Sturgeon (Acipenser schrenckii) spinal cord peptides: Antioxidative and acetylcholinesterase inhibitory efficacy and mechanisms.

Providing antioxidants and targeting acetylcholinesterase (AChE) are key strategies in treating neurocognitive dysfunction. In this study, bioactive sturgeon (Acipenser schrenckii) spinal cord peptides (SSCPs) with antioxidant and AChE inhibitory potency were extracted and separated from sturgeon spinal cord by enzymatic hydrolysis and ultrafiltration, and targeted peptide PGGW was screened via computer simulated molecular docking. Further, the molecular dynamic interactions of the PGGW with superoxide dismutase (SOD) and AChE were analyzed, and the protective effect of PGGW on glutamate-induced PC12 cells in vitro was evaluated. The <3 kDa fraction of SSCPs displays the most potent antioxidative efficacy (1 mg/mL, DPPH•: 89.07%, ABTS+: 76.35%). Molecular dynamics simulation showed that PGGW was stable within AChE and tightly bound to residues SER203, PHE295, ILE294 and TRP236. When combined with SOD, the indole group of PGGW was stuck inside SOD, but the tail chain PGG fluctuated greatly outside. Surface plasmon resonance demonstrated that PGGW has a high binding affinity for AChE (KD = 1.4 mM) and 0.01 mg/mL PGGW provided good protection against glutamate-induced apoptosis. The findings suggest a promising strategy for drug research on neurodegenerative diseases.

Platinum nanowires/MXene nanosheets/porous carbon ternary nanocomposites for in situ monitoring of dopamine released from neuronal cells.

Dopamine is an important neurotransmitter in the body and closely related to many neurodegenerative diseases. Therefore, the detection of dopamine is of great significance for the diagnosis and treatment of diseases, screening of drugs and unraveling of relevant pathogenic mechanisms. However, the low concentration of dopamine in the body and the complexity of the matrix make the accurate detection of dopamine challenging. Herein, an electrochemical sensor is constructed based on ternary nanocomposites consisting of one-dimensional Pt nanowires, two-dimensional MXene nanosheets, and three-dimensional porous carbon. The Pt nanowires exhibit excellent catalytic activity due to the abundant grain boundaries and highly undercoordinated atoms; MXene nanosheets not only facilitate the growth of Pt nanowires, but also enhance the electrical conductivity and hydrophilicity; and the porous carbon helps induce significant adsorption of dopamine on the electrode surface. In electrochemical tests, the ternary nanocomposite-based sensor achieves an ultra-sensitive detection of dopamine (S/N = 3) with a low limit of detection (LOD) of 28 nM, satisfactory selectivity and excellent stability. Furthermore, the sensor can be used for the detection of dopamine in serum and in situ monitoring of dopamine release from PC12 cells. Such a highly sensitive nanocomposite sensor can be exploited for in situ monitoring of important neurotransmitters at the cellular level, which is of great significance for related drug screening and mechanistic studies.

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).

N6-methyldeoxyadenosine modification difference contributes to homocysteine-induced mitochondrial perturbation in rat hippocampal primary neurons and PC12 cells.

Elevated homocysteine (Hcy) levels are detrimental to neuronal cells and contribute to cognitive dysfunction in rats. Mitochondria plays a crucial role in cellular energy metabolism. Interestingly, the damaging effects of Hcy in vivo and in vitro conditions exhibit distinct results. Herein, we aimed to investigate the effects of Hcy on mitochondrial function in primary neurons and PC12 cells and explore the underlying mechanisms involved. The metabolic intermediates of Hcy act as methyl donors and play important epigenetic regulatory roles. N6-methyldeoxyadenosine (6 mA) modification, which is enriched in mitochondrial DNA (mtDNA), can be mediated by methylase METTL4. Our study suggested that mitochondrial perturbation caused by Hcy in primary neurons and PC12 cells may be attributable to mtDNA 6 mA modification difference. Hcy could activate the expression of METTL4 within mitochondria to facilitate mtDNA 6 mA status, and repress mtDNA transcription, then result in mitochondrial dysfunction.

Design, Synthesis, and Biological Evaluation of Novel Cinnamide Derivatives as Neuroprotective Agents for the Treatment of Cerebral Ischemia.

