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The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

The potential neurotoxicity from an increasing number of drugs and untested environmental chemicals creates a need to develop reliable and efficient in vitro methods for identifying chemicals that may adversely affect the nervous system. An important process in neurodevelopment is neurite outgrowth, which can be affected by developmental neurotoxicity. Currently, neurite outgrowth assays rely mainly on staining, which requires multiple sample processing steps, particularly washing steps, that may introduce variation and limit throughput. Here, we describe a neurite outgrowth assay that uses induced pluripotent stem cell (iPSC)-derived human cortical glutamatergic neurons and/or spinal motor neurons labeled with green fluorescent protein (GFP) to test compounds in a high-content and high-throughput format. This method enables live and time-lapse imaging of GFP-labeled neurons using an assay plate that is continuously imaged at multiple times after chemical treatment. In this article, we describe how to thaw frozen GFP-labeled neurons, culture them, treat them with a compound of interest, and analyze neurite outgrowth using a high-content imaging platform. In this assay, GFP-labeled iPSC-derived human neurons represent a promising tool for identifying and prioritizing compounds with potential developmental neurotoxicity for further hazard characterization. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Thawing and seeding of iPSC-derived neurons Basic Protocol 2: Compound plate preparation and treatment of neurons Basic Protocol 3: High-content imaging and analysis.

BackgroundProstatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn’t been explored.AimTo determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT.MethodsMajor pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling.OutcomesWe measured neurite length, branching, specific neuron populations and apoptosis.ResultsEx vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss.Clinical TranslationThese findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT.Strengths and LimitationsThis is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo.ConclusionUnirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions.

The translation elongation factor-1, alpha-2 (eEF1A2) plays an important role in protein synthesis. Mutations in this gene have been described in individuals with neurodevelopmental disorders. Here, we silenced the expression of eEFA2 in human SH-SY5Y neuroblastoma cells and observed its roles in neuronal proliferation and differentiation upon induction with retinoic acid.eEF1A2 were silenced using siRNA transfection. Cell proliferation was qualitatively evaluated by Ki-67 immunocytochemistry. Neuronal differentiation was induced with retinoic acid for 3, 5, 7 and 10 days. Neurite length was measured. The expression of microtubule-associated protein 2 (MAP2) was analyzed by western blotting. Tyrosine hydroxylase expression was visualized by immunofluorescence. Cytotoxicity to a neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and western blotting of cleaved caspase-3.eEF1A2 knockdown suppressed the proliferative activity of undifferentiated SH-SY5Y cells as shown by decreased Ki-67 immunostaining. Upon retinoic acid-induction, differentiated neurons with eEF1A2 knockdown exhibited shorter neurite length than untransfected cells, which was associated with the reduction of tyrosine hydroxylase and suppression of MAP2 at 10 days of differentiation. eEF1A2 knockdown decreased the survival of neurons, which was clearly observed in undifferentiated and short-term differentiated cells. Upon treatment with MPP+, cells with eEF1A2 knockdown showed a further reduction in cell survival and an increase of cleaved caspase-3 protein.Our results suggest that eEF1A2 may be required for neuronal proliferation and differentiation of SH-SY5Y cells. Increased cell death susceptibility against MPP+ in eEF1A2-knockdown neurons may imply the neuroprotective role of eEF1A2.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show thatMllt11expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss ofMllt11in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, andMllt11loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENTThe regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

Altres ajuts: acords transformatius de la UAB Peripheral nerve regeneration is limited after injury, especially in humans, due to the large distance the axons have to grow in the limbs. This process is highly dependent on the expression of neuroinflammatory factors produced by macrophages and glial cells. Given the importance of the epigenetic BET proteins on inflammation, we aimed to ascertain if BET inhibition may have an effect on axonal outgrowth. For this purpose, we treated female mice with JQ1 or vehicle after sciatic nerve crush injury and analyzed target reinnervation. We also used dorsal root ganglion (DRG) culture explants to analyze the effects of direct BET inhibition or treatment with conditioned medium from BET-inhibited macrophages. We observed that although JQ1 produced an enhancement of IL-4, IL-13, and GAP43 expression, it did not have an effect on sensory or motor reinnervation after crush injury in vivo. In contrast, JQ1 reduced neurite growth when interacting directly with DRG neurons ex vivo, whereas conditioned medium from JQ1-treated macrophages promoted neurite outgrowth. Therefore, BET-inhibited macrophages secrete pro-regenerative factors that induce neurite outgrowth, and that may counteract the direct inhibition of BET proteins in neurons in vivo. Finally, we observed an activation of the STAT6 pathway in DRG explants treated with conditioned medium from JQ1-treated macrophages. In conclusion, this study demonstrates that BET protein inhibition in macrophages provides a mechanism to enhance axonal outgrowth. However, specific targeting of BET proteins to macrophages will be needed to efficiently enhance functional recovery after nerve injury.

