Neurite Length Research Articles

Therapeutic Potential of Fosgonimeton, a Small-Molecule Positive Modulator of the Neurotrophic HGF/MET Pathway, in Neurodegenerative Conditions (S14.008)

<h3>Objective:</h3> Evaluate the neurotrophic and neuroprotective effects of fosgonimeton, a small-molecule positive modulator of hepatocyte growth factor (HGF)/MET. <h3>Background:</h3> HGF signaling through the MET receptor promotes neural survival and function, making it a compelling target to combat neurodegeneration. We developed a series of small-molecule positive modulators of HGF/MET, including fosgonimeton, to target this neurotrophic system. <h3>Design/Methods:</h3> To demonstrate the neurotrophic effects of fosgonimeton, primary neurons were treated with the active metabolite of fosgonimeton (fosgo-AM) and assessed for synaptic count and neurite outgrowth. To evaluate neuroprotective effects, primary neurons were treated with fosgo-AM before exposure to relevant neurodegenerative insults, namely oxidative stress (hydrogen peroxide [H₂O₂]), neuroinflammation (lipopolysaccharide [LPS]), excitotoxicity (glutamate), and mitochondrial dysfunction (1-methyl-4-phenylpyridinium [MPP+]). The effects of fosgo-AM on protein aggregation in primary neuron cultures were evaluated via levels of phosphorylated tau (p-tau) induced by amyloid β (Aβ) toxicity in cortical neurons and α-synuclein aggregation induced by 6-hydroxydopamine (6-OHDA) in dopaminergic neurons. In vivo, the therapeutic usefulness of fosgonimeton was assessed for LPS-induced cognitive impairment in mice and 6-OHDA–induced motor deficits in rats. <h3>Results:</h3> In vitro, fosgo-AM significantly increased the number of synapses and neurite length in hippocampal and cortical neurons, respectively. Fosgo-AM was neuroprotective of cortical neurons subjected to oxidative, neuroinflammatory, excitotoxic, and mitochondrial challenges. Significant reduction in the number of disease biomarkers was observed with fosgo-AM treatment, including Aβ-induced p-tau accumulation in cortical neurons and 6-OHDA–induced α-synuclein aggregation in dopaminergic neurons. In vivo, fosgonimeton administration significantly attenuated LPS-induced cognitive impairment and 6-OHDA–induced motor deficits in rodent models. <h3>Conclusions:</h3> These results, from in vitro and in vivo systems, consistently support the broad application of fosgonimeton to neurodegenerative conditions. Collectively, our observations highlight the therapeutic potential of HGF/MET modulation in the management of neurodegenerative diseases that include AD and PD, supporting further clinical exploration of this approach. <b>Disclosure:</b> Dr. Reda has received personal compensation for serving as an employee of Athira Pharma, Inc. . Dr. Reda has stock in Athira Pharma, Inc. . Dr. Taylor has received personal compensation for serving as an employee of Athira Pharma, Inc. Dr. Taylor has stock in Athira Pharma, Inc.. Dr. Taylor has received intellectual property interests from a discovery or technology relating to health care. Dr. Johnston has received personal compensation for serving as an employee of Athira Pharma, Inc. Dr. Johnston has stock in Athira Pharma, Inc. An immediate family member of Dr. Johnston has stock in Athira Pharma, Inc. Dr. Johnston has received intellectual property interests from a discovery or technology relating to health care. Dr. Berthiaume has received personal compensation for serving as an employee of Athira Pharma. Dr. Berthiaume has stock in Athira Pharma. An immediate family member of Dr. Berthiaume has stock in Athira Pharma. Dr. Setti has received personal compensation for serving as an employee of Athira Pharma. Dr. Setti has stock in Athira Pharma. Dr. Church has received personal compensation for serving as an employee of Athira Pharma. Dr. Church has stock in Athira Pharma. Dr. Church has received intellectual property interests from a discovery or technology relating to health care.

Crocetin Regulates Functions of Neural Stem Cells to Generate New Neurons for Cerebral Ischemia Recovery.

