Samples were first fixed in 4% PFA (1x PBS) overnight at 4°C and were subsequently washed three times with 1x PBS. Next, cells were permeabilized with 0.3% Triton X-100 (1x PBS) for 10 min at room temperature, followed by three washes with 1x PBS for 10 min each. After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58. During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min. The samples were submerged in 200 µl of hybridization buffer (4ml 100% formamide, 0.5 ml 20x SSC, 1 ml BSA fraction V, 0.5ml RVC (20 mM), 1ml NaPO4 (0.1 M), 3 ml nuclease-free water) and 27 µl of the probe mixture was added to each sample and incubated for 1 hour at 60°C. After probe hybridization, the samples were washed twice with 50% formamide (2x SSC) for 20 min each at 65°C and once more with 40% formamide (1x SSC) for 10 min at 60°C. The remaining formamide was removed by briefly washing with 1x SSC three times. A final crosslinking step was performed by first incubating the samples with 1x Tris-Glycine for 5 minutes followed by a 5 min incubation in 4% PFA. Samples were washed three times with 1x PBS, stained with DAPI, and imaged using a Zeiss LSM 800 confocal microscope.

To confirm that reduced C9ORF72 protein levels are sufficient to cause neurodegeneration, we used CRISPR/Cas9-mediated genome editing to introduce a frameshift mutation into one or both alleles of C9ORF72 in control iPSCs (Fig. 2e and Supplementary Fig. 4e). qPCR showed that targeting one allele reduced C9ORF72 transcript levels due to nonsense-mediated decay and transcript levels were more severely reduced in homozygous mutant cells (Supplementary Fig. 4f). Frameshift mutations also decreased C9ORF72 protein expression (Supplementary Fig. 4g, 5c). RNA sequencing of flow-purified Hb9::RFP+ iMNs showed that targeting C9ORF72 did not significantly alter the expression of the top 10 genes with predicted off-target sites for the CRISPR guide RNA (Supplementary Fig. 4h and Supplementary Table 7). In addition, expression levels of the 20 genes nearest C9ORF72 on chromosome 9 were largely unperturbed in either the C9ORF72+/− and C9ORF72−/− iMNs, indicating that this approach specifically inactivated C9ORF72 (Supplementary Fig. 4i).
With the four components of a chemical heat pump (two solid-gas reactors, an evaporator and a condenser), a cycle of the double-effect type can be applied to continuous refrigeration. The performance of this process is analysed, allowing the infinite sink temperature and the couples of reactive salts to be used, which depend on the production temperature envisaged, to be selected. The results are ... [Show full abstract]Read more
The GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for about 10% of each disease worldwide 1–4. In the central nervous system (CNS), neurons and microglia express the highest levels of C9ORF72 5, suggesting that C9ORF72 acts in part cell autonomously and effects in neurons are a key source of disease etiology. Studies showing that the repeat expansion generates neurotoxic species including nuclear RNA foci 6–8, RNA/DNA G-quadruplexes 9, and dipeptide repeat proteins (DPRs) 10–12 have oriented the field towards a therapeutic focus on blocking the toxicity of these products 6–8,13,14. However, these strategies have not fully rescued the degeneration of patient-derived neurons 7,13. Moreover, tandem GGGGCC repeats are transcribed from over 80 other genomic locations within human spinal motor neurons (Supplementary Tables 1 and 2), yet genetic studies have not linked repeat expansions in these regions to ALS/FTD. In addition, hexanucleotide repeat-mediated toxicity in mice requires supraphysiological expression levels or a specific genetic background 14–16. These observations suggest that there are additional pathogenic triggers caused by repeat expansion within C9ORF72.
Immunostaining revealed that C9ORF72+/− and C9ORF72−/− iMNs contained elevated levels of NMDA (NR1) and AMPA (GLUR1) receptors on neurites and dendritic spines compared to control iMNs under basal conditions (Fig. 4a, c, d and Supplementary Fig. 5b and 10a, c-e, g, h, j, k). In addition, control iMNs treated with C9ORF72-specific ASOs displayed increased numbers of NMDA and AMPA receptors in their neurites (Supplementary Fig. 10l, m). C9ORF72 patient iMNs (n=3 patients) also showed elevated NR1 and GLUR1 levels compared to controls (n=3 controls), and forced expression of C9ORF72 isoform B reduced glutamate receptor levels in patient iMNs (n=3 patients) to that of controls (n=3 controls) (Fig. 4a-c and Supplementary Fig. 10a-h). mRNA levels of NR1 (GRIN1) and GLUR1 (GRIA1) were not elevated in flow-purified C9ORF72+/− iMNs, indicating that increased transcription could not explain the increased glutamate receptor levels (Supplementary Fig. 10n).
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