scholarly journals Axonal TAU sorting requires the C-terminus of TAU but is independent of ANKG and TRIM46 enrichment at the AIS

2020 ◽  
Author(s):  
M. Bell ◽  
S. Bachmann ◽  
J. Klimek ◽  
F. Langerscheidt ◽  
H. Zempel
Keyword(s):  
Cell Model ◽  
Low Cost ◽  
Neuroblastoma Cells ◽  
Neuronal Cell ◽  
Neuronal Polarity ◽  
Primary Neurons ◽  
C Terminus ◽  
Human Neurons ◽  
Mature Neuron ◽  
Axonal Protein

AbstractSomatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimer’s disease and related tauopathies. Cultured rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming and/or require the sacrification of animals. In this study, we evaluated four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells, in comparison to mouse primary neurons, and tested their TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild type TAU, similar to primary neurons. We demonstrate that axonal TAU enrichment requires the presence of the C-terminal half, as a C-terminus-lacking construct (N-term-TAUHA) is not axonally enriched in both neuronal cell models. Moreover, SH-SY5Y-derived neurons do not show formation of a classical axon initial segment (AIS), indicated by the lack of Ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results suggest i) that SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting, which is readily accessible at low cost and without animal need, and that ii) the mechanisms of axonal TAU targeting require the TAU C-terminal half but are independent of ANKG or TRIM46 enrichment at the proximal axon.

scholarly journals An evaluation of the molecular mode of action of trans-resveratrol in the Porphyromonas gingivalis lipopolysaccharide challenged neuronal cell model

2020 ◽  
Author(s):  
Bojlul Bahar ◽  
Sim K. Singhrao
Keyword(s):  
Porphyromonas Gingivalis ◽  
Pathway Analysis ◽  
Mode Of Action ◽  
Cell Model ◽  
Neuroblastoma Cells ◽  
Neuronal Cell ◽  
Nutrient Utilization ◽  
Membrane Receptors ◽  
Gene Markers ◽  
Metabolic Inflammation

AbstractPorphyromonas gingivalis triggers a range of innate immune responses in the host that may contribute to the development of periodontitis and dementing diseases including Alzheimer’s disease (AD). This study aimed to assess the mode of action of trans-resveratrol in modulating the P. gingivalis lipopolysaccharide (PgLPS) induced metabolic inflammation in a neuronal cell model. Confluent IMR-32 neuroblastoma cells were treated with trans-resveratrol from Polygonum cuspidatum in the presence or absence of PgLPS. The abundance of messenger ribo-nucleic acid (mRNA) transcripts of a panel of 92 genes was quantitatively assessed through targeted transcriptome profiling technique and the biochemical pathways affected were identified through Ingenuity Pathway Analysis. Gene expression analysis revealed that trans-resveratrol down-regulated the mRNA of multiple gene markers including growth factors, transcription factors, kinases, trans-membrane receptors, cytokines and enzymes that were otherwise activated by PgLPS treatment of IMR-32 neuroblastoma cells. Pathway analysis demonstrated that the cellular oxidative stress caused by the activation of phosphoinositide-3-kinase/Akt1 (PI3K/Akt1) pathway that leads to the production of reactive oxygen species (ROS), chronic inflammatory response induced by the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway and nutrient utilization pathways were favourably modulated by trans-resveratrol in the PgLPS challenged IMR-32 cells. This study demonstrates the potential of trans-resveratrol as a bioactive compound with multiple modes of intracellular action further supporting its therapeutic application in neuroinflammatory diseases.

scholarly journals The Novel Alpha-2 Adrenoceptor Inhibitor Beditin Reduces Cytotoxicity and Huntingtin Aggregates in Cell Models of Huntington’s Disease

2021 ◽  
Vol 14 (3) ◽  
pp. 257
Author(s):  
Elisabeth Singer ◽  
Lilit Hunanyan ◽  
Magda M. Melkonyan ◽  
Jonasz J. Weber ◽  
Lusine Danielyan ◽  
...  
Keyword(s):  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Neurodegenerative Disorder ◽  
Cell Model ◽  
Resonance Energy Transfer ◽  
Neuronal Cell ◽  
Resonance Energy ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Cell Models

