Increased Neurite Outgrowth Research Articles

Three-dimensional-printed polycaprolactone/polypyrrole conducting scaffolds for differentiation of human olfactory ecto-mesenchymal stem cells into Schwann cell-like phenotypes and promotion of neurite outgrowth.

Implantation of a suitable nerve guide conduit (NGC) seeded with sufficient Schwann cells (SCs) is required to improve peripheral nerve regeneration efficiently. Given the limitations of isolating and culturing SCs, using various sources of stem cells, including mesenchymal stem cells (MSCs) obtained from nasal olfactory mucosa, can be desirable. Olfactory ecto-MSCs (OE-MSCs) are a new population of neural crest-derived stem cells that can proliferate and differentiate into SCs and can be considered a promising autologous alternative to produce SCs. Regardless, a biomimetic physicochemical microenvironment in NGC such as electroconductive substrate can affect the fate of transplanted stem cells, including differentiation toward SCs and nerve regeneration. Therefore, in this study, the effect of 3D printed polycaprolactone (PCL)/polypyrrole (PPy) conductive scaffolds on differentiation of human OE-MSCS into functional SC-like phenotypes was investigated. Biological evaluation of 3D printed scaffolds was examined by in vitro culturing the OE-MSCs on samples surfaces, and conductivity showed no effect on increased cell attachment, proliferation rate, viability, and distribution. In contrast, immunocytochemical staining and real-time polymerase chain reaction analysis indicated that 3D structures coated with PPy could provide a favorable microenvironment for OE-MSCs differentiation. In addition, it was found that differentiated OE-MSCs within PCL/PPy could secrete the highest amounts of nerve growth factor and brain-derived neurotrophic factor neurotrophic factors compared to pure PCL and 2D culture. After co-culturing with PC12 cells, a significant increase in neurite outgrowth on PCL/PPy conductive scaffold seeded with differentiated OE-MSCs. These findings indicated that 3D printed PCL/PPy conductive scaffold could support differentiation of OE-MSCs into SC-like phenotypes to promote neurite outgrowth, suggesting their potential for neural tissue engineering applications.

Resveratrol-Selenium Nanoparticles Alleviate Neuroinflammation and Neurotoxicity in a Rat Model of Alzheimer's Disease by Regulating Sirt1/miRNA-134/GSK3β Expression.

Alzheimer's disease (AD) is a brain disorder associated with a gradual weakening in neurocognitive functions, neuroinflammation, and impaired signaling pathways. Resveratrol (RSV) has neuroprotective properties, but with low bioavailability, and low solubility in vivo. Selenium (Se) is an essential micronutrient for brain function. Thus, this study aimed to evaluate the role of formulated RSV-Se nanoparticles (RSV-SeNPs) on neurochemical and histopathological approaches associated with the AD model in rats induced by Aluminum chloride (AlCl3) at a dose of 100mg/kg/day for 60days. RSV-SeNPs supplementation attenuates the impaired oxidative markers and mitochondrial dysfunction. The ameliorative effect of RSV-SeNPs on cholinergic deficits was associated with clearance of amyloid β (Aβ). Furthermore, activation of phosphatidylinositol 3 kinase (PI3K) deactivates glycogen synthase kinase 3 beta (GSK-3β)-mediated tau hyperphosphorylation. Additionally, RSV-SeNPs downregulate signal transducer and activator of transcription (STAT3) expression as well as interleukin-1β (IL-1β) levels, therefore alleviating neuroinflammation in AD. Moreover, RSV-SeNPs upregulate the expression of Sirtuin-1 (SIRT1) and lower that of microRNA-134, consequently increasing neurite outgrowth. Eventually, the obtained results showed that nano-formulation of resveratrol with selenium maximized the therapeutic potential of RSV against Alzheimer's disease not only by their antioxidant but also by anti-inflammatory effect improving the neurocognitive function and modulating the signaling pathways.

