Epoxiconazole (EPX) is a very effective fungicide of the triazole family. Given its wide spectrum of use, the increased application of this pesticide may represent a serious risk on human health. ...Previous studies have found that EPX is cytotoxic to cells, although the exact mechanism remains elusive. In particular, the effect on the nervous system is poorly elucidated. Here we evaluated the implication of oxidative stress in the neurotoxicity and studied its apoptotic mechanism of action. We demonstrated that the treatment by EPX reduces the viability of cells in a dose dependent manner with an IC50 of 50 μM. It also provokes the reduction of cell proliferation. EPX could trigger arrest in G1/S phase of cell cycle with low doses, however with IC50, it induced an accumulation of F98 cells in G2/M phase. Moreover, EPX induced cytoskeleton disruption as evidenced by immunocytochemical analysis. It provoked also DNA fragmentation in a concentration dependent manner. The EPX induced apoptosis, which was observed by morphological changes and by positive Annexin V FITC-PI staining concurrent with a depolarization of mitochondria. Furthermore, the cell mortality provoked by EPX was significantly reduced by pretreatment with Z-VAD-FMK, a caspase inhibitor. Moreover, N-acetylcysteine (NAC) strongly restores cell viability that has been inhibited by EPX. The results of these findings highlight the implication of ROS generation in the neurotoxicity induced by EPX, indicating that the production of ROS is the main cause of the induction of apoptosis probably via the mitochondrial pathway.
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•EPX induces cytotoxic effects in F98 glioma cells.•EPX produces cell cycle arrest, cytoskeleton disruption, DNA damage.•EPX affects mitochondrial function and induces apoptosis via caspases dependent signaling pathway in F98 cells.•EPX provokes ROS generation and lipid peroxidation.
The replacement of injured neurons by the selective stimulation of neural stem cells in situ represents a potential therapeutic strategy for the treatment of neurodegenerative diseases. The peptide ...NFL-TBS.40-63 showed specific interactions towards neural stem cells of the subventricular zone. The aim of our work was to produce a NFL-based drug delivery system able to target neural stem cells through the selective affinity between the peptide and these cells. NFL-TBS.40-63 (NFL) was adsorbed on lipid nanocapsules (LNC) whom targeting efficiency was evaluated on neural stem cells from the subventricular zone (brain) and from the central canal (spinal cord). NFL-LNC were incubated with primary neural stem cells in vitro or injected in vivo in adult rat brain (right lateral ventricle) or spinal cord (T10). NFL-LNC interactions with neural stem cells were different depending on the origin of the cells. NFL-LNC showed a preferential uptake by neural stem cells from the brain, while they did not interact with neural stem cells from the spinal cord. The results obtained in vivo correlate with the results observed in vitro, demonstrating that NFL-LNC represent a promising therapeutic strategy to selectively deliver bioactive molecules to brain neural stem cells.
The drug delivery system NFL-LNC is able to target in vitro and in vivo neural stem cells of the subventricular zone of the brain. Display omitted
Abstract The discovery of adult neurogenesis drastically changed the therapeutic approaches of central nervous system regenerative medicine. The stimulation of this physiologic process can increase ...memory and motor performances in patients affected by neurodegenerative diseases. Neural stem cells contribute to the neurogenesis process through their differentiation into specialized neuronal cells. In this review, we describe the most important methods developed to restore neurological functions via neural stem cell differentiation. In particular, we focused on the role of nanomedicine. The application of nanostructured scaffolds, nanoparticulate drug delivery systems, and nanotechnology-based real-time imaging has significantly improved the safety and the efficacy of neural stem cell-based treatments. This review provides a comprehensive background on the contribution of nanomedicine to the modulation of neurogenesis via neural stem cell differentiation.
