Neuroprotective Activity of a Non-Covalent Imatinib+TP10 Conjugate in HT-22 Neuronal Cells In Vitro
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PROVIDER: S-EPMC11207969 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
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Project description:Zinc is a trace metal vital for various functions in nerve cells, although the effect of zinc deficiency on neuronal autophagy remains unclear. This study aimed to elucidate whether zinc deficiency induced by treatment with N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, affects and alters autophagy activity. In cell viability assays, TPEN showed cytotoxicity in HT-22 cells. TPEN treatment also increased LC3-II levels and the ratio of LC3-II to LC3-I. Western blot analysis showed that phospho-AMP-activated protein kinase levels and the ratio of phospho-AMP-activated protein kinase to total AMP-activated protein kinase increased. Protein levels of the mammalian target of rapamycin and sirtuin 1 decreased following TPEN treatment. When TPEN-treated HT-22 cells were cotreated with autophagy inhibitors, 3-methyladenine (1 mM), or bafilomycin A1 (3 nM), the TPEN-induced decrease in cell viability was exacerbated. Cotreatment with chloroquine (10 μM) partially restored cell viability. The study showed that zinc deficiency induces autophagy and may be cytoprotective in neurons. We expect our results to add a new perspective to our understanding of the neuronal pathology related to zinc deficiency.
| S-EPMC9369147 | biostudies-literature
Project description:Background: MicroRNAs (miRNAs) have an increasing functional role in some neurodegenerative diseases. Autophagy, the degradation of bulk protein in the cytoplasm, is the quality control function of protein and has a protective role in the survival of neural cells. miR-433 may play a regulatory role in neurodegenerative diseases. Many aspects underlying the mechanism of miR-433 in neural development and neurodegeneration are not clear. Methods: In this study, we established stable cell lines expressing miR-433 by infecting mouse hippocampal neural cell line (HT-22) cells with rLV-miR-433 and the control rLV-miR. Pre-miR-433 expression was analyzed using polymerase chain reaction (PCR). Mature miR-433 expression was measured using quantitative PCR (qPCR). The effect of miR-433 overexpression on cell proliferation was determined using a CCK-8 assay and flow cytometry. RNA interference was used to analyze the function of Cdk12 in mediating the effect of miR-433 on cell proliferation. The effect of miR-433 overexpression on cell apoptosis was determined by flow cytometry. Autophagy-related genes Atg4a, LC3B, and Beclin-1 were determined using qPCR, Western blot, or immunofluorescence. In addition, RNA interference was used to analyze the effect of Atg4a on the induction of autophagy. TargetScan 7.2 was used to predict the target genes of miR-433, and Smad9 was determined using qPCR. Results: Our results indicated that miR-433 increased the expression of Atg4a and induced autophagy by increasing the expression of LC3B-Ⅱ and Beclin-1 in an Atg4a-dependent manner. In addition, miR-433 upregulated the expression of Cdk12 and inhibited cell proliferation in a Cdk12-dependent manner and promoted apoptosis in HT-22 cells under the treatment of 10-hydroxycamptothecin. Conclusion: The results of our study suggest that miR-433 may regulate neuronal growth by promoting autophagy and attenuating cell proliferation. This might be a potential therapeutic intervention in neurodegenerative diseases.
| S-EPMC7768889 | biostudies-literature
Project description:Angiogenin (ANG) is a human ribonuclease that is compromised in patients with amyotrophic lateral sclerosis (ALS). ANG also promotes neovascularization, and can induce hemorrhage and encourage tumor growth. The causal neurodegeneration of ALS is associated with reactive oxygen species, which are also known to elicit the oxidative cleavage of carbon-boron bonds. We have developed a synthetic boronic acid mask that restrains the ribonucleolytic activity of ANG. The masked ANG does not stimulate endothelial cell proliferation but protects astrocytes from oxidative stress. By differentiating between the two dichotomous biological activities of ANG, this strategy could provide a viable pharmacological approach for the treatment of ALS.
