Structured illumination microscopy