosomal miR-20b-5p derived from hypothermia-treated microglia promotes neurite outgrowth and synapse recovery after traumatic brain injury

Mild hypothermia has been proven to inhibit microglia activation after TBI. Exosomal microRNA derived from microglia played a critical role in promoting neurite outgrowth and synapse recovery. Here, we aimed to investigate the role of microRNAs in microglial exosomes after hypothermia treatment on neuronal regeneration after TBI. For in vitro study, stretch-injured neurons were co-cultured with microglial exosomes. For in vivo study, C57BL/6 mice were under controlled cortical impact and injected with microglial exosomes. The results showed that MG-LPS-EXOHT increased the number of dendrite branches and total length of dendrites both in vitro and in vivo, elevated the expression levels of PSD-95 and GluR1 in stretch-injured neurons, and increased spine density in the pericontusion region. Moreover, MG-LPS-EXOHT improved motor function and motor coordination. A high-throughput sequencing showed that miR-20b-5p was upregulated in MG-LPS-EXOHT. Elevating miR-20b-5p promoted neurite outgrowth and synapse recovery of injured neurons both in vitro and in vivo. Following mechanistic study demonstrated that miR-20b-5p might promote neurite outgrowth and synapse recovery by directly targeting PTEN and activating PI3K-AKT pathway. In conclusion, mild hypothermia could modify the microRNA prolife of exosomes derived from LPS activated BV2 cells. Furthermore, high level of microglial exosomal miR-20b-5p induced by mild hypothermia could transfer into injured neurons and promote neurite outgrowth and synapse recovery after TBI via activating the PI3K-AKT pathway by suppressing PTEN expression.

Role of SOCS and VHL Proteins in Neuronal Differentiation and Development.

The basic helix-loop-helix factors play a central role in neuronal differentiation and nervous system development, which involve the Notch and signal transducer and activator of transcription (STAT)/small mother against decapentaplegic signaling pathways. Neural stem cells differentiate into three nervous system lineages, and the suppressor of cytokine signaling (SOCS) and von Hippel-Lindau (VHL) proteins are involved in this neuronal differentiation. The SOCS and VHL proteins both contain homologous structures comprising the BC-box motif. SOCSs recruit Elongin C, Elongin B, Cullin5(Cul5), and Rbx2, whereas VHL recruits Elongin C, Elongin B, Cul2, and Rbx1. SOCSs form SBC-Cul5/E3 complexes, and VHL forms a VBC-Cul2/E3 complex. These complexes degrade the target protein and suppress its downstream transduction pathway by acting as E3 ligases via the ubiquitin-proteasome system. The Janus kinase (JAK) is the main target protein of the E3 ligase SBC-Cul5, whereas hypoxia-inducible factor is the primary target protein of the E3 ligase VBC-Cul2; nonetheless, VBC-Cul2 also targets the JAK. SOCSs not only act on the ubiquitin-proteasome system but also act directly on JAKs to suppress the Janus kinase-signal transduction and activator of transcription (JAK-STAT) pathway. Both SOCS and VHL are expressed in the nervous system, predominantly in brain neurons in the embryonic stage. Both SOCS and VHL induce neuronal differentiation. SOCS is involved in differentiation into neurons, whereas VHL is involved in differentiation into neurons and oligodendrocytes; both proteins promote neurite outgrowth. It has also been suggested that the inactivation of these proteins may lead to the development of nervous system malignancies and that these proteins may function as tumor suppressors. The mechanism of action of SOCS and VHL involved in neuronal differentiation and nervous system development is thought to be mediated through the inhibition of downstream signaling pathways, JAK-STAT, and hypoxia-inducible factor-vascular endothelial growth factor pathways. In addition, because SOCS and VHL promote nerve regeneration, they are expected to be applied in neuronal regenerative medicine for traumatic brain injury and stroke.

Abstract 36: Astrocyte-derived Small Extracellular Vesicles Mediate Astrocyte-neuron Communication To Promote Neurite Outgrowth After Stroke In Astrocyte-vesicle Reporter Mice

