No influence of LOTUS overexpression in brain atrophy and cortical cavitation in the chronic phase after ischemic stroke.

<p>(<b>a</b>) Representative coronal sections stained with cresyl violet of brain in WT and LOTUS-Tg mice 19 weeks after MCAO. Arrowheads indicate edge of stroke lesion. (<b>b</b>) Infarct area (mm²) in each slice and volume (mm³) in WT and LOTUS-Tg mice. (Infarct area: 2-way ANOVA; NS. Data are mean ± S.E.M. n = 9 per group, Infarct volume: Unpaired <i>t</i> test; NS. Data are mean ± S.E.M. n = 9 per group) (<b>c</b>) Left, cortical width index; maximum width from midpoint to edge of non-infarcted (A) and infarcted (B) hemispheres. Right, dorsal view of WT and LOTUS-Tg mouse brains. White arrowheads indicate infarct region. (<b>d</b>) Quantitative analysis of cortical width index. (Unpaired <i>t</i> test; NS. Data are mean ± S.E.M. n = 9 per group) Bar indicates 1 mm. NS, not significant.</p

LOTUS overexpression accelerates neuronal plasticity after focal brain ischemia in mice

<div><p>Nogo receptor-1 (NgR1) and its ligands inhibit neuronal plasticity and limit functional recovery after brain damage such as ischemic stroke. We have previously shown that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated signaling. Here, we investigated whether LOTUS enhances neuronal plasticity and functional recovery after brain focal ischemia in adult mice. Focal ischemic infarcts were induced in wild-type and LOTUS-overexpressing transgenic mice via middle cerebral artery occlusion. Endogenous LOTUS expression was increased in brain and cervical spinal cord of the contralateral side of ischemia in the chronic phase after brain ischemia. LOTUS overexpression accelerated midline-crossing axonal sprouting from the contralateral side to the ipsilateral side of ischemia in the medullar reticular formation and gray matter of denervated cervical spinal cord. Importantly, LOTUS overexpression improved neurological score highly correlated with laterality ratio of corticoreticular fibers of the medulla oblongata, indicating that LOTUS overexpression may overcome the inhibitory environment induced by NgR1 signaling for damaged motor pathway reconstruction after ischemic stroke. Thus, our data suggest that LOTUS overexpression accelerates neuronal plasticity in the brainstem and cervical spinal cord after stroke and LOTUS administration is useful for future therapeutic strategies.</p></div

Schematic diagram showing enhancement of the midline-crossing fibers by LOTUS overexpression after MCAO.

<p>The midline-crossing corticoreticular tract (CRT) and corticospinal tract (CST) indicated by the red dashed lines from non-ischemic CST after MCAO in LOTUS-Tg mice. RST: reticulospinal tract.</p

Behavioral outcome after MCAO.

<p>Behavioral outcome after MCAO assessed by neurological scoring according to Bederson et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184258#pone.0184258.ref032" target="_blank">32</a>]. Note that significant improvement at 12 and 16 weeks of LOTUS-Tg mice is shown. (2-way repeated-measures ANOVA with Tukey multiple comparison; ***<i>p</i> < 0.001, ^†<i>p</i> < 0.05, ^††<i>p</i> < 0.01. Data are mean ± S.E.M. n = 9 per group).</p

LOTUS, Nogo-A and NgR1 expression in WT and LOTUS-Tg mice.

<p>(<b>a</b>) Protein (20 μg) from lysates of each animal in pre-stroke, 6, and 16 weeks after MCAO was analyzed. (<b>b</b>-<b>d</b>) Quantitative analysis of expression level in LOTUS, Nogo-A, and NgR1. Immunoblots were normalized with β-actin. (<b>b</b>) Increases in LOTUS expression in the non-ischemic side of both WT and LOTUS-Tg mice 6 weeks after MCAO were found. (<b>c</b>, <b>d</b>) No significant difference was seen in Nogo-A and NgR1 expression. (2-way ANOVA with Tukey post hoc analysis; **<i>p</i> < 0.01. Data are mean ± S.E.M. n = 3).</p

Neuronal remodeling in cortico-spinal fibers.

<p>(<b>a</b>) Representative photomicrographs of the gray matter of the cervical spinal cord in frontal sections of WT mouse and LOTUS-Tg mouse, showing increased midline-crossing fibers (black arrowheads) in LOTUS-Tg mice after MCAO. Insets of the spinal cord scheme indicating the position of the photomicrograph. Images in right panel are shown at higher magnification of indicated dashed boxes. Scale bars indicate 500 μm. (<b>b, c</b>) The number of midline-crossing fibers of WT and LOTUS-Tg mice at the C4-5 and C6-7 levels of the cervical spinal cord. (2-way ANOVA with Tukey post hoc analysis; **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ^†<i>p</i> < 0.05. Data are interquartile range. n = 9 per group).</p

Correlation between axonal remodeling and behavioral recovery.

<p>The behavioral improvement assessed by Bederson’s neurological score was highly correlated with the laterality ratio of corticoreticular fibers of the medulla oblongata (R = 0.81, <i>p</i> < 0.001, Spearman’s Rank-Order Correlation).</p

Neuronal remodeling in cortico-reticular fibers.

<p>(<b>a</b>) Representative photomicrographs of the medulla oblongata in frontal sections of WT and LOTUS-Tg mice, showing increased midline (dashed lines)-crossing corticoreticular fibers (black arrows) from non-ischemic CST in LOTUS-Tg mice after MCAO without influencing uncrossed corticoreticular tact (white arrows on dashed lines). Insets of the scheme indicating the position of the photomicrograph. Images in right panel are shown at higher magnification of indicated dashed boxes. Scale bars indicate 500 μm. (<b>b, c</b>) Percentage of uncrossed and midline-crossing fibers from CST to RF (2-way ANOVA with Tukey post hoc analysis; *<i>p</i> < 0.05, ***<i>p</i> < 0.001, ^†<i>p</i> < 0.05. Data are interquartile range. n = 9 per group). (<b>d</b>) Laterality ratio comparing WT and LOTUS-Tg mice. (Unpaired <i>t</i> test, *<i>p <</i> 0.05. Data are mean ± S.E.M. n = 9).</p

Additional file 2: Table S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Molecular and clinical characteristics of Cohort 1 (n = 758). Table S2. Molecular and clinical characteristics of GBM cohort (n = 453). Table S3. Univariate and multivariate Cox regression analyses for Group A (IDH mutated-TERT mutated) tumors in Cohort 1 (n = 155). Table S4. Univariate and multivariate Cox regression analyses for Group B (IDH mutated-TERT wild-type) tumors in Cohort 1 (n = 131). Table S5. Univariate and multivariate Cox regression analyses for Group C (IDH wild-type-TERT wild-type) tumors in Cohort 1 (n = 237). Table S6. Univariate and multivariate Cox regression analyses for Group D (IDH wild-type-TERT mutated) tumors in Cohort 1 (n = 235). Table S7. Univariate and multivariate Cox regression analyses for GBM in Cohort 1 (n = 260). Table S8. Univariate and multivariate Cox regression analyses for GBM in Cohort 2 (n = 193). Table S9. Background of combined GBM cohort stratified by TERT and MGMT status (n = 453). Table S10. Survival time and WHO grade in each molecular subgroup of Cohort 1 (n = 758). (XLSX 254 kb

Additional file 3: Figure S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Distributions of molecular alterations according to histology in Cohort 1. Figure S2. Kaplan-Meier analysis for Group A cases stratified by 1p/19q status. Figure S3. Kaplan-Meier analyses for GBM cases in Cohorts 1 and 2. (PPTX 172 kb