| S-EPMC5418131 | biostudies-literature
Project description:In vitro excitotoxic cell death experiments can be considered a screening model of stroke to evaluate the neuroprotective property of specific compounds. Survival of neurons following excitotoxicity is influenced by the neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor). Here, a novel 12 amino-acid peptide [AYKSYVRALPLL (TUF1)] with a high level of evolutionary conservation was assessed for its neuroprotective property in an in vitro model of glutamate-induced N-methyl-D-aspartic acid receptor hyperactivation and excitotoxicity. This peptide shares 100% homology to the conserved motif (SYVRAL) of the neurotrophic factors, which is found in numerous US patents. Following exposure to toxic levels of glutamate (500 µM), cultured primary rat forebrain neurons treated with TUF1 showed a dose-dependent survival rate compared with untreated neurons. The neuroprotective effect was blocked by p75 neurotrophic receptor (p75) inhibitor (MC192), but not by tyrosine kinase receptor inhibitor (K252a) or N-methyl-D-aspartic acid receptor antagonists (MK801 and D-amino-5-phosphonovaleric acid). Serine to alanine substitution that abolishes p75 interaction showed a loss of neuroprotective effect. Collectively, the findings showed that TUF1 can protect cultured primary cortical neurons from excitotoxic cell death through the p75-dependent pathway. Given that TUF1 is derived from TMEM35 (NACHO), which is required for the assembly and expression of nicotinic acetylcholine receptors, mechanism of TUF1 action may involve organization of nicotinic acetylcholine receptor and p75 neurotrophin receptor to modulate neuronal responses, including Ca signaling, to cytotoxic events. Unlike nerve growth factor, which requires a pre-insult exposure, TUF1 has neuroprotective properties even with post-insult administration, making it a potential target for therapeutic development in mitigating neuronal damage due to stroke and brain injury.
| S-EPMC6380445 | biostudies-literature
Project description:The mechanisms by which ethanol damages the developing and adult central nervous system (CNS) remain unclear. Activity-dependent neuroprotective protein (ADNP) is a glial protein that protects the CNS against a wide array of insults and is critical for CNS development. NAPVSIPQ (NAP), a potent active fragment of ADNP, potentiated axon outgrowth in cerebellar granule neurons by activating the sequential tyrosine phosphorylation of Fyn kinase and the scaffold protein Crk-associated substrate (Cas). Pharmacological inhibition of Fyn kinase or expression of a Fyn kinase siRNA abolished NAP-mediated axon outgrowth. Concentrations of ethanol attained after social drinking blocked NAP-mediated axon outgrowth (IC(50) = 17 mM) by inhibiting NAP activation of Fyn kinase and Cas. These findings identify a mechanism for ADNP regulation of glial-neuronal interactions in developing cerebellum and a pathogenesis of ethanol neurotoxicity.
| S-EPMC2604983 | biostudies-literature
Project description:Ferroptosis is a type of programmed cell death dependent on iron. It is different from other forms of cell death such as apoptosis, classic necrosis and autophagy. Ferroptosis is involved in many neurodegenerative diseases. The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood. To test its toxicity, glutamate (1.25-20 mM) was applied to HT-22 cells for 12 to 48 hours. The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate. Cells were cultured with 3-12 μM ferrostatin-1, an inhibitor of ferroptosis, for 12 hours before exposure to glutamate. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity. Damage to cell structures was observed under light and by transmission electron microscopy. The release of lactate dehydrogenase was detected by the commercial kit. Reactive oxygen species were measured by flow cytometry. Glutathione peroxidase activity, superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit. Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction. Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis. Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells, improving the survival rate, reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure. However, it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells. Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity. It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor (erythroid-derived)-like 2 in glutamate-injured HT-22 cells. Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp (OMe)-fluoromethyl ketone (2-8 μM), autophagy inhibitor 3-methyladenine (100-400 μM) or necrosis inhibitor necrostatin-1 (10-40 μM) had no effect on glutamate induced cell damage. However, the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death. Thus, the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.