Neural interfaces using biocompatible scaffolds provide crucial properties, such as cell adhesion, structural support, and mass transport, for the functional repair of nerve injuries and neurodegenerative diseases. Neural stimulation has also been found to be effective in promoting neural regeneration. This work provides a generalized strategy to integrate photoacoustic (PA) neural stimulation into hydrogel scaffolds using a nanocomposite hydrogel approach. Specifically, polyethylene glycol (PEG)-functionalized carbon nanotubes (CNT), highly efficient photoacoustic agents, are embedded into silk fibroin to form biocompatible and soft photoacoustic materials. We show that these photoacoustic functional scaffolds enable nongenetic activation of neurons with a spatial precision defined by the area of light illumination, promoting neuron regeneration. These CNT/silk scaffolds offered reliable and repeatable photoacoustic neural stimulation, and 94% of photoacoustic-stimulated neurons exhibit a fluorescence change larger than 10% in calcium imaging in the light-illuminated area. The on-demand photoacoustic stimulation increased neurite outgrowth by 1.74-fold in a rat dorsal root ganglion model, when compared to the unstimulated group. We also confirmed that promoted neurite outgrowth by photoacoustic stimulation is associated with an increased concentration of neurotrophic factor (BDNF). As a multifunctional neural scaffold, CNT/silk scaffolds demonstrated nongenetic PA neural stimulation functions and promoted neurite outgrowth, providing an additional method for nonpharmacological neural regeneration.

Oriented arrays of nanofibers are ubiquitous in nature and have been widely used in recreation of the biological functions such as bone and muscle tissue regenerations. However, it remains a challenge to produce nanofiber arrays with a complex organization by using current fabrication techniques such as electrospinning and extrusion. In this work, we propose a method to fabricate the complex organization of nanofiber structures templated by a spatially varying ordered liquid crystal host, which follows the pattern produced by a maskless projection display system. By programming the synchronization of the rotated polarizer and projected segments with different shapes, various configurations of nanofiber organization ranging from a single to two-dimensional lattice of arbitrary topological defects are created in a deterministic manner. The nanofiber arrays can effectively guide and promote neurite outgrowth. The application of nanofibers with arced profiles and topological defects on neural tissue organization is also demonstrated. This finding, combined with the versatility and programmability of nanofiber structures, suggests that they will help solve challenges in nerve repair, neural regeneration, and other related tissue engineering fields.

We have reported that nicotine has a neurotrophic action on peripheral adrenergic nerves in vivo, which is mediated by α7 nicotinic acetylcholine receptors (nAChRs). To clarify the possible mechanisms, the present study further investigated the effect of nicotine on neurite outgrowth in tyrosine hydroxylase (TH)-positive superior cervical ganglia (SCG) cells isolated from neonatal rats in vitro. Nicotine at low concentrations (0.01–0.3 mM) increased the number of neurite outgrowths in TH-immunopositive SCG cells, while high concentrations of nicotine (1–10 mM) gradually reduced it, and only 10 mM nicotine was markedly inhibited compared to the control. A 100 μM of nicotine-induced increase in neurite numbers depended on the exposure time and was inhibited by treatment with the nAChR antagonist hexamethonium (Hex) and α7 nAChR antagonist α-bungarotoxin (α-Bgtx). The nicotine (10 mM)-induced a significant decrease in neurite outgrowth in SCG, which was perfectly canceled by Hex to the control level but not by α-Bgtx. These results suggest that nicotine has a regulatory neurotrophic action mediated by both α7 nAChR and other subtypes in TH-positive SCG cells of rats.

Acrylamide (AA) is a neurotoxicant that causes synaptic impairment in distal axons. We previously found that developmental exposure to AA decreased proliferation of late-stage neural progenitor cells (NPCs) in the hippocampal neurogenesis of the dentate gyrus (DG) in rats. To investigate whether hippocampal neurogenesis is similarly affected by AA exposure in a general toxicity study, AA was administered to 7-week-old male rats via oral gavage at dosages of 0, 5, 10, and 20 mg/kg for 28 days. In the subgranular zone (SGZ) and granule cell layer, AA decreased the densities of doublecortin-positive

After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication. ispartof: RNA BIOLOGY vol:18 issue:sup2 pages:832-855 ispartof: location:United States status: published

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in interorgan cross talk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin β4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in the plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular cross talk.

