Inhibition Of Neurite Outgrowth Research Articles

Sensitivity of CNN image analysis to multifaceted measurements of neurite growth

Quantitative analysis of neurite growth and morphology is essential for understanding the determinants of neural development and regeneration, however, it is complicated by the labor-intensive process of measuring diverse parameters of neurite outgrowth. Consequently, automated approaches have been developed to study neurite morphology in a high-throughput and comprehensive manner. These approaches include computer-automated algorithms known as 'convolutional neural networks' (CNNs)—powerful models capable of learning complex tasks without the biases of hand-crafted models. Nevertheless, their complexity often relegates them to functioning as 'black boxes.' Therefore, research in the field of explainable AI is imperative to comprehend the relationship between CNN image analysis output and predefined morphological parameters of neurite growth in order to assess the applicability of these machine learning approaches. In this study, drawing inspiration from the field of automated feature selection, we investigate the correlation between quantified metrics of neurite morphology and the image analysis results from NeuriteNet—a CNN developed to analyze neurite growth. NeuriteNet accurately distinguishes images of neurite growth based on different treatment groups within two separate experimental systems. These systems differentiate between neurons cultured on different substrate conditions and neurons subjected to drug treatment inhibiting neurite outgrowth. By examining the model's function and patterns of activation underlying its classification decisions, we discover that NeuriteNet focuses on aspects of neuron morphology that represent quantifiable metrics distinguishing these groups. Additionally, it incorporates factors that are not encompassed by neuron morphology tracing analyses. NeuriteNet presents a novel tool ideally suited for screening morphological differences in heterogeneous neuron groups while also providing impetus for targeted follow-up studies.

In vitro bioassays for monitoring drinking water quality of tap water, domestic filtration and bottled water

BackgroundLocation-specific patterns of regulated and non-regulated disinfection byproducts (DBPs) were detected in tap water samples of the Barcelona Metropolitan Area. However, it remains unclear if the detected DBPs together with undetected DPBs and organic micropollutants can lead to mixture effects in drinking water.ObjectiveTo evaluate the neurotoxicity, oxidative stress response and cytotoxicity of 42 tap water samples, 6 treated with activated carbon filters, 5 with reverse osmosis and 9 bottled waters. To compare the measured effects of the extracts with the mixture effects predicted from the detected concentrations and the relative effect potencies of the detected DBPs using the mixture model of concentration addition.MethodsMixtures of organic chemicals in water samples were enriched by solid phase extraction and tested for cytotoxicity and neurite outgrowth inhibition in the neuronal cell line SH-SY5Y and for cytotoxicity and oxidative stress response in the AREc32 assay.ResultsUnenriched water did not trigger neurotoxicity or cytotoxicity. After up to 500-fold enrichment, few extracts showed cytotoxicity. Disinfected water showed low neurotoxicity at 20- to 300-fold enrichment and oxidative stress response at 8- to 140-fold enrichment. Non-regulated non-volatile DBPs, particularly (brominated) haloacetonitriles dominated the predicted mixture effects of the detected chemicals and predicted effects agreed with the measured effects. By hierarchical clustering we identified strong geographical patterns in the types of DPBs and their association with effects. Activated carbon filters did not show a consistent reduction of effects but domestic reverse osmosis filters decreased the effect to that of bottled water.Impact statementBioassays are an important complement to chemical analysis of disinfection by-products (DBPs) in drinking water. Comparison of the measured oxidative stress response and mixture effects predicted from the detected chemicals and their relative effect potencies allowed the identification of the forcing agents for the mixture effects, which differed by location but were mainly non-regulated DBPs. This study demonstrates the relevance of non-regulated DBPs from a toxicological perspective. In vitro bioassays, in particular reporter gene assays for oxidative stress response that integrate different reactive toxicity pathways including genotoxicity, may therefore serve as sum parameters for drinking water quality assessment.

Multiple Metabolites Derived from Mushrooms and Their Beneficial Effect on Alzheimer's Diseases.

