Wnt5a Promotes Axon Elongation in Coordination with the Wnt-Planar Cell Polarity Pathway.

The establishment of neuronal polarity, involving axon specification and outgrowth, is critical to achieve the proper morphology of neurons, which is important for neuronal connectivity and cognitive functions. Extracellular factors, such as Wnts, modulate diverse aspects of neuronal morphology. In particular, non-canonical Wnt5a exhibits differential effects on neurite outgrowth depending upon the context. Thus, the role of Wnt5a in axon outgrowth and neuronal polarization is not completely understood. In this study, we demonstrate that Wnt5a, but not Wnt3a, promotes axon outgrowth in dissociated mouse embryonic cortical neurons and does so in coordination with the core PCP components, Prickle and Vangl. Unexpectedly, exogenous Wnt5a-induced axon outgrowth was dependent on endogenous, neuronal Wnts, as the chemical inhibition of Porcupine using the IWP2- and siRNA-mediated knockdown of either Porcupine or Wntless inhibited Wnt5a-induced elongation. Importantly, delayed treatment with IWP2 did not block Wnt5a-induced elongation, suggesting that endogenous Wnts and Wnt5a act during specific timeframes of neuronal polarization. Wnt5a in fibroblast-conditioned media can associate with small extracellular vesicles (sEVs), and we also show that these Wnt5a-containing sEVs are primarily responsible for inducing axon elongation.

Nogo Receptor Antagonist LOTUS Promotes Neurite Outgrowth through Its Interaction with Teneurin-4.

Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo receptor-1 (NgR1). We previously reported that lateral olfactory tract usher substance (LOTUS) functions as an endogenous antagonist for NgR1 in forming neuronal circuits in the developing brain and improving axonal regeneration in the adult injured CNS. However, another molecular and cellular function of LOTUS remains unknown. In this study, we found that cultured retinal explant neurons extend their neurites on the LOTUS-coating substrate. This action was also observed in cultured retinal explant neurons derived from Ngr1-deficient mouse embryos, indicating that the promoting action of LOTUS on neurite outgrowth may be mediated by unidentified LOTUS-binding protein(s). We therefore screened the binding partner(s) of LOTUS by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis and pull-down assay showed that LOTUS interacts with Teneurin-4 (Ten-4), a cell adhesion molecule. RNAi knockdown of Ten-4 inhibited neurite outgrowth on the LOTUS substrate in retinoic acid (RA)-treated Neuro2A cells. Furthermore, a soluble form of Ten-4 attenuates the promoting action on neurite outgrowth in cultured retinal explant neurons on the LOTUS substrate. These results suggest that LOTUS promotes neurite outgrowth by interacting with Ten-4. Our findings may provide a new molecular mechanism of LOTUS to contribute to neuronal circuit formation in development and to enhance axonal regeneration after CNS injury.

Standardization of a Novel Semi-Automatic Software for Neurite Outgrowth Measurement.

Effective live-imaging techniques are crucial to assess neuronal morphology in order to measure neurite outgrowth in real time. The proper measurement of neurite outgrowth has been a long-standing challenge over the years in the neuroscience research field. This parameter serves as a cornerstone in numerous in vitro experimental setups, ranging from dissociated cultures and organotypic cultures to cell lines. By quantifying the neurite length, it is possible to determine if a specific treatment worked or if axonal regeneration is enhanced in different experimental groups. In this study, the aim is to demonstrate the robustness and accuracy of the Incucyte Neurotrack neurite outgrowth analysis software. This semi-automatic software is available in a time-lapse microscopy system which offers several advantages over commonly used methodologies in the quantification of the neurite length in phase contrast images. The algorithm masks and quantifies several parameters in each image and returns neuronal cell metrics, including neurite length, branch points, cell-body clusters, and cell-body cluster areas. Firstly, we validated the robustness and accuracy of the software by correlating its values with those of the manual NeuronJ, a Fiji plug-in. Secondly, we used the algorithm which is able to work both on phase contrast images as well as on immunocytochemistry images. Using specific neuronal markers, we validated the feasibility of the fluorescence-based neurite outgrowth analysis on sensory neurons in vitro cultures. Additionally, this software can measure neurite length across various seeding conditions, ranging from individual cells to complex neuronal nets. In conclusion, the software provides an innovative and time-effective platform for neurite outgrowth assays, paving the way for faster and more reliable quantifications.

Analysis of Microtubule Polymerization During Axon Outgrowth Using Fluorescently Labeled Microtubule Plus-End Tracking Proteins.

The study of microtubules arrangements and dynamics during axon outgrowth and pathfinding has gained scientific interest during the last decade, and numerous technical resources for its visualization and analysis have been implemented. In this chapter, we describe the cell culture protocols of embryonic cortical and retinal neurons, the methods for transfecting them with fluorescent reporters of microtubule polymerization, and the procedures for time-lapse imaging and quantification in order to study microtubule dynamics during axon morphogenesis.

