Altered transcription factor trafficking in oxidatively-stressed neuronal cells.

Age-related neurodegenerative diseases are associated with alterations in gene expression in affected neurons. One of the mechanisms that could account for this is altered subcellular localization of transcription factors, which has been observed in human post-mortem brains of each of the major neurodegenerative diseases, including Parkinson's disease (PD). The specific mechanisms are yet to be elucidated; however a potential mechanism involves alterations in nuclear transport. In this study, we examined the nucleocytoplasmic trafficking of select transcription factors in response to a PD-relevant oxidative injury, 6-hydroxydopamine (6OHDA). Utilizing a well-established model of ligand-regulated nucleocytoplasmic shuttling, the glucocorticoid receptor, we found that 6OHDA selectively impaired nuclear import through an oxidative mechanism without affecting nuclear export or nuclear retention. Interestingly, impaired nuclear import was selective as Nrf2 (nuclear factor E2-related factor 2) nuclear localization remained intact in 6OHDA-treated cells. Thus, oxidative stress specifically impacts the subcellular localization of some but not all transcription factors, which is consistent with observations in post-mortem PD brains. Our data further implicate a role for altered microtubule dependent trafficking in the differential effects of 6OHDA on transcription factor import. Oxidative disruption of microtubule-dependent nuclear transport may contribute to selective declines in transcriptional responses of aging or diseased dopaminergic cells.

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule.

BACKGROUND: Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. RESULTS: L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. CONCLUSION: Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

Ischemic Stroke Treatment Trials: Neuroimaging Advancements and Implications.

There have been significant advancements in the treatment of acute ischemic stroke in the last 2 decades. Recent trials have placed a significant emphasis on minimizing the time from symptom onset to stroke treatment by reperfusion therapies, which decreases the cerebral infarct volume and improves clinical outcomes. These clinical advances have paralleled and been aided by advances in neuroimaging. However, controversy remains regarding how much time should be spent on neuroimaging evaluation versus expediting patient treatment. In this review article, we examine the key endovascular stroke trials published in the past 25 years, and we briefly highlight the failures and successes of endovascular stroke trials performed in the past 4 years. We also discuss the advantages and disadvantages of using time from symptom onset versus neuroimaging in determining endovascular stroke therapy candidacy.

Decreased SIRT2 activity leads to altered microtubule dynamics in oxidatively-stressed neuronal cells: implications for Parkinson's disease.

The microtubule (MT) system is important for many aspects of neuronal function, including motility, differentiation, and cargo trafficking. Parkinson's disease (PD) is associated with increased oxidative stress and alterations in the integrity of the axodendritic tree. To study dynamic mechanisms underlying the neurite shortening phenotype observed in many PD models, we employed the well-characterized oxidative parkinsonian neurotoxin, 6-hydroxydopamine (6OHDA). In both acute and chronic sub-lethal settings, 6OHDA-induced oxidative stress elicited significant alterations in MT dynamics, including reductions in MT growth rate, increased frequency of MT pauses/retractions, and increased levels of tubulin acetylation. Interestingly, 6OHDA decreased the activity of tubulin deacetylases, specifically sirtuin 2 (SIRT2), through more than one mechanism. Restoration of tubulin deacetylase function rescued the changes in MT dynamics and prevented neurite shortening in neuron-differentiated, 6OHDA-treated cells. These data indicate that impaired tubulin deacetylation contributes to altered MT dynamics in oxidatively-stressed cells, conferring key insights for potential therapeutic strategies to correct MT-related deficits contributing to neuronal aging and disease.

Nuclear transport, oxidative stress, and neurodegeneration.

Trafficking of transcription factors between the cytoplasm and the nucleus is an essential aspect of signal transduction, which is particularly challenging in neurons due to their highly polarized structure. Disruption in the subcellular localization of many proteins, including transcription factors, is observed in affected neurons of human neurodegenerative diseases. In these diseases, there is also growing evidence supporting alterations in nuclear transport as potential mechanisms underlying the observed mislocalization of proteins. Oxidative stress, which plays a key pathogenic role in these diseases, has also been associated with significant alterations in nuclear transport. After providing an overview of the major nuclear import and export pathways and discussing the impact of oxidative injury on nuclear trafficking of proteins, this review synthesizes emerging evidence for altered nuclear transport as a possible mechanism in the pathogenesis of neurodegenerative diseases. Potential strategies to overcome such deficits are also discussed.

Automated time-lapse microscopy and high-resolution tracking of cell migration.

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in "scratch" or "wound healing" assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.