Many neurons undergo apoptosis after ischemic stroke. In the brain, neurogenesis has the potential for neuronal replacement and can be activated by external conditions to repair the injury. Crocetin (CRO), naturally extracted from the plant saffron, acts as a neuroprotective agent for ischemic stroke. However, the underlying mechanism remains unknown. In this work, the effect of CRO on neural stem cell (NSC) behaviors and subventricular zone neurogenesis is investigated. Initially, NSCs are incubated with different concentrations of CRO to detect the cell proliferation and differentiation in vitro. Second, ischemic stroke induced rats are treated with CRO using nimodipine (NMDP) as a comparison. The behavioral functions, infarcted volume, and apoptotic Nissl bodies of rats are noticeably improved after CRO-treatment, comparable to those of NMDP. In addition, the increased regional cerebral blood flow and promoted neuronal differentiation are achieved by CRO-treatment. Brain tissue examination shows significantly increased neuronal regeneration in the focal ischemic injury area. Meanwhile, the length of neurites is prolonged, indicating that CRO could potentially promote neurite extension to enhance cell-cell communication. These findings demonstrate that CRO facilitated the neuronal differentiation of NSCs by activating subventricular zone neurogenesis in damaged cortex and striatum sites to repair ischemic stroke.

Promising application of a novel biomaterial, light chain of silk fibroin combined with NT3, in repairment of rat sciatic nerve defect injury

Autologous nerve transplantation is the gold standard for treating peripheral nerve defects, but it is associated with defects such as insufficient donor and secondary injury. Artificial nerve guidance conduits (NGCs) are now considered promising alternatives for bridging long nerve gaps, although exploring new biomaterials to construct NGCs remains challenging. Silk fibroin (SF) has good biocompatibility and can self-assemble in aqueous solutions. However, the lack of proximal neurotrophic factors after nerve injury is a major concern, leading to incomplete nerve regeneration. In this study, NT-3, a neurotrophin that promotes neuronal survival and differentiation, was bound to the light chain of silk fibroin (FIBL) in two ways: one was directly bound to FIBL (FIBL-NT3) and the other was a polypeptides-linker (FIBL-Linker-NT3). The design aimed to take advantage of silk fiber's character of self-assembly of heavy-light chains and test whether a flexible linker with NT3 molecule is easy to be a NT3 dimer, the active form. In vitro studies indicated that FIBL-Linker-NT3 combined with SF membranes promoted axon growth in adult rat dorsal root ganglion (DRG) neurons. Then we tested if FIBL-Linker-NT3 could self-assemble with the SF heavy chain (SFH). DTT (Dithiothreitol) was used to break the disulfide bonds between the SF light and heavy chains, and the light-chain protein was removed via dialysis. SFH was assembled using FIBL-Linker-NT3, as evidenced by the western blotting results that showed a high molecular band corresponding to SFH-FIBL-Linker-NT3. Chitosan scaffolds have been identified to provide a suitable microenvironment, so a chitosan/SF-FIBL-Linker-NT3 conduit was also constructed. Nerve transplantation of this conduit was evaluated in vivo in a rat sciatic nerve defect model. Immunohistochemical assays showed that the chitosan/SF-FIBL-Linker-NT3 group was superior to the chitosan/PBS, SF, PBS + FIBL-Linker-NT3 groups in nerve regeneration. In addition, the chitosan/SF-FIBL-Linker-NT3 conduit-transplanted group exhibited better recovery in terms of neurite length, sciatic functional index value, sensitivity to heat, time on the rotarod, wet weight ratio, cross-sectional area, compound muscle action potential, number of myelin layers, and myelin thickness in the nerve. Taking together, our study identified that FIBL-Linker-NT3 could promote axonal growth and regeneration in vivo and in vitro and is a promising candidate biomaterial for artificial NGCs.

Automated algorithm development to assess survival of human neurons using longitudinal single-cell tracking: Application to synucleinopathy.

The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.