Mushrooms with edible and medicinal potential have received widespread attention because of their diverse biological functions, nutritional value, and delicious taste, which are closely related to their rich active components. To date, many bioactive substances have been identified and purified from mushrooms, including proteins, carbohydrates, phenols, and vitamins. More importantly, molecules derived from mushrooms show great potential to alleviate the pathological manifestations of Alzheimer's disease (AD), which seriously affects the health of elderly people. Compared with current therapeutic strategies aimed at symptomatic improvement, it is particularly important to identify natural products from resource-rich mushrooms that can modify the progression of AD. This review summarizes recent investigations of multiple constituents (carbohydrates, peptides, phenols, etc.) isolated from mushrooms to combat AD. In addition, the underlying molecular mechanisms of mushroom metabolites against AD are discussed. The various mechanisms involved in the antiAD activities of mushroom metabolites include antioxidant and anti-neuroinflammatory effects, apoptosis inhibition, and stimulation of neurite outgrowth, etc. This information will facilitate the application of mushroom-derived products in the treatment of AD. However, isolation of new metabolites from multiple types of mushrooms and further in vivo exploration of the molecular mechanisms underlying their antiAD effect are still required.

Axonal growth inhibitors and their receptors in spinal cord injury: from biology to clinical translation.

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelin-associated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19 (that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the RhoA/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Abstract 2513: Distinct cancer-intrinsic mechanisms mediate nerve recruitment/outgrowth versus perineural invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) features abundant perineural invasion (PNI). Intra-tumoral nerves play critical roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown—hindering the development of therapies targeting this key pathological process. To address this gap, we performed whole transcriptome digital spatial profiling on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched regions with and without PNI in primary PDAC specimens (n=31 patients) and independently measured gene expression from cancer cells, fibroblasts, and nerves. We undertook a differential gene expression analysis comparing malignant cells in PNI-present and -absent regions. We quantified the growth kinetics of dorsal root ganglia (DRG) sensory neurons cultured with exogenous candidate proteins, which validated that some candidates augment (e.g., Lgals1) and others inhibit (e.g., Sema3b) neurite outgrowth. Next, we mapped our previously discovered malignant cell programs onto the epithelial segments and observed a significant enrichment of the mesenchymal, basal-like, and neural-like progenitor (NRP) programs versus depletion of the classical program in PNI-present regions. To determine the effects of malignant subtype on nerve outgrowth, we engineered isogenic KrasG12D/+;Trp53FL/FL;Rosa26-dCas9-VPR (KP;dCas9-VPR) organoids to overexpress the master transcription factors (TFs) for each malignant subtype (e.g., Gata6 for classical, Glis3 for NRP). We then performed the DRG neuronal outgrowth assay using conditioned media from each subtype-specific organoid line versus an off-target control and observed that the classical line suppressed neurite outgrowth, the mesenchymal and basal-like lines were neutral, and the NRP line enhanced neurite outgrowth dynamics comparable to the Ngf positive control. Taken together, our findings suggest that the mechanisms underlying nerve recruitment/outgrowth and perineural invasion may be partly decoupled. To further test this hypothesis, we are performing transwell invasion assays comparing KP;dCas9-VPR cancer cell lines that overexpress each of the candidate PNI-associated genes and malignant subtype TFs. We anticipate that this study will transform our understanding of how cancer cells and the peripheral nervous system collaborate and guide prioritization for therapeutic intervention in the burgeoning cancer neuroscience field. Citation Format: William L. Hwang, Jennifer Su, Carina Shiau, Peter L. Wang, Jimmy A. Guo, Nicole A. Lester, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Joseph M. Beechem, Andrew J. Aguirre, Lei Zheng, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Distinct cancer-intrinsic mechanisms mediate nerve recruitment/outgrowth versus perineural invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2513.

Nogo-A Mediated Endoplasmic Reticulum Stress During Myocardial Ischemic-Reperfusion Injury in Diabetic Rats.

