RESUMEN

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease. Previously, we identified midnolin (MIDN) as a genetic risk factor for PD. Although MIDN copy number loss increases the risk of PD, the molecular function of MIDN remains unclear. To investigate the role of MIDN in PD, we established monoclonal Midn knockout (KO) PC12 cell models. Midn KO inhibited neurite outgrowth and neurofilament light chain (Nefl) gene expression. Although MIDN is mainly localized in the nucleus, it does not encode DNA-binding domains. We therefore hypothesized that MIDN might bind to certain transcription factors and regulate gene expression. Of the candidate transcription factors, we focused on early growth response 1 (EGR1) because it is required for neurite outgrowth and its target genes are downregulated by Midn KO. An interaction between MIDN and EGR1 was confirmed by immunoprecipitation. Surprisingly, although EGR1 protein levels were significantly increased in Midn KO cells, the binding of EGR1 to the Nefl promoter and resulting transcriptional activity were downregulated as measured by luciferase assay and chromatin immunoprecipitation quantitative real-time polymerase chain reaction. Overall, we identified the MIDN-dependent regulation of EGR1 function. This mechanism may be an underlying reason for the neurite outgrowth defects of Midn KO PC12 cells.

RESUMEN

BACKGROUND: Atrazine (ATR), a commonly used herbicide, is linked to dopaminergic neurotoxicity, which may cause symptoms resembling Parkinson's disease (PD). This study aims to reveal the molecular regulatory networks responsible for ATR exposure and its effects on dopaminergic neurotoxicity based on an integration strategy. METHODS: Our approach involved network toxicology, construction of protein-protein interaction (PPI) networks, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, as well as molecular docking techniques. Subsequently, we validated the predicted results in PC12 cells in vitro. RESULTS: An integrated analysis strategy indicating that 5 hub targets, including mitogen-activated protein kinase 3 (Mapk3), catalase (Cat), heme oxygenase 1 (Hmox1), tumor protein p53 (Tp53), and prostaglandin-endoperoxide synthase 2 (Ptgs2), may play a crucial role in ATR-induced dopaminergic injury. Molecular docking indicated that the 5 hub targets exhibited certain binding activity with ATR. Cell counting kit-8 (CCK8) results illustrated a dose-response relationship in PC12 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) displayed notable changes in the expression of hub targets mRNA levels, with the exception of Mapk3. Western blotting results suggested that ATR treatment in PC12 cells resulted in an upregulation of the Cat, Hmox1, and p-Mapk3 protein expression levels while causing a downregulation in Tp53, Ptgs2, and Mapk3. CONCLUSION: Our findings indicated that 5 hub targets identified could play a vital role in ATR-induced dopaminergic neurotoxicity in PC12 cells. These results provide preliminary support for further investigation into the molecular mechanism of ATR-induced toxicity.

Asunto(s)

Atrazina , Neuronas Dopaminérgicas , Herbicidas , Simulación del Acoplamiento Molecular , Atrazina/toxicidad , Animales , Células PC12 , Ratas , Herbicidas/toxicidad , Neuronas Dopaminérgicas/efectos de los fármacos , Mapas de Interacción de Proteínas , Dopamina/metabolismo

RESUMEN

OBJECTIVE: This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide (LPS) and to examine the effects of astragaloside IV on key targets using high-throughput sequence technology and bioinformatics analyses. METHODS: PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25, 0.5, 0.75, 1, and 1.25 mg/mL for 24 h. Cell morphology was evaluated, and cell survival rates were calculated. A neurocyte inflammatory model was established with LPS treatment, which reached a 50% cell survival rate. PC12 cells were treated with 0.01, 0.1, 1, 10, or 100 µmol/L astragaloside IV for 24 h. The concentration of astragaloside IV that did not affect the cell survival rate was selected as the treatment group for subsequent experiments. NOS activity was detected by colorimetry; the expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting. The differentially expressed genes (DEGs) between the groups were screened using a second-generation sequence (fold change>2, P<0.05) with the following KEGG enrichment analysis, RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells. RESULTS: The viability of PC12 cells was not altered by treatment with 0.01, 0.1, or 1 µmol/L astragaloside IV for 24 h (P>0.05). However, after treatment with 0.5, 0.75, 1, or 1.25 mg/mL LPS for 24 h, the viability steadily decreased (P<0.01). The mRNA and protein expression levels of ERCC2, XRCC4, XRCC2, TNF-α, IL-1ß, TLR4, NOS, and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h (P<0.01); however, these changes were reversed when PC12 cells were pretreated with 0.01, 0.1, or 1 µmol/L astragaloside IV in PC12 cells and then treated with 1 mg/mL LPS for 24 h (P<0.05). Second-generation sequencing revealed that 1026 genes were upregulated, while 1287 genes were downregulated. The DEGs were associated with autophagy, TNF-α, interleukin-17, MAPK, P53, Toll-like receptor, and NOD-like receptor signaling pathways. Furthermore, PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2, CCL11, CCL7, MMP3, and MMP10, which are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results. CONCLUSION: LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage. astragaloside IV plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.