A challenge in current stem cell therapies for Parkinson's disease (PD) is controlling neuronal outgrowth from the substantia nigra towards the targeted area where connectivity is required in the striatum. Here we present progress towards controlling directional neurite extensions through the application of iron-oxide magnetic nanoparticles (MNPs) labelled neuronal cells combined with a magnetic array generating large spatially variant field gradients (greater than 20 T m −1 ). We investigated the viability of this approach in both two-dimensional and organotypic brain slice models and validated the observed changes in neurite directionality using mathematical models. Results showed that MNP-labelled cells exhibited a shift in directional neurite outgrowth when cultured in a magnetic field gradient, which broadly agreed with mathematical modelling of the magnetic force gradients and predicted MNP force direction. We translated our approach to an ex vivo rat brain slice where we observed directional neurite outgrowth of transplanted MNP-labelled cells from the substantia nigra towards the striatum. The improved directionality highlights the viability of this approach as a remote-control methodology for the control and manipulation of cellular growth for regenerative medicine applications. This study presents a new tool to overcome challenges faced in the development of new therapies for PD.

The correct wiring of a neural network requires neuron to integrate an incredible repertoire of cues found in their extracellular environment. The astonishing efficiency of this process plays a pivotal role in the correct wiring of the brain during development and axon regeneration. Biologically inspired micro- and nanostructured substrates have been shown to regulate axonal outgrowth. In parallel, several studies investigated graphene's potential as a conductive neural interface, able to enhance cell adhesion, neurite sprouting and outgrowth. Here, we engineered a 3D single- to few-layer fuzzy graphene morphology (3DFG), 3DFG on a collapsed Si nanowire (SiNW) mesh template (NT-3DFGc), and 3DFG on a noncollapsed SiNW mesh template (NT-3DFGnc) as neural-instructive materials. The micrometric protruding features of the NWs templates dictated neuronal growth cone establishment, as well as influencing axon elongation and branching. Furthermore, neurons-to-graphene coupling was investigated with comprehensive view of integrin-mediated contact adhesion points and plasma membrane curvature processes.

Fucosylated chondroitin sulfate (FCS) from the sea cucumber

Mechanical properties of the extracellular environment modulate axon outgrowth. Growth cones at the tip of extending axons generate traction force for axon outgrowth by transmitting the force of actin filament retrograde flow, produced by actomyosin contraction and F-actin polymerization, to adhesive substrates through clutch and cell adhesion molecules. A molecular clutch between the actin filament flow and substrate is proposed to contribute to cellular mechanosensing. However, the molecular identity of the clutch interface responsible for mechanosensitive growth cone advance is unknown. We previously reported that mechanical coupling between actin filament retrograde flow and adhesive substrates through the clutch molecule shootin1a and the cell adhesion molecule L1 generates traction force for axon outgrowth and guidance. Here, we show that cultured mouse hippocampal neurons extend longer axons on stiffer substrates under elastic conditions that correspond to the soft brain environments. We demonstrate that this stiffness-dependent axon outgrowth requires actin-adhesion coupling mediated by shootin1a, L1, and laminin on the substrate. Speckle imaging analyses showed that L1 at the growth cone membrane switches between two adhesive states: L1 that is immobilized and that undergoes retrograde movement on the substrate. The duration of the immobilized phase was longer on stiffer substrates; this was accompanied by increases in actin-adhesion coupling and in the traction force exerted on the substrate. These data suggest that the interaction between L1 and laminin is enhanced on stiffer substrates, thereby promoting force generation for axon outgrowth.

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10−7 M, 10−8 M, and 10−9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10−7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

Iron is an essential cofactor for several metabolic processes, including the generation of ATP in mitochondria, which is required for axonal function and regeneration. However, it is not known how mitochondria in long axons, such as those in sciatic nerves, acquire iron in vivo. Because of their close proximity to axons, Schwann cells are a likely source of iron for axonal mitochondria in the PNS. Here we demonstrate the critical role of iron in promoting neurite growth in vitro using iron chelation. We also show that Schwann cells express the molecular machinery to release iron, namely, the iron exporter, ferroportin (Fpn) and the ferroxidase ceruloplasmin (Cp). In Cp KO mice, Schwann cells accumulate iron because Fpn requires to partner with Cp to export iron. Axons and Schwann cells also express the iron importer transferrin receptor 1 (TfR1), indicating their ability for iron uptake. In teased nerve fibers, Fpn and TfR1 are predominantly localized at the nodes of Ranvier and Schmidt-Lanterman incisures, axonal sites that are in close contact with Schwann cell cytoplasm. We also show that lack of iron export from Schwann cells in Cp KO mice reduces mitochondrial iron in axons as detected by reduction in mitochondrial ferritin, affects localization of axonal mitochondria at the nodes of Ranvier and Schmidt-Lanterman incisures, and impairs axonal regeneration following sciatic nerve injury. These finding suggest that Schwann cells contribute to the delivery of iron to axonal mitochondria, required for proper nerve repair. SIGNIFICANCE STATEMENT This work addresses how and where mitochondria in long axons in peripheral nerves acquire iron. We show that Schwann cells are a likely source as they express the molecular machinery to import iron (transferrin receptor 1), and to export iron (ferroportin and ceruloplasmin [Cp]) to the axonal compartment at the nodes of Ranvier and Schmidt-Lanterman incisures. Cp KO mice, which cannot export iron from Schwann cells, show reduced iron content in axonal mitochondria, along with increased localization of axonal mitochondria at Schmidt-Lanterman incisures and nodes of Ranvier, and impaired sciatic nerve regeneration. Iron chelation in vitro also drastically reduces neurite growth. These data suggest that Schwann cells are likely to contribute iron to axonal mitochondria needed for axon growth and regeneration.