Among the three isoforms encoded by neurite outgrowth inhibitor proteins has been intensely investigated as a central nervous system inhibitor. Although neurite outgrowth inhibitor protein-A (Nogo-A) expression is increased in plasma of patients who have experienced a coronary heart disease, its role in heart disease is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in diabetic myocardial ischemia reperfusion (MI/R) injury conditions. Accelerated Nogo-A and MI/R injury in diabetic rats was attenuated by tauroursodeoxycholic acid treatment and knockdown of Nogo-A per se is sufficient to decrease endoplasmic reticulum (ER) stress as well as prevents cardiomyocyte apoptosis. We hypothesized that decreased Nogo-A levels might reducing diabetic MI/R injury. Nogo-A interacted with C/EBP homologous protein, suggesting a role for Nogo-A in ER stress during diabetic MI/R. In conclusion, Nogo-A mediated ER stress plays a major role in diabetic MI/R injury, and pathologically altered Nogo-A expression mediates diabetic MI/R injury, suggesting Nogo-A as a novel target for the treatment of diabetic MI/R injury in clinical settings.

Helicobacter pylori outer membrane vesicles induce astrocyte reactivity through nuclear factor-κappa B activation and cause neuronal damage in vivo in a murine model

BackgroundHelicobacter pylori (Hp) infects the stomach of 50% of the world’s population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro.MethodsPurified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability.ResultsUrease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVβ3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons.ConclusionsOMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.

Pyruvate dehydrogenase beta subunit (Pdhb) promotes peripheral axon regeneration by regulating energy supply and gene expression

Key metabolic enzymes not only regulate Glucose, lipid, amino acid metabolism to serve the cellular energy needs, but also modulate noncanonical or nonmetabolic signaling pathway such as gene expression, cell-cycle progression, DNA repair, apoptosis and cell proliferation in regulating the pathologic progression of disease. However, the role of glycometabolism in peripheral nerve axon regeneration is little known. In this study, we investigated the expression of Pyruvate dehydrogenase E1(PDH), a key enzyme linking glycolysis and the tricarboxylic acid (TCA) cycle, with qRT-PCR and found that pyruvate dehydrogenase beta subunit (Pdhb) is up-regulated at the early stage during peripheral nerve injury. The knockdown of Pdhb inhibits neurite outgrowth of primary DRG neurons in vitro and restrains axon regeneration of sciatic nerve after crush injury. Pdhb overexpression promoting axonal regeneration is reversed by knockdown of Monocarboxylate transporter 2(Mct2), a transporter involved in the transport and metabolism of lactate, indicating Pdhb promoting axon regeneration depends on lactate for energy supply. Given the nucleus-localization of Pdhb, further analysis revealed that Pdhb enhances the acetylation of H3K9 and affecting the expression of genes involved in arachidonic acid metabolism and Ras signaling pathway, such as Rsa-14-44 and Pla2g4a, thereby promoting axon regeneration. Collectively, our data indicates that Pdhb is a positive dual modulator of energy generation and gene expression in regulating peripheral axon regeneration.

Nogo-A and LINGO-1: Two Important Targets for Remyelination and Regeneration

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that causes progressive neurological disability in most patients due to neurodegeneration. Activated immune cells infiltrate the CNS, triggering an inflammatory cascade that leads to demyelination and axonal injury. Non-inflammatory mechanisms are also involved in axonal degeneration, although they are not fully elucidated yet. Current therapies focus on immunosuppression; however, no therapies to promote regeneration, myelin repair, or maintenance are currently available. Two different negative regulators of myelination have been proposed as promising targets to induce remyelination and regeneration, namely the Nogo-A and LINGO-1 proteins. Although Nogo-A was first discovered as a potent neurite outgrowth inhibitor in the CNS, it has emerged as a multifunctional protein. It is involved in numerous developmental processes and is necessary for shaping and later maintaining CNS structure and functionality. However, the growth-restricting properties of Nogo-A have negative effects on CNS injury or disease. LINGO-1 is also an inhibitor of neurite outgrowth, axonal regeneration, oligodendrocyte differentiation, and myelin production. Inhibiting the actions of Nogo-A or LINGO-1 promotes remyelination both in vitro and in vivo, while Nogo-A or LINGO-1 antagonists have been suggested as promising therapeutic approaches for demyelinating diseases. In this review, we focus on these two negative regulators of myelination while also providing an overview of the available data on the effects of Nogo-A and LINGO-1 inhibition on oligodendrocyte differentiation and remyelination.