Asunto(s)

Antiinflamatorios , Supervivencia Celular , Reparación del ADN , Lipopolisacáridos , Saponinas , Triterpenos , Animales , Células PC12 , Ratas , Lipopolisacáridos/farmacología , Triterpenos/farmacología , Saponinas/farmacología , Antiinflamatorios/farmacología , Supervivencia Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos

RESUMEN

Controlling biomolecular-cell interactions is crucial for the design of scaffolds for tissue engineering (TE). Regenerated silk fibroin (RSF) has been extensively used as TE scaffolds, however, RSF showed poor attachment of neuronal cells, such as rat pheochromocytoma (PC12) cells. In this work, amphiphilic peptides containing a hydrophobic isoleucine tail (I3) and laminin or fibronectin derived peptides (IKVAV, PDSGR, YIGSR, RGDS and PHSRN) were designed for promoting scaffold-cell interaction. Three of them (I3KVAV, I3RGDS and I3YIGSR) can self-assemble into nanofibers, therefore, were used to enhance the application of RSF in neuron TE. Live / dead assays revealed that the peptides exhibited negligible cytotoxicity against PC12 cells. The specific interaction between PC12 cells and the peptides were investigate using atomic force microscopy (AFM). The results indicated a synergistic effect in the designed peptides, promoting cellular attachment, proliferation and morphology changes. In addition, AFM results showed that co-assembling peptides I3KVAV and I3YIGSR possesses the best regulation of proliferation and attachment of PC12 cells, consistent with immunofluorescence staining results. Moreover, cell culture with hydrogels revealed that a mixture of peptides I3KVAV and I3YIGSR can also promote 3D neurites outgrowth. The approach of combining two different self-assembling peptides shows great potential for nerve regeneration applications.

Asunto(s)

Nanofibras , Proyección Neuronal , Péptidos , Seda , Andamios del Tejido , Animales , Células PC12 , Ratas , Nanofibras/química , Andamios del Tejido/química , Proyección Neuronal/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Seda/química , Proliferación Celular/efectos de los fármacos , Fibroínas/química , Fibroínas/farmacología , Ingeniería de Tejidos/métodos , Adhesión Celular/efectos de los fármacos

RESUMEN

INTRODUCTION: The proline-rich decapeptide 10c (Bj-PRO-10c; ENWPHPQIPP) from the Bothrops jararaca snake modulates argininosuccinate synthetase (AsS) activity to stimulate L-arginine metabolite production and neuroprotection in the SH-SY5Y cell line. The relationships between structure, interactions with AsS, and neuroprotection are little known. We evaluated the neuroprotective effects of Bj-PRO-10c and three other PROs (Bn-PRO-10a, Bn-PRO-10c > Bn-PRO-10a-MK > Bn-PRO-10a. The structure of PROs and their correlations with enzyme activity revealed that histidine (H5) and glutamine (Q7) in Bj-PRO-10c potentiated their affinity for AsS. CONCLUSIONS: Our investigation provides the first insights into the structure and molecular interactions of PROs with AsS, which could possibly further their neuropharmacological applications.