MP27-01 TARGETED DEPLETION OF FIDGETIN-LIKE 2 (FL2) PROMOTES NERVE REGENERATION AND IMPROVES BOTH ERECTILE AND BLADDER FUNCTION OUTCOMES IN A RODENT MODEL OF RADICAL PROSTATECTOMY

You have accessJournal of UrologyCME1 Apr 2023MP27-01 TARGETED DEPLETION OF FIDGETIN-LIKE 2 (FL2) PROMOTES NERVE REGENERATION AND IMPROVES BOTH ERECTILE AND BLADDER FUNCTION OUTCOMES IN A RODENT MODEL OF RADICAL PROSTATECTOMY Lisa Baker, Moses Tar, Yi Wang, David Sharp, Sylvia Suadicani, and Kelvin Davies Lisa BakerLisa Baker More articles by this author , Moses TarMoses Tar More articles by this author , Yi WangYi Wang More articles by this author , David SharpDavid Sharp More articles by this author , Sylvia SuadicaniSylvia Suadicani More articles by this author , and Kelvin DaviesKelvin Davies More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Damage to the cavernous nerves (CN) during radical prostatectomy (RP) commonly results in urogenital dysfunction, which includes bladder and erectile dysfunction (ED). We recently identified fidgetin-like 2 (FL2) as a negative regulator of microtubule dynamics which when depleted in nerve cells in vitro accelerates axonal outgrowth, and in an in vivo model of RP promotes nerve regeneration and improves erectile function outcomes. We have expanded upon these studies to determine if FL2 depletion might also improve bladder function outcomes following CN injury. METHODS: To study the effects of FL2 depletion on axon growth and on the microtubule (MT) cytoskeleton within the axon shaft in vitro, dissociated dorsal root ganglion (DRG) neurons were transduced with AAV5 containing a plasmid encoding FL2 or scrambled small hairpin RNA (shRNA) and a GFP reporter. Five-days after treatment, neurons were assessed for neurite growth, fixed and immunostained for MT. As a model of CN injury, rats underwent CN transection. At the site and time of injury, control- or FL2-siRNA was applied. Voiding function was evaluated 1 week post-injury, using the voided stain on paper (VSOP) method. At 2 weeks CN regeneration was assessed visually and erectile function was assessed by cavernosometry. RESULTS: In vitro FL2 knockdown in neurons enhanced average neurite length and growth rate. Animals treated withFL2-siRNA had significantly lower voiding frequency and higher voided volume per micturition when compared to animals treated with control-siRNA. There was visible regeneration of the CN in 7 out of 8 animals treated with FL2-siRNA (with no observed regeneration in animals treated with control-siRNA). In animals in which there was sufficient CN regeneration to allow electrostimulation, FL2-siRNA treated animals had significantly improved erectile function compared to controls. CONCLUSIONS: FL2-depletion after CN injury results in visible regeneration of the CN, with improved erectile and bladder function outcomes. Our work suggests that depleting FL2 promotes nerve regeneration through directly enhancing the growth rate of injured axons. Overall, we have identified FL2 as a promising therapeutic target for promoting CN regeneration and functional recovery of urogenital function after iatrogenic nerve injury resulting from the surgical procedures of RP. Source of Funding: NIH/NIDDK © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e362 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lisa Baker More articles by this author Moses Tar More articles by this author Yi Wang More articles by this author David Sharp More articles by this author Sylvia Suadicani More articles by this author Kelvin Davies More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of sodium channels in Alzheimer’s disease

Alzheimer disease (AD), depicted by the formation of amyloid β-protein plaque(Aβ), has been recognized for dementia and epilepsy. The pathological mechanism of AD is complicated and it remains unclear. Currently, most therapies are focusing on elimination of Aβ plaque and preventing the breakdown of acetylcholine to prevent neuron cell death, however, most of these drugs fail to improve cognitive functions in AD patients. Therefore, the development of novel therapies for AD is in urgent need. Emerging evidences show that cellular sodium (Na+) signals play a significant role in neurol physiology. Aβ oligomer upregulates the expression of Nav1.6, leading to cognition loss, epilepsy, and malfunction of neuron development (shorter neurite length is observed during Aβ-interfered neuron development), indicating that sodium channel may be a target for treating AD. This review summarizes current development of molecular mechanisms linking sodium dysregulation with AD pathologies and discusses potential therapies for AD by correcting Na+ disruption.

The Combined Effects of Topography and Stiffness on Neuronal Differentiation and Maturation Using a Hydrogel Platform

Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2-10 µm) were patterned onto gels with varying stiffness (6.1-110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of β-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.

