Rapamycin attenuates development of posttraumatic epilepsy in the CCI model.

<p>Representative EEG tracings of seizures (A) and interictal epileptiform abnormalities (B). (C) Rapamycin treatment significantly decreased the development of PTE following TBI (*p<0.05 by Mantel-Cox log-rank test).</p

Controlled cortical impact (CCI) injury model in mice.

<p>(A) Impactor tip of an electromagnetic CCI device. (B) Schematic diagram of sites of craniotomy, cortical injury, and EEG electrode placements. (C) Cresyl violet-stained coronal section documenting typical 2 mm CCI injury, which leads to damage through the depth of the neocortex but leaves the underlying hippocampus grossly intact. *cortical injury site.</p

Effect of Rapamycin on Posttraumatic Epilepsy (PTE) in the CCI Model.

<p>Effect of Rapamycin on Posttraumatic Epilepsy (PTE) in the CCI Model.</p

Rapamycin transiently reduces mossy fiber sprouting following TBI.

<p>(A–D) Timm staining shows mossy fiber sprouting from control mice (Ctrl+Veh, A), and vehicle-treated TBI mice (TBI+Veh, B) and rapamycin-treated TBI mice (TBI+Rap, C) five weeks after CCI. Panels A1, B1 and C1 are higher magnification of boxed regions in panels A, B and C, respectively. Quantitative analysis demonstrates a significant increase in Timm score in vehicle-treated TBI mice compared to control mice and a significant decrease in Timm score in rapamycin-treated compared to vehicle-treated TBI mice (D). (E–H) At sixteen weeks after CCI (12 weeks after rapamycin was stopped), Timm score in rapamycin-treated TBI mice increased back to similar levels of vehicle-treated TBI mice. *p<0.05 by one-way ANOVA, n = 6 mice per group.</p

Rapamycin blocks mTORC1 activation induced by TBI.

<p>(A, B) Rapamycin treatment, initiated one hour after CCI injury and continued daily at 6 mg/kg, inhibited mTORC1 activation at both 6 hr and 3 d following CCI, as reflected by the P-S6/S6 ratio. (C, D) Daily rapamycin treatment for 4 weeks continued to inhibit mTOR activity. (E, F) After rapamycin was stopped, mTOR activity returned to control levels. *p<0.05 vs. Ctrl+Veh; ^#p<0.05 vs. TBI+Veh at the same time point by two-way repeated measures ANOVA. n = 6 mice per group.</p

Rapamycin reduces neuronal degeneration in hippocampus following TBI.

<p>Representative sections of Fluoro-Jade B staining in different regions of hippocampus of control mice (Ctrl, A–C), vehicle-treated TBI mice (TBI+Veh, D–F) and rapamycin-treated TBI mice (TBI+Rap, G–I) three days after CCI are shown. Abundant Fluoro-Jade B positive neurons are seen in vehicle-treated TBI mice in CA1, CA3 and DG, but to a lesser degree in rapamycin-treated TBI mice. Quantitative analysis showed a significant decrease in Fluoro-Jade B positive cells in rapamycin-treated compared to vehicle-treated TBI mice (J–L). *p<0.05 by one-way ANOVA, n = 6 mice per group.</p

Rapamycin inhibits increased P-4EBP1, but not P-STAT3, expression induced by TBI.

<p>For comparison with P-S6, the phosphorylation of another downstream mTORC1 target (4EBP1) and a non-mTORC1 mediated phosphorylation pathway (JAK-STAT) was assessed following CCI. (A) P-4EBP1 was elevated following CCI injury and was inhibited by rapamycin. (B) In contrast, P-STAT3 was increased after CCI, but was not inhibited by rapamycin. *p<0.05 vs. Ctrl+Veh; ^#p<0.05 vs. TBI+Veh at the same time point by two-way repeated measures ANOVA. n = 6 mice per group.</p

Knockdown of endogenous Nischarin expression promotes neurite outgrowth in Neuro-2a cells.

<p>Neuro-2a cells were transfected with Nis-shRNA1-4 or control-shRNA. Nischarin expression was determined by real time quantitative RT-PCR <b>(A)</b> and western blot assay <b>(B)</b>. Expression of the Nischarin gene and protein after transfection with Nis-shRNA1-4 decreased to some degree. The knockdown efficiency of Nis-shRNA-3 could reach up to ~60% inhibition. <b>(C)</b> The Nischarin signal came back to ~90% level when loading double amount of the lysates. Asterisks indicate a significant difference compared with control-shRNA (*<i>p</i><0.05, n = 3). <b>(D)</b> Representative images of Neuro-2a cells 48 h after transfection with Nis-shRNA-2, Nis-shRNA-3 or control-shRNA. Cells were differentiated with 20 μM retinoic acid in DMEM for 24 h. For the analysis of the neurite length, pictures were taken with an inverted microscope from five fields of view per well. The neurite length for each individual cell was determined by manual tracing and measured using NIH ImageJ software. Neurites were defined as a process with lengths equivalent to one diameters of a cell body. The percentage of neurite-bearing cells was calculated from the total number of counted cells (n = 3, ~1200 cells measured). Scale bar: 20 μm. Suppressing expression of Nischarin increased the number of neurite-bearing cells <b>(E)</b>, the mean length of the longest neurite <b>(F)</b>, and the total neurite length <b>(G)</b>. Asterisks indicate significant differences from the control shRNA cells (*<i>p</i><0.05). Data presented are the mean ± SEM.</p

Cellular expression pattern of Nischarin in neurons.

<p><b>(A, B)</b> Western blots revealed the expression of Nischarin protein in neuronal cell lines (PC-12 and Neuro-2a), neurons and astrocytes (n = 3). Expression levels of Nischarin were quantified by normalization against the housekeeping gene GAPDH. <b>(C)</b> Immunofluorescence staining was performed on pure cortical neuron cultures (top panel) and mixed cultures of neurons and astrocytes (bottom panel). Strong staining for Nischarin (green) co-localized with Map-2 (red) in the perinuclear region (arrowheads) and the dendrites of cortical neurons. No co-localization of Nischarin (green) and GFAP (red) was observed in the cytoplasm of astrocytes (arrow). Scale bars: 20 μm.</p

Knockdown of Nischarin expression promotes dendrite elongation in cortical neurons.

<p><b>(A)</b> Cortical neurons were infected with lentiviral control-shRNA or Nis-shRNA-3 at 7 DIV and dendrite morphology was examined at 10 DIV. Scale bar: 20 μm. <b>(B)</b> Number of dendritic intersections within 100 μm of the cell body determined by Sholl analysis. Suppressing Nischarin expression increases the mean length of the longest dendrite <b>(C)</b> and total length of dendrites per cell <b>(D)</b>. Asterisks indicate a significant difference from control shRNA cells (*<i>p</i><0.05, **<i>p</i><0.01, n = 3, ~60 neurons measured). Data presented are the mean ± SEM.</p