BACKGROUND: Ischemic stroke, the most common type of cerebrovascular accident, is a major cause of severe disability among adults worldwide. Although there has been progress in interventions for ischemic stroke in the past decades, there is no effective treatment to prevent brain damage in acute ischemic stroke. Therefore, it is urgent to develop novel neuroprotective agents with a wide therapeutic time window to provide a better prognosis for ischemic stroke patients. OBJECTIVE: The current study aimed to synthesize novel derivatives with substituent cinnamide scaffolds, evaluate biological activity, and obtain neuroprotective agents. METHODS: The target compounds were synthesized using classical methods of medicinal chemistry. The neuroprotective effects in vitro against Glu-induced neurotoxicity injury were evaluated in PC12 cells by MTT assay. The cell apoptosis was analyzed by flow cytometer. The proteins were detected by western blotting. The neuroprotective activities in vivo were determined in two in vivo models of global and focal cerebral ischemia. RESULTS: Among the title compounds, 9t, 9u, 9y, and 9z exhibited good neuroprotection in vivo and in vitro, which were selected and further studied to determine their mechanism of action. 9t, 9u, 9y and 9z protected PC12 cells against glutamate-induced apoptosis in a dose-dependent manner via caspase-3 pathway. Moreover, the four compounds significantly reduced brain infarct area and exhibited excellent neuroprotective activities in the in vivo MCAO model. CONCLUSION: Compounds 9t, 9u, 9y, and 9z, as potent neuroprotective agents with anti- neurotoxicity activity in vitro and anticerebral infarction efficacy in vivo, might serve as a useful molecular tool for further physiology and pathophysiology function studies, leading to potential clinical therapeutic agents for ischemic injury.

Study on the Neuroprotective Effects of Eight Iridoid Components Using Cell Metabolomics.

Iridoid components have been reported to have significant neuroprotective effects. However, it is not yet clear whether the efficacy and mechanisms of iridoid components with similar structures are also similar. This study aimed to compare the neuroprotective effects and mechanisms of eight iridoid components (catalpol (CAT), genipin (GE), geniposide (GEN), geniposidic acid (GPA), aucubin (AU), ajugol (AJU), rehmannioside C (RC), and rehmannioside D (RD)) based on corticosterone (CORT)-induced injury in PC12 cells. PC12 cells were randomly divided into a normal control group (NC), model group (M), positive drug group (FLX), and eight iridoid administration groups. Firstly, PC12 cells were induced with CORT to simulate neuronal injury. Then, the MTT method and flow cytometry were applied to evaluate the protective effects of eight iridoid components on PC12 cell damage. Thirdly, a cell metabolomics study based on ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was performed to explore changes in relevant biomarkers and metabolic pathways following the intervention of administration. The MTT assay and flow cytometry analysis showed that the eight iridoid components can improve cell viability, inhibit cell apoptosis, reduce intracellular ROS levels, and elevate MMP levels. In the PCA score plots, the sample points of the treatment groups showed a trend towards approaching the NC group. Among them, AU, AJU, and RC had a weaker effect. There were 38 metabolites (19 metabolites each in positive and negative ion modes, respectively) identified as potential biomarkers during the experiment, among which 23 metabolites were common biomarkers of the eight iridoid groups. Pathway enrichment analysis revealed that the eight iridoid components regulated the metabolism mainly in relation to D-glutamine and D-glutamate metabolism, arginine biosynthesis, the TCA cycle, purine metabolism, and glutathione metabolism. In conclusion, the eight iridoid components could reverse an imbalanced metabolic state by regulating amino acid neurotransmitters, interfering with amino acid metabolism and energy metabolism, and harmonizing the level of oxidized substances to exhibit neuroprotective effects.

In vitro drug screening models derived from different PC12 cell lines for exploring Parkinson's disease based on electrochemical signals of catecholamine neurotransmitters.

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.

Effects of latroeggtoxin-VI on dopamine and α-synuclein in PC12 cells and the implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by death of dopaminergic neurons leading to dopamine deficiency, excessive α-synuclein facilitating Lewy body formation, etc. Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin discovered from the eggs of spider L. tredecimguttatus, was previously found to promote the synthesis and release of PC12 cells, showing a great potential as a drug candidate for PD. However, the relevant mechanisms have not been understood completely. The present study explored the mechanism underlying the effects of LETX-VI on dopamine and α-synuclein of PC12 cells and the implications for PD. RESULTS: After PC12 cells were treated with LETX-VI, the level of dopamine was significantly increased in a dose-dependent way within a certain range of concentrations. Further mechanism analysis showed that LETX-VI upregulated the expression of tyrosine hydroxylase (TH) and L-dopa decarboxylase to enhance the biosynthesis of dopamine, and downregulated that of monoamine oxidase B to reduce the degradation of dopamine. At the same time, LETX-VI promoted the transport and release of dopamine through modulating the abundance and/or posttranslational modification of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT). While the level of dopamine was increased by LETX-VI treatment, α-synuclein content was reduced by the spider toxin. α-Synuclein overexpression significantly decreased the dopamine level and LETX-VI efficiently alleviated the inhibitory action of excessive α-synuclein on dopamine. In the MPTP-induced mouse model of PD, application of LETX-VI ameliorated parkinsonian behaviors of the mice, and reduced the magnitude of MPTP-induced α-synuclein upregulation and TH downregulation. In addition, LETX-VI displayed neuroprotective effects by inhibiting MPTP-induced decrease in the numbers of TH-positive and Nissl-stained neurons in mouse brain tissues. CONCLUSIONS: All the results demonstrate that LETX-VI promotes the synthesis and release of dopamine in PC12 cells via multiple mechanisms including preventing abnormal α-synuclein accumulation, showing implications in the prevention and treatment of PD.