Huntington’s disease (HD) is a monogenetic neurodegenerative disorder characterized by the accumulation of polyglutamine-expanded huntingtin (mHTT). There is currently no cure, and therefore disease-slowing remedies are sought to alleviate symptoms of the multifaceted disorder. Encouraging findings in Alzheimer’s and Parkinson’s disease on alpha-2 adrenoceptor (α2-AR) inhibition have shown neuroprotective and aggregation-reducing effects in cell and animal models. Here, we analyzed the effect of beditin, a novel α2- adrenoceptor (AR) antagonist, on cell viability and mHTT protein levels in cell models of HD using Western blot, time-resolved Foerster resonance energy transfer (TR-FRET), lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) cytotoxicity assays. Beditin decreases cytotoxicity, as measured by TUNEL staining and LDH release, in a neuronal progenitor cell model (STHdh cells) of HD and decreases the aggregation propensity of HTT exon 1 fragments in an overexpression model using human embryonic kidney (HEK) 293T cells. α2-AR is a promising therapeutic target for further characterization in HD models. Our data allow us to suggest beditin as a valuable candidate for the pharmaceutical manipulation of α2-AR, as it is capable of modulating neuronal cell survival and the level of mHTT.

On the Application of Cultured Neuronal Cell Lines in Neurotoxicological Studies: Cytotoxicity of Acrylamide

1983 ◽  
Vol 11 (3) ◽  
pp. 135-145
Author(s):  
Erik Walum
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Neuroblastoma Cells ◽  
Neuronal Cell ◽  
Leucine Incorporation ◽  
Biochemical Process ◽  
Neurite Retraction ◽  
Mouse Neuroblastoma ◽  
Neuronal Cell Lines

Summary Acrylamide, a well known neurotoxic compound, was used in a first evaluation of cultured mouse neuroblastoma cells as an alternative to animal models for neurotoxicological studies. Hence, the effects of acrylamide on the growth, size, morphology and leucine incorporation of three neuroblastoma (41A3, N18 and N1E115), one neuroblastoma x glioma hybrid (NG108CC15), two glioma (138MG and C6) and two fibroblast (RLF and RMC) cell lines were studied. It was found that the concentration of acrylamide needed to inhibit the growth by 50% in 24 hr was similar in all cell lines, i.e. around 2 x 10-4g/ml culture medium. In the two cell lines, N1E115 and NG108CC15, acrylamide at this concentration caused neurite retraction and at higher concentrations (5 x 10-4g/ml) a decrease in cell viability. In a concentration range of 5 x 10-5 - 5 x 10-4g/ml acrylamide did not affect cell size, or at 2 x 10-4g/ml incorporation of leucine into trichloroacetic acid precipitable material. It is suggested that acrylamide interferes with a biochemical process common to all the tested cells, but of greater importance in differentiated nerve cells than in others. Whether this process is consistent with the in vivo target for the neurotoxic action of acrylamide remains to be unravelled.

scholarly journals Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin

2007 ◽  
Vol 192 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Fang Cai ◽  
Armen V Gyulkhandanyan ◽  
Michael B Wheeler ◽  
Denise D Belsham
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Cell Model ◽  
Neuronal Cell ◽  
Cell Types ◽  
Glucose Sensing ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

Abstract 3115: Low-Level Laser Therapy Protects Against OGD-induced Neurotoxicity Of Primary Cultured Mouse Cortical Neurons

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Zhanyang Yu ◽  
Ning Liu ◽  
Eng H Lo ◽  
Thomas J McCarthy ◽  
Xiaoying Wang
Keyword(s):  
Laser Therapy ◽  
Mitochondrial Function ◽  
Cortical Neurons ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Low Level Laser Therapy ◽  
Primary Neurons ◽  
Novel Therapy ◽  
Low Level ◽  
Mtt Reduction

