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PC-12 cells have been widely used as a neuronal line study model in many biosensing devices, mainly due to the neurogenic characteristics acquired after differentiation, such as high level of secreted neurotransmitter, neuron morphology characterized by neurite outgrowth, and expression of ion and neurotransmitter receptors. For understanding the pathophysiology processes involved in brain disorders, PC-12 cell line is extensively assessed in neuroscience research, including studies on neurotoxicity, neuroprotection, or neurosecretion. Various analytical technologies have been developed to investigate physicochemical processes and the biosensors based on optical and electrochemical techniques, among others, have been at the forefront of this development. This article summarizes the application of different biosensors in PC-12 cell cultures and presents the modern approaches employed in neuronal networks biosensing.

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their effective administration to reach the target organ or target cells at therapeutic concentrations. Currently, the nose-to-brain delivery with nanoformulations specifically designed for intranasal administration is a strategy widely investigated with the goal to reach the brain while bypassing the BBB. To produce nanosystems suitable to study both in vitro and/or in vivo cells trafficking for potential nose-to-brain delivery route, we prepared and characterized two types of fluorescent poly(ethylene glycol)-methyl-ether-block-poly(lactide-co-glycolide) (PLGA–PEG) nanoparticles (PNPs), i.e., Rhodamine B (RhB) dye loaded- and grafted- PNPs, respectively. The latter were produced by blending into the PLGA–PEG matrix a RhB-labeled polyaspartamide/polylactide graft copolymer to ensure a stable fluorescence during the time of analysis. Photon correlation spectroscopy (PCS), UV-visible (UV-vis) spectroscopies, differential scanning calorimetry (DSC), atomic force microscopy (AFM) were used to characterize the RhB-loaded and RhB-grafted PNPs. To assess their potential use for brain targeting, cytotoxicity tests were carried out on olfactory ensheathing cells (OECs) and neuron-like differentiated PC12 cells. Both PNP types showed mean sizes suitable for nose-to-brain delivery (<200 nm, PDI < 0.3) and were not cytotoxic toward OECs in the concentration range tested, while a reduction in the viability on PC12 cells was found when higher concentrations of nanomedicines were used. Both the RhB-labelled NPs are suitable drug carrier models for exploring cellular trafficking in nose-to-brain delivery for short-time or long-term studies.

Hesperidin as a natural flavonoid has a wide range of beneficial properties such as anti-oxidant, anti-cancer, and anti-inflammatory effects. However, low water solubility and low bioavailability have limited its use in clinic. Selenium nanoparticles have attracted great interest in recent years due to their unique characteristics and anti-oxidant activates. Therefore, in this study, we synthesized hesperidin-loaded selenium nanoparticles stabilized by chitosan (Hesp-SeNPs@CS) and investigated its protective effects against glutamate-induced toxicity in PC12 cells. The physicochemical properties of the nanoparticles were evaluated using various techniques. The MTT and intracellular reactive oxygen species (ROS) assay were performed to evaluate the cell viability and ROS levels, respectively. In addition, flow cytometry was used to determine the cell cycle arrest and apoptosis rate in PC12 cells. Our results showed that, Hesp-SeNPs@CS nanoparticles have a 210.6 nm diameter and a negative zeta potential of -15.9 mV. The results of MTT assay revealed that glutamate significantly decreased cell viability by about 50% at a concentration of 20 mM. However, hesperidin at the concentration of 62.5 µM and Hesp-SeNPs@CS at the concentrations of 62.5 and 125 μg/ml significantly decreased the glutamate-induced cell toxicity, sub-G1 cell cycle arrest and apoptosis rate in PC12 cells. In addition, in the groups treated with hesperidin and Hesp-SeNPs@CS, the intracellular ROS induced by glutamate showed a significant decrease compared to glutamate-treated group. Taken together, these results revealed that Hesp-SeNPs@CS has protective effects against glutamate-induced cytotoxicity and oxidative stress damage in PC12 cells that merit further investigation.

