Inhibition Of Outgrowth Research Articles

Exploiting a tumor softening targeted bomb for mechanical gene therapy of chemoresistant Triple-Negative breast cancer

Triple-negative breast cancer (TNBC)-the most aggressive and refractory subtype of breast cancer-plagues many patients. The tumor physical microenvironment (TPME) is characterized by altered stromal composition and extracellular matrix (ECM), which may contribute to tissue stiffness and compromise therapeutic efficacy. Here, based on clinical data analysis and preclinical model validation, we discover that chemoresistant TNBC tends to have a stiffer TPME with a more aggressive genomic landscape, in which lysyl oxidase (LOX) exerts pivotal effects. Anlotinib (An), a multi-targeted tyrosine kinase inhibitor, has great promising potential in the treatment of TNBC. In this regard, we developed a tumor-softening nanobomb (An&siLOX@NPs) that remodels TPME by co-delivering small interfering siRNAs silencing LOX and the anti-angiogenic drug, anlotinib (An). The improved cytosolic delivery of siLOX enables TPME remodeling via reducing deposition and cross-linking between collagen and fibronectin, thereby promoting enrichment of An to improve its therapeutic outcomes. In chemoresistant TNBC, we confirm the An&siLOX@NPs nanobomb-mediated mechanical gene therapy results in obvious inhibition of local tumor outgrowth and prolongs survival. Additionally, softened TPME reactivates intratumoral immune milieu to bolster sensitivity to anti-PD-L1 immunotherapy, completely curing 33.3% of the metastatic tumors, further generating a synergistic anti-tumor efficacy. Collectively, our mechanical gene therapy strategy represents a highly efficient, safe, and versatile anti-drug resistance modality, which not only can solve the snag problem of TNBC treatment but also provides a new demonstration for other tumor treatments.

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).

Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Abstract 7260: An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance

Abstract Clinical use of recombinant interleukin (IL)-12 and -2 therapy has been hindered due to unfavorable kinetics and toxicity associated with systemic exposure. Durable targeted delivery to the tumor microenvironment (TME) is a promising therapeutic strategy to overcome these clinical limitations, prompting the development of KB707, a replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding human IL-12 and -2, for the treatment of solid tumors. Prior studies in mice demonstrated that local vector-encoded cytokine administration enhanced murine IL-12/IL-2 expression while limiting systemic exposure compared to conventional protein therapy. In a syngeneic, B16-F10 melanoma mouse model, intratumoral murine KB707-equivalent treatment led to improved survival in a unilateral tumor model and an abscopal effect in a bilateral tumor model. Additionally, the majority of previously treated animals survived rechallenge with B16-F10 in the absence of further therapy, indicative of systemic anti-tumor immune memory. Together, these results suggested that vector-mediated delivery of IL-12 and -2 could elicit both local and systemic anti-tumor immune responses. To elucidate the underlying changes to the immune landscape, B16-F10 tumor-bearing animals were treated intratumorally with vector or vehicle control on day 0 and were sacrificed on day 7; a second cohort of tumor-bearing animals received two vector doses (days 0 and 7) and were sacrificed on days 8, 10, 12, or 14. Immune cell profiling of samples taken from the tumor, tumor draining lymph node (dLN), spleen, and whole blood were analyzed by flow cytometry, and serum cytokine analysis was conducted by multiplex ELISA. Flow cytometric analysis of tumors revealed that vector treatment resulted in increased frequencies of proliferating and activated effector CD8+ and CD4+ T cells, peaking 24 hours post-second dose. These results correlated with high levels of the proinflammatory cytokines IFNγ and TNFα in the serum of vector recipient animals. The blood, spleens, and dLNs also showed increased frequencies of activated effector CD8+ and CD4+ T cells, peaking 24-72 hours post-second dose. Interestingly, although IL-2 alone has been shown to promote immune suppression and tumor outgrowth by mediating regulatory T cell (Treg) expansion and infiltration in certain contexts, the frequency of Tregs in treated vs. control tumors was substantially reduced after first and repeat dosing. Together, these results support a mechanism by which vector-driven local expression of IL-12 and IL-2 can expand and activate effector T cells both in the TME and systemically while simultaneously repressing Tregs, consistent with the previously described inhibition of primary tumor outgrowth, abscopal effects on secondary tumors, and prevention of recurrence observed with this therapeutic approach. Citation Format: Dana M. Previte, Trevor J. Parry, Suma M. Krishnan. An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7260.