Effect of Sei and Fin whale Müller glia in the survival and neurite growth of RGCs in vitro

AbstractPurposeMüller cells are crucial in retinal homeostasis. In zebrafish and lower vertebrates, spontaneous retinal regeneration was observed. In the mammalian retina, there is no such evidence. However, recent studies have been demonstrated that Müller cells promote the survival and the neurite outgrowth of RGCs in vitro. Here, the effect of Müller cells from two of the largest mammals in the world, Fin and Sei whales, was analyzed on RGCs survival and neurite outgrowth.MethodsThe retinas of beached Balaenoptera physalus and Balaenoptera borealis whales, 24 hr post‐mortem, were studied. Cultures of Müller cells were grown and, once whale Müller cells reached confluence, adult rat RGCs were seeded on Müller cells. The cells were cultured during six days. The number and length of the neurites of the RGCs were quantified. The conditioned media (CM) from whale Müller cells was analysed by mass spectrometry to identify and quantify proteins. Results were compared to the CM from pig Müller cells. NELL2, SEMA3F, CD56, NRCAM, OGN and PEDF were selected as candidate factors to promote neurite outgrowth and tested in pure rat RGCs cultures.ResultsRGCs survival increased 60% when cultured with whale Müller cells. The length of the neurites (>200 μm) increased 90% and the length of the longest neurites (>1000 μm) increased more than 500%. Proteomic analysis from whale Müller cells CM showed that the function of the 10% of the most represented proteins detected was related to neurite outgrowth, compared to only 2% of the proteins in pig CM. The selected proteins induced an increase in the percentage of RGCs with long neurites (>200 μm) and an increase of 200% in RGCs with very long neurites (>1000 μm).ConclusionsWhale Müller cells increase RGCs neurite growth, suggesting that whale Müller cells secrete a combination of neurotrophic factors promoting regeneration of RGCs. Besides, the selected proteins from the CM increase neurite outgrowth, confirming the capacity of the factors secreted by whale Müller cells to regenerate RGCs.Supported by ELKARTEK (KK‐2019/00086), MINECO‐Retos (PID2019‐111139RB‐I00) Grupos UPV/EHU (GIU2018/50).

Nanogel-mediated delivery of oncomodulin secreted from regeneration-associated macrophages promotes sensory axon regeneration in the spinal cord

Preconditioning nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these macrophages influence the neuronal capacity of axon regeneration remains elusive. We report that oncomodulin (ONCM) is produced from the regeneration-associated macrophages and strongly influences regeneration of DRG sensory axons. We also attempted to promote sensory axon regeneration by nanogel-mediated delivery of ONCM to DRGs.Methods: In vitro neuron-macrophage interaction model and preconditioning sciatic nerve injury were used to verify the necessity of ONCM in preconditioning injury-induced neurite outgrowth. We developed a nanogel-mediated delivery system in which electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) enabled a controlled release of ONCM.Results: Sciatic nerve injury upregulated ONCM in DRG macrophages. ONCM in macrophages was necessary to produce pro-regenerative macrophages in the in vitro model of neuron-macrophage interaction and played an essential role in preconditioning-induced neurite outgrowth. ONCM increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. Increasing extracellularly secreted ONCM in DRGs sufficiently enhanced the capacity of neurite outgrowth. Intraganglionic injection of REPL-NG/ONCM complex allowed sustained ONCM activity in DRG tissue and achieved a remarkable long-range regeneration of dorsal column sensory axons beyond spinal cord lesion.Conclusion: NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.

CHL1-Deficient and Wild-Type Male Mice do Not Differ in Locomotor Recovery from Spinal Cord Injury.

CHL1 is a close homolog of L1, a cell adhesion molecule that plays major roles in neural and tumor cell functions. We had found that young adult female mice deficient in CHL1 recovered better than their wild-type female littermates after thoracic Spinal Cord Injury (SCI). This observation was surprising, because CHL1 increases neurite outgrowth in vitro. Injury of adult mouse central and peripheral nervous systems upregulate CHL1 expression in neurons and astrocytes, consistent with CHL1's pro-active, homophilic interaction between CHL1 surface molecules in wild-type mice. After SCI, CHL1 expression was observed to increase in the glial scar, areas of axonal regrowth and remodeling of neural circuits. These observations were made only in females, and we therefore sought to analyze SCI in CHL1-deficient male mice. We now show that CHL1-deficient males did not recover better or worse than their male wild-type littermates. Primary and secondary lesion volumes were similar in the two genotypes, as seen in female mice which were studied in parallel with male mice. Assessment of peripheral leukocytes showed a significant increase in numbers of blood neutrophils at 24 h after SCI in males, but not in females. Lymphocyte numbers in mutant males increased slightly, but numbers of lymphocytes or monocytes did not differ significantly between males or females. These results indicate that CHL1-deficient males and females differ in the number of neutrophils but not lymphocytes or monocytes, suggesting that the difference between males and females is unlikely due to differences in leukocytes.