Understanding the molecular mechanism of neuronal differentiation is important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is prerequisite for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the major transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin (mTOR) complex 2 (mTORC2)/Akt signalling to phosphorylate CREB; however, the precise molecular mechanism of this event has not been fully understood. In this manuscript, we show that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases. PP6 knockdown suppressed mTORC2/Akt/CREB signalling and results in failure of neurite outgrowth. SIN1 is a unique component of mTORC2 that enhances mTORC2 activity towards Akt when it is in dephosphorylated form. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signalling.

Isorhamnetin-3

ADP-ribosylation factor 6 (ARF6) is a small GTPase that has a variety of neuronal functions including stimulating neurite outgrowth, a crucial process for the establishment and maintenance of neural connectivity. As impaired and atrophic neurites are often observed in various brain injuries and neurological diseases, understanding the intrinsic pathways that stimulate neurite outgrowth may provide insights into developing strategies to trigger the reconnection of injured neurons. The neuronal adaptor FE65 has been shown to interact with ARF6 and potentiate ARF6-mediated neurite outgrowth. However, the precise mechanism that FE65 activates ARF6 remains unclear, as FE65 does not possess a guanine nucleotide exchange factor (GEF) domain/function. Here, we show that FE65 interacts with the ARF6 GEF, namely the ARF nucleotide-binding site opener (ARNO). Moreover, a complex consisting of ARNO, ARF6 and FE65 is detected. Notably, FE65 potentiates the stimulatory effect of ARNO on ARF6-mediated neurite outgrowth, and the effect of FE65 is abrogated by an FE65 mutation that disrupts FE65–ARNO interaction. Additionally, the intramolecular interaction for mediating the autoinhibited conformation of ARNO is attenuated by FE65. Moreover, FE65 potentiates the effects of wild-type ARNO, but not the monomeric mutant, suggesting an association between FE65 and ARNO dimerization. Collectively, we demonstrate that FE65 binds to and activates ARNO and, consequently, potentiates ARF6-mediated neurite outgrowth.

With no lysine kinase 1 (WNK1) phosphorylates and activates STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1) to regulate ion homeostasis in the kidney. Mutations in WNK1 result in dysregulation of the WNK1-SPAK/OSR1 pathway and cause pseudohypoaldosteronism type II (PHAII), a form of hypertension. WNK1 is also involved in the autosomal recessive neuropathy, hereditary sensory and autonomic neuropathy type II (HSANII). Mutations in a neural-specific splice variant of WNK1 (HSN2) cause HSANII. However, the mechanisms underlying HSN2 regulation in neurons and effects of HSN2 mutants remain unclear. Here, we found that HSN2 regulated neurite outgrowth through OSR1 activation and glycogen synthase kinase 3β (GSK3β). Moreover, HSN2-OSR1 and HSN2-GSK3β signalling induced expression of LIM homeobox 8 (Lhx8), which is a key regulator of cholinergic neural function. The HSN2-OSR1/GSK3β-LHX8 pathway is therefore important for neurite outgrowth. Consistently, HSN2 mutants reported in HSANII patients suppressed SPAK and OSR1 activation and LHX8 induction. Interestingly, HSN2 mutants also suppressed neurite outgrowth by preventing interaction of between wild-type HSN2 and GSK3β. These results indicate that HSN2 mutants cause dysregulation of neurite outgrowth via GSK3β in the HSN2 and/or WNK1 pathways.

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-ssociated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiensspecific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit., Communications Biology, 5 (1), art. no. 935

The functional importance of neuronal differentiation of the transmembrane proteins’ plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

Neurite outgrowth is a fundamental process in neurons that produces extensions and, consequently, neural connectivity. Neurite damage and atrophy are observed in various brain injuries and disorders. Understanding the intrinsic pathways of neurite outgrowth is essential for developing strategies to stimulate neurite regeneration. Insulin is a pivotal hormone in the regulation of glucose homeostasis. There is increasing evidence for the neurotrophic functions of insulin, including the induction of neurite outgrowth. However, the associated mechanism remains elusive. Here, we demonstrate that insulin potentiates neurite outgrowth mediated by the small GTPases ADP-ribosylation factor 6 (ARF6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) through the neuronal adaptor FE65. Moreover, insulin enhances atypical protein kinase Cι/λ (PKCι/λ) activation and FE65 phosphorylation at serine 459 (S459) in neurons and mouse brains. In vitro and cellular assays show that PKCι/λ phosphorylated FE65 at S459. Consistently, insulin potentiates FE65 S459 phosphorylation only in the presence of PKCι/λ. Phosphomimetic studies show that an FE65 S459E mutant potently activates ARF6, Rac1, and neurite outgrowth. Notably, this phosphomimetic mutation enhances the FE65-ARF6 interaction, a process that promotes ARF6-Rac1-mediated neurite outgrowth. Likewise, insulin treatment and PKCι/λ overexpression potentiate the FE65-ARF6 interaction. Conversely, PKCι/λ knockdown suppresses the stimulatory effect of FE65 on ARF6-Rac1-mediated neurite outgrowth. The effect of insulin on neurite outgrowth is also markedly attenuated in PKCι/λ knockdown neurons, in the presence and absence of FE65. Our findings reveal a novel mechanism linking insulin with ARF6-Rac1-dependent neurite extension through the PKCι/λ-mediated phosphorylation of FE65.