Abstract A074: Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages

Abstract Metastatic breast cancer can recur years after primary tumor resection and adjuvant therapy and appears to arise from quiescent disseminated tumor cells (QDTC) that persist at distant sites and resist conventional therapies. To date there are no treatments to target QDTCs. Previously, the fibrotic-like microenvironment enriched with Type I collagen (Col-I) was shown to be required for the switch of QDTC to metastatic outgrowth in lungs of mice. Here, we examined whether soluble mediators secreted by ex-vivo generated pro-resolving CD11blow macrophages (CM-Mres) will reinstate resolution of inflammation thus inhibiting the establishment of the fibrotic-like-niche and in turn prevent the emergence of QDTC to metastatic outgrowth. Our findings indicate that CM-Mres promoted immune silencing of alveolar macrophages at the metastatic site where dormant DTCs reside. Immune silencing is one of the hallmarks of resolution of inflammation. Furthermore, CM-Mres inhibited the establishment of a fibrotic-like niche resulting in the inhibition of the metastatic outgrowth in vitro and in vivo. This was due to inhibition of fibroblasts differentiation to myofibroblasts independent of TGFbeta1 canonical signaling and abolishment of Col-I expression. Furthermore, CM-Mres deactivated myofibroblasts as part of the resolution process by inducing an increase in reactive oxygen species (ROS) via activation of NADPH oxidase. This in turn induced DNA damage leading to Go/G1 arrest and intrinsic apoptosis. Similarly, apoptosis mediated by oxidative stress was induced by CM-Mres in dormant and outbreaking QDTCs. Taken together our results demonstrate for the first time that by reinstating resolution of inflammation via physiological mediators, secreted by pro-resolving macrophages, can prevent metastatic outgrowth of QDTCs at their permissive site. Since conventional therapies fail to eradicate dormant DTCs and their transition to clinical metastasis, our findings hold great promise in the quest to identify novel strategies to prevent or treat recurrent metastatic disease. Citation Format: Odelya Gilon, Keren Weidenfeld, Hadell Samara, Yonatan Feuermann, Sagie Schif-Zuck, Sergei Butenko, Edmond Sabo, Amiram Ariel, Dalit Barkan. Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A074.

Neuroprotective effect of olive leaf extract in the mouse neuroblastoma cell line (NB2a): A good model to evaluate the neuroprotective effects of natural products

Chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, whose prevalence increases with aging, cause serious health problems with inadequate treatment options. Long-term exposure to low doses of pesticides like chlorpyrifos has been linked to an increased risk of developing neurodegenerative diseases. This study assessed the effects of olive leaf extract (OLE) on cell viability and neurite outgrowth inhibition induced by chlorpyrifos (25 µM) using the mouse NB2a neuroblastoma cell line. High concentrations of OLE as 100 to 3000 µg/ml reduced cell viability, likely due to its anticancer properties. On the other hand, lower concentrations of OLE as 3, 10, 30 and 100 µg/ml significantly reversed the inhibition of neurite outgrowth caused by chlorpyrifos exposure, demonstrating a neuroprotective effect. In conclusion, the regular consumption of OLE may effectively slow or prevent the advancement of neurodegenerative diseases linked to long-term exposure to low-dose organophosphate pesticides. Furthermore, the chlorpyrifos-induced moderate neurotoxicity model can be employed as a valuable screening method for evaluating the neuroprotective potential of natural products, including various plant extracts.