RESUMEN

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Accumulation of ß-amyloid (Aß) in the brain has been recognized as a key factor in the onset and progression of Alzheimer's disease (AD).The accumulation of Aß in the brain catalyzes the production of reactive oxygen species (ROS), which in turn triggers oxidative damage to cellular components such as DNA, lipids, and proteins. In the present study, we investigated the protective effect of Ganoderic acid A (GA.A) against Aß42-induced apoptosis in PC12 cells. Changes in mitochondrial membrane potential indicated that GA.A treats mitochondrial dysfunction by decreasing Aß42 deposition and inhibiting neural protofiber tangle formation. Changes in intracellular Ca2+ and caspase-3 indicated that GA.A reduced mitochondrial damage by Aß42 in PC12 cells, thereby decreasing ROS accumulation and reducing Aß protofiber-induced cytotoxicity. These features suggest that GA.A has great potential as an effective neuroprotective drug in the treatment of Alzheimer's disease.

Asunto(s)

Péptidos beta-Amiloides , Apoptosis , Lanosterol , Potencial de la Membrana Mitocondrial , Mitocondrias , Fármacos Neuroprotectores , Fragmentos de Péptidos , Especies Reactivas de Oxígeno , Animales , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/metabolismo , Células PC12 , Ratas , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fragmentos de Péptidos/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Apoptosis/efectos de los fármacos , Lanosterol/farmacología , Lanosterol/análogos & derivados , Calcio/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Ácidos Heptanoicos

RESUMEN

AIMS: This study investigates the neuroprotective effects of lipidized analogues of 2-SS-CART(61-102) derived from anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript peptide (CARTp) in light of the link between obesity, its comorbidities, and the development of Alzheimer's disease. METHODS: We introduce novel lipidized analogues derived from 2-SS-CART(61-102), a specific analogue of natural CART(61-102), with two disulfide bridges. Using hypothermic PC12 cells, we tested the effect of the most potent analogues on Tau phosphorylation. We further described the anorexigenic and neuroprotective potential of subcutaneously (SC) injected lipidized CARTp analogue in a mouse model with prediabetes and obesity induced by neonatal monosodium glutamate (MSG) administration. RESULTS: Compared to the non-lipidized 2-SS-CART(61-102), all lipidized analogues exhibited a potent binding affinity to PC12 cells and enhanced in vitro stability in rat plasma. Two most potent lipidized analogues attenuated hypothermia-induced Tau hyperphosphorylation at multiple epitopes. Subsequently, chronic SC treatment with palm-2-SS-CART(61-102) significantly decreased body weight and food intake, improved metabolic parameters, decreased level of pTau and increased neurogenesis in hippocampi of obese MSG mice. CONCLUSION: Our unique CARTp analogue palm-2-SS-CART(61-102) shows promise as a potent anti-obesity and neuroprotective agent.

Asunto(s)

Proteínas del Tejido Nervioso , Fármacos Neuroprotectores , Obesidad , Glutamato de Sodio , Animales , Masculino , Ratones , Ratas , Depresores del Apetito/farmacología , Modelos Animales de Enfermedad , Lípidos/química , Lípidos/sangre , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Células PC12 , Fosforilación/efectos de los fármacos , Proteínas tau/metabolismo

RESUMEN

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

Asunto(s)

Escherichia coli , Proteínas Ligadas a GPI , Proteínas Recombinantes , Animales , Células PC12 , Ratas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Neuropéptidos/genética , Neuropéptidos/química , Neuropéptidos/metabolismo , Enteropeptidasa/metabolismo , Enteropeptidasa/genética , Enteropeptidasa/química , Cromatografía de Afinidad , Proyección Neuronal/efectos de los fármacos , Cromatografía en Gel , Expresión Génica

RESUMEN

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP's known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson's disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP(1-27) glucoside retains the known neurotrophic activity of native PACAP(1-27)in vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP(1-27) lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by âˆ¼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need.

