A Combination Therapy of 17ß-Estradiol and Memantine Is More Neuroprotective Than Monotherapies in an Organotypic Brain Slice Culture Model of Traumatic Brain Injury.

Combination therapies are a promising therapeutic option for traumatic brain injury (TBI) owing to the clinical failure of monotherapy treatments, such as progesterone. Organotypic hippocampal slice cultures (OHSCs) from Sprague-Dawley rats were subjected to an in vitro TBI, and the neuroprotective effects of 17ß-estradiol (E2) or memantine (MEM) monotherapies were quantified. Several combination treatments at different concentrations of both drugs were tested, with 100 pM of E2 and 10 µM of MEM statistically and significantly reducing cell death over either monotherapy when administered immediately after injury. This combination was also significantly neuroprotective when administered 1 h postinjury, possibly supporting future in vivo studies. Further, we hypothesized that this synergy could be the result of MEM blocking a potentially deleterious effect of E2, specifically E2 enhancement of N-methyl-D-aspartate (NMDA) currents. Evoked electrophysiological responses in OHSCs were potentiated by E2 treatment, whereas this potentiation was significantly reduced by MEM. In conclusion, a combination therapy of E2 and memantine was significantly more neuroprotective than both monotherapy treatments, and this synergy may be the result of MEM blocking a deleterious E2-mediated enhancement of NMDA receptors.

GPR30 activation is neither necessary nor sufficient for acute neuroprotection by 17ß-estradiol after an ischemic injury in organotypic hippocampal slice cultures.

In this study, we investigated the role of GPR30 in 17ß-estradiol- (E2) mediated neuroprotection after an ischemic injury in an organotypic hippocampal slice culture (OHSC) model. We report that after oxygen-glucose deprivation (OGD), a physiological concentration of 100 pM E2 provided the greatest significant reduction in cell death while supra-physiological levels were less effective. The canonical estrogen receptor (ER) inhibitor ICI 182,780 completely abrogated the therapeutic effect of E2 while the GPR30 antagonist G-15 effected a slight but not significant reduction in neuroprotection. Only supra-physiological levels of E2 led to significantly increased phosphorylation of Akt and Erk which are well known downstream effects of GPR30 activation. We conclude that GPR30 activation may facilitate acute E2 mediated neuroprotection after OGD, but is neither necessary nor sufficient.

Ferroptosis: an iron-dependent form of nonapoptotic cell death.

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.

Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1-S6K pathway signaling.

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.

CellProfiler: free, versatile software for automated biological image analysis.

Careful visual examination of biological samples is quite powerful, but many visual analysis tasks done in the laboratory are repetitive, tedious, and subjective. Here we describe the use of the open-source software, CellProfiler, to automatically identify and measure a variety of biological objects in images. The applications demonstrated here include yeast colony counting and classifying, cell microarray annotation, yeast patch assays, mouse tumor quantification, wound healing assays, and tissue topology measurement. The software automatically identifies objects in digital images, counts them, and records a full spectrum of measurements for each object, including location within the image, size, shape, color intensity, degree of correlation between colors, texture (smoothness), and number of neighbors. Small numbers of images can be processed automatically on a personal computer and hundreds of thousands can be analyzed using a computing cluster. This free, easy-to-use software enables biologists to comprehensively and quantitatively address many questions that previously would have required custom programming, thereby facilitating discovery in a variety of biological fields of study.