(A) ATF4 (♦) and ATF4 (▴) cortical neurons were treated with 10 mM HCA

At the indicated time points, cells were trypsinized, washed, and pelleted. Reduced glutathione (GSH) was determined in the cell pellets using HPLC electrochemical detection. Data are from three separate cultures, and each data point was measured in duplicate. Graph depicts mean ± SD. (B) Schematic overview over cysteine uptake and glutathione synthesis and their inhibition. (C) ATF4 and ATF4 cortical neurons were treated with a vehicle control (shown as C) or 200 μM BSO. 24 h later, cell viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from five separate experiments for each group ( = 46 ATF4 and 58 ATF4). *, P < 0.05 from untreated ATF4 cultures by the Kruskal-Wallis test followed by Dunn's multiple comparisons test. The difference between treated and untreated ATF4 neurons was not significant (n.s.). (D) Live/dead assay displaying untreated and BSO-treated ATF4 and ATF4 neurons. Bar, 50 μm. (E) Protein expression of γ-GCS does not differ between ATF4 and ATF4 cortical neurons. Cytoplasmic extracts were separated using gel electrophoresis and immunodetected using an antibody against γ-GCS. Total eIF2α was monitored as a loading control. (F) ATF4 (♦) and ATF4 (▪) cortical neurons were treated with 200 μM BSO. At the indicated time points, cells were trypsinized, washed, and pelleted. GSH was determined in the cell pellets using HPLC electrochemical detection. Data are from three separate cultures, and each data point was measured in duplicate. Graph depicts mean ± SD. (G) ATF4 cortical neurons were infected with GFP (♦), ATF4WT (▪), and ATF4ΔRK (▴) adenoviruses at an MOI of 100. At the indicated time points after infection, cells were trypsinized, washed, and pelleted. GSH was determined in the cell pellets using HPLC electrochemical detection. The graph depicts mean ± SD calculated from three separate experiments for each group, and each data point was measured in duplicate. The value obtained from noninfected neurons was arbitrarily defined as 100%. (H) ATF4 cortical neurons were infected with GFP and ATF4WT adenoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C), 10 mM HCA, 10 μM BHA, or a combination of both. The graph depicts mean (compared with control) ± SD calculated from five separate experiments for each group ( = 29). *, P < 0.05 from untreated neurons overexpressing ATF4WT by the Kruskal-Wallis test followed by Dunn's multiple comparisons test. The difference between neurons overexpressing ATF4WT treated with BHA alone and neurons overexpressing ATF4WT treated with both BHA and HCA was not significant (n.s.). (I) Live/dead assay. Bar, 50 μm.<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

(A, top) Sagittal sections of ATF4 (left) and ATF4 (right) brains stained with cresyl violet

Bar, 3,000 μm. (A, middle) Ventral view of large cerebral blood vessels of representative ATF4 (left) and ATF4 (right) mice that were perfused with India ink. Note the higher degree of tortuosity of the MCA in the brain from the ATF4 mouse. Bar, 3,000 μm. (A, bottom) Representative microscopic views of brain sections from ATF4 and ATF4 mice that were immunostained for the endothelial cell–specific marker CD31 (green). Bar, 100 μm. (B) Representative brain sections at 4 d after MCAo from ATF4 ( = 5) and ATF4 ( = 6) mice from rostral to caudal stained with cresyl violet to determine the infarct area. Bar, 1,000 μm. (C) The infarct area in ATF4 (♦) and ATF4 (▪) brains was measured in 12 sequential sections taken from ATF4 ( = 5) and ATF4 ( = 6) mice at rostral to caudal regular intervals. Graph depicts mean ± SD. (D) The infarct volume was assessed by adding the infarct volumes based on the infarct area in each section. Graph depicts mean ± SD. *, P < 0.0001 by the test. (E) Scoring of neurological deficit was assessed at different time points of recovery in ATF4 ( = 5) and ATF4 ( = 4) mice. Graph depicts mean ± SD. *, P < 0.05 by the Mann-Whitney test. (F) Inclined plane test at different time points after stroke in ATF4 ( = 5) and ATF4 ( = 4) mice. The test measured the time a mouse managed to hold itself on an inclined glass plate angled at 50° before sliding down. Graph depicts mean ± SD. *, P < 0.05 by the test. (G) Hanging wire test at different time points after stroke in ATF4 ( = 5) and ATF4 ( = 4) mice. The hanging wire test determined the time it took an animal to cross a distance of 45 cm on a freely hanging narrow metal bar. Graph depicts mean ± SD. *, P < 0.05 by the test.<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

(A) Cortical neuronal cultures (1 d in vitro) were treated with a vehicle control (shown as C), 10 mM of the glutamate analogue HCA, 1 μg/ml arginase, 1 μM thapsigargin, 1 μg/ml arginase and 10 mM HCA, or 10 mM HCA and 1 μM thapsigargin