Taurine Promotes Differentiation and Maturation of Neural Stem/Progenitor Cells from the Subventricular Zone via Activation of GABAA Receptors

Neurogenesis, the formation of new neurons in the brain, occurs throughout the lifespan in the subgranular zone of the dentate gyrus and subventricular zone (SVZ) lining the lateral ventricles of the mammal brain. In this process, gamma-aminobutyric acid (GABA) and its ionotropic receptor, the GABAA receptor (GABAAR), play a critical role in the proliferation, differentiation, and migration process of neural stem/progenitor cells (NPC). Taurine, a non-essential amino acid widely distributed throughout the central nervous system, increases the proliferation of SVZ progenitor cells by a mechanism that may involve GABAAR activation. Therefore, we characterized the effects of taurine on the differentiation process of NPC expressing GABAAR. Preincubation of NPC-SVZ with taurine increased microtubule-stabilizing proteins assessed with the doublecortin assay. Taurine, like GABA, stimulated a neuronal-like morphology of NPC-SVZ and increased the number and length of primary, secondary, and tertiary neurites compared with control NPC of the SVZ. Furthermore, neurite outgrowth was prevented when simultaneously incubating cells with taurine or GABA and the GABAAR blocker, picrotoxin. Patch-clamp recordings revealed a series of modifications in the NPCs’ passive and active electrophysiological properties exposed to taurine, including regenerative spikes with kinetic properties similar to the action potentials of functional neurons.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

Evaluation of Per- and Polyfluoroalkyl Substances (PFAS) In Vitro Toxicity Testing for Developmental Neurotoxicity.

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

Electrohydrodynamic Printing of Microfibrous Architectures with Cell-Scale Spacing for Improved Cellular Migration and Neurite Outgrowth.

Electrohydrodynamic (EHD) printing provides unparalleled opportunities in fabricating microfibrous architectures to direct cellular orientation. However, it faces great challenges in depositing orderly microfibers with cell-scale spacing due to inherent fiber-fiber electrostatic interactions. Here a finite element method is established to analyze the electrostatic forces induced on the EHD-printed microfibers and the relationship between the fiber diameter and spacing for parallel deposition of EHD-printed microfibers is revealed theoretically and experimentally. It is found that uniform fiber arrangement can be achieved when the fiber spacing is five times larger than the fiber diameter. This finding enables the successful printing of parallel fibrous architectures with a fiber diameter of 4.9 ± 0.1µm and a cell-scale fiber spacing of 25.6 ± 1.9µm. The resultant microfibrous architectures exhibit unique capability to direct cellular alignment and enhance cellular density and migration as the fiber spacing decreases from 100 to 25µm. The EHD-printed parallel microfibers with cell-scale spacing are found to improve the outgrowth length of neurites and accelerate the migration of Schwann cells from Dorsal Root Ganglion spheres, which facilitate the formation of densely-arranged and highly-aligned cellular constructs. The presented method is promising to produce biomimetic microfibrous architectures for functional nerve regeneration.

IPSC‐based modeling of THD recapitulates disease phenotypes and reveals neuronal malformation

Tyrosine hydroxylase deficiency (THD) is a rare genetic disorder leading to dopaminergic depletion and early‐onset Parkinsonism. Affected children present with either a severe form that does not respond to L‐Dopa treatment (THD‐B) or a milder L‐Dopa responsive form (THD‐A). We generated induced pluripotent stem cells (iPSCs) from THD patients that were differentiated into dopaminergic neurons (DAn) and compared with control‐DAn from healthy individuals and gene‐corrected isogenic controls. Consistent with patients, THD iPSC‐DAn displayed lower levels of DA metabolites and reduced TH expression, when compared to controls. Moreover, THD iPSC‐DAn showed abnormal morphology, including reduced total neurite length and neurite arborization defects, which were not evident in DAn differentiated from control‐iPSC. Treatment of THD‐iPSC‐DAn with L‐Dopa rescued the neuronal defects and disease phenotype only in THDA‐DAn. Interestingly, L‐Dopa treatment at the stage of neuronal precursors could prevent the alterations in THDB‐iPSC‐DAn, thus suggesting the existence of a critical developmental window in THD. Our iPSC‐based model recapitulates THD disease phenotypes and response to treatment, representing a promising tool for investigating pathogenic mechanisms, drug screening, and personalized management.