| S-EPMC6921338 | biostudies-literature
Project description:Kaempferia parviflora Wall. ex Baker (KP) or "Kra-chai-dam" has been shown to exhibit several pharmacological effects including anti-inflammation, antimicrobial, and sexual-enhancing activity. The objectives of this study included an investigation of the effect of KP rhizome extract against glutamate-induced toxicity in mouse hippocampal HT-22 neuronal cells, determination of the underlying mechanism of neuroprotection, and an evaluation of the effect of KP extract on the longevity of Caenorhabditis elegans. HT-22 cells were co-treated with glutamate (5 mM) and KP extract (25, 50, and 75 μg/mL) for 14 h. Cell viability, intracellular reactive oxygen species (ROS) assay, fluorescence-activated cell sorting (FACS) analysis, and Western blotting were performed. The longevity effect of KP extract on C. elegans was studied by lifespan measurement. In HT-22 cells, co-treatment of glutamate with KP extract significantly inhibited glutamate-mediated cytotoxicity and decreased intracellular ROS production. Additionally, the glutamate-induced apoptosis and apoptotic-inducing factor (AIF) translocation were blocked by KP extract co-treatment. Western blot analysis also demonstrated that KP extract significantly diminished extracellular signal-regulated kinase (ERK) phosphorylation induced by glutamate, and brain-derived neurotrophic factor (BDNF) was recovered to the control. Moreover, this KP extract treatment prolonged the lifespan of C. elegans. Altogether, this study suggested that KP extract possesses both neuroprotective and longevity-inducing properties, thus serving as a promising candidate for development of innovative health products.
| S-EPMC8066628 | biostudies-literature
Project description:N-Acyl indolines 4 are potent, non-covalent Notum inhibitors developed from a covalent virtual screening hit 2a. The lead compounds were simple to synthesise, achieved excellent potency in a biochemical Notum-OPTS assay and restored Wnt signalling in a cell-based TCF/LEF reporter assay. Multiple high resolution X-ray structures established a common binding mode of these inhibitors with the indoline bound centred in the palmiteolate pocket with key interactions being aromatic stacking and a water mediated hydrogen bond to the oxyanion hole. These N-acyl indolines 4 will be useful tools for use in vitro studies to investigate the role of Notum in disease models, especially when paired with a structurally related covalent inhibitor (e.g. 4w and 2a). Overall, this study highlights the designed switch from covalent to non-covalent Notum inhibitors and so illustrates a complementary approach for hit generation and target inhibition.
| S-EPMC10626578 | biostudies-literature
Project description:Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503.The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907.WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity.These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.
| S-EPMC2014685 | biostudies-other
Project description:The digestive enzymes-polyphenolic compounds (PCs) interactions behind the inhibition of these enzymes have not been completely studied. The existing studies have mainly analyzed polyphenolic extracts and reported inhibition percentages of catalytic activities determined by UV-Vis spectroscopy techniques. Recently, pure PCs and new methods such as isothermal titration calorimetry and circular dichroism have been applied to describe these interactions. The present review focuses on PCs structural characteristics behind the inhibition of digestive enzymes, and progress of the used methods. Some characteristics such as molecular weight, number and position of substitution, and glycosylation of flavonoids seem to be related to the inhibitory effect of PCs; also, this effect seems to be different for carbohydrate-hydrolyzing enzymes and proteases. The digestive enzyme-PCs molecular interactions have shown that non-covalent binding, mostly by van der Waals forces, hydrogen binding, hydrophobic binding, and other electrostatic forces regulate them. These interactions were mainly associated to non-competitive type inhibitions of the enzymatic activities. The present review emphasizes on the digestive enzymes such as ?-glycosidase (AG), ?-amylase (PA), lipase (PL), pepsin (PE), trypsin (TP), and chymotrypsin (CT). Existing studies conducted in vitro allow one to elucidate the characteristics of the structure-function relationships, where differences between the structures of PCs might be the reason for different in vivo effects.
| S-EPMC6154557 | biostudies-literature