Asunto(s)

Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Animales , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/administración & dosificación , Masculino , Ratas , Ratones , Células PC12 , Ratones Endogámicos C57BL , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Modelos Animales de Enfermedad , Glicósidos/farmacología , Glicósidos/uso terapéutico , Glicósidos/administración & dosificación , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Neuritas/efectos de los fármacos , Neuritas/patología

RESUMEN

Rapid and accurate in situ determination of dopamine is of great significance in the study of neurological diseases. In this work, poly (3,4-ethylenedioxythiophene): poly (styrenesulfonic acid) (PEDOT: PSS)/graphene oxide (GO) fibers were fabricated by an effective method based on microfluidic wet spinning technology. The composite microfibers with stratified and dense arrangement were continuously prepared by injecting PEDOT: PSS and GO dispersion solutions into a microfluidic chip. PEDOT: PSS/GO fiber microelectrodes with high electrochemical activity and enhanced electrochemical oxidation activity of dopamine were constructed by controlling the structure composition of the microfibers with varying flow rate. The fabricated fiber microelectrode had a low detection limit (4.56 nM) and wide detection range (0.01-8.0 µM) for dopamine detection with excellent stability, repeatability, and reproducibility. In addition, the PEDOT: PSS/GO fiber microelectrode prepared was successfully used for the detection of dopamine in human serum and PC12 cells. The strategy for the fabrication of multi-component fiber microelectrodes is a new and effective approach for monitoring the intercellular neurotransmitter dopamine and has high potential as an implantable neural microelectrode.

Asunto(s)

Dopamina , Grafito , Microelectrodos , Poliestirenos , Células PC12 , Dopamina/sangre , Humanos , Ratas , Animales , Poliestirenos/química , Grafito/química , Límite de Detección , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Tiofenos/química , Dispositivos Laboratorio en un Chip , Polímeros

RESUMEN

We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription.

RESUMEN

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid beta (Aß) plaques in the brain. Aß1-42 is the main component of Aß plaque, which is toxic to neuronal cells. Si nanowires (Si NWs) have the advantages of small particle size, high specific surface area, and good biocompatibility, and have potential application prospects in suppressing Aß aggregation. In this study, we employed the vapor-liquid-solid (VLS) growth mechanism to grow Si NWs using Au nanoparticles as catalysts in a plasma-enhanced chemical vapor deposition (PECVD) system. Subsequently, these Si NWs were transferred to a phosphoric acid buffer solution (PBS). We found that Si NWs significantly reduced cell death in PC12 cells (rat adrenal pheochromocytoma cells) induced by Aß1-42 oligomers via double staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and fluorescein diacetate/propyl iodide (FDA/PI). Most importantly, pre-incubated Si NWs largely prevented Aß1-42 oligomer-induced PC12 cell death, suggesting that Si NWs exerts an anti-Aß neuroprotective effect by inhibiting Aß aggregation. The analysis of Fourier Transform Infrared (FTIR) results demonstrates that Si NWs reduce the toxicity of fibrils and oligomers by intervening in the formation of ß-sheet structures, thereby protecting the viability of nerve cells. Our findings suggest that Si NWs may be a potential therapeutic agent for AD by protecting neuronal cells from the toxicity of Aß1-42.

Asunto(s)

Péptidos beta-Amiloides , Nanocables , Fármacos Neuroprotectores , Silicio , Animales , Ratas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Nanocables/química , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/farmacología , Agregado de Proteínas/efectos de los fármacos , Silicio/química

RESUMEN

Aim: To explore the differential genes in Parkinson's disease (PD) through a preliminary GEO database, and to investigate the possible mechanisms. Materials and Methods: The PD differentially expressed genes (DEGs) were analyzed by the microarray method. Then, these DEGs were applied to KEGG and GO analyses to predict the related signaling pathways and molecular functions. Comparison of GRAMD1C expression levels in the putamen of normal and Parkinson's patients by bioinformatic analysis. PC12 cells were cultured to construct a 6-hydroxydopamine (6-OHDA)-induced Parkinson's cell model. RT-qPCR was performed to detect the efficiency of GRAMD1C siRNA. MTT assay was conducted to examine the proliferation of cells. Then, the apoptosis of each group of cells was measured by flow cytometry. Western blot was carried out to determine the expression of apoptosis-related proteins. Results: Through bioinformatics, GRAMD1C was confirmed to be one of the most significantly upregulated genes in PD. Furthermore, GRAMD1C was notably enhanced in the PD patients and 6-OHDA-induced PC12 cells. Besides, 6-OHDA stimulation significantly reduced PC12 cell proliferation, and it reverted with the GRAMD1C siRNA. Moreover, the flow cytometry results showed that knockdown of GRAMD1C could effectively reduce the high apoptosis rate of PC12 cells induced by 6-OHDA treatment. Similarly, western blot results found that 6-OHDA stimulation markedly increased the expression levels of Bax and Caspase 3Caspase 3 and decreased the Bcl-2 expression in PC12 cells, and GRAMD1C knockdown reversed these changes. Conclusion: GRAMD1C is upregulated in PD, and may affect the PD process through the apoptotic pathway.