Three rare spirocyclohexadienone-type neolignans, magnoflorins A-C (1-3), and three known analogs (4-6), were isolated from the leaves of Magnolia liliiflora. Magnoflorin D (4) was obtained from natural resources for the first time. The chemical structures and absolute configurations of 1-4 were elucidated through detailed analysis of HR-ESI-MS, IR

Protein kinases are responsible for protein phosphorylation and are involved in important signal transduction pathways; however, a considerable number of poorly characterized kinases may be involved in neuronal development. Here, we considered cyclin G-associated kinase (GAK) as a candidate regulator of neurite outgrowth and synaptogenesis by examining the effects of the selective GAK inhibitor SGC-GAK-1. SGC-GAK-1 treatment of cultured neurons reduced neurite length and decreased synapse number and phosphorylation of neurofilament 200-kDa subunits relative to the control. In addition, the related kinase inhibitor erlotinib, which has distinct specificity and potency from SGC-GAK-1, had no effect on neurite growth, unlike SGC-GAK-1. These results suggest that GAK may be physiologically involved in normal neuronal development, and that decreased GAK function and the resultant impaired neurite outgrowth and synaptogenesis may be related to neurodevelopmental disorders.

Objective To investigate the effect of Fasudil on H

It has been suggested that the intelligence quotient of children born to pregnant women taking 1000 mg or more of valproic acid per day is lower than that of children born to pregnant women taking other antiepileptic drugs. However, the mechanism whereby intelligence quotient is decreased in children exposed to valproic acid during the fetal period has not yet been elucidated. Therefore, we used the human neuroblastoma cell line SH-SY5Y to evaluate the effects of antiepileptic drugs containing valproic acid on nerve cells. We assessed the anti-proliferative effects of drugs in these cells via WST-8 colorimetric assay, using the Cell Counting Kit-8. We also quantified drug effects on axonal elongation from images using ImageJ software. We also evaluated drug effects on mRNA expression levels on molecules implicated in nervous system development and folic acid uptake using real-time PCR. We observed that carbamazepine and lamotrigen were toxic to SH-SY5Y cells at concentrations >500 μM. In contrast, phenytoin and valproic acid were not toxic to these cells. Carbamazepine, lamotrigen, phenytoin, and valproic acid did not affect axonal outgrowth in SH-SY5Y cells. Sodium channel neuronal type 1a (SCN1A) mRNA expression-level ratios increased when valproic acid was supplemented to cells. The overexpression of SCN1A mRNA due to high valproic acid concentrations during the fetal period may affect neurodevelopment. However, since detailed mechanisms have not yet been elucidated, it is necessary to evaluate it by comparing cell axon elongation and SCN1A protein expression due to high-concentration valproic acid exposure.

Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons. Supplementary Information The online version contains supplementary material available at 10.1007/s12640-021-00364-8.

Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.

Evidence suggests that n-3 polyunsaturated fatty acids may act as activators of the Nrf2 antioxidant pathway. The antioxidant response, in turn, promotes neuronal differentiation and neurite outgrowth. Nrf2 has recently been suggested to be a cell intrinsic mediator of docosohexanoic acid (DHA) signaling. In the current study, we assessed whether DHA-mediated axodendritic development was dependent on activation of the Nrf2 pathway and whether Nrf2 protected from agrochemical-induced neuritic retraction. Expression profiling of the DHA-enriched Fat-1 mouse brain relative to wild type showed a significant enrichment of genes associated with neuronal development and neuronal projection and genes associated with the Nrf2-transcriptional pathway. Moreover, we found that primary cortical neurons treated with DHA showed a dose-dependent increase in Nrf2 transcriptional activity and Nrf2-target gene expression. DHA-mediated activation of Nrf2 promoted neurite outgrowth and inhibited oxidative stress-induced neuritic retraction evoked by exposure to agrochemicals. Finally, we provide evidence that this effect is largely dependent on induction of the Nrf2-target gene NAD(P)H: (quinone acceptor) oxidoreductase 1 (NQO1), and that silencing of either Nrf2 or Nqo1 blocks the effects of DHA on the axodendritic compartment. Collectively, these data support a role for the Nrf2-NQO1 pathway in DHA-mediated axodendritic development and protection from agrochemical exposure.