The Conditioning Lesion Response in Dorsal Root Ganglion Neurons Is Inhibited in Oncomodulin Knock-Out Mice.

Regeneration can occur in peripheral neurons after injury, but the mechanisms involved are not fully delineated. Macrophages in dorsal root ganglia (DRGs) are involved in the enhanced regeneration that occurs after a conditioning lesion (CL), but how macrophages stimulate this response is not known. Oncomodulin (Ocm) has been proposed as a proregenerative molecule secreted by macrophages and neutrophils, is expressed in the DRG after axotomy, and stimulates neurite outgrowth by DRG neurons in culture. Wild-type (WT) and Ocm knock-out (KO) mice were used to investigate whether Ocm plays a role in the CL response in DRG neurons after sciatic nerve transection. Neurite outgrowth was measured after 24 and 48 h in explant culture 7 d after a CL. Sciatic nerve regeneration was also measured in vivo 7 d after a CL and 2 d after a subsequent sciatic nerve crush. The magnitude of the increased neurite outgrowth following a CL was significantly smaller in explants from Ocm KO mice than in explants from WT mice. In vivo after a CL, increased regeneration was found in WT animals but not in KO animals. Macrophage accumulation and levels of interleukin-6 (IL-6) mRNA were measured in axotomized DRG from WT and Ocm KO animals, and both were significantly higher than in sham-operated ganglia. At 6 h after axotomy, Il-6 mRNA was higher in WT than in Ocm KO mice. Our data support the hypothesis that Ocm plays a necessary role in producing a normal CL response and that its effects possibly result in part from stimulation of the expression of proregenerative macrophage cytokines such as IL-6.

Derivation of a CRISPR genome edited stem cell line containing a risk variant in TTC3.

We identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in all 11 Alzheimer disease (AD) individuals in a non-Hispanic white late onset Alzheimer disease (LOAD) family (mean AAO=75.7 years, Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious by five in silico algorithms and extremely rare in the gnomAD database (allele frequency=3.231x10-5 ). Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with the AD neuropathology. To understand the mechanism by which the TTC3 alteration may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed to examine cellular consequences in neuronal cells, which can otherwise only be collected postmortem. Starting with an iPSC line derived from a healthy 45 year old male donor of European ancestry, ASE-9203, genome editing was performed by introducing an sgRNA cloned into the Cas9 containing TLCV2 plasmid (Addgene) simultaneously with a ssODN template. An independent homozygous edited iPSC clone was isolated and verified by Sanger sequencing to carry the TTC3 variant, thus creating an isogenic pair of iPSCs. The stem cells were differentiated into forebrain neurons for 70 days and evaluated. Quantitative PCR analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as day 30 and day 70 differentiated neurons. Since there is evidence that modulation of TTC3 affects neurite growth (Berto, et al, 2007), morphological measures of axon formation were assessed in differentiating neuronal cultures using the Incucyte Zoom. Preliminary studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. Levels of amyloid beta 40 and 42 released from day 65 edited and unedited neurons were not significantly different. Results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3 will enable us to determine how this genetic alteration which may contribute to specific cellular phenotypes and, on a broader scale, LOAD risk.

Endophenotype-based in silico network medicine discovery combined with insurance record data mining identifies sildenafil as a candidate drug for Alzheimer's disease.

We developed an endophenotype disease module-based methodology for Alzheimer's disease (AD) drug repurposing and identified sildenafil as a potential disease risk modifier. Based on retrospective case-control pharmacoepidemiologic analyses of insurance claims data for 7.23 million individuals, we found that sildenafil usage was significantly associated with a 69% reduced risk of AD (hazard ratio = 0.31, 95% confidence interval 0.25-0.39, P<1.0×10-8). Propensity score stratified analyses confirmed that sildenafil is significantly associated with a decreased risk of AD across all four drug cohorts we tested (diltiazem, glimepiride, losartan and metformin) after adjusting age, sex, race, and disease comorbidities. We also found that sildenafil increases neurite growth and decreases phospho-tau expression in AD patient-induced pluripotent stem cells-derived neuron models, supporting mechanistically its potential beneficial effect in Alzheimer's disease. The association between sildenafil use and decreased incidence of AD does not establish causality or its direction, which requires a randomized clinical trial approach.

