Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells

Datki, Zsolt; Juhász, Anna; Gálfi, Márta; Soós, Katalin; Papp, Rita; Zádori, Dénes; Penke, Botond

doi:10.1016/j.brainresbull.2003.09.011

Cited by 147 publications

(132 citation statements)

References 30 publications

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“…Some authors question the use of the MTT assay as a reporter of A␤-mediated cytotoxicity [70]. In contrast, according to other researchers, the MTT assay, generally shows a good correlation with other viability tests and in vivo results [21]. Therefore, even though inhibition of MTT reduction represents a controversial indicator of A␤-mediated cell injury, other corroborating evidence was added in this study.…”

Section: Discussionmentioning

confidence: 54%

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts

Cecchi

Fiorillo

Baglioni

et al. 2007

Neurobiology of Aging

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Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimer's disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of A␤ 1-40 and A␤ 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

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Section: Discussionmentioning

confidence: 54%

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts

Cecchi

Fiorillo

Baglioni

et al. 2007

Neurobiology of Aging

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“…5), and the cells were incubated for 48 h. 10 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) labeling reagent was added to each well, and the samples were incubated for a further 4 h. 100 l of solubilization mixture (10% SDS, 0.01 M HCl) were added to each well, and the samples were incubated overnight. Absorbance of blue formazan at 570 nm was measured with a plate reader (28).…”

Section: Methodsmentioning

confidence: 99%

“…Cell Viability Measured by MTT Reduction and DNA Fragmentation-The effect of amyloid structures on the oxidative metabolism of IMR-32 cells was evaluated by MTT assay, in which the metabolic activity of the cells was assessed by their ability to cleave MTT to a water-insoluble formazan (28). The cell viability decreased with increasing protein concentrations reaching a maximum toxic effect of ϳ30 and ϳ15% in the presence of 64 M EL amyloid aggregates formed at pH 4.5 and 2.0, respectively (Fig.…”

Section: Table I Molecular Dimensions Of El-soluble Amyloid Oligomersmentioning

confidence: 99%

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Mališauskas

Östman

Darinskas

et al. 2005

Journal of Biological Chemistry

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In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57°C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.The molecular basis of the pathogenicity of amyloid aggregates is a central theme in understanding the causes of a wide range of amyloid-related diseases, including Alzheimer's, Parkinson's, prion diseases, type II diabetes, and familial amyloidotic polyneuropathy (1-4). There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver (5, 6). In neurodegenerative diseases, by contrast, there is no clear correlation between the amount of amyloid deposition and the clinical severity of disease. Significant cognitive impairment of Alzheimer's patients was observed without noticeable amyloid deposits in the brain, although the level of readily soluble amyloid oligomeric assemblies in the Alzheimer's brain was found to be greatly elevated (7,8).The evidence is accumulating that prefibrillar aggregates are cytotoxic both in vivo and in vitro (4,8,9), although this question is still open to debate. Moreover, aggregates of the proteins, which are not related to clinical amyloidoses, such as human ␣-lactalbumin (10), SH3 domain, or HypF-N protein, have also been found to be cytotoxic, which implies a common mechanism for cytotoxicity of misfolded proteins (11). By contrast, it has been shown that the mature amyl...

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“…Retinoic acid was used to induce neuronal differentiation (Fig. 1A) similar to primary cell cultures (Uberti et al, 1997;Datki et al, 2003), which was controlled by immunohistochemistry with MAP2 (Sigma-Aldrich) and anti-tau Alz50 (gift from Dr. Peter Davies) antibodies. FLAG epitope-tagged wild-type human SphK1 (hSphK1) cDNA and hSphK1 containing the G82D mutation, subcloned into pcDNA3* vector (Pitson et al, 2000), were used for stable transfection in SH-SY5Y cells.…”

Section: Methodsmentioning

confidence: 99%

Critical Role for Sphingosine Kinase-1 in Regulating Survival of Neuroblastoma Cells Exposed to Amyloid-β Peptide

Gomez‐Brouchet¹,

Pchejetski²,

Brizuela³

et al. 2007

Mol Pharmacol

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We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid ␤ (A␤) peptide (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Upon incubation with A␤, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited A␤-induced cell death. SphK1 overexpression impaired the cytotoxicity of A␤, whereas SphK1 silencing by RNA interference mimicked A␤-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against A␤ toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-␤1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of A␤-treated cells.

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Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells

Cited by 147 publications

References 30 publications

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Critical Role for Sphingosine Kinase-1 in Regulating Survival of Neuroblastoma Cells Exposed to Amyloid-β Peptide

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