Cerebrospinal Fluid with ALS inhibits Sonic H

ALS CSF inhibits Shh biological activity in vitro.

(A) Standard curves with R2 demonstrate a dose-dependent response of Shh-LTII cells to human recombinant Shh (rShh) and purmorphamine (Purm) (0.3 μM). Shh-LTII cells were cultured with rShh and Purm and developed with Promega Dual-Glo Luciferase Assay System, assaying for firefly (FFL) and Renilla (RL) luciferase-driven luminescence measured as luminescence light unit (LLU). Data are expressed as mean ± SD and shown as arbitrary units (AU) where FFL was normalized to RL (LLU FFL/RL). * and ** indicate significant differences from media alone for rShh and Purm, correspondingly, based on a one way ANOVA analysis with Tukey post-hoc test (p < 0.05). 

(B) The inhibitory effect of ALS CSF on Shh biological activity measured in Shh-LTII cells is shown. Fully confluent Shh-LTII cells were incubated either with CSF alone, half-diluted with reduced-serum medium as a baseline (white bar), or in the presence of rShh (0.3 μg/ml) (grey bar) or Purm (0.3 μM) (black bar) and assayed with Promega. Data are expressed as mean ± SD and shown as AU. * and *** indicate significant differences between a baseline, rShh, and Purm in neurological controls (Neuro) or normal controls (Controls), respectively, using one way ANOVA with Tukey or Newman-Keuls post-hoc tests (p < 0.05). 

(C) The inhibitory effect of Shh antagonist cyclopamine (Cycl) is shown in Shh-LTII cells cultured with hrShh and Purm and developed with Promega as described in detail. Data are expressed as mean ± SD and shown as AU with significance value illustrated. 

(D) The inhibitory effect of ALS CSF shown in (B) is expressed as fold increase over baseline for each sample (AU stimulated/AU unstimulated), demonstrating a significantly increased luminescence in response to rShh and Purm treatment in presence of CSF from controls, but not ALS patients. Data analysis was performed using one way ANOVA with Tukey post-hoc test. Displayed p values indicate significant differences between groups, and N.S. indicates non-significant differences. 

(E) The inhibitory effect of ALS CSF observed in Shh-LTII cells (B, C) was repeated in NSC-34-Gli cells cultured in half-diluted CSF from controls and ALS patients. Data are expressed as mean ± SD and shown as AU with significance indicated on a graph. 

(F) Nuclear expression of Gli1, Sufu, and Gli2 proteins is shown in immunofluorescently stained NSC-34-Gli cells cultured for 72 hours in a serum-reduced medium with a half-diluted pooled CSF from Neuro and ALS groups. Data are expressed as mean ± SEM, with significance value included, and shown as a relative nuclear intensity analyzed per cell (ImageJ) over manually contoured nuclear (DAPI) area. Reduced Gli1 and Sufu nuclear translocation in ALS CSF-treated cells are shown, in contrast to elevated Gli2 nuclear expression. 

(G) Representative immunofluorescence images demonstrating Gli1 (green), Sufu (red), and Gli2 (red) distribution in NSC-34-Gli cells treated either with CSF from neurological controls (Neuro panel) or ALS group (ALS panel) using DAPI (blue) as a nuclear stain. Single or merged images are shown with a scale bar depicted in a bottom right image.   

TNF-α is a significant inhibitor of Shh biological activity in vitro.

The inhibitory effect of human recombinant TNF-α (hr TNF-α) is shown based on luminescence assay and linear regression plots are shown with corresponding p values. (A-C) Shh-LTII cells were cultured without or with hrTNF-α alone (0–125 ng/ml), or in the presence of Purm (0.8 μM) (D-F), and (G-I) NSC-34-Gli cells were cultured without or with hrTNF-α alone. Luminescence output was quantified 48–72 hours later with Promega. Individual data points are shown as arbitrary units (AU) (A,D,G) where FFL was normalized to RL (LLU FFL/RL), as well as raw data points for FFL(B,E,H) and RL (C,F,I).