Multi-Target Effects of Novel Synthetic Coumarin Derivatives Protecting Aβ-GFP SH-SY5Y Cells against Aβ Toxicity.

Alzheimer’s disease (AD) is a common neurodegenerative disease presenting with progressive memory and cognitive impairments. One of the pathogenic mechanisms of AD is attributed to the aggregation of misfolded amyloid β (Aβ), which induces neurotoxicity by reducing the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TRKB) and increasing oxidative stress, caspase-1, and acetylcholinesterase (AChE) activities. Here, we have found the potential of two novel synthetic coumarin derivatives, ZN014 and ZN015, for the inhibition of Aβ and neuroprotection in SH-SY5Y neuroblastoma cell models for AD. In SH-SY5Y cells expressing the GFP-tagged Aβ-folding reporter, both ZN compounds reduced Aβ aggregation, oxidative stress, activities of caspase-1 and AChE, as well as increased neurite outgrowth. By activating TRKB-mediated extracellular signal-regulated kinase (ERK) and AKT serine/threonine kinase 1 (AKT) signaling, these two ZN compounds also upregulated the cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of ZN014 and ZN015. A parallel artificial membrane permeability assay showed that ZN014 and ZN015 could be characterized as blood–brain barrier permeable. Our results suggest ZN014 and ZN015 as novel therapeutic candidates for AD and demonstrate that ZN014 and ZN015 reduce Aβ neurotoxicity via pleiotropic mechanisms.

Experimental Epileptogenesis in a Cell Culture Model of Primary Neurons from Rat Brain: A Temporal Multi-Scale Study.

Understanding seizure development requires an integrated knowledge of different scales of organization of epileptic networks. We developed a model of “epilepsy-in-a-dish” based on dissociated primary neuronal cells from neonatal rat hippocampus. We demonstrate how a single application of glutamate stimulated neurons to generate spontaneous synchronous spiking activity with further progression into spontaneous seizure-like events after a distinct latency period. By computational analysis, we compared the observed neuronal activity in vitro with intracranial electroencephalography (EEG) data recorded from epilepsy patients and identified strong similarities, including a related sequence of events with defined onset, progression, and termination. Next, a link between the neurophysiological changes with network composition and cellular structure down to molecular changes was established. Temporal development of epileptiform network activity correlated with increased neurite outgrowth and altered branching, increased ratio of glutamatergic over GABAergic synapses, and loss of calbindin-positive interneurons, as well as genome-wide alterations in DNA methylation. Differentially methylated genes were engaged in various cellular activities related to cellular structure, intracellular signaling, and regulation of gene expression. Our data provide evidence that a single short-term excess of glutamate is sufficient to induce a cascade of events covering different scales from molecule- to network-level, all of which jointly contribute to seizure development.

Modulation of Vagal Sensory Neurons via High Mobility Group Box-1 and Receptor for Advanced Glycation End Products: Implications for Respiratory Viral Infections

Vagal sensory neurons contribute to the symptoms and pathogenesis of inflammatory pulmonary diseases through processes that involve changes to their morphological and functional characteristics. The alarmin high mobility group box-1 (HMGB1) is an early mediator of pulmonary inflammation and can have actions on neurons in a range of inflammatory settings. We hypothesized that HMGB1 can regulate the growth and function of vagal sensory neurons and we set out to investigate this and the mechanisms involved. Culturing primary vagal sensory neurons from wildtype mice in the presence of HMGB1 significantly increased neurite outgrowth, while acute application of HMGB1 to isolated neurons under patch clamp electrophysiological investigation produced inward currents and enhanced action potential firing. Transcriptional analyses revealed the expression of the cognate HMGB1 receptors, Receptor for Advanced Glycation End products (RAGE) and Toll-like Receptor 4 (TLR4), in subsets of vagal sensory neurons. HMGB1-evoked growth and electrophysiological responses were significantly reduced in primary vagal sensory neurons harvested from RAGE deficient mice and completely absent in neurons from RAGE/TLR4 double deficient mice. Immunohistochemical analysis of vagal sensory neurons collected from mice after intranasal infection with murine pneumovirus or influenza A virus (IAV), or after intratracheal administration with the viral mimetic PolyI:C, revealed a significant increase in nuclear-to-cytoplasm translocation of HMGB1 compared to mock-inoculated mice. Neurons cultured from virus infected wildtype mice displayed a significant increase in neurite outgrowth, which was not observed for neurons from virus infected RAGE or RAGE/TLR4 deficient mice. These data suggest that HMGB1 can enhance vagal sensory neuron growth and excitability, acting primarily via sensory neuron RAGE. Activation of the HMGB1-RAGE axis in vagal sensory neurons could be an important mechanism leading to vagal hyperinnervation and hypersensitivity in chronic pulmonary disease.

