Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Duquesne University

Postmortem tissue from patients with neurodegeneration exhibits protein-misfolding stress and reduced proteasome activity. This hallmark burden of proteotoxic stress has led to the term "proteinopathies" for neurodegenerative diseases. Proteinopathies may also be exacerbated by previous insults, according to the two hit hypothesis of accelerated neurodegeneration. In order to model the response to two successive insults in a high-throughput fashion, we exposed the neuronal cell line N2a to two hits of the proteasome inhibitor MG132 and performed three unbiased viability assays. MG132 toxicity was synergistically exacerbated following sequential hits provided the first hit was high enough to be toxic. This accelerated viability loss was apparent by (1) a nuclear and cytoplasmic stain (DRAQ5+Sapphire), (2) immunocytochemistry for a cytoskeletal marker (α-tubulin), and (3) ATP levels (Cell Titer Glo). Ubiquitin-conjugated proteins were raised by toxic, but not subtoxic MG132, and were thus correlated with toxicity exacerbation at higher doses. We hypothesized that levels of autophagic and antioxidant defenses would be reduced with toxic, but not subtoxic MG132, explaining their differential impact on a second hit. However, proteins involved in chaperone-mediated autophagy were raised by toxic MG132, not reduced. Furthermore, inhibiting autophagy enhanced the toxicity of both subtoxic and toxic MG132 as well as of dual hits, suggesting that autophagic removal of cellular debris protected against proteasome inhibition. Two toxic hits of MG132 synergistically decreased the antioxidant glutathione. The glutathione precursor N-acetyl cysteine reversed this glutathione loss and prevented the toxic response to dual hits by all three assays. Dietary supplementation with N-acetyl cysteine benefits Alzheimer's patients and is currently undergoing clinical trials in Parkinson's disease. The present report is the first demonstration that this versatile compound is protective against synergistic loss of viability as well as of glutathione following unrelenting, sequential hits of proteotoxic stress as may occur in the diseased brain.

Rescue from a two hit, high-throughput model of neurodegeneration with N-acetyl cysteine.

Neurodegeneration is characterized by an accumulation of misfolded proteins in neurons. It is less well appreciated that glia often also accumulate misfolded proteins. However, glia are highly plastic and may adapt to stress readily. Endogenous adaptations to stress can be measured by challenging stressed cells with a second hit and then measuring viability. For example, subtoxic stress can elicit preconditioning or tolerance against second hits. However, it is not known if severe stress that kills half the population can elicit endogenous adaptations in the remaining survivors. Glia, with their resilient nature, offer an ideal model in which to test this new hypothesis. The present study is the first demonstration that astrocytes surviving one LC50 hit of the proteasome inhibitor MG132 were protected against a second MG132 hit. ATP loss in response to the second hit was also prevented. MG132 caused compensatory rises in stress-sensitive heat shock proteins. However, stressed astrocytes exhibited an even greater rise in ubiquitin-conjugated proteins upon the second hit, illustrating the severity of the proteotoxicity and verifying the continued impact of MG132. Despite this stress, MG132-pretreated astrocytes were completely prevented from losing glutathione with the second hit. Furthermore, inhibiting glutathione synthesis rendered astrocytes sensitive to the second hit, unmasking the cumulative impact of two hits by removal of an endogenous adaptation. These findings suggest that stressed astrocytes become progressively harder to kill by virtue of antioxidant defenses. Such plasticity may permit astrocytes under severe stress to better support neurons and help explain the protracted nature of neurodegeneration.

Astrocyte plasticity revealed by adaptations to severe proteotoxic stress.

In Parkinson's and Alzheimer's diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H2O2. The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein aggregations through the cerebrum may reflect differential responses to low level protein-misfolding stress but also reveal impressive compensatory adaptations in allocortex.

Jessica M. Posimo

Division of Pharmaceutical Sciences,
Mylan School of Pharmacy,
Division of Pharmaceutical Sciences, Mylan School of Pharmacy

Duquesne University

Viability Assays for Cells in Culture

Jessica M. Posimo

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Duquesne University

Viability Assays for Cells in Culture

Division of Pharmaceutical Sciences,
Mylan School of Pharmacy,
Division of Pharmaceutical Sciences, Mylan School of Pharmacy