Background: Low level light (or laser) therapy (LLLT) has been studied and practiced for promoting wound healing, reducing pain, inflammation, and ischemic tissue damage. Recently, a series of experimental and clinical investigations have suggested that LLLT may be a novel therapy against hypoxic/ischemic brain damage. A clinical trial of LLLT therapy for ischemic stroke is now on going. However, the molecular mechanism of LLLT-conferred neuroprotection remains poorly defined. In this study, we tested our hypothesis that LLLT may attenuate impairments of mitochondrial function induced by hypoxic/ischemic insults in primary cultured mouse cortical neurons. Method: At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. One 810-nm LLLT treatment was applied for 2 minutes at 2 hr after reoxygenation. Neurotoxicity was measured after 20 hr after reoxygenation by LDH release assay. We also measured MTT reduction and mitochondria membrane potential (MMP) at 2 hr after LLLT treatment as markers of mitochondrial function. Results: The neurotoxicity study showed that 4 hr OGD plus 20 hr reoxygenation caused 33.8± 3.4% neuronal cell death, while LLLT treatment significantly reduced the neuronal death rate to 23.6± 2.9% (30.2% reduction, n=6, p smaller than 0.05). Mitochondrial functional assays showed OGD decreased MTT reduction to 75.9± 2.68%, but LLLT treatment significantly rescued MTT reduction to 87.6±4.55% (15.4% improvement, n=6, p smaller than 0.05). Furthermore, after OGD, MMP was reduced to 48.9±4.39%, while LLLT treatment significantly ameliorated this reduction to 89.6± 13.9% (83% improvement, n=4, p smaller than 0.05) compared to normoxic controls. Conclusion: The present study suggests that LLLT treatment is protective against OGD-induced neurotoxicity of primary neurons and that this protection may be conferred through preservation or rescue of mitochondrial function.

Proteasome or calpain inhibition does not alter cellular tau levels in neuroblastoma cells or primary neurons

2005 ◽  
Vol 7 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Maile R. Brown ◽  
Vimala Bondada ◽  
Jeffery N. Keller ◽  
Jeffery Thorpe ◽  
James W. Geddes
Keyword(s):  
Neuroblastoma Cells ◽  
Primary Neurons ◽  
Calpain Inhibition

A complex consisting of pp60c-src/pp60c-srcN and a 38 kDa protein is highly enriched in growth cones from differentiated SH-SY5Y neuroblastoma cells

1992 ◽  
Vol 103 (1) ◽  
pp. 233-243
Author(s):  
G. Meyerson ◽  
K.H. Pfenninger ◽  
S. Pahlman
Keyword(s):  
Growth Cone ◽  
Gel Electrophoresis ◽  
Neuroblastoma Cells ◽  
Growth Cones ◽  
Cell Periphery ◽  
Primary Neurons ◽  
Reducing Conditions ◽  
Oligomeric Complex ◽  
Nerve Growth Cones

Nerve growth cones of primary neurons are highly enriched in the proto-oncogene product pp60c-src. In order to investigate this molecule further in growing neuronal cells, growth cone and cell body fractions were prepared from human SH-SY5Y neuroblastoma cells differentiated neuronally in vitro under the influence of phorbol ester. The fractions were characterized ultrastructurally and by biochemical criteria. The neuronal (pp60c-srcN) and the fibroblastic (pp60c-src) forms of pp60src are slightly enriched and activated in the growth cones relative to the perikarya. Immunoprecipitates of pp60src from differentiated SH-SY5Y growth cones contain at least four phosphoproteins in addition to pp60src. One of these, pp38, migrates as a 100–140 kDa complex with pp60src under non-reducing conditions of gel electrophoresis. The pp38/pp60src complex is not easily detected in non-differentiated SH-SY5Y cells or perikarya of differentiated SH-SY5Y cells, but it is highly enriched in the growth cone preparation. These data suggest that growth-cone pp60src exists in a disulfide-linked oligomeric complex. The complex appears to be assembled only in the cell periphery and may be dependent upon neuronal differentiation.

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