Identification of a novel pathway in sporadic Amyotrophic Lateral Sclerosis mediated by the long non-coding RNA ZEB1-AS1

BackgroundDeregulation of transcription in the pathogenesis of sporadic Amyotrophic Lateral Sclerosis (sALS) is taking central stage with RNA-sequencing analyses from sALS patients tissues highlighting numerous deregulated long non-coding RNAs (lncRNAs). The oncogenic lncRNA ZEB1-AS1 is strongly downregulated in peripheral blood mononuclear cells of sALS patients. In addition, in cancer-derived cell lines, ZEB1-AS1 belongs to a negative feedback loop regulation with hsa-miR-200c, acting as a molecular sponge for this miRNA. The role of the lncRNA ZEB1-AS1 in sALS pathogenesis has not been characterized yet, and its study could help identifying a possible disease-modifying target. Methodsthe implication of the ZEB1-AS1/ZEB1/hsa-miR-200c/BMI1 pathway was investigated in multiple patients-derived cellular models (patients-derived peripheral blood mononuclear cells and induced pluripotent stem cells-derived neural stem cells) and in the neuroblastoma cell line SH-SY5Y, where its function was inhibited via RNA interference. Molecular techniques such as Real Time PCR, Western Blot and Immunofluorescence were used to assess the pathway dysregulation. ResultsOur results show a dysregulation of a signaling pathway involving ZEB1-AS1/hsa-miR-200c/β-Catenin in peripheral blood mononuclear cells and in induced pluripotent stem cells-derived neural stem cells from sALS patients. These results were validated in vitro on the cell line SH-SY5Y with silenced expression of ZEB1-AS1. Moreover, we found an increase for ZEB1-AS1 during neural differentiation with an aberrant expression of β-Catenin, highlighting also its aggregation and possible impact on neurite length. ConclusionsOur results support and describe the role of ZEB1-AS1 pathway in sALS and specifically in neuronal differentiation, suggesting that an impairment of β-Catenin signaling and an alteration of the neuronal phenotype are taking place.

Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures.

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features. Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age. Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated β-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age. Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Closing the Gap between the Auditory Nerve and Cochlear Implant Electrodes: Which Neurotrophin Cocktail Performs Best for Axonal Outgrowth and Is Electrical Stimulation Beneficial?

Neurotrophins promote neurite outgrowth of auditory neurons and may help closing the gap to cochlear implant (CI) electrodes to enhance electrical hearing. The best concentrations and mix of neurotrophins for this nerve regrowth are unknown. Whether electrical stimulation (ES) during outgrowth is beneficial or may direct axons is another open question. Auditory neuron explant cultures of distinct cochlear turns of 6-7 days old mice were cultured for four days. We tested different concentrations and combinations of BDNF and NT-3 and quantified the numbers and lengths of neurites with an advanced automated analysis. A custom-made 24-well electrical stimulator based on two bulk CIs served to test different ES strategies. Quantification of receptors trkB, trkC, p75NTR, and histological analysis helped to analyze effects. We found 25 ng/mL BDNF to perform best, especially in basal neurons, a negative influence of NT-3 in combined BDNF/NT-3 scenarios, and tonotopic changes in trk and p75NTR receptor stainings. ES largely impeded neurite outgrowth and glia ensheathment in an amplitude-dependent way. Apical neurons showed slight benefits in neurite numbers and length with ES at 10 and 500 µA. We recommend BDNF as a potent drug to enhance the man-machine interface, but CIs should be better activated after nerve regrowth.

Phosphorylation mutation impairs the promoting effect of spastin on neurite outgrowth without affecting its microtubule severing ability.

Spastin, a microtubule-severing enzyme, is known to be important for neurite outgrowth. However, the role of spastin post-translational modification, particularly its phosphorylation regulation in neuronal outgrowth, remains unclear. This study aimed to investigate the effects of eliminating spastin phosphorylation on the neurite outgrowth of rat hippocampal neurons. To accomplish this, we constructed a spastin mutant with eleven potential phosphorylation sites mutated to alanine. The phosphorylation levels of the wildtype spastin (WT) and the mutant (11A) were then detected using Phos-tag SDS-PAGE. The spastin constructs were transfected into COS7 cells for the observation of microtubule severing, and into rat hippocampal neurons for the detection of neuronal outgrowth. The results showed that compared to the spastin WT, the phosphorylation levels were significantly reduced in the spastin 11A mutant. The spastin mutant 11A impaired its ability to promote neurite length, branching, and complexity in hippocampal neurons, but did not affect its ability to sever microtubules in COS7 cells. In conclusion, the data suggest that mutations at multiple phosphorylation sites of spastin do not impair its microtubule cleavage ability in COS7 cells, but reduce its ability to promote neurite outgrowth in rat hippocampal neurons.