PD62-03 SCHWANN CELL CO-CULTURE PROMOTES NEURITOGENESIS AND SURVIVAL IN IRRADIATED PELVIC NEURONS

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (PD62)1 Sep 2021PD62-03 SCHWANN CELL CO-CULTURE PROMOTES NEURITOGENESIS AND SURVIVAL IN IRRADIATED PELVIC NEURONS Joshua Randolph, Elena Pak, Bridget Koontz, and Johanna Hannan Joshua RandolphJoshua Randolph More articles by this author , Elena PakElena Pak More articles by this author , Bridget KoontzBridget Koontz More articles by this author , and Johanna HannanJohanna Hannan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002099.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostatic radiation therapy (RT) damages the major pelvic ganglia (MPG) and nerves leading to erectile dysfunction (ED). Schwann cells (SCs) are known to facilitate neuron repair following mechanical injury, but their role in RT-induced neuron injury and repair has not been established. This study investigates the effects of co-culturing MPG neurons with SCs after ex vivo RT. METHODS: SCs were isolated by collecting MPGs from male Sprague-Dawley rats (n=12) which were digested in collagenase/dispase, and grown in SC medium. SCs were plated on poly-L lysine-coated coverslips and incubated to confluence for 24 hours. Confluent SCs were irradiated (0 or 8 Gy) prior to plating of neurons. Additional MPGs (n=18) were irradiated (0 or 8 Gy) and digested to isolate neurons. Dissociated neurons were plated alone or on SC-coated coverslips to create 6 experimental groups (n=3/grp): 1) CON MPG, 2) RT MPG, 3) CON SC+CON MPG, 4) RT MPG+CON SC, 5) CON MPG+RT SC, and 6) RT MPG+RT SC. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 protein (myelinating SC marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and TUNEL to assess neurite length, branching, specific neuron populations and apoptosis. RESULTS: As previously demonstrated, ex vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons. In the current study, co-culturing RT MPG+CON SC increased neurite outgrowth compared to all other groups (p<0.001). Neurite branching was markedly reduced in the RT MPG+RT SC co-culture, but unchanged in other groups. The proportion of apoptotic neurons was markedly greater in RT MPG neurons, but when co-cultured with CON SC RT-induced neuronal apoptosis was prevented (p<0.01). The proportion of adrenergic TH positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and co-culture with CON SCs was unable to prevent nitrergic loss. CONCLUSIONS: RT increases MPG neuron death and decreases pro-erectile nitrergic neurons. CON SC co-culture appears to stimulate neurite growth and restore branching networks in RT MPGs. While CON SCs significantly reduced apoptosis in RT MPG neurons, they were unable to prevent RT-induced nitrergic neuron loss. These data suggest that healthy SCs may promote survival and growth in RT-injured MPG neurons. Further in vivo studies are required to confirm whether administration or activation of SCs can help prevent RT-induced neuronal injury and ED. Source of Funding: Urology Care Foundation’s Herbert Brendler, MD., Summer Medical Student Fellowship; Sexual Medicine Society of North America, Research Grant © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1073-e1073 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joshua Randolph More articles by this author Elena Pak More articles by this author Bridget Koontz More articles by this author Johanna Hannan More articles by this author Expand All Advertisement Loading ...