Phosphate Transporter OsPT4, Ubiquitinated by E3 Ligase OsAIRP2, Plays a Crucial Role in Phosphorus and Nitrogen Translocation and Consumption in Germinating Seed

Phosphorus (P) and nitrogen (N) are essential macronutrients necessary for plant growth and development. OsPT4 is a high-affinity phosphate (Pi) transporter that has a positive impact on nutrient uptake and seed development. In this study, the expression patterns of different Pi transporter genes in germinating seeds were determined, and the relative expression of OsPT4 was induced in Pi-deficient seeds and gradually increased with the passage of germination time. The analysis of P, N, Pi, and amino acid concentrations in germinating seeds of OsPT4 mutants showed that the OsPT4 mutation caused P and N retention and a continuous reduction in multiple amino acid concentrations in germinating seeds. Transcriptome analysis and qRT-PCR results also indicated that the OsPT4 mutation inhibits the expression of genes related to P and N transportation and amino acid synthesis in germinating seeds. In addition, the paraffin section and TUNEL assay of OsPT4 mutant germinating seeds suggests that OsPT4 mutation causes programmed cell death (PCD) delayed in the aleurone layer and inhibition of leaf outgrowth. Moreover, we also found that OsPT4 was ubiquitinated by OsAIRP2, which is a C3HC4-type RING E3 Ub ligase. Our studies illustrate that OsPT4 plays a crucial role in P and N collaborative translocation and consumption in germinating seeds. It also provides a theoretical basis for the molecules and physiological mechanisms of P and N cross-talk under suppressed Pi uptake conditions.

Heat shock factor 1 promotes neurite outgrowth and suppresses inflammation in the severed spinal cord of geckos.

The low intrinsic growth capacity of neurons and an injury-induced inhibitory milieu are major contributors to the failure of sensory and motor functional recovery following spinal cord injury. Heat shock transcription factor 1 (HSF1), a master regulator of the heat shock response, plays neurogenetic and neuroprotective roles in the damaged or diseased central nervous system. However, the underlying mechanism has not been fully elucidated. In the present study, we used a gecko model of spontaneous nerve regeneration to investigate the potential roles of gecko HSF1 (gHSF1) in the regulation of neurite outgrowth and inflammatory inhibition of macrophages following spinal cord injury. gHSF1 expression in neurons and microglia at the lesion site increased dramatically immediately after tail amputation. gHSF1 overexpression in gecko primary neurons significantly promoted axonal growth by suppressing the expression of suppressor of cytokine signaling-3, and facilitated neuronal survival via activation of the mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases and phosphatidylinositol 3-kinase/protein kinase B pathways. Furthermore, gHSF1 efficiently inhibited the macrophage-mediated inflammatory response by inactivating IkappaB-alpha/NF-kappaB signaling. Our findings show that HSF1 plays dual roles in promoting axonal regrowth and inhibiting leukocyte inflammation, and provide new avenues of investigation for promoting spinal cord injury repair in mammals.

BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

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.

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.

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.

Monitoring Mixture Effects of Neurotoxicants in Surface Water and Wastewater Treatment Plant Effluents with Neurite Outgrowth Inhibition in SH-SY5Y Cells.

Cell-based assays covering environmentally relevant modes of action are widely used for water quality monitoring. However, no high-throughput assays are available for testing developmental neurotoxicity of water samples. We implemented an assay that quantifies neurite outgrowth, which is one of the neurodevelopmental key events, and cell viability in human neuroblastoma SH-SY5Y cells using imaging techniques. We used this assay for testing of extracts of surface water collected in agricultural areas during rain events and effluents from wastewater treatment plants (WWTPs), where more than 200 chemicals had been quantified. Forty-one chemicals were tested individually that were suspected to contribute to the mixture effects among the detected chemicals in environmental samples. Sample sensitivity distributions indicated higher neurotoxicity for surface water samples than for effluents, and the endpoint of neurite outgrowth inhibition was six times more sensitive than cytotoxicity in the surface water samples and only three times more sensitive in the effluent samples. Eight environmental pollutants showed high specificity, and those ranged from pharmaceuticals (mebendazole and verapamil) to pesticides (methiocarb and clomazone), biocides (1,2-benzisothiazolin-3-one), and industrial chemicals (N-methyl-2-pyrrolidone, 7-diethylamino-4-methylcoumarin, and 2-(4-morpholinyl)benzothiazole). Although neurotoxic effects were newly detected for some of our test chemicals, less than 1% of the measured effects were explained by the detected and toxicologically characterized chemicals. The neurotoxicity assay was benchmarked against other bioassays: activations of the aryl hydrocarbon receptor and the peroxisome proliferator-activated receptor were similar in sensitivity, highly sensitive and did not differ much between the two water types, with surface water having slightly higher effects than the WWTP effluent. Oxidative stress response mirrored neurotoxicity quite well but was caused by different chemicals in the two water types. Overall, the new cell-based neurotoxicity assay is a valuable complement to the existing battery of effect-based monitoring tools.