Preparation of recombinant neuritin protein.

Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli. After optimizing induction conditions and screening for high expression, a neuritin recombinant protein with purity exceeding 85 % was obtained through Ni-affinity chromatography. Subsequently, unlabeled neuritin with a molecular weight of 11 kDa was obtained through the enzymatic cleavage of the His label using an enterokinase. Furthermore, a neuritin recombinant protein with purity exceeding 95 % was obtained using gel chromatography. Functional investigations revealed that neurite outgrowth of PC12 cells was stimulated by the isolated neuritin. This study establishes a method to obtain active and unlabeled neuritin protein, providing a foundation for subsequent research on its biological functions.

l-DOPA receptor GPR143 inhibits neurite outgrowth via L-type calcium channels in PC12 cells.

The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s).

Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.

Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT7 Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells.

There is some evidence that the serotonin receptor subtype 7 (5-HT7) could be new therapeutic target for neuroprotection. The aim of this study was to compare the neuroprotective and neurite outgrowth potential of new 5-HT7 receptor agonists (AH-494, AGH-238, AGH-194) with 5-CT (5-carboxyamidotryptamine) in human neuroblastoma SH-SY5Y cells. The results revealed that 5-HT7 mRNA expression was significantly higher in retinoic acid (RA)-differentiated cells when compared to undifferentiated ones and it was higher in cell cultured in neuroblastoma experimental medium (DMEM) compared to those placed in neuronal (NB) medium. Furthermore, the safety profile of compounds was favorable for all tested compounds at concentration used for neuroprotection evaluation (up to 1 µM), whereas at higher concentrations (above 10 µM) the one of the tested compounds, AGH-194 appeared to be cytotoxic. While we observed relatively modest protective effects of 5-CT and AH-494 in UN-SH-SY5Y cells cultured in DMEM, in UN-SH-SY5Y cells cultured in NB medium we found a significant reduction of H2O2-evoked cell damage by all tested 5-HT7 agonists. However, 5-HT7-mediated neuroprotection was not associated with inhibition of caspase-3 activity and was not observed in RA-SH-SY5Y cells exposed to H2O2. Furthermore, none of the tested 5-HT7 agonists altered the damage induced by 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium ion (MPP +) and doxorubicin (Dox) in UN- and RA-SH-SY5Y cells cultured in NB. Finally we showed a stimulating effect of AH-494 and AGH-194 on neurite outgrowth. The obtained results provide insight into neuroprotective and neurite outgrowth potential of new 5-HT7 agonists.

Calycosin promotes axon growth by inhibiting PTPRS and alleviates spinal cord injury.

Our former studies have identified the alleviating effect of Calycosin (CA) on spinal cord injury (SCI). In this study, our purpose is to explore the influence of CA on SCI from the perspective of promoting axon growth. The SCI animal model was constructed by spinal cord compression, wherein rat primary cortex neuronal isolation was performed, and the axonal growth restriction cell model was established via chondroitin sulfate proteoglycan (CSPG) treatment. The expressions of axon regeneration markers were measured via immunofluorescent staining and western blot, and the direct target of CA was examined using silver staining. Finally, the expression of the protein tyrosine phosphatase receptor type S (PTPRS) was assessed using western blot. CA treatment increased neuronal process outgrowth and the expressions of axon regeneration markers, such as neurofilament H (NF-H), vesicular glutamate transporter 1 (vGlut1), and synaptophysin (Syn) in both SCI model rats and CSPG-treated primary cortical neurons, and PTPRS levels were elevated after SCI induction. In addition, PTPRS was the direct target of CA, and according to in vivo findings, exposure to CA reduced the PTPRS content. Furthermore, PTPRS overexpression inhibited CA's enhancement of axon regeneration marker content and neuronal axon lengths. CA improves SCI by increasing axon development through regulating PTPRS expression.

Characterization of HSP90 expression and function following CNS injury.

Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair.

Identification of Proteolysis Targeting Chimeras (PROTACs) for Lysine Demethylase 5 and Their Neurite Outgrowth-Promoting Activity.

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

BACKGROUND: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). METHODS: Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). RESULTS: Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. CONCLUSIONS: Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Neurite outgrowth promotion in PC12 cells by 24(R)-ethyllophenol from Morus alba.

We examined the mechanism by which 24(R)-ethyllophenol (MAB28) isolated from the branches of Morus alba caused neurite outgrowth in rat pheochromocytoma cells (PC12). MAB28 significantly promoted neurite outgrowth to a similar degree as the positive control, nerve growth factor (NGF). After incubation with MAB28 in PC12 cells, phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and cyclic AMP response element-binding protein was detected, but the time course of phosphorylation was different from that induced by NGF. The expression of chloride intracellular channel protein 3 (CLIC3) was significantly decreased by MAB28. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), an outward rectifying chloride channel inhibitor, significantly promoted neurite outgrowth in PC12 cells. These data suggested that MAB28 could induce neurite outgrowth by downregulating CLIC3 expression.