Molecular Characterization and Function of the Nogo-66 Receptor (NgR1) Gene in the Chinese Tree Shrew

Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.

Data-driven approach to integrating genomic and behavioral preclinical traumatic brain injury research.

Understanding recovery from TBI is complex, involving multiple systems and modalities. The current study applied modern data science tools to manage this complexity and harmonize large-scale data to understand relationships between gene expression and behavioral outcomes in a preclinical model of chronic TBI (cTBI). Data collected by the Moody Project for Translational TBI Research included rats with no injury (naïve animals with similar amounts of anesthetic exposure to TBI and sham-injured animals), sham injury, or lateral fluid percussion TBI, followed by recovery periods up to 12 months. Behavioral measures included locomotor coordination (beam balance neuroscore) and memory and cognition assessments (Morris water maze: MWM) at multiple timepoints. Gene arrays were performed using hippocampal and cortical samples to probe 45,610 genes. To reduce the high dimensionality of molecular and behavioral domains and uncover gene-behavior associations, we performed non-linear principal components analyses (NL-PCA), which de-noised the data. Genomic NL-PCA unveiled three interpretable eigengene components (PC2, PC3, and PC4). Ingenuity pathway analysis (IPA) identified the PCs as an integrated stress response (PC2; EIF2-mTOR, corticotropin signaling, etc.), inflammatory factor translation (PC3; PI3K-p70S6K signaling), and neurite growth inhibition (PC4; Rho pathways). Behavioral PCA revealed three principal components reflecting the contribution of MWM overall speed and distance, neuroscore/beam walk, and MWM platform measures. Integrating the genomic and behavioral domains, we then performed a 'meta-PCA' on individual PC scores for each rat from genomic and behavioral PCAs. This meta-PCA uncovered three unique multimodal PCs, characterized by robust associations between inflammatory/stress response and neuroscore/beam walk performance (meta-PC1), stress response and MWM performance (meta-PC2), and stress response and neuroscore/beam walk performance (meta-PC3). Multivariate analysis of variance (MANOVA) on genomic-behavioral meta-PC scores tested separately on cortex and hippocampal samples revealed the main effects of TBI and recovery time. These findings are a proof of concept for the integration of disparate data domains for translational knowledge discovery, harnessing the full syndromic space of TBI.

MS/MS molecular networking-guided in-depth profiling of triterpenoid saponins from the fruit of Eleutherococcus senticosus and their neuroprotectivity evaluation.

Eleutherococcus senticosus fruit (ESF) is a natural health supplement resource that has been extensively applied as a tonic for the nervous system. The structures and neural bioactivities of triterpenoid saponins (TS), which are the major constituents of ESF, have not been comprehensively analyzed thus far. We conducted a complete in-depth MS/MS molecular networking (MN)-based targeted analysis of TS from the crude extract of ESF and investigated its neuroprotective value. An MS/MS MN-guided strategy was used to rapidly present a series of precursor ions (PIs) of TS in a compound cluster as TS-targeted information used in the discovery and characterization of TS. In addition, a prepared TS-rich fraction of ESF was assayed for its restraining effects on β-amyloid-induced inhibition of neurite outgrowth. A total of 87 TS were discovered using a PI tracking strategy, 28 of which were characterized as potentially undescribed structures according to their high-resolution MS values. Furthermore, the TS-rich fraction can significantly reduce β-amyloid-induced damage to neural networks by promoting the outgrowth of neurites and axons. Our findings reveal the richness of TS in ESF and will accelerate their application in the treatment of neurodegenerative diseases.