Melatonin Mediates Axillary Bud Outgrowth by Improving Nitrogen Assimilation and Transport in Rice.

Melatonin plays an important role in plant resistance to biotic and abiotic stresses. However, whether melatonin is involved in the regulation of plant architecture, such as the formation of axillary bud outgrowth or tillering, in rice remains unknown. Here, we found that different concentrations of melatonin influenced axillary bud outgrowth in rice, and moderate melatonin concentrations also alleviated the inhibition of axillary bud outgrowth in the presence of high concentrations of basic amino acids lysine and arginine. Furthermore, transcriptome analysis demonstrated that genes involved in nitrogen metabolism and phytohormone signal transduction pathways may affect axillary bud outgrowth, which is regulated by melatonin. We determined that the differentially expressed genes glutamine synthetase OsGS2 and amino acid transporter OsAAP14, which are involved in nitrogen metabolism and are regulated by melatonin and basic amino acids, were the key regulators of axillary bud outgrowth in rice. In addition, we validated the functions of OsGS2 and OsAAP14 using rice transgenic plants with altered axillary bud outgrowth and tillers. Taken together, these results suggest that melatonin mediates axillary bud outgrowth by improving nitrogen assimilation and transport in rice.

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14–22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Water Treadmill Training Ameliorates Neurite Outgrowth Inhibition Associated with NGR/RhoA/ROCK by Inhibiting Astrocyte Activation following Spinal Cord Injury.

Spinal cord injury (SCI) often results in damage to or degeneration of axons. Crosstalk between astrocytes and neurons plays a pivotal role in neurite outgrowth following SCI. Rehabilitative training is a recognized method for the treatment of SCI, but the specific mechanism underlying its effect on axonal outgrowth in the central nervous system (CNS) has not yet been determined. A total of 190 adult male SD rats weighing 200–250 g were randomly divided into eight groups for use as animal models of SCI. Rats were subjected to water treadmill training (TT) for 7 or 14 d. The Basso-Beattie-Bresnahan (BBB) motor function scale, hematoxylin-eosin (HE) staining, Nissl staining, Western blotting, and immunofluorescence were used to measure tissue morphology and the degree of neurological deficit and to determine quantitative expression and accurate localization of the corresponding proteins. We found that TT decreased tissue structure damage and improved functional recovery. TT also promoted the regeneration of neurons and reduced SCI-induced apoptosis SCI around the lesion, as well as significantly increasing the expression of GAP43 and NF200 after SCI. In addition, TT significantly inhibited the injury-induced increase in the expression of proinflammatory factors. Moreover, TT reduced the activation of astrocytes and microglia, accompanied by the reduced expression of C3d and increased expression of S100A10. Finally, TT effectively reduced the level of chondroitin sulfate proteoglycan (CSPG) surrounding the lesion and inhibited the NGR/RhoA/ROCK signaling pathway in neurons after SCI. Overall, we found that TT played a novel role in recovery from SCI by promoting axonal outgrowth associated with NGR/RhoA/ROCK signaling by inhibiting astrocyte activation after SCI.

Glycan-binding Properties of Basic Whey Protein Lactoferrin and Its Application in Nerve Regenerative Medicine

The basic whey protein lactoferrin (LF) is a glycosaminoglycan (GAG)-binding protein that plays a role in innate immunity and is expected to be a potential biopharmaceutical because of its diverse functions. LF binds to heparan sulfate, a type of GAG on the cell surface, and is involved in neuroprotective and anti-cancer activities. Recently, it was found that human LF (hLF) binds to chondroitin sulfate E (CS-E), which inhibits neuronal axon outgrowth and neutralizes CS-E-induced axon outgrowth inhibition. LFs are potential therapeutic targets for GAG-related diseases, such as cancer, neurodegenerative diseases, and infectious diseases. This article outlines the general properties of LF and discusses the binding properties of hLF to CS-E and its application.

Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.