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

Extraction, rapid preparation and neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir.

Searching for new anti-ischemic stroke (anti-IS) drugs has always been a hot topic in the pharmaceutical industry. Natural products are an important source of discovering anti-IS drugs. The aim of the present study is to extract, rapidly prepare and explore the neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir., which is a kind of Tibetan medicine with a clear anti-IS effect. The results showed that 95% ethanol was the optimal extraction solvent. A three-step rapid preparation method for texasin was successfully established, with a purity of 99.2%. Texasin at the concentration of 25-100 µM had no effect on the viability of normal cultured PC12 cells; 12.5 and 25 µM texasin could enhance the viability of PC12 cells damaged by oxygen and glucose deprivation/reoxygenation (OGD/R), and their effects are comparable to the positive drug edaravone at the concentration of 50 µM. Compared with the normal group, the expression of Bcl-2 protein in OGD/R-injured PC12 cells was downregulated (p < 0.01), and that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins were upregulated (p < 0.01, p < 0.001). Compared with the OGD/R group, 25 µM texasin could upregulate the expression of Bcl-2 protein (p < 0.01), and downregulate that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins (p < 0.01, p < 0.001). The 7-OH and 1-O of texasin formed H-bonds with residues Cys891 of the hinge ß-strand of PERK, which is crucial for kinase inhibitors. The above results suggest that the method established in the present study achieved rapid preparation of high-purity texasin. Texasin might inhibit neuronal apoptosis via the regulation of endoplasmic reticulum stress PERK/eIF2α/ATF4/CHOP signalling pathway to exert a protective effect on OGD/R-injured PC12 cells. Aiding by molecular docking, texasin was assumed to be a potential PERK inhibitor.

Effects of latroeggtoxin-VI on dopamine and α-synuclein in PC12 cells and the implications for Parkinson's disease

Background Parkinson's disease (PD) is characterized by death of dopaminergic neurons leading to dopamine deficiency, excessive α-synuclein facilitating Lewy body formation, etc. Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin discovered from the eggs of spider L. tredecimguttatus, was previously found to promote the synthesis and release of PC12 cells, showing a great potential as a drug candidate for PD. However, the relevant mechanisms have not been understood completely. The present study explored the mechanism underlying the effects of LETX-VI on dopamine and α-synuclein of PC12 cells and the implications for PD. Results After PC12 cells were treated with LETX-VI, the level of dopamine was significantly increased in a dose-dependent way within a certain range of concentrations. Further mechanism analysis showed that LETX-VI upregulated the expression of tyrosine hydroxylase (TH) and L-dopa decarboxylase to enhance the biosynthesis of dopamine, and downregulated that of monoamine oxidase B to reduce the degradation of dopamine. At the same time, LETX-VI promoted the transport and release of dopamine through modulating the abundance and/or posttranslational modification of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT). While the level of dopamine was increased by LETX-VI treatment, α-synuclein content was reduced by the spider toxin. α-Synuclein overexpression significantly decreased the dopamine level and LETX-VI efficiently alleviated the inhibitory action of excessive α-synuclein on dopamine. In the MPTP-induced mouse model of PD, application of LETX-VI ameliorated parkinsonian behaviors of the mice, and reduced the magnitude of MPTP-induced α-synuclein upregulation and TH downregulation. In addition, LETX-VI displayed neuroprotective effects by inhibiting MPTP-induced decrease in the numbers of TH-positive and Nissl-stained neurons in mouse brain tissues. Conclusions All the results demonstrate that LETX-VI promotes the synthesis and release of dopamine in PC12 cells via multiple mechanisms including preventing abnormal α-synuclein accumulation, showing implications in the prevention and treatment of PD.

Enhancing the Study of Quantal Exocytotic Events: Combining Diamond Multi-Electrode Arrays with Amperometric PEak Analysis (APE) an Automated Analysis Code.

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.