Gold nanostructures and a Nafion modified screen-printed carbon electrode (Nafion/AuNS/SPCE) were developed to assess the cell viability of Parkinson’s disease (PD) cell models. The electrochemical measurement of cell viability was reflected by catecholamine neurotransmitter (represented by dopamine) secretion capacity, followed by a traditional tetrazolium-based colorimetric assay for confirmation. Due to the  capacity to synthesize, store, and release catecholamines as well as their unlimited homogeneous proliferation, and ease of manipulation, pheochromocytoma (PC12) cells were used for PD cell modeling. Commercial low-differentiated and highly-differentiated PC12 cells, and home-made nerve growth factor (NGF) induced low-differentiated PC12 cells (NGF-differentiated PC12 cells) were included in the modeling. This approach achieved sensitive and rapid determination of cellular modeling and intervention states. Notably, among the three cell lines, NGF-differentiated PC12 cells displayed the enhanced neurotransmitter secretion level accompanied with attenuated growth rate, incremental dendrites in number and length that were highly resemble with neurons. Therefore, it was selected as the PD-tailorable modeling cell line. In short, the electrochemical sensor can be used to sensitively determine the biological function of neuron-like PC12 cells with negligible destruction and to explore the protective and regenerative impact of various substances on nerve cell model. Graphical Abstract

Anisomycin, a ribotoxic compound, is an efficient inhibitor of eukaryotic translation: at toxic concentrations, it interferes with the function of ribosomal peptidyl transferase, blocks protein synthesis, and ultimately leads to apoptosis. The process is accompanied by the activation of various cellular stress mechanisms. Subinhibitory anysomycin concentrations, in contrast, do not inhibit translation and cause apoptosis, but still activate certain stress pathways. The present study aimed to compare the signaling effects of toxic (1 µg/mL) and non-toxic (10 ng/mL) anisomycin treatment in PC12 cells. In addition, the role of the p53 tumor suppressor protein in these processes was explored, using a PC12 cell line expressing a dominant inhibitory p53 protein. Apoptosis-mediating events (PKR cleavage; eIF2α phosphorylation; activation of caspase 3, 8, and 9 enzymes) were caused by high, but not low, anisomycin concentration in a p53-dependent manner. MAPK pathways (JNK, p38 MAPK, ERK) were stimulated by non-toxic anisomycin treatment, with a more complex p53 involvement. The apoptotic response of cells appeared to be supported by exosomal paracrine signaling.

The aim of the present study was to investigate the expression levels of microRNA(miR)-4722-5p and miR-615-3p in Alzheimer's disease (AD) and their diagnostic value. Blood samples were collected from 33 patients with AD and 33 healthy controls, and an β-amyloid (Aβ)25-35-induced PC12 cell model was also established. The relative mRNA expression levels of miR-4722-5p and miR-615-3p were detected using reverse transcription-quantitative PCR. The correlations between the mRNA expression levels of the two miRNAs and the mini-mental state examination (MMSE) scores were analyzed, and the receiver operating characteristic curve was used to assess the diagnostic value of miR-4722-5p and miR-615-3p in AD. Functional enrichment analysis of the miRNA target genes was performed using The Database for Annotation, Visualization and Integrated Discovery database and the R language analysis package. The mRNA expression levels of miR-4722-5p and miR-615-3p were increased in patients with AD and the Aβ25-35-induced PC12 cell model. The mRNA expression levels of miR-4722-5p and miR-615-3p were negatively correlated with MMSE scores, and the combination of the two miRNAs for AD had an improved diagnostic value than that of each miRNA alone. The results of Gene Ontology (GO) enrichment analysis showed that the target genes of miR-4722-5p were found in the cytoplasm and cytosol, and were mainly involved in protein folding and cell division. The molecular functions included protein binding and GTPase activator activity. The results of Kyoto Encyclopedia of Genes and Genomes analysis showed that miR-4722-5p was associated with the regulation of dopaminergic synapses and mTOR signaling pathways. GO enrichment analysis also revealed that the target genes of miR-615-3p were located in the nucleus and cytoplasm, were involved in the regulation of transcription and protein phosphorylation, and were associated with protein binding, metal ion binding and transcription factor activity. The target genes of miR-615-3p played important roles in the regulation of the Ras and FoxO signaling pathways. In conclusion, miR-4722-5p and miR-615-3p may be potential biomarkers in the early diagnosis of AD.