Due to the increasing number of drugs and untested environmental compounds introduced into commercial use, there is recognition for a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. One process that has been implicated in neurodevelopment is neurite outgrowth; the disruption of which can result in adverse outcomes that persist later in life. Here, we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. The assay was optimized for use in a 1536-well plate format. Then, we used this assay to screen a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 hours and then treated with compounds at 11 concentrations ranging from 1.56 nM to 92 μM for 24 and 48 hours. Effects of compounds on neurite outgrowth were evaluated by quantifying total neurite length, number of segments and maximum neurite length per cell. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2’,4,4’-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization.

Spinal cord injury (SCI) leads to permanent loss of motor and sensory function due to the complex mechanisms of the external microenvironment and internal neurobiochemistry that restrict neuronal plasticity and axonal regeneration. Chemokine CXCL12 was verified in regulating the development of central nervous system (CNS) and repairing of CNS disease. In the present study, CXCL12 was downregulated in the spinal cord after SCI. SCI also induced gliosis and loss of synapse. Intrathecal treatment of CXCL12 promoted the functional recovery of SCI by inducing the formation of neuronal connections and suppressing glia scar. To confirm whether CXCL12 promoted synapse formation and functional neuronal connections, the primary cortical neurons were treated with CXCL12 peptide, the synapse was examined using Immunofluorescence staining and the function of synapse was tested using a whole-cell patch clamp. The results indicated that CXCL12 peptide promoted axonal elongation, branche formation, dendrite generation and synaptogenesis. The electrophysiological results showed that CXCL12 peptide increased functional connections among neurons. Taken together, the present study illustrated an underlying mechanism of the development of SCI and indicated a potential approach to facilitate functional recovery of spinal cord after SCI.

Although conductive bioelectronic interfaces (BEIs) can allow neural cell culturing while providing electrical stimulation (ES) to the nervous system, there are few simple approaches for the preparation of conductive BEIs with topographical features designed for cell manipulation. In this study, we developed a facile method for fabricating microwrinkled poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) films through spin-coating onto pre-elongated polydimethylsiloxane substrates. The microwrinkles of our PEDOT:PSS films pre-elongated by 20 and 40% had average widths of 6.47 ± 1.49 and 5.39 ± 1.53 μm, respectively. These microwrinkled PEDOT:PSS films promoted the directional ordering of neurite outgrowth of PC12 cells and displayed favorable biocompatibility and outstanding electrochemical properties for long-term ES treatment. When using this BEI platform, the level of PC12 gene expression of Neun was enhanced significantly after 5 days of culturing in differentiation media and under ES, in line with the decreased expression of early phase markers. Therefore, such readily fabricated microwrinkled PEDOT:PSS films are promising candidates for use as BEIs for tissue regenerative medicine.

Separating the immunosuppressive activity of FK506 (1) from its neurotrophic activity is required to develop FK506 analogues as drugs for the treatment of neuronal diseases. Two new FK506 analogues, 9-deoxo-36,37-dihydro-prolylFK506 (2) and 9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506 (3) containing a proline moiety instead of the pipecolate ring at C-1 and modifications at the C-9/C-31 and C-36-C-37 positions, respectively, were biosynthesized, and their biological activities were evaluated. The proline substitution in 9-deoxo-36,37-dihydroFK506 and 9-deoxo-31-O-demethyl-36,37-dihydroFK506 reduced immunosuppressive activity by more than 120-fold, as previously observed. Compared with FK506 (1), 2 and 3 exhibited ∼1.2 × 105- and 2.2 × 105-fold reductions in immunosuppressive activity, respectively, whereas they retained almost identical neurite outgrowth activity. Furthermore, these compounds significantly increased the strength of synaptic transmission, confirming that replacement of the pipecolate ring with a proline is critical to reduce the strong immunosuppressive activity of FK506 (1) while enhancing its neurotrophic activity.

Methylmercury (MeHg) is a chemical substance that causes adverse effects on fetal development. However, the molecular mechanisms by which environmental MeHg affects fetal development have not been clarified. Recently, it has been suggested that the toxic effects of chemicals on fetal development are related alterations in epigenetics, such as DNA methylation and histone modification. In order to analyze the epigenetic effects of low-level MeHg exposure on neuronal development, we evaluated neuronal development both in vivo and in vitro. Pregnant mice (C57BL/6J) were orally administrated 3 mg/kg of MeHg once daily from embryonic day 12-14. Fetuses were removed on embryonic day 19 and brain tissues were collected. LUHMES cells were treated with 1 nM of MeHg for 6 days and collected on the last day of treatment. In both in vivo and in vitro samples, MeHg significantly suppressed neurite outgrowth. Decreased acetylated histone H3 (AcH3) levels and increased histone deacetylase (HDAC) 3 and HDAC6 levels were observed in response to MeHg treatment in both in vivo and in vitro experiments. In addition, increased DNA methylation and DNA methyltransferase 1 (DNMT1) levels were observed in both in vivo and in vitro experiments. The inhibition of neurite outgrowth resulting from MeHg exposure was restored by co-treatment with DNMT inhibitor or HDAC inhibitors. Our results suggest that neurological effects such as reduced neurite outgrowth due to low-level MeHg exposure result from epigenetic changes, including a decrease in AcH3 via increased HDAC levels and an increase in DNA methylation via increased DNMT1 levels.