Assessing the combination of magnetic field stimulation, iron oxide nanoparticles, and aligned electrospun fibers for promoting neurite outgrowth from dorsal root ganglia in vitro

Magnetic fiber composites combining superparamagnetic iron oxide nanoparticles (SPIONs) and electrospun fibers have shown promise in tissue engineering fields. Controlled grafting of SPIONs to the fibers post-electrospinning generates biocompatible magnetic composites without altering desired fiber morphology. Here, for the first time, we assess the potential of SPION-grafted scaffolds combined with magnetic fields to promote neurite outgrowth by providing contact guidance from the aligned fibers and mechanical stimulation from the SPIONs in the magnetic field. Neurite outgrowth from primary rat dorsal root ganglia (DRG) was assessed from explants cultured on aligned control and SPION-grafted electrospun fibers as well as on non-grafted fibers with SPIONs dispersed in the culture media. To determine the optimal magnetic field stimulation to promote neurite outgrowth, we generated a static, alternating, and linearly moving magnet and simulated the magnetic flux density at different areas of the scaffold over time. The alternating magnetic field increased neurite length by 40% on control fibers compared to a static magnetic field. Additionally, stimulation with an alternating magnetic field resulted in a 30% increase in neurite length and 62% increase in neurite area on SPION-grafted fibers compared to DRG cultured on PLLA fibers with untethered SPIONs added to the culture media. These findings demonstrate that SPION-grafted fiber composites in combination with magnetic fields are more beneficial for stimulating neurite outgrowth on electrospun fibers than dispersed SPIONs. Statement of significanceAligned electrospun fibers improve axonal regeneration by acting as a passive guidance cue but do not actively interact with cells, while magnetic nanoparticles can be remotely manipulated to interact with neurons and elicit neurite outgrowth. Here, for the first time, we examine the combination of magnetic fields, magnetic nanoparticles, and aligned electrospun fibers to enhance neurite outgrowth. We show an alternating magnetic field alone increases neurite outgrowth on aligned electrospun fibers. However, combining the alternating field with magnetic nanoparticle-grafted fibers does not affect neurite outgrowth compared to control fibers but improves outgrowth compared to freely dispersed magnetic nanoparticles. This study provides the groundwork for utilizing magnetic electrospun fibers and magnetic fields as a method for promoting axonal growth.

Aging Human Endothelial Cells Affect Neuronal Viability and Function

It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer's disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for neurodegenerative disease through increased inflammation and cellular dysfunction. Despite the fact that aging is a key risk factor in neurodegenerative disease, preclinical studies frequently model age-related diseases using young rodents. In this study we used replicative senescence, a standard model of cell senescence that permits the study of cells as they progress to senescence to examine the differential effects of senescent vs. non-senescent primary human endothelial cells on a human neuronal cell line. We hypothesize that senescent (Sen) endothelial cells (ECs), which accumulate with aging, create a pro-inflammatory environment that adversely affects viability and function of neurons. First, we validated the replicative senescence process in human-derived ECs by quantifying β-galactosidase activity, a marker for cellular senescence, and the protein expression of Lamin B1, which decreases with senescence. After determining senescence phenotypes by passage number, ECs at varying stages of cellular senescence (EP: early passage, ES: early senescence, LS: late senescence) were co-cultured with LUHMES (Lund Human Mesencephalic cell line - dopamine-like neurons) to assess neuronal cell viability by measuring release of lactate dehydrogenase (LDH) into the culture medium and neurite outgrowth. Nitric oxide and extracellular vesicles released into the culture medium were also evaluated as indicators of an inflammatory environment. Co-culturing ECs with LUHMES decreased LUHMES cell viability as evidenced by an increase in LDH in co-cultures of LUHMES LS EC and by a decreased number of nuclei in LUHMES co-cultured with any of the EC types (EP, ES, LS). Co-culture with any of the EC types increased the area labeled by MAP2 and phospho-Neurofilament in LUHMES at 24h, indicating increased neurite outgrowth. At 48h, increased neurite outgrowth was observed in LUHMES+EP ECs and LUHMES+ES ECs co-cultures but not in the LUHMES + LP ECs. Nitric oxide was not increased by co-culture of LUHMES with any of the EC subtypes. The amount of extracellular vesicles as quantified by measuring the activity of acetylcholinesterase (AChE) was increased in co-cultures compared to LUHMES controls, but did not significantly differ from EC controls. In summary, ES ECs promote neurite outgrowth whereas LS ECs promote neurodegeneration. These findings suggest the importance of EC aging on neuronal cell morphology and viability, both parameters affected in age-related dementia, including Alzheimer's disease.