Highly Efficient Singlet Oxygen Generation by BODIPY-Ruthenium(II) Complexes for Promoting Neurite Outgrowth and Suppressing Tau Protein Aggregation.

Singlet oxygen (1O2) has been recently identified as a key molecule against toxic Aβ aggregation, which is associated with the currently incurable Alzheimer's disease (AD). However, limited research has studied its efficiency against tau protein aggregation, the other major hallmark of AD. Herein, we designed and synthesized boron-dipyrromethene (BODIPY)-ruthenium conjugates and isolated three isomers. Under visible-light irradiation, the ε isomer can be photoactivated and efficiently generate singlet oxygen. Particularly, the complex demonstrated successful results in attenuating tauopathy─an appreciable decrease to 43 ± 2% at 100 nM. The photosensitizer was further found to remarkably promote neurite outgrowth and significantly increased the length and number of neurites in nerve cells. As a result of effective photoinduced singlet oxygen generation and proactive neurite outgrowth, the hybrid design has great potential for therapeutics for Alzheimer's disease.

SOD1G93A Astrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motor neuron (MN) degeneration. Astrocytes surrounding MNs are known to modulate ALS progression. When cocultured with astrocytes overexpressing the ALS-linked mutant Cu/Zn superoxide dismutase (SOD1G93A) or when cultured with conditioned medium from SOD1G93A astrocytes, MN survival is reduced. The exact mechanism of this neurotoxic effect is unknown. Astrocytes secrete extracellular vesicles (EVs) that transport protein, mRNA, and microRNA species from one cell to another. The size and protein markers characteristic of exosomes were observed in the EVs obtained from cultured astrocytes, indicating their abundance in exosomes. Here, we analyzed the microRNA content of the exosomes derived from SOD1G93A astrocytes and evaluated their role in MN survival. Purified MNs exposed to SOD1G93A astrocyte-derived exosomes showed reduced survival and neurite length compared to those exposed to exosomes derived from non-transgenic (non-Tg) astrocytes. Analysis of the miRNA content of the exosomes revealed that miR-155-5p and miR-582-3p are differentially expressed in SOD1G93A exosomes compared with exosomes from non-Tg astrocytes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicates that miR-155-5p and miR-582-3p predicted targets are enriched in the neurotrophin signaling pathway. Importantly, when levels of miR-155-5p were reduced by incubation with a specific antagomir, SOD1G93A exosomes did not affect MN survival or neurite length. These results demonstrate that SOD1G93A-derived exosomes are sufficient to induce MN death, and miRNA-155-5p contributes to this effect. miRNA-155-5p may offer a new therapeutic target to modulate disease progression in ALS.

Human Olfactory Ecto-mesenchymal Stem Cells Displaying Schwann-cell-like Phenotypes and Promoting Neurite Outgrowth in Vitro.

Strategies of Schwann cell (SC) transplantation for regeneration of peripheral nerve injury involve many limitations. Stem cells can be used as alternative cell source for differentiation into Schwann cells. Given the high potential of neural crest-derived stem cells for the generation of multiple cell lineages, in this research, we considered whether olfactory ectomesenchymal stem cells (OE-MSCs) derived from neural crest can spontaneously differentiate into SC lineage. OE-MSCs were isolated from human nasal mucosa and characterized by the mesenchymal and neural crest markers. The cells were cultured in glial growth factors-free medium and further investigated in terms of the phenotypic and functional properties. Immunocytochemical staining and real-time PCR analysis indicated that the cultured OE-MSCs expressed SCs markers, SOX10, p75, S100, GFAP and MBP, differentiation indicative. It was found that the cells could secrete neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Furthermore, after co-cultured with PC12, the mean neurite length was enhanced by OE-MSCs. The findings indicated that OE-MSCs could be differentiated spontaneously into SC-like phenotypes, suggesting their applications for transplantation in peripheral nerve injuries.

Low dose of lithium carbonate as a neurites length enhancer in dopaminergic cells

Low dose of lithium carbonate as a neurites length enhancer in dopaminergic cells