As an integral part of the neuron-glia system, astrocytes directly contact and communicate to neurons. Small extracellular vesicles (sEVs) are essential mediators of intercellular communication for both physiological and pathological processes. Using astrocyte-sEV reporter mice, we investigated how endogenous astrocyte-derived sEVs communicate to neurons after ischemic stroke. The reporter mice were generated by crossing a CD63-green fluorescent protein (GFP) floxed mouse line with a GFAP-Cre ERT2 mouse line. Mice were subjected to permanent middle cerebral artery occlusion (n=10). To activate Cre recombinase, tamoxifen was injected daily for 4 days starting 1 day after stroke. Animals were euthanized 14 days after stroke. Coronal brain and spinal cord slices were used for immunofluorescent staining with antibodies against GFAP for astrocytes and SMI32 for neurons. Confocal microscopic imaging analysis showed that ischemia dramatically (p&lt;0.01) increased GFP signal intensity in the brain peri-infarct area (58.4±3.6 A.U.) and in the stroke-impaired gray matter of the spinal cord (42.9±2.8 A.U.), compared to that in normal brain (31.7±1.8 A.U) and spinal cord (34.3±2.7 A.U). Neuronal internalization of astrocyte-sEVs measured as colocalization of GFP with SMI32 neurons in the brain and spinal cord was concomitantly and significantly increased in the stroke mice compared with that in normal mice (stroke brain 0.14±0.01% vs 0.07±0.01% in normal, and spinal cord 0.10±0.01% vs 0.06±0.01% in normal, p&lt;0.05), which was confirmed by electron microscopy. In addition, GFP positive sEVs isolated from ischemic brain of the reporter mice significantly promoted neurite outgrowth of primary cultured mouse embryonic cortical neurons, compared to sEVs isolated from non-ischemic brain (p&lt;0.001). Collectively, our data for the first time provide in vivo evidence that endogenous astrocyte-derived sEVs communicate with neurons under non-ischemic condition, while stroke robustly augments this communication, and thereby promotes neurite remodeling during stroke recovery.

The coupling between calcium channel activity/calcium oscillation and neuritogenesis in cortical neurons.

L-type CaV1 channels transduce membrane excitability into dynamic Ca2+signals, such as autonomous Ca2+ oscillations, which regulate gene transcription in the nucleus critical to neurite outgrowth. Such signaling axis is defined as the coupling between CaV1 activity or Ca2+ oscillation and neurite outgrowth (i.e., CaV1-neuritogenesis coupling). In cortical neurons, stand-alone CaV1-encoded DCT (distal C-terminus) peptides are unveiled to inhibit the CaV1-neuritogenesis coupling, in contrast to the documented role of DCT as nuclear transcription factors to promote neurite outgrowth. When present in the cytosol, DCT peptides downregulate CaV1 activity, Ca2+ influx, pCREB, c-Fos, etc. Importantly, the potency of inhibition is tightly correlated with the affinity of DCT binding CaV1, quantitatively demonstrated by a series of DCT peptides of native origins (Yang 2022 Communications Biology). Autonomous Ca2+ oscillations play key roles in neural development. Specific CaV1 antagonists including DCT peptides are able to abolish oscillations, leading to their effects on neuritogenesis. Aiming at the Ca2+ oscillation-neuritogenesis coupling, with neuron-compatible GCaMP-X that our lab recently developed, we have achieved chronic GCaMP-X imaging which overcomes the toxicity of long/high expression known to conventional GCaMP. Chronic GCaMP-X imaging (∼1 month) of virus-infected cortical neurons recapitulated the tight correlation between spontaneous Ca2+ activity and neuronal development/health with quantitative insights. In support, in vivo imaging from whisker responses and spontaneous Ca2+ slow waves in live mice also demonstrated that GCaMP-X causing less toxicity outperformed GCaMP (Geng 2022 eLife).In summary, the CaV1 activity/Ca2+ oscillation-neuritogenesis coupling and its regulations/perturbations have been examined in vitro and in vivo by DCT peptides and GCaMP-X probes (versus GCaMP), both of which are expected to promote methodology development as well as mechanistic understanding.

Pharmacological Ischemic Conditioning with Roxadustat Does Not Affect Pain-Like Behaviors but Mitigates Sudomotor Impairment in a Murine Model of Deep Hind Paw Incision.

The involvement of hypoxic response mechanisms in local functional impairments in surgical wounds is unclear. In the present study, we characterized tissue hypoxia in surgical wounds and investigated the role of pharmacological ischemic conditioning (PIC) using roxadustat, an oral prolyl hydroxylase domain enzyme inhibitor, in postoperative local functional impairments in a murine model of deep hind paw incision. Male BALB/cAJcl mice aged 9-13 weeks were used in all experiments. Plantar skins of mice that underwent surgical incision were subjected to immunohistochemistry to localise tissue hypoxia. Pain-like behaviours and sudomotor function were compared between mice treated with 6-week perioperative PIC and control mice. The effects of PIC were examined in vitro by immunocytochemistry using sympathetically differentiated PC12 cells and in vivo by immunohistochemistry using plantar skins collected on postoperative day 21. Prominent tissue hypoxia was detected within axons in the nerve bundles underneath surgical wounds. Six-week perioperative PIC using roxadustat failed to ease spontaneous pain-like behaviors; however, it mitigated local sudomotor impairment postoperatively. Upregulation of sympathetic innervation to the eccrine glands was observed in the PIC-treated skins collected on postoperative day 21, in accordance with the in vitro study wherein roxadustat promoted neurite growth of sympathetically differentiated PC12 cells. This study suggests that tissue hypoxia is involved in the pathogenesis of local sudomotor dysfunction associated with surgical trauma. Targeting the hypoxic response mechanisms with PIC may be of therapeutic potential in postsurgical local sympathetic impairments that can be present in complex regional pain syndrome.