Inhibition of Sirtuin2 (SIRT2) protein rescues the α-synuclein toxicity in vitro and in vivo models of Parkinson’s disease (PD). Thioacetyl group can structurally mimic the acetyl group and restrain the deacetylating p53 reaction by SIRT2. This work evaluated the biological activity of designed pentapeptides inhibitor containing N-thioacetyl-lysine against SIRT2. Pentapeptide by introducing thioacetyl-lysine as an inhibitor of SIRT2 was screened by molecular docking and synthesized by solid phase method. The inhibition of pure recombinant SIRT2 as well as SIRT2 in serum of PD patients by peptide was done by fluorescent activity assay. The inhibition of SIRT2 was assessed in PC12 cell line by measuring acetylated α-tubulin level. The peptide YKK(ε-thioAc)AM and HRK(ε-thioAc)AM were found to be SIRT2 inhibitors by molecular docking. However, YKK(ε-thioAc)AM was more specific towards SIRT2 than SIRT1 (Sirtuin1). It inhibited recombinant SIRT2 by IC50 value of 0.15 µM and KD values 9.92 × 10−8/M. It also inhibited serum SIRT2 of PD. It increased the acetylation of α-tubulin in PC12 neuroblastoma cells which is essential for maintaining the microtubular cell functions of brain. It can be concluded that novel peptide YKK(ε-thioAc)AM may be a platform for therapeutic agent for Parkinson’s disease targeting SIRT2.Graphic abstract

Heat stroke (HS) is a life-threatening condition that leads to neuronal injury, particularly in the prefrontal cortex, though its mechanisms remain unclear. In this study, we established a rat HS model and observed significant inflammatory responses and neuronal pyroptosis in the prefrontal cortex 6 h post-heat exposure, with the injury severity increasing over time. Mechanistically, HS activated the caspase-1/GSDMD-dependent pyroptosis pathway through NLRP3 inflammasome activation, resulting in IL-1β and IL-18 release. Additionally, HS caused a marked increase in homocysteine (Hcy) levels in both the serum and the prefrontal cortex, accompanied by reduced expression of the Hcy metabolic enzymes MTHFR and CSE, suggesting Hcy metabolism disruption. In vitro, Hcy induced pyroptosis in PC12 cells, elevating IL-1β, IL-18, and LDH levels. Notably, the NLRP3 inhibitor MCC950 mitigated this effect by reducing IL-18 and LDH release. Reducing Hcy in vivo alleviated neuronal pyroptosis and counteracted the YTHDF2-mediated decrease in NLRP3 mRNA m6A modification. Hcy reduced global m6A modification, YTHDF2 expression, and NLRP3 m6A modification in PC12 cells. This study reveals that the activation of a novel m6A-YTHDF2-NLRP3 pathway by Hcy underlies HS-induced neuronal injury, suggesting potential therapeutic targets for HS intervention.

MicroGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As μG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.

The bacterial antibiotic anisomycin is known to induce apoptosis by activating several mitogen-activated protein kinases and by inhibiting protein synthesis. In this study, the influence of p53 protein on the apoptosis-inducing effect of anisomycin was investigated. The effect of protein synthesis-inhibiting concentration of anisomycin on apoptotic events was analyzed using Western blot, DNA fragmentation, and cell viability assays in wild-type PC12 and in mutant p53 protein expressing p143p53PC12 cells. Anisomycin stimulated the main apoptotic pathways in both cell lines, but p143p53PC12 cells showed lower sensitivity to the drug than their wild-type counterparts. Anisomycin caused the activation of the main stress kinases, phosphorylation of the p53 protein and the eukaryotic initiation factor eIF2α, proteolytic cleavage of protein kinase R, Bid, caspase-9 and -3. Furthermore, anisomycin treatment led to the activation of TRAIL and caspase-8, two proteins involved in the extrinsic apoptotic pathway. All these changes were stronger and more sustained in wtPC12 cells. In the presence of the dominant inhibitory p53 protein, p53- dependent genes involved in the regulation of apoptosis may be less transcribed and this can lead to the decrease of apoptotic processes in p143p53PC12 cells.