Plasma proteins were separated by SDS-PAGE, in mini-gel format

in SOD1 mice. The first study crossed Perk haplo-insufficient mice with transgenic G85R-SOD1 mice. These Perk haplo-insufficient SOD1 mice displayed accelerated disease. This suggested that diminished capacity to respond to ER stress is deleterious. Next they developed a SOD1 transgenic mouse harboring a Gadd34 mutation. The mutation retarded its capacity for dephosphorylation of the eIF2 alpha subunit. These mice had a delayed and less severe disease phenotype. These results suggested that treatment with a drug that could also reduce GADD34 activity might be protective. Salubrinal, another small molecule Gadd34 inhibitor has been reported as efficacious in SOD1 mice. However, it has a poor in vivo pharmacology profile. Guanabenz, a central acting drug with similar inhibitory effects on Pp1/ Gadd34, was tested in SOD1 mice by the Roos lab and the Feng Lab at doses of 8 mg/kg or 4 mg/kg every other day respectively. Both labs reported significant extension PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19722344 of survival. Additionally, guanabenz treatment of 4 mg/kg three times weekly demonstrated efficacy in a mouse model of prion disease. The experiments and results detailed in this report were mostly completed before reports of efficacy by Jiang et al and Wang et al. and were catalyzed by the 2011 findings that guanabenz interferes with eIf2alpha dephosphorylation. Herein we report the effects of guanabenz on viability of SOD1 mouse fibroblasts exposed to BQ 123 price tunicamycin induced cell stress. Further, we demonstrate guanabenz PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723429 dependent effects on levels of UPR related transcripts and proteins from G93A-SOD1 spinal cord. Finally, we demonstrate disease acceleration by guanabenz in G93A-SOD1 mice by two treatment regimens: 1) 4.5 mg/kg/day by osmotic minipump continuous infusion and 2) a repeat of the 4 mg/kg qod protocol reported by the Feng Lab. Results Guanabenz treatment enhances viability of HeLa cells that have been stressed by tunicamycin. Tunicamycin is a mixture of homologous nucleoside antibiotics that inhibits N-linked glycosylation and reproducibly induces the UPR in cells that are exposed to it. Here we 2 / 15 Guanabenz Treatment Accelerates Disease in a SOD1 Mouse Model of ALS Fig 1. WST1 absorbance as an indicator of cell viability in a tunicamycin challenge assay using fibroblasts derived from G93A-SOD1 mice. WST absorbance indicator a tunicamycin challenge assay using fibroblasts derived from non-transgenic mice. Tunicamycin EC50s as a function of guanabenz concentration in G93A-SOD1 and non-transgenic mouse fibroblasts. doi:10.1371/journal.pone.0135570.g001 repeated these experiments using primary fibroblasts derived from tail tips from G93A-SOD1 mice and in TTF derived from littermates that harbor no mutant transgenes. These primary cells were used to take into account the role of SOD1 over-expression on the UPR induction and whether that would influence effects by guanabenz on the pathways. Similarly to the reported HeLa cell results, guanabenz treatment at low micromolar concentrations enhanced viability against tunicamycin induced cell death in fibroblasts in both WT TTFs and SOD1 TTFs at 3 and 10 uM concentrations and shifted the EC50 of tunicamycin significantly. Guanabenz treatment shifted tunicamycin EC50 values higher to a greater extent in nontransgenic fibroblast experiments than in the G93A-SOD1 fibroblast experiments. The cell viability assay that was used relies on the cleavage of water soluble tetrazolium salt 1 to formazan by mitochondrial dehydrogenase enzymes