, 2013). We selected 26 target genes (Table S16) and tested Trichostatin A in vivo their expression in C9ORF72 autopsied CNS tissue against non-ALS control tissue using nanostring gene expression methodologies. Sixteen of the target genes tested were also aberrantly expressed in C9ORF72 ALS patient tissue (Figures 5C, S7A, and S7B), of which seven showed the same direction of dysregulation

(up or down) when compared to iPSNs (Figure 5D). These genes could be potential candidates for the future development of a pharmacodynamic biomarker to monitor C9ORF72 therapy in human CSF and/or blood. Glutamate toxicity has been shown to play a major role in ALS as sporadic ALS and C9ORF72 patients exhibit a loss of astroglial glutamate transporter 1 (GLT-1/EAAT2), which buffers synaptic glutamate thus preventing excitotoxicity (Lin et al., 1998, Rothstein

et al., 1995 and Renton et al., 2011). Notably, C9ORF72 iPSNs express glutamate receptors (GluR2), NMDA receptors (NR2B), and postsynaptic marker protein postsynaptic density protein-95 (PSD95), comparable to control iPSNs, thus suggesting that these cells form functional synapses and are capable of responding to glutamate-induced excitotoxicity (Figure 6A). To 5-Fluoracil solubility dmso determine whether the C9ORF72 mutation leads to an altered physiology in iPSC neurons, we explored sensitivity to glutamate excitotoxicity. Glutamate treatment resulted in a dose- and time-dependent increase of cell death in control and C9ORF72 iPSN cultures as determined by cellular propidium iodide (PI) uptake (Sattler et al., 1997 and Sattler et al., 1999) (Figures 6B–6D and S8). Notably, C9ORF72 ALS iPSNs are almost 100-fold more sensitive to glutamate treatment, as toxicity at 3 μM glutamate in healthy control iPSNs was comparable to death at 100 μM

glutamate in C9ORF72 iPSNs (Figures 6B and S8). Glutamate-induced cell death was blocked by inhibitors of glutamate receptors and calcium channels (MK-801, 10 μM; CNQX, 10 μM; nimodipine, 2 μM; Sattler until et al., 1999), confirming that C9ORF72 iPSN cell death was glutamate-receptor dependent (Figure 6E). To test whether the sequestration of ADARB2 plays a role in the observed glutamate susceptibility, we knocked down ADARB2 levels in healthy control iPSNs via siRNA treatment (Figure 6F) and investigated their susceptibility to glutamate-mediated cell death. Interestingly, a loss of about 50% of ADARB2 RNA significantly enhances control iPSC neurons’ susceptibility to glutamate (30 μM at 4 hr) to levels similar to those observed in C9ORF72 iPSNs (Figures 6F and 6G). This suggests that a partial loss of ADARB2 via sequestration to GGGGCC RNA might play a role in C9ORF72-mediated RNA toxicity. If an RNA-dominant pathogenic mechanism exists for C9ORF72 ALS, it could be a candidate for antisense oligonucleotide therapeutics.