Regenerative medicine is a promising approach to treat neurodegenerative diseases by replacing degenerating cells like neurons or oligodendrocytes. Targeting human neural stem cells directly in the ...brain is a big challenge in such a strategy. The neurofilament derived NFL-TBS.40-63 peptide has recently been introduced as a novel tool to target neural stem cells. Previous studies showed that this peptide can be internalized by rat neural stem cells in vitro and in vivo, which coincided with lower proliferation and self-renewal capacity and increase of differentiation. In this study, we analyzed the uptake and potential effects of the NFL-TBS.40-63 peptide on human neural stem cells isolated from human fetuses. We showed that the peptide inhibits proliferation and the ability to produce neurospheres in vitro, which is consistent with an increase in cell adhesion and differentiation. These results confirm that the peptide could be a promising molecule to target and manipulate human neural stem cells and thus could serve as a strategic tool for regenerative medicine.
Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable ...analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.
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•Buruli ulcer caused by Mycobacterium ulcerans is characterized by painless lesions•Mycolactone produced by M. ulcerans induces hypoesthesia without nerve damage•Pathway characterization pinpoints AT2R receptors as targets for mycolactone•Model provides insight into implementation of analgesia in natural systems
The absence of pain in Buruli ulcer, despite extensive skin lesions, is ensured by the toxin mycolactone secreted by Mycobacterium ulcerans through the triggering of a specific angiotensin (AT2R) pathway.
Purpose
Current preclinical therapeutic strategies involving nanomedicine require increasingly sophisticated nanosystems and the characterization of the complexity of such nanoassemblies is becoming ...a major issue. Accurate characterization is often the factor that can accelerate the translational approaches of nanomedicines and their pharmaceutical development to reach the clinic faster. We conducted a case study involving the adsorption of the NFL-TBS.40–63 (NFL) peptide (derived from neurofilaments) to the surface of lipid nanocapsules (LNCs) (a combined nanosystem used to target glioblastoma cells) to develop an analytical approach combining the separation and the quantification in a single step, leading to the characterization of the proportion of free peptide and thus the proportion of peptide adsorbed to the lipid nanocapsule surface.
Methods
LNC suspensions, NFL peptide solution and LNC/NFL peptide mixtures were characterized using a Size-Exclusion Chromatography method (with a chromatographic apparatus). In addition, this method was compared to centrifugal-filtration devices, currently used in literature for this case study.
Results
Combining the steps for separation and characterization in one single sequence improved the accuracy and robustness of the data and led to reproducible results. Moreover the data deviation observed for the centrifugal-filtration devices demonstrated the limits for this increasingly used characterization approach, explained by the poor separation quality and highlighting the importance for the method optimization. The high potential of the technique was shown, proving that H-bond and/or electrostatic interactions mediate adsorption of the NFL peptide to the surface of LNCs.
Conclusions
Used only as a characterization tool, the process using chromatographic apparatus is less time and solvent consuming than classical Size-Exclusion Chromatography columns only used for separation. It could be a promising tool for the scientific community for characterizing the interactions of other combinations of nanosystems and active biological agents.
Graphical abstract
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Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood–brain barrier (BBB), which limits the ...delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40–63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood–brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.
Epoxiconazole is one of the most commonly used fungicides in the world. The exposition of humans to pesticides is mainly attributed to its residue in food or occupational exposure in agricultural ...production. Because of its lipophilic character, Epoxiconazole can accumulate in the brain Heusinkveld et al. (2013) 1. Consequently, it is urgent to explore efficient strategies to prevent or treat Epoxiconazole-related brain damages. The use of natural molecules commonly found in our diet represents a promising avenue. Flavonoids belong to a major sub-group compounds possessing powerful antioxidant activities based on their different structural and sterical properties 2. We choose to evaluate Myricetin, a flavonoid with a wide spectrum of pharmacological effects, for its possible protective functions against Epoxiconazole-induced toxicities. The cytotoxicity induced by this fungicide was evaluated by the cell viability, cell cycle arrest, ROS generation, antioxidant enzyme activities, and Malondialdehyde production, as previously described in Hamdi et al., 2019 3. The apoptosis was assessed through the evaluation of the mitochondrial transmembrane potential (ΔΨm), caspases activation, DNA fragmentation, cytoskeleton disruption, nuclear condensation, appearance of sub-G0/G1 peak (fragmentation of the nucleus) and externalization of Phosphatidylserine. This study indicates that pre-treatment of F98 cells with Myricetin during 2 h before Epoxiconazole exposure significantly increased the survival of cells, restored DNA synthesis of the S phase, abrogated the ROS generation, regulated the activities of Catalase (CAT) and Superoxide Dismutase (SOD), and reduced the MDA level. The loss of mitochondrial membrane potential, DNA fragmentation, cytoskeleton disruption, chromatin condensation, Phosphatidylserine externalization, and Caspases activation were also reduced by Myricetin. Together, these findings indicate that Myricetin is a powerful natural product able to protect cells from Epoxiconazole-induced cytotoxicity and apoptosis.