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntington's disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal length in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases total neuronal length and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance total neuronal length through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

Alzheimer's disease (AD) is an incurable neurodegenerative disease. Repairing damaged nerves and promoting nerve regeneration are key ways to relieve AD symptoms. However, due to the lack of effective strategies to deliver nerve growth factor (NGF) to the brain, achieving neuron regeneration is a major challenge for curing AD. Herein, a ROS-responsive ruthenium nanoplatform (R@NGF-Se-Se-Ru) drug delivery system for AD management by promoting neuron regeneration and Aβ clearance was investigated. Under near-infrared (NIR) irradiation, nanoclusters have good photothermal properties, which can effectively inhibit the aggregation of Aβ and disaggregate Aβ fibrils. Interestingly, the diselenide bond in the nanoclusters is broken, and the nanoclusters are degraded into small ruthenium nanoparticles in the high reactive oxygen species (ROS) environment of the diseased area. Besides, NGF can promote neuronal regeneration and repair damaged nerves. Furthermore, R@NGF-Se-Se-Ru efficiently crosses the blood-brain barrier (BBB) owing to the covalently grafted target peptides of RVG (R). In vivo studies demonstrate that R@NGF-Se-Se-Ru nanoclusters decrease Aβ deposits, inhibit Aβ-induced cytotoxicity, and promote neurite outgrowth. The study confirms that promoting both Aβ clearance and neuron regeneration is an important therapeutic target for anti-AD drugs and provides a novel insight for AD therapy.

To investigate the therapeutic efficacy of Scutellarin (SCU) on neurite growth and neurological functional recovery in neonatal hypoxic-ischemic (HI) rats. Primary cortical neurons were cultured to detect the effect of SCU on cell viability of neurons under oxygen-glucose deprivation (OGD). Double immunofluorescence staining of Tuj1 and TUNEL then observed the neurite growth and cell apoptosis in vitro,and double immunofluorescence staining of NEUN and TUNEL was performed to examine the neuronal apoptosis and cell apoptosis in brain tissues after HI in vivo. Pharmacological efficacy of SCU was also evaluated in HI rats by neurobehavioral tests, triphenyl tetrazolium chloride staining, Hematoxylin and eosin staining and Nissl staining. Astrocytes and microglia expression in damaged brain tissues were detected by immunostaining of GFAP and Iba1. A quantitative real-time polymerase chain reaction and western blot were applied to investigate the genetic expression changes and the protein levels of autophagy-related proteins in the injured cortex and hippocampus after HI. We found that SCU administration preserved cell viability, promoted neurite outgrowth and suppressed apoptosis of neurons subjected to OGD both in vitroand in vivo. Meanwhile, 20 mg/kg SCU treatment improved neurological functions and decreased the expression of astrocytes and microglia in the cortex and hippocampus of HI rats. Additionally, SCU treatment depressed the elevated levels of autophagy-related proteins and the p75 neurotrophin receptor (p75NTR) in both cortex and hippocampus. This study demonstrated the potential therapeutic efficacy of SCU by enhancing neurogenesis and restoring long-term neurological dysfunctions, which might be associated with p75NTR depletion in HI rats. Refereed/Peer-reviewed

Diabetic neuropathy is referred to as a subsequential and debilitating complication belonging to type 1 and type 2 diabetes mellitus. It is a heterogeneous group of disorders with a particularly complex pathophysiology and also includes multiple forms, ranging from normal discomfort to death. The evaluation of diabetic neuropathy is associated with hyperglycemic responses, resulting in an alteration in various metabolic pathways, including protein kinase C pathway, polyol pathway and hexosamine pathway in Schwann and glial cells of neurons. The essential source of neuronal destruction is analogous to these respective metabolic pathways, thus identified as potential therapeutic targets. These pathways regulating therapeutic medications may be used for diabetic neuropathy, however, only target specific drugs could have partial therapeutic activity. Various antidiabetic medications have been approved and marketed, which possess the therapeutic ability to control hyperglycemia and ameliorate the prevalence of diabetic neuropathy. Among all antidiabetic medications, incretin therapy, including Glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors, are the most favorable medications for the management of diabetes mellitus and associated peripheral neuropathic complications. Besides enhancing glucose-evoked insulin release from pancreatic β-cells, these therapeutic agents also play a vital role to facilitate neurite outgrowth and nerve conduction velocity in dorsal root ganglion. Furthermore, incretin therapy also activates cAMP and ERK signalling pathways, resulting in nerve regeneration and repairing. These effects are evidently supported by a series of preclinical data and investigations associated with these medications. However, the literature lacks adequate clinical trial outcomes related to these novel antidiabetic medications. The manuscript emphasizes the pathogenesis, current pharmacological approaches and vivid description of preclinical and clinical data for the effective management of diabetic neuropathy.

