scholarly journals A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258420
Author(s):  
Ryohei Tanaka-Kanegae ◽  
Koichiro Hamada
Keyword(s):  
Cell Line ◽  
Neuroblastoma Cell ◽  
Cholinergic Neurons ◽  
Mechanisms Of Action ◽  
Neuroblastoma Cell Line ◽  
Ache Inhibitor ◽  
Vitro Assay ◽  
Food Ingredients ◽  
High Doses

Background Cholinergic neurons utilize choline (Ch) to synthetize acetylcholine (ACh) and contain a high-affinity Ch transporter, Ch acetyltransferase (ChAT), ACh receptors, and acetylcholinesterase (AChE). As the depletion or malfunction of each component of the cholinergic system has been reported in patients with dementia, many studies have sought to evaluate whether treatment candidates affect each of the cholinergic components. The associated changes in the cholinergic components may be reflected by intra- or extra-cellular ACh levels, with an increase in extracellular ACh levels occurring following AChE inhibition. We hypothesized that increases in intracellular ACh levels can be more sensitively detected than those in extracellular ACh levels, thereby capturing subtle effects in the cholinergic components other than AChE. The objective of this study was to test this hypothesis. Methods We developed an in vitro model to measure both extracellular and intracellular ACh levels using the human cholinergic neuroblastoma cell line, LA-N-2, which have been reported to express Ch transporter, ChAT, muscarinic ACh receptor (mAChR), and AChE. With this model, we evaluated several drug compounds and food constituents reported to improve cholinergic function through various mechanisms. In addition, we conducted western blotting to identify the subtype of mAChR that is expressed on the cell line. Results Our cell-based assay system was capable of detecting increases in extracellular ACh levels induced by an AChE inhibitor at relatively high doses, as well as increases in intracellular ACh levels following the administration of lower AChE-inhibitor doses and an mAChR agonist. Moreover, increases in intracellular ACh levels were observed even after treatment with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. In addition, we revealed that LA-N-2 cells expressed mAChR M2. Conclusion The findings support our hypothesis and indicate that the developed assay model can broadly screen compounds from drugs to food ingredients, with varying strengths and mechanisms of action, to develop treatments for ACh-relevant phenomena, including dementia and aging-related cognitive decline.

scholarly journals Inhibition of the SK-N-MC human neuroblastoma cell line in vivo and in vitro by a novel nutrient mixture

2013 ◽  
Vol 29 (5) ◽  
pp. 1714-1720 ◽  
Author(s):  
M. WAHEED ROOMI ◽  
TATIANA KALINOVSKY ◽  
NUSRATH W. ROOMI ◽  
ALEKSANDRA NIEDZWIECKI ◽  
MATTHIAS RATH
Keyword(s):  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Human Neuroblastoma Cell Line

scholarly journals Camptothecin Regulates Microglia Polarization and Exerts Neuroprotective Effects via Activating AKT/Nrf2/HO-1 and Inhibiting NF-κB Pathways In Vivo and In Vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Dewei He ◽  
Shoupeng Fu ◽  
Ang Zhou ◽  
Yingchun Su ◽  
Xiyu Gao ◽  
...  
Keyword(s):  
Cell Line ◽  
Inflammatory Responses ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Mechanism Study ◽  
Antitumor Effects ◽  
Neuroprotective Effects ◽  
Microglia Polarization

Microglia, the main immune cells in the brain, participate in the innate immune response in the central nervous system (CNS). Studies have shown that microglia can be polarized into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. Accumulated evidence suggests that over-activated M1 microglia release pro-inflammatory mediators that damage neurons and lead to Parkinson’s disease (PD). In contrast, M2 microglia release neuroprotective factors and exert the effects of neuroprotection. Camptothecin (CPT), an extract of the plant Camptotheca acuminate, has been reported to have anti-inflammation and antitumor effects. However, the effect of CPT on microglia polarization and microglia-mediated inflammation responses has not been reported. In our study we found that CPT improved motor performance of mice and reduced the loss of neurons in the substantia nigra (SN) of the midbrain in LPS-injected mice. In the mechanism study, we found that CPT inhibited M1 polarization of microglia and promotes M2 polarization via the AKT/Nrf2/HO-1 and NF-κB signals. Furthermore, CPT protected the neuroblastoma cell line SH-SY5Y and dopaminergic neuron cell line MN9D from damage mediated by microglia activation. In conclusion, our results demonstrate that CPT regulates the microglia polarization phenotype via activating AKT/Nrf2/HO-1 and inhibiting NF-κB pathways, inhibits neuro-inflammatory responses, and exerts neuroprotective effects in vivo and in vitro.

Functional Changes in Potassium Conductances of the Human Neuroblastoma Cell Line SH-SY5Y During In Vitro Differentiation

1998 ◽  
Vol 79 (2) ◽  
pp. 648-658 ◽  
Author(s):  
Patrizia Tosetti ◽  
Vanni Taglietti ◽  
Mauro Toselli
Keyword(s):  
Steady State ◽  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Human Neuroblastoma Cell Line ◽  
Differentiated Cells ◽  
Voltage Dependent