RESUMEN

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo , Cobre , Factor de Crecimiento Nervioso , Péptidos Cíclicos , Factor A de Crecimiento Endotelial Vascular , Células PC12 , Animales , Ratas , Factor de Crecimiento Nervioso/farmacología , Factor de Crecimiento Nervioso/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cobre/metabolismo , Cobre/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ionóforos/farmacología , Proteínas de Transporte de Catión/metabolismo , Receptor trkA/metabolismo

RESUMEN

The sigma-1 receptor (σ-1R), a chaperone protein located at the mitochondria-associated membrane (MAM) of the endoplasmic reticulum, can interact with and modify the signaling pathways of various proteins, thereby modulating many disease pathologies, including Alzheimer's disease (AD). The σ-1R ligand dipentylammonium (DPA) was analyzed for its anti-AD properties using PC12 cells (in vitro) and Caenorhabditis elegans (in vivo) models along with molecular docking (in silico) analysis. DPA at 1 and 10 µM concentrations was able to significantly potentiate NGF-induced neurite growth length by 137.7 ± 12.0 and 187.8 ± 16.4, respectively, when compared to the control 76.9 ± 7.4. DPA also regulated neurite damage caused by Aß(25-35) treatment in differentiated PC12 cells by improving cell viability and neurite length. In C. elegans, DPA could significantly extend the median and maximum lifespan of Aß transgenic strain CL2006 without impacting wild-type nematodes. Additionally, it could significantly reduce the paralysis phenotype of another Aß transgenic strain, CL4176, thereby improving the overall health in AD pathogenesis. This effect depended on σ-1R, as DPA could not modulate the lifespan of σ-1R mutant TM3443. This was further confirmed using agonist PRE084 and antagonist BD1047, wherein the agonist alone could extend the lifespan of CL2006, while the antagonist suppressed the effect of DPA in CL2006. Interestingly, neither had an TM3443. Further, molecular docking analysis showed that DPA had a similar binding affinity as that of PRE084, BD1047 and pentazocine against the σ-1R receptor in humans and C. elegans, which collectively suggests the anti-AD properties of DPA.

Asunto(s)

Enfermedad de Alzheimer , Compuestos de Amonio , Etilenodiaminas , Fármacos Neuroprotectores , Receptores sigma , Animales , Ratas , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Receptor Sigma-1 , Caenorhabditis elegans , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ligandos , Simulación del Acoplamiento Molecular , Animales Modificados Genéticamente/metabolismo , Técnicas de Cultivo de Célula , Péptidos beta-Amiloides/metabolismo , Receptores sigma/metabolismo

RESUMEN

Neurotrophins (NGF, BDNF, NT3, NT4) can decrease cell death, induce differentiation, as well as sustain the structure and function of neurons, which make them promising therapeutic agents for the treatment of neurodegenerative disorders. However, neurotrophins have not been very effective in clinical trials mostly because they cannot pass through the blood-brain barrier owing to being high-molecular-weight proteins. Thus, neurotrophin-mimic small molecules, which stimulate the synthesis of endogenous neurotrophins or enhance neurotrophic actions, may serve as promising alternatives to neurotrophins. Small-molecular-weight natural products, which have been used in dietary functional foods or in traditional medicines over the course of human history, have a great potential for the development of new therapeutic agents against neurodegenerative diseases such as Alzheimer's disease. In this contribution, a variety of natural products possessing neurotrophic properties such as neurogenesis, neurite outgrowth promotion (neuritogenesis), and neuroprotection are described, and a focus is made on the chemistry and biology of several neurotrophic natural products.