24 h later, cell viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from three separate experiments for each group ( = 25). *, P < 0.05 from HCA-treated cultures by the Kruskal-Wallis test followed by Dunn's multiple comparisons test. (B) Live/dead assay of cortical neuronal cultures (2 d in vitro). Live cells were detected by uptake and trapping of calcein-AM (green fluorescence). Dead cells failed to trap calcein but were freely permeable to the highly charged DNA intercalating dye ethidium homodimer (red fluorescence). Bar, 50 μm. (C) Treatment with 10 mM HCA (shown as H) and 1 μg/ml arginase (shown as A) or 1 μM thapsigargin (shown as T), alone or in combination with HCA, increases the expression of ATF4 in cultured cortical neurons as compared with vehicle-treated control (shown as C). Cells were harvested at the indicated time points, and nuclear extracts were separated using gel electrophoresis and immunodetected using an antibody against ATF4. YY1 was monitored as a loading control. The immunoblot is a representative example of three experiments.<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

(A) Representative immunocytochemistry of ATF4 and ATF4 cortical neurons infected with the adenoviral constructs ATF4WT and ATF4ΔRK

Neurons were stained with antibodies against myc (green) and MAP2 (red), and were counterstained with Hoechst dye (blue). Bar, 50 μm. (B) Whole-cell extracts obtained from both ATF4 and ATF4 neurons infected with GFP, ATF4WT, and ATF4ΔRK were separated using gel electrophoresis and immunodetected using an antibody directed against the myc tag. Total eIF2α was monitored as a loading control. (C) ATF4 cortical neurons were infected with GFP, ATF4WT, and ATF4ΔRK adenoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C) or 10 mM HCA. 24 h later, cell viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from four separate experiments for each group ( = 45). P < 0.05 by the Kruskal-Wallis test followed by Dunn's multiple comparisons test from untreated ATF4WT-overexpressing neurons (*) and from HCA-treated neurons overexpressing GFP (§). (D) Live/dead assay. Bar, 50 μm. (E) ATF4 cortical neurons were infected with GFP, ATF4WT, and ATF4ΔRK adenoviruses at an MOI of 100. 24 h after infection, neurons were treated with vehicle control (shown as C) or 10 mM HCA. 24 h later, cell viability was determined using the MTT assay. The graph depicts mean (compared with control) ± SD calculated from four separate experiments for each group ( = 28). P < 0.05 by the Kruskal-Wallis test followed by Dunn's multiple comparisons test from untreated ATF4WT-overexpressing neurons (*) and from HCA-treated neurons overexpressing GFP (§). (F) Live/dead assay. Bar, 50 μm.<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

(A) Heatmap with cluster dendrogram of the differentially expressed genes (log2 fold change vs

control) at a false discovery rate of 5%. Unsupervised clustering groups samples by genotype and by treatment. Genes are in rows and samples are in columns. Column color coding is as follows: red, ATF4 versus ATF4 untreated neurons; blue, HCA-treated ATF4 versus untreated ATF4 neurons; and green, HCA-treated ATF4 versus untreated ATF4 neurons. (B) The number of genes that are up- (red) and down-regulated (green). Shown are three different contrasts: ATF4 versus ATF4 untreated neurons, HCA-treated ATF4 versus untreated ATF4 neurons, and HCA-treated ATF4 versus untreated ATF4 neurons. The complete list of differentially expressed genes is available in Table S3 (available at ). ANOVA FDR, analysis of variance false discovery rate; C, control.<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

(A) EMSA performed with 10 μg of dialyzed nuclear extracts from HT22 cells transfected with ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP, respectively

Extracts were incubated with a radioactively labeled WT oligonucleotide containing the TRB3 promoter binding site or with a mutant oligonucleotide. Binding of ATF4WT to the TRB3WT promoter binding site was confirmed by supershift analysis (arrow) performed with an antibody (Ab) directed against ATF4. (B) Overexpressed ATF4WT protein occupies its putative binding site within the TRB3 promoter in HT22 cells, as shown by chromatin immunoprecipitation assay. HT22 cells were transfected with ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP. An anti-myc antibody was used to precipitate the proteins in nuclear extracts of cross-linked HT22 cells. Coprecipitated DNA fragments were detected using PCR with a set of primers specific for the ATF4 binding site in the TRB3 promoter, yielding a 190-bp product. A representative example of three experiments is shown. (C) HT22 cells were transfected with the expression plasmids for ATF4WT, mutant ATF4 (ATF4ΔRK), or GFP. The cells were cotransfected with either a luciferase reporter vector containing the 33-bp ATF4WT binding site (33 bp WT), a reporter vector containing a mutant form of this binding site (33 bp MUT), or empty vector (pGL3 basic). In parallel, the transfection mix contained a plasmid expressing Renilla to allow normalization for transfection efficiency. The value for empty pGL3 cotransfected with GFP was arbitrarily defined as 1. Shown are ratios of luciferase and Renilla activities (mean ± SD for three independent experiments; each data point was performed in duplicate).<p><b>Copyright information:</b></p><p>Taken from "ATF4 is an oxidative stress–inducible, prodeath transcription factor in neurons in vitro and in vivo"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1227-1242.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373852.</p><p></p