We previously reported that 2-O-α-D-glucopyranosyl-6-O-octanoyl-L-ascorbic acid, having a C

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

We recently showed that membrane progesterone receptor α (mPRα/PAQR7) promotes pro-regenerative effects in Schwann cell-like adipose stem cells (SCL-ASC), an alternative model to Schwann cells for the promotion of peripheral nerve regeneration. In this study, we investigated how mPRα activation with the mPR-specific agonist Org OD 02-0 in SCL-ASC affected regenerative parameters in two neuronal cell lines, IMR-32 and SH-SY-5Y. In a series of conditioned medium experiments, we found that mPR activation of SCL-ASC led to increased neurite outgrowth, protection from cell death and increased expression of peripheral nerve regeneration markers (CREB3, ATF3, GAP43) in neuronal cell lines. These effects were stronger than the ones observed with the conditioned medium from untreated SCL-ASC. The addition of Org OD 02-0 to the untreated cell medium mimicked the effects of mPR activation of SCL-ASC on cell death, but not on neurite outgrowth. Therefore, the effect of Org OD 02-0 on neurite outgrowth is SCL-ASC-dependent, while its effect on cell survivability is likely due to the direct activation of mPRs on neuronal cells. SCL-ASC transfection with mPRα siRNA showed that this isoform is responsible for the beneficial effect on neurite outgrowth. Further experiments showed that SCL-ASC-dependent outcomes likely involved the release of BDNF and IGF-2 from these cells. The beneficial mPRα effect on neurite outgrowth was confirmed in co-culture conditions. These findings strengthen the hypothesis that mPRα could play a pro-regenerative role in SCL-ASC and be a therapeutic target for the promotion of peripheral nerve regeneration.

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the

A peripheral nerve injury results in disruption of the fiber that usually protects axons from the surrounding environment. Severed axons from the proximal nerve stump are capable of regenerating, but axons are exposed to a completely new environment. Regeneration recruits cells that produce and deposit key molecules, including growth factor proteins and fibrils in the extracellular matrix (ECM), thus changing the chemical and geometrical environment. The regenerating axons thus surf on a newly remodeled micro-landscape. Strategies to enhance and control axonal regeneration and growth after injury often involve mimicking the extrinsic cues that are found in the natural nerve environment. Indeed, nano- and micropatterned substrates have been generated as tools to guide axons along a defined path. The mechanical cues of the substrate are used as guides to orient growth or change the direction of growth in response to impediments or cell surface topography. However, exactly how axons respond to biophysical information and the dynamics of axonal movement are still poorly understood. Here we use anisotropic, groove-patterned substrate topography to direct and enhance sensory axonal growth of whole mouse dorsal root ganglia (DRG) transplanted ex vivo. Our results show significantly enhanced and directed growth of the DRG sensory fibers on the hemi-3D topographic substrates compared to a 0 nm pitch, flat control surface. By assessing the dynamics of axonal movement in time-lapse microscopy, we found that the enhancement was not due to increases in the speed of axonal growth, but to the efficiency of growth direction, ensuring axons minimize movement in undesired directions. Finally, the directionality of growth was reproduced on topographic patterns fabricated as fully 3D substrates, potentially opening new translational avenues of development incorporating these specific topographic feature sizes in implantable conduits in vivo.

Alzheimer’s disease (AD)—one of the most common neurodegenerative diseases worldwide—impairs cognition, memory, and language ability and causes dementia. However, AD pathogenesis remains poorly elucidated. Recently, a potential link between AD and circular RNAs (circRNAs) has been uncovered, but only a few circRNAs that might be involved in AD have been identified. Here, we systematically investigated circRNAs in the APP/PS1 model mouse brain through deep RNA sequencing. We report that circRNAs are markedly enriched in the brain and that several circRNAs exhibit differential expression between wild-type and APP/PS1 mice. We characterized one abundant circRNA, circTulp4, derived from Intron1 of the gene Tulp4. Our results indicate that circTulp4 predominantly localizes in the nucleus and interacts with U1 small nuclear ribonucleoprotein particle (snRNP) and RNA polymerase II to modulate the transcription of its parental gene, Tulp4, and thereby regulate the function of the nervous system, and might participate in the development of AD.