Chemical chaperones ameliorate neurodegenerative disorders in Derlin-1-deficient mice via improvement of cholesterol biosynthesis

There are no available therapies targeting the underlying molecular mechanisms of neurodegenerative diseases. Although chaperone therapies that alleviate endoplasmic reticulum (ER) stress recently showed promise in the treatment of neurodegenerative diseases, the detailed mechanisms remain unclear. We previously reported that mice with central nervous system-specific deletion of Derlin-1, which encodes an essential component for ER quality control, are useful as models of neurodegenerative diseases such as spinocerebellar degeneration. Cholesterol biosynthesis is essential for brain development, and its disruption inhibits neurite outgrowth, causing brain atrophy. In this study, we report a novel mechanism by which chemical chaperones ameliorate brain atrophy and motor dysfunction. ER stress was induced in the cerebella of Derlin-1 deficiency mice, whereas the administration of a chemical chaperone did not alleviate ER stress. However, chemical chaperone treatment ameliorated cholesterol biosynthesis impairment through SREBP-2 activation and simultaneously relieved brain atrophy and motor dysfunction. Altogether, these findings demonstrate that ER stress may not be the target of action of chaperone therapies and that chemical chaperone-mediated improvement of brain cholesterol biosynthesis is a promising novel therapeutic strategy for neurodegenerative diseases.

Microtubule retrograde flow retains neuronal polarization in a fluctuating state.

In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array has been regarded as stationary. Using live-cell imaging of neurons in culture or in brain slices, combined with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation, thereby inhibiting neurite growth. The motor protein dynein fuels this process. Shortly after axon formation, microtubule retrograde flow slows down in the axon, reducing microtubule density cycles and enabling axon extension. Thus, keeping neurites short is an active process. Microtubule retrograde flow is a previously unknown type of cytoskeletal dynamics, which changes the hitherto axon-centric view of neuronal polarization.

Extracellular calcium induces neurite outgrowth through calcium-sensing receptors in PC12 HS cells

PC12 cells have been used extensively to study neuronal differentiation and neurite outgrowth. However, the effects of extracellular Ca2+ on PC12 cells have not yet been sufficiently evaluated. In this study, we investigated the effect of extracellular calcium using PC12 HS cells, which have high sensitivity to nerve growth factor (NGF). The addition of Ca2+ (1 mM) to the medium induced neurite outgrowth and increased the intracellular calcium concentration. In contrast, the calcium-sensing receptor antagonist NPS2143 suppressed neurite outgrowth and increased the intracellular calcium concentration, suggesting that extracellular calcium promotes neurite outgrowth through calcium-sensing receptors. An increase in extracellular calcium concentration increased phosphorylation of Akt and cyclic AMP response element-binding protein (CREB). Semi-quantitative RT-PCR showed that elevated extracellular calcium increased the mRNA expression of brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor (PDGF), which are regulated by CREB activation. Our results suggest that elevated extracellular calcium can promote the phosphorylation of Akt and CREB through calcium-sensing receptors, resulting in the promotion of neurites. PC12 HS cells are a useful model to investigate the regulation of extracellular calcium via calcium-sensing receptors in neuronal cells.

WNK1/HSN2 mediates neurite outgrowth and differentiation via a OSR1/GSK3β-LHX8 pathway

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.

Myelin-associated glycoprotein alters the neuronal secretome and stimulates the release of TGFβ and proteins that affect neural plasticity.

Myelin-associated glycoprotein (MAG) and Nogo inhibit neurite outgrowth by binding to receptors such as NgR1, PirB and LRP1, and they have also been shown to induce phosphorylation of Smad2, a key intermediate in the transforming growth factor β (TGFβ) signalling pathway. In this study, we determined that MAG and Nogo do not transactivate the TGFβ receptor through their canonical receptors or discoidin domain receptor 1, which we identified as a novel receptor for MAG and Nogo. Instead, MAG and Nogo promoted Smad2 phosphorylation by stimulating secretion of TGFβ. Proteomic analysis of the neuronal secretome revealed that MAG also regulated the secretion of proteins that affect central nervous system plasticity-inducing the secretion of S100A6, septin-7 and neurofascin 186, while inhibiting the secretion of frataxin, MAP6, syntenin-1 and GAP-43. This represents a novel function for MAG that has broad implications for the treatment for spinal cord injury.

GPHR-mediated acidification of the Golgi lumen is essential for cholesterol biosynthesis in the brain.