Asunto(s)

Productos Biológicos , Humanos , Productos Biológicos/farmacología , Factores de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Neurogénesis , Diferenciación Celular/fisiología

RESUMEN

During recent decades, the application of zirconium dioxide nanoparticles (ZrO2-NP) has been expanded in various fields ranging from medicine to industry. It has been shown that ZrO2-NP has the potential to cross the blood-brain barrier (BBB) and induce neurotoxicity. In the current study, we investigated the in vivo neurotoxicity, as well as, the cellular mechanism of ZrO2-NP toxicity on two neuronal-like cell lines, PC12 and N2a. PC12 and N2a cells were exposed to increasing concentrations of ZrO2-NP (0-2000 µg/ml) for 48 h. The apoptotic effect of ZrO2-NP was determined using annexin V/propidium iodide double staining (by flow cytometry), and western blot analysis of relative apoptotic proteins, including caspase-3, caspase-9, bax, and bcl2. Based on our results, ZrO2-NP at concentrations of 250-2000 µg/mL increased both early and late-stage apoptosis in a concentration-dependent manner. Moreover, the expressions of cleaved-caspase-3 and -9 proteins and the bax/bcl2 ratio were significantly increased. In addition, oral administration of ZrO2-NP (50 mg/kg) to male Wistar rats for 28 days led to the loss of neuronal cells in the cerebral cortex. Taken together, our findings highlighted the role of apoptosis on cytotoxicity induced by ZrO2-NP.

Asunto(s)

Nanopartículas , Proteínas Proto-Oncogénicas c-bcl-2 , Circonio , Ratas , Masculino , Animales , Caspasa 3 , Proteína X Asociada a bcl-2/metabolismo , Ratas Wistar , Células PC12 , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis , Neuronas , Supervivencia Celular

RESUMEN

SNAP25 (synaptosome-associated protein of 25 kDa) is a core SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein; and the interaction between SNAP25 and other SNARE proteins is essential for synaptic vesicle exocytosis. Identified as a SNAP25 interacting protein, SIP30 (SNAP25 interacting protein at 30 kDa) has been shown to modulate neuropathic pain behavior, and is potentially involved in the cellular process of vesicle exocytosis. Previous study demonstrated that using a vesicle secretion assay in PC12 cells, anti-SIP30 siRNA reduced vesicle exocytosis. We investigated vesicle exocytosis from PC12 cells with FM1-43 fluorescence dye, and demonstrated that anti-SIP30 siRNA reduced the pool of releasable vesicles and the rate of vesicle exocytosis, without affecting the endocytosis and recycling of the exocytosed vesicles. The results show that SIP30 is involved in vesicle exocytosis, suggesting a potential mechanism of SIP30 modulation of neuropathic pain.

RESUMEN

OBJECTIVE: Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Despite extensive research, no definitive cure or effective disease-modifying treatment for PD exists to date. Therefore, the identification of novel therapeutic agents with neuroprotective properties is of utmost importance. Here, we aimed to investigate the potential neuroprotective effects of Carpesii fructus extract (CFE) in both cellular and Caenorhabditis elegans (C. elegans) models of PD. METHODS: The neuroprotective effect of CFE in H2O2- or 6-OHDA-induced PC-12 cells and α-synuclein-overexpressing PC-12 cells were investigated by determining the cell viability, mitochondrial damage, reactive oxygen species (ROS) production, apoptosis, and α-synuclein expression. In NL5901, BZ555, and N2 worms, the expression of α-synuclein, motive ability, the viability of dopaminergic neurons, lifespan, and aging-related phenotypes were investigated. The signaling pathway was detected by Western blotting and validated by employing small inhibitors and RNAi bacteria. RESULTS: In cellular models of PD, CFE significantly attenuated H2O2- or 6-OHDA-induced toxicity, as evidenced by increased cell viability and reduced apoptosis rate. In addition, CFE treatment suppressed ROS generation and restored mitochondrial membrane potential, highlighting its potential as a mitochondrial protective agent. Furthermore, CFE reduced the expression of α-synuclein in wide type (WT)-, A53T-, A30P-, or E46K-α-synuclein-overexpressing PC-12 cells. Our further findings reveal that CFE administration reduced α-synuclein expression and improved its induced locomotor deficits in NL5901 worms, protected dopaminergic neurons against 6-OHDA-induced degeneration in BZ555 worms, extended lifespan, delayed aging-related phenotypes, and enhanced the ability of stress resistance in N2 worms. Mechanistic studies suggest that the neuroprotective effects of CFE may involve the modulation of the MAPK signaling pathway, including ERK, JNK, and p38, whereas the interference of these pathways attenuated the neuroprotective effect of CFE in vitro and in vivo. CONCLUSION: Overall, our study highlights the potential therapeutic value of CFE as a neuroprotective agent in the context of PD. Furthermore, elucidation of the active compounds of CFE will provide valuable insights for the development of novel therapeutic strategies for PD.