A proposed mechanism of pretreatment with Myricetin in alleviating Epoxiconazole-induced neurotoxicty. The protective effect is mainly attributed to its ability to reduce intracellular reactive oxygen levels, modulating the antioxidant enzymes activity, reducing malondialdhyde level, stabilizing the cytoskeleton organization and mitochondrial membrane potential Finally, Myricetin pretreatment prevented the increase of DNA fragmentation, caspases-3 and 9 activities and DNA condensation. Display omitted
•Myricetin prevents Epoxiconazole-induced cytotoxicity in F98cells.•Cell pre-treatment with Myricetin reduces Epoxiconazole-induced Oxidative Stress.•Dietary Myricetin alleviates Epoxiconazole-induced Programmed Cell Death.
Neural stem cells (NSC) are located in restricted areas of the central nervous system where they self-renew or differentiate into neurons, astrocytes or oligodendrocytes. The stimulation of ...endogenous NSC differentiation is one of the most promising therapeutic approaches to restore neurological function in patients affected by neurodegenerative diseases. Endogenous NSC of the subventricular zone (SVZ) can be selectively targeted by lipid nanocapsules (LNC) coated with the peptide NFLTBS.40-63 (NFL-LNC) after intra-lateral ventricular injection in the brain. NFL-LNC can potentially deliver active compounds to SVZ-NSC and thus promote their differentiation to treat neurodegenerative diseases. The aim of this work was to induce endogenous NSC differentiation by specifically delivering retinoic acid (RA) to SVZ-NSC via NFL-LNC. RA was successfully encapsulated into NFL-LNC and RA-NFL-LNC were incubated with primary rat SVZ-NSC. In vitro, RA-NFL-LNC decreased the number of nestin+ (NSC marker) cells and neurospheres compared to controls and increased the number of GalC+ (oligodendrocytic marker) cells. Then, RA-NFL-LNC were injected in the right lateral ventricle of a lysolecithin-induced rat focal white matter lesion model to evaluate their impact on oligodendrocyte repopulation and remyelination. RA-NFL-LNC significantly increased the percentage of mature oligodendrocytes, stimulating oligodendrogenesis, nearly to the pre-lesion levels. Thus, RA-NFL-LNC represent a promising nanomedicine to be further investigated in the treatment of demyelinating diseases.
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•NFL-lipid nanocapsules (NFL-LNC) efficiently encapsulate retinoic acid (RA).•Primary neural stem cells differentiate towards the oligodendrocyte lineage after incubation with RA-NFL-LNC.•RA-NFL-LNC promote oligodendrocyte repopulation in focal white matter lesioned rats.•RA-NFL-LNC is a promising therapeutic system for the treatment of demyelinating diseases.
Intermediate filaments (IFs) of the nervous system, including neurofilaments, α-internexin, glial fibrillary acidic protein, synemin, nestin, peripherin and vimentin, are finely expressed following ...elaborated cell, tissue and developmental specific patterns. A common characteristic of several neurodegenerative diseases is the abnormal accumulation of neuronal IFs in cell bodies or along the axon, often associated with impairment of the axonal transport and degeneration of neurons. In this review, we also present several perturbations of IF metabolism and organization associated with neurodegenerative disorders. Such modifications could represent strong markers of neuronal damages. Moreover, recent data suggest that IFs represent potential biomarkers to determine the disease progression or the differential stages of a neuronal disorder. Finally, recent investigations on IF expression and function in cancer provide evidence that they may be useful as markers, or targets of brain tumours, especially high-grade glioma. A better knowledge of the molecular mechanisms of IF alterations, combined to neuroimaging, is essential to improve diagnosis and therapeutic strategies of such neurodegenerative diseases and glioma.
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