The Golgi pH regulator (GPHR) is essential for maintaining the function and morphology of the Golgi apparatus through the regulation of luminal acidic pH. Abnormal morphology of the Golgi apparatus is associated with neurodegenerative diseases. Here, we found that knockout of GPHR in the mouse brain led to morphological changes in the Golgi apparatus and neurodegeneration, which included brain atrophy, neuronal cell death, and gliosis. Furthermore, in the GPHR knockout mouse brain, transcriptional activity of sterol regulatory element-binding protein 2 (SREBP2) decreased, resulting in a reduction in cholesterol levels. GPHR-deficient cells exhibited suppressed neurite outgrowth, which was recovered by exogenous expression of the active form of SREBP2. Our results show that GPHR-mediated luminal acidification of the Golgi apparatus maintains proper cholesterol levels and, thereby, neuronal morphology.

Sel1l May Contributes to the Determinants of Neuronal Lineage and Neuronal Maturation Regardless of Hrd1 via Atf6-Sel1l Signaling.

The endoplasmic reticulum (ER) is the primary site of intracellular quality control involved in the recognition and degradation of unfolded proteins. A variety of stresses, including hypoxia and glucose starvation, can lead to accumulation of unfolded proteins triggering the ER-associated degradation (ERAD) pathway. Suppressor Enhancer Lin12/Notch1 Like (Sel1l) acts as a "gate keeper" in the quality control of de novo synthesized proteins and complexes with the ubiquitin ligase Hrd1 in the ER membrane. We previously demonstrated that ER stress-induced aberrant neural stem cell (NSC) differentiation and inhibited neurite outgrowth. Inhibition of neurite outgrowth was associated with increased Hrd1 expression; however, the contribution of Sel1l remained unclear. To investigate whether ER stress is induced during normal neuronal differentiation, we semi-quantitatively evaluated mRNA expression levels of unfolded protein response (UPR)-related genes in P19 embryonic carcinoma cells undergoing neuronal differentiation in vitro. Stimulation with all-trans retinoic acid (ATRA) for 4days induced the upregulation of Nestin and several UPR-related genes (Atf6, Xbp1, Chop, Hrd1, and Sel1l), whereas Atf4 and Grp78/Bip were unchanged. Small-interfering RNA (siRNA)-mediated knockdown of Sel1l uncovered that mRNA levels of the neural progenitor marker Math1 (also known as Atoh1) and the neuronal marker Math3 (also known as Atoh3 and NeuroD4) were significantly suppressed at 4days after ATRA stimulation. Consistent with this result, Sel1l silencing significantly reduced protein levels of immature neuronal marker βIII-tubulin (also known as Tuj-1) at 8days after induction of neuronal differentiation, whereas synaptogenic factors, such as cell adhesion molecule 1 (CADM1) and SH3 and multiple ankyrin repeat domain protein 3 (Shank3) were accumulated in Sel1l silenced cells. These results indicate that neuronal differentiation triggers ER stress and suggest that Sel1l may facilitate neuronal lineage through the regulation of Math1 and Math3 expression.

Adhesion Energy Modulation Acts as an NGF Receptor Activator for Neuronal Differentiation in NGF‐Free Medium

Neuronal repair is promoted after a nerve injury by chemical and physical stimuli from their environment. Among them, local adhesion energy gradients generated on surfaces trigger cone formation and neurite outgrowth without any nerve growth factor (NGF) addition. In this study, the molecular mechanisms leading to neuronal differentiation after stimulation via energy gradients are investigated. For this purpose, PC12 cells are cultured on n-[3-(trimethoxysilyl)propyl] ethylendiamine (EDA) and n-hexyl trimethoxysilane (HTMS), two functionalized surfaces possessing local energy gradients but chemically different. These surfaces similarly trigger neurite and growth cone formation after 3 days in culture. Gene expression of PI3/Akt quantified through a microarray and the action of a specific inhibitor of the corresponding pathway reveal that PI3/Akt signaling pathway is induced and activated on these surfaces in the same way as NGF does after binding on its TrkA receptor. The biological downstream effectors of this signaling pathway are also upregulated, thereby promoting cell survival, growth cone formation, and neurite outgrowth. EDA and HTMS surfaces act as an ongoing stimulus of the TrkA receptor as the addition of 100 nM K252a, its specific inhibitor, leads to the inhibition of neurite growth. Taken together, these results suggest that functionalized surface with energy surface gradients could be useful to promote nerve repair after an injury.