Asunto(s)

Fármacos Neuroprotectores , Enfermedad de Parkinson , Animales , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Oxidopamina/toxicidad , Peróxido de Hidrógeno/toxicidad , Peróxido de Hidrógeno/metabolismo , Neuronas Dopaminérgicas/metabolismo , Modelos Animales de Enfermedad

RESUMEN

OBJECTIVE To study the protective effect of saikosaponin b2(SSb2)on corticosterone(CORT)induced PC12 cell injury and its mechanism.METHODS ① PC12 cells were divided into the cell control group(24 h of culture with RPMI-1640 medium),CORT group(24 h of culture with CORT 100-800 μmol·L-1)and SSb2 group(24 h of culture with SSb2 1.5625,3.125,6.25,12.5,25,50 and 100 μmol·L-1).MTT assay was used to detect the cell survival rate.②PC12 cells were divided into the cell control group(24 h of culture with RPMI 1640 medium),model group(24 h of culture with CORT 400 μmol·L-1),and model+SSb2 group(3 h pretreatment with SSb2 1.5625,3.125,6.25,12.5 and 25 μmol·L-1,removal of the supernatant before cells were co-incubated with CORT 400 μmol·L-1 and corresponding concentrations of SSb2 for 24 h).MTT assay was used to detect the cell survival rate while micro-plate assay was used to detect the lactate dehydrogenase(LDH)leakage rate of PC12 cells.③PC12 cells were divided into the cell control group,model group and model+SSb2 12.5 μmol·L-1 group.AnnexinV-FITC/PI flow cytometry assay was used to detect PC12 cell apoptosis,ultra-perfor-mance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)cell metabonomics was used to detect metabolic profile changes and colorimetric assay was employed to detect the glutamic acid content and glutaminase activity in PC12 cells.RESULTS Compared with the cell control group,the cell viability decreased to(55±6)%(P<0.01)when the concentration of CORT was 400 μmol·L-1.When the concentration of SSb2 was higher than 50 μmol·L-1,there was significant toxicity to PC12 cells(P<0.01).②Compared with the cell control group,the cell survival rate was signif-icantly decreased(P<0.01),while the release rate of LDH was significantly increased(P<0.01)in the model group.Compared with the model group,the cell survival rate significantly increased(P<0.05,P<0.01),while the LDH release rate significantly decreased(P<0.01)in the model+SSb2 group.③ Com-pared with the cell control group,cell apoptosis was significantly increased in the model group(P<0.05).Compared with the model group,cell apoptosis was significantly decreased(P<0.05)in the model+ SSb2 group.Metabolomics results show that SSb2 significantly back-regulated nine differential metabo-lites of glutamate,creatine,N-acetylaspartate,L-tyrosine,citric acid,L-isoleucine,lactic acid,glutamine and choline.Further network analysis of the key metabolites regulated by SSb2 yielded five major metabolic pathways:D-glutamine and D-glutamate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,alanine,aspartate and glutamate metabolism,tyrosine metabolism and arginine biosynthesis.Compared with the cell control group,the content of glutamate and activity of glutaminase were significantly decreased in the model group(P<0.01).Compared with the model group,the content of glutamate(P<0.01)and activity of glutaminase(P<0.05)were significantly increased in the model+SSb2 group.CONCLUSION SSb2 has a neuroprotective effect on CORT-injured PC12 cells,and the mechanism of which is related to inhibition of apoptosis and regulation of metabolic disorders.