Derivation of Multivariate Syndromic Outcome Metrics for Consistent Testing across Multiple Models of Cervical Spinal Cord Injury in Rats

<div><p>Spinal cord injury (SCI) and other neurological disorders involve complex biological and functional changes. Well-characterized preclinical models provide a powerful tool for understanding mechanisms of disease; however managing information produced by experimental models represents a significant challenge for translating findings across research projects and presents a substantial hurdle for translation of novel therapies to humans. In the present work we demonstrate a novel ‘syndromic’ information-processing approach for capitalizing on heterogeneous data from diverse preclinical models of SCI to discover translational outcomes for therapeutic testing. We first built a large, detailed repository of preclinical outcome data from 10 years of basic research on cervical SCI in rats, and then applied multivariate pattern detection techniques to extract features that are conserved across different injury models. We then applied this translational knowledge to derive a data-driven multivariate metric that provides a common ‘ruler’ for comparisons of outcomes across different types of injury (NYU/MASCIS weight drop injuries, Infinite Horizons (IH) injuries, and hemisection injuries). The findings revealed that each individual endpoint provides a different view of the SCI syndrome, and that considering any single outcome measure in isolation provides a misleading, incomplete view of the SCI syndrome. This limitation was overcome by taking a novel multivariate integrative approach for leveraging complex data from preclinical models of neurological disease to identify therapies that target multiple outcomes. We suggest that applying this syndromic approach provides a roadmap for translating therapies for SCI and other complex neurological diseases.</p> </div

Standardized digital locomotor analysis for evaluating recovery after spinal cord injury in rodents.

<p><b><i>A,</i></b> Digital footprint analysis allows objective quantification of many correlated outcomes including: <b><i>B,</i></b> Stride-length for each limb; <b><i>C,</i></b> Print area for each limb; <b><i>D,</i></b> Distribution of limb use reflected as the absolute deviation from the pre-injury baseline (i.e. deviation from ∼25% recruitment for each limb).</p

Histological outcomes.

<p><b><i>A,</i></b> Tissue sparing measures in SCI research are typically taken at the lesion center as determined by the largest extent of the lesion ellipsoid. Although specific methods for quantification may vary across studies, typical measures include lesion size, <b><i>B,</i></b> gray matter (GM) sparing, <b><i>C,</i></b> white matter (WM) sparing, <b><i>D,</i></b> total sparing (GM+WM), <b><i>E,</i></b> total tissue area (GM+WM+debris), <b><i>F,</i></b> motorneuron number. Scale bar, 100 µm. Since the compiled dataset was limited to unilateral injuries (hemisections or hemicontusions), all measures are represented as a percentage of the contralateral, spared hemicord. The quantified area is illustrated in red on a representative example. The representative example was taken from the subject closest to the group mean for lesion size across the study’s 159 subjects.</p

Multivariate analysis of the SCI syndrome using data from two research sites.

<p><b><i>A,</i></b> Heat map of the bivariate correlation matrix, indicating all cross-correlations between behavioral and histological outcomes sorted in a randomized fashion. Blue indicates negative relationships and red indicates positive relationships. Heat reflects magnitude of Pearson correlation (r). <b><i>B,</i></b> Zoomed view of a small portion of the correlation matrix showing the interrelationships between a subset of outcomes. <b><i>C,</i></b> Principal components analysis (PCA) by eigenvalue decomposition was used to reduce the correlation matrix to synthetic multivariate variables known as principal components (PCs). PCs reflect clustered variance shared by numerous outcome measures. PC identities are indicated by significant PC loadings (arrows, loadings |>.40|). Each loading is equivalent to a Pearson correlation between individual outcomes and the PC. Loading magnitude is indicated by arrow width and heat (blue reflects negative and red reflects positive relationships). Exact loading values are shown next to each arrow. See Fig. S1 for non-significant loadings. <b><i>D,</i></b> Plot of individual subjects (N = 159) in the 3D multivariate syndrome space described by PC1-3. <b><i>E–G</i></b>, 2D plots of PC1-3 on their own axes. Significant differences: <b><i>E,</i></b>*<i>P</i><.05 from sham, ** <i>P</i><.05 from 75 kdyn and sham, §<i>P</i><.05 from all groups except 6.25 mm. <b><i>F,</i></b> *<i>P</i><.05 from sham, **<i>P</i><.05 from all groups but sham, ***<i>P</i><.05 from sham, 75 kdyn, 100 kdyn and hemisection. §<i>P</i><.05 from all other groups. <b><i>G,</i></b> *<i>P</i><.05 from sham, ** <i>P</i><.05 from 75 and 100 kdyn.</p