Effects of Oxytocin Analogs on Neurite Outgrowth and Cellular Signaling in Human SH‐SY5Y Neuroblastoma Cell Line

Introduction In the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G-protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression. Methods The human neuroblastoma derived SH-SY5Y cell line demonstrates morphological characteristics of neurons including neurites, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8-AM to measure ligand-induced Ca2+mobilization. Sigmoidal dose-response curves for OT analogs were generated using three-parameter nonlinear regression analysis in GraphPad Prism 8.3 software. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of the consensus mammalian OT sequence (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly; Leu8-OT) and a new world monkey analog (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Pro-Gly; Pro8-OT) for 24 hours, fixed and stained with phalloidin and DAPI. The Nikon A1R confocal microscope was used to collect 60X images of GFP-expressing neurons assessed for effects on neurite outgrowth using the Image J package FIJI with the NeuronJ plugin. Results In SH-SY5Y cells, both OT analogs were more potent than AVP at inducing Ca2+ mobilization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L-371,257. Additionally, both OT analogs show a dose-dependent increase in neurite outgrowth. Conclusions Together, these data suggest that OTR mediated signaling pathways lead to neurite outgrowth in SH-SY5Y cells. Integrative studies of behavior, genetics and ligand-receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.

Anacardium Occidentale L. Leaf Extracts Protect Against Glutamate/H2O2-Induced Oxidative Toxicity and Induce Neurite Outgrowth: The Involvement of SIRT1/Nrf2 Signaling Pathway and Teneurin 4 Transmembrane Protein.

Neurodegenerative diseases are linked to neuronal cell death and neurite outgrowth impairment that are often caused by oxidative stress. Natural products, which have neuroprotective against oxidative stress and neurite outgrowth inducing activity, could be potential candidates for alternative treatment of neurodegenerative diseases. This study aims to investigate the neuroprotective effects and neuritogenesis properties of Anacardium occidentale leaf extracts in cultured neuronal (HT22 and Neuro-2a) cells. We found gallic acid, catechin and quercetin as the main compounds in A. occidentale extracts. The extracts have a protective effect against glutamate/H2O2-mediated oxidative stress-induced cell toxicity. The gene expression of cellular antioxidant enzymes (SODs, GPx and, GSTs) were up-regulated by this treatment. The treatment also triggered SIRT, Nrf2 proteins as well as the mRNA transcriptions of relevant anti-oxidation genes (NQO1, GCLM, and EAAT3). We demonstrated that the extracts promote antioxidant defense in neuronal cells via the SIRT1/Nrf2 signaling pathway. Moreover, the extracts increase neurite outgrowth and Ten-4 expression in Neuro-2a cells. However, the neuritogenesis properties did not occur, when Ten-4 expression was knocked down by corresponding siRNA. These results suggest that the leaf extracts have an interesting neuritogenesis and neuroprotective potential against glutamate/H2O2-mediated toxicity and could be a potential therapeutic candidate for neurodegenerative diseases.

Potential of N-trans feruloyl tyramine from Lycium barbarum fruit extract on neurogenesis and neurotrophins; targeting TrkA/ERK/CREB signaling pathway

This study was aimed to evaluate the potential neurotrophic effect of Lycium barbarum fruit extract (LB-E) via increased neurite outgrowth and synaptic formation, and its neurotrophic and neuroprotective efficacy was also checked in vitro. LB-E exerted neurotrophic effects by regulating TrkA/ERK/CREB signaling. In nervous system, LB-E promoted neuronal survival and differentiation by increasing neurotrophic factor secretion in C6 glioma cells. Interestingly, LB-E increased the expressed levels of synaptophysin and GAP-43 in CNS cell lines. It also subsequently increased the number of ciliated cells and length of cilia in N1 hypothalamic neuronal cells induced by CNTF and displayed the enhanced dendrite length in primary cortical neurons. We investigated N-trans feruloyl tyramine (NTFT) from LB-E by LC-MS/MS, and confirmed NTFT as a biologically active ingredient showing the neurotrophic effect. These results suggested that NTFT from LB-E may be beneficial in the treatment of age-related brain diseases caused by the deficiency of neurotrophins.