Tosetti, Patrizia, Vanni Taglietti, and Mauro Toselli. Functional changes in potassium conductances of the human neuroblastoma cell line SH-SY5Y during in vitro differentiation. J. Neurophysiol. 79: 648–658, 1998. The electrophysiological properties of voltage-dependent outward currents were investigated under voltage-clamp conditions in the human neuroblastoma cell line SH-SY5Y before and after in vitro differentiation with retinoic acid, by using the whole cell variant of the patch-clamp technique. Voltage steps to depolarizing potentials from a holding level of −90 mV elicited, in both undifferentiated and differentiated cells, outward potassium currents that were blocked by tetraethylammonium, but were unaffected by 4-aminopyridine, cadmium, and by shifts of the holding potentials to −40 mV. These currents activated rapidly and inactivated slowly in a voltage-dependent manner. In undifferentiated cells the threshold for current activation was about −30 mV, with a steady-state half activation potential of 19.5 mV. Maximum conductance was 4.3 nS and mean conductance density was 0.34 mS/cm2. Steady-state half inactivation potential was −13.8 mV and ∼10% of the current was resistant to inactivation. Both activation and inactivation kinetics were voltage dependent. In differentiated cells the threshold for current activation was about −20 mV, with a half potential for steady-state activation of 37.0 mV. Maximum conductance was 15.2 nS and mean conductance density was 0.78 mS/cm2. Steady-state half inactivation potential was −9.7 mV and ∼37% of the current was resistant to inactivation. Both activation and inactivation kinetics were voltage dependent. This diversity in potassium channel properties observed between undifferentiated and differentiated cells was related to differences in cell excitability. Under current-clamp conditions, the action potential repolarization rate in differentiated cells was about threefold faster than that of the abortive action potentials elicitable in undifferentiated cells. Furthermore, during prolonged stimulation, trains of spikes could be generated in some differentiated cells but not in undifferentiated cells.

scholarly journals HLXB9 Gene Expression, and Nuclear Location during In Vitro Neuronal Differentiation in the SK-N-BE Neuroblastoma Cell Line

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e105481 ◽  
Author(s):  
Claudia Giovanna Leotta ◽  
Concetta Federico ◽  
Maria Violetta Brundo ◽  
Sabrina Tosi ◽  
Salvatore Saccone
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Neuronal Differentiation ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Nuclear Location

scholarly journals Suppression of Progranulin Expression Leads to Formation of Intranuclear TDP-43 Inclusions In Vitro: A Cell Model of Frontotemporal Lobar Degeneration

2019 ◽  
Vol 78 (12) ◽  
pp. 1124-1129
Author(s):  
Jiuling Zhu ◽  
Ning Wang ◽  
Xianan Li ◽  
Xiaojing Zheng ◽  
Junli Zhao ◽  
...  
Keyword(s):  
Cell Line ◽  
Frontotemporal Lobar Degeneration ◽  
Cell Model ◽  
Neuroblastoma Cell ◽  
Brain Regions ◽  
Neuroblastoma Cell Line ◽  
Null Alleles ◽  
Intranuclear Inclusions ◽  
A Cell

Abstract Mutations in the GRN gene coding for progranulin (PGRN) are responsible for many cases of familial frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein 43 (TDP-43)-positive inclusions (FTLD-TDP). GRN mutations create null alleles resulting in decreased progranulin protein or haploinsufficiency. FTLD-TDP with GRN mutations is characterized by lentiform neuronal intranuclear inclusions that are positive for TDP-43 in affected brain regions. In this study, by stably expressed short hairpin RNA, we established a neuroblastoma cell line with decreased PGRN level. This cell line reveals TDP-43-positive intranuclear inclusions. In addition, replacement with purified PGRN protein restores normal TDP-43 nuclear distribution. This cell model can be valuable for the study of the role of PGRN in the pathogenesis in FTLD-TDP.

scholarly journals Histidyl-Proline Diketopiperazine Isomers as Multipotent Anti-Alzheimer Drug Candidates

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 737 ◽  
Author(s):  
Hasan Turkez ◽  
Ivana Cacciatore ◽  
Mehmet Enes Arslan ◽  
Erika Fornasari ◽  
Lisa Marinelli ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Lymphocytes ◽  
Neuroblastoma Cell ◽  
Disease Model ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Gene Expressions ◽  
Damage Potential ◽  
Human Neuroblastoma

Cyclic dipeptides administered by both parenteral and oral routes are suggested as promising candidates for the treatment of neurodegeneration-related pathologies. In this study, we tested Cyclo (His-Pro) isomers (cHP1-4) for their anti-Alzheimer potential using a differentiated human neuroblastoma cell line (SH-SY5Y) as an Alzheimer’s disease (AD) experimental model. The SH-SY5Y cell line was differentiated by the application of all-trans retinoic acid (RA) to obtain mature neuron-like cells. Amyloid-beta 1-42 (Aβ1-42) peptides, the main effector in AD, were administered to the differentiated cell cultures to constitute the in vitro disease model. Next, we performed cell viability analyses 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays) to investigate the neuroprotective concentrations of cyclodipeptides using the in vitro AD model. We evaluated acetylcholinesterase (AChE), α- and β-secretase activities (TACE and BACE1), antioxidant potency, and apoptotic/necrotic properties and performed global gene expression analysis to understand the main mechanism behind the neuroprotective features of cHP1-4. Moreover, we conducted sister chromatid exchange (SCE), micronucleus (MN), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) analyses to evaluate the genotoxic damage potential after applications with cHP1-4 on cultured human lymphocytes. Our results revealed that cHP1-4 isomers provide a different degree of neuroprotection against Aβ1-42-induced cell death on the in vitro AD model. The applications with cHP1-4 isomers altered the activity of AChE but not the activity of TACE and BACE1. Our analysis indicated that the cHP1-4 increased the total antioxidant capacity without altering total oxidative status levels in the cellular AD model and that cHP1-4 modulated the alterations of gene expressions by Aβ1-42 exposure. We also observed that cHP1-4 exhibited noncytotoxic and non-genotoxic features in cultured human whole blood cells. In conclusion, cHP1-4 isomers, especially cHP4, have been explored as novel promising therapeutics against AD.

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