Asporin and CD109, expressed in the injured neonatal spinal cord, attenuate axonal re-growth in vitro.

Axonal regeneration is restricted in adults and causes irreversible motor dysfunction following spinal cord injury (SCI). In contrast, neonates have prominent regenerative potential and can restore their neural function. Although the distinct cellular responses in neonates have been studied, how they contribute to neural recovery remains unclear. To assess whether the secreted molecules in neonatal SCI can enhance neural regeneration, we re-analyzed the previously performed single-nucleus RNA-seq (snRNA-seq) and focused on Asporin and Cd109, the highly expressed genes in the injured neonatal spinal cord. In the present study, we showed that both these molecules were expressed in the injured spinal cords of adults and neonates. We treated the cortical neurons with recombinant Asporin or CD109 to observe their direct effects on neurons in vitro. We demonstrated that these molecules enhance neurite outgrowth in neurons. However, these molecules did not enhance re-growth of severed axons. Our results suggest that Asporin and CD109 influence neurites at the lesion site, rather than promoting axon regeneration, to restore neural function in neonates after SCI.

Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

The dynamic TRPV2 ion channel proximity proteome reveals functional links of calcium flux with cellular adhesion factors NCAM and L1CAM in neurite outgrowth.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

Thrombopoietin exerts a neuroprotective effect by inhibiting the suppression of neuronal proliferation and axonal outgrowth in intrauterine growth restriction rats.

Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but the mechanism is unclear. In this study, we analyzed the structure of the cerebral cortex using IUGR model rats generated through a reduced uterine perfusion pressure operation. IUGR rats exhibited thinner cerebral white matter and enlarged lateral ventricles compared with control rats. Expression of neuron cell markers, Satb2, microtubule-associated protein (MAP)-2, α-tubulin, and nestin was reduced in IUGR rats, indicating that neurons were diminished at various developmental stages in IUGR rats, from neural stem cells to mature neurons. However, there was no increase in apoptosis in IUGR rats. Cells positive for Ki67, a marker of cell proliferation, were reduced in neurons and all glial cells of IUGR rats. In primary neuron cultures, axonal elongation was impaired under hypoxic culture conditions mimicking the intrauterine environment of IUGR infants. Thus, in IUGR rats, chronic hypoxia in utero suppresses the proliferation of neurons and glial cells as well as axonal elongation, resulting in cortical thinning and enlarged lateral ventricles. Thrombopoietin (TPO), a platelet growth factor, inhibited the decrease in neuron number and promoted axon elongation in primary neurons under hypoxic conditions. Intraperitoneal administration of TPO to IUGR rats resulted in increases in the number of NeuN-positive cells and the area coverage of Satb2. In conclusion, suppression of neuronal proliferation and axonal outgrowth in IUGR rats resulted in cortical thinning and enlargement of lateral ventricles. TPO administration might be a novel therapeutic strategy for treating brain dysmaturation in IUGR infants.

Promotion of neurite outgrowth by 3,5,7,3',4'-pentamethoxyflavone is mediated through ERK signaling pathway in Neuro2a cells.

In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.

Natural products from Odontonema strictum promote neurite outgrowth in neuronal PC12 cells.

The leaves of Odontonema strictum, a tropical plant used for its antihypertensive properties, are rich in nutrients and biologically active phytochemicals, such as ß-sitosterol, stigmasterol, umuravumbolide, deacetylumuravumbolide, dideacetylboronolide, deacetylboronolide, verbascoside, and isoverbascoside. In addition, its roots are rich in ß-sitosterol, stigmasterol, and the iridoid glycoside ß-O-methyl-unedoside. Ingestion of the roots was reported to have a sedative effect in a dog was previously reported on a dog eating the roots of this plant. In the present study, we report for the first time the cell proliferation- and neurite outgrowth-promoting effects in PC12 neuronal cells of the isolated organic compounds and crude extracts from O. strictum. Pituitary adenylate cyclase-activating peptide (PACAP) and quercetin were used as positive controls. At the concentration of 0.2 µg/mL, ß-sitosterol was more potent than quercetin and displayed the same activity (>45 µm/cell) as PACAP (100 nM). At a low concentration (0.04 µg/mL), verbascoside and isoverbascoside showed the strongest neurite outgrowth-promoting effect (neurite length of 30 to 35 µm/cell). Our results indicate that phytomedicines made from O. strictum may be useful in preventing neurodegenerative diseases.