Characterization of Mice Ubiquitously Overexpressing Human 15-Lipoxygenase-1: Effect of Diabetes on Peripheral Neuropathy and Treatment with Menhaden Oil

To rigorously explore the role of omega-3 polyunsaturated fatty acids (PUFA) in the treatment of diabetic peripheral neuropathy (DPN), we have created a transgenic mouse utilizing a Cre-lox promoter to control overexpression of human 15-lipoxygenase-1 (15-LOX-1). In this study, we sought to determine the effect of treating type 2 diabetic wild-type mice and transgenic mice ubiquitously overexpressing 15-LOX-1 with menhaden oil on endpoints related to DPN. Wild-type and transgenic mice on a C57Bl/6J background were divided into three groups. Two of each of these groups were used to create a high-fat diet/streptozotocin model for type 2 diabetes. The remaining mice were control groups. Four weeks later, one set of diabetic mice from each group was treated with menhaden oil for twelve weeks and then evaluated using DPN-related endpoints. Studies were also performed using dorsal root ganglion neurons isolated from wild-type and transgenic mice. Wild-type and transgenic diabetic mice developed DPN as determined by slowing of nerve conduction velocity, decreased sensory nerve fibers in the skin and cornea, and impairment of thermal and mechanical sensitivity of the hindpaw compared to their respective control mice. Although not significant, there was a trend for the severity of these DPN-related deficits to be less in the diabetic transgenic mice compared to the diabetic wild-type mice. Treating diabetic wild-type and transgenic mice with menhaden oil improved the DPN-related endpoints with a trend for greater improvement or protection by menhaden oil observed in the diabetic transgenic mice. Treating dorsal root ganglion neurons with docosahexanoic acid but not eicosapentaenoic acid significantly increased neurite outgrowth with greater efficacy observed with neurons isolated from transgenic mice. Targeting pathways that will increase the production of the anti-inflammatory metabolites of omega-3 PUFA may be an efficacious approach to developing an effective treatment for DPN.

Hepatocyte growth factor induces pErk and pSTAT3 (Ser 727) to promote mitochondrial activity and neurite outgrowth in primary dorsal root ganglion cultures.

Hepatocyte growth factor (HGF) promotes the neurite outgrowth of sensory neurons in developmental stages, but its role in injured peripheral nerves in adult mice remains largely been unexplored. In this study, we investigated the role of HGF in the regeneration of injured peripheral nerves using cultured dorsal root ganglions (DRGs). When cells were treated with HGF protein, the length of the neurite was increased 1.4-fold compared to the untreated control group. HGF greatly increased the level of phosphorylated STAT3 at serine 727 [pSTAT3 (Ser 727)], thereby translocating the protein to the mitochondria. HGF treatment increased the activity of mitochondrial complex I. When DRGs were cultured in the presence of U0126, a pharmacological inhibitor of Erk, the HGF-mediated increase in neurite outgrowth and the level of pSTAT3 (Ser 727) were both suppressed. Taken together, these results suggest that the HGF/c-met pathway might promote neurite outgrowth by controlling mitochondrial activity through the HGF/Erk/STAT3 axis.

Box Jellyfish (Cnidaria, Cubozoa) Extract Increases Neuron's Connection: A Possible Neuroprotector Effect.

Neurodegenerative diseases are one of the major causes of death worldwide, characterized by neurite atrophy, neuron apoptosis, and synapse loss. No effective treatment has been indicated for such diseases so far, and the search for new drugs is being increased in the last years. Animal venoms' secretion/venom can be an alternative for the discovery of new molecules, which could be the prototype for a new treatment. Here, we present the biochemical characterization and activity of the extract from the box jellyfish Chiropsalmus quadrumanus (Cq) on neurites. The Cq methanolic extract was obtained and incubated to human SH-SY5Y neurons, and neurite parameters were evaluated. The extract was tested in other cell types to check its cytotoxicity and was submitted to biochemical analysis by mass spectrometry in order to check its composition. We could verify that the Cq extract increased neurite outgrowth length and branching junctions, amplifying the contact between SH-SY5Y neurons, without affecting cell body and viability. The extract action was selective for neurons, as it did not cause any effects on other cell types, such as tumor line, nontumor line, and red blood cells. Moreover, mass spectrometry analysis revealed that there are no proteins but several low molecular mass compounds and peptides. Three peptides, characterized as cryptides, and 14 low molecular mass compounds were found to be related to cytoskeleton reorganization, cell membrane expansion, and antioxidant/neuroprotective activity, which act together to increase neuritogenesis. After this evaluation, we conclude that the Cq extract is a promising tool for neuronal connection recovery, an essential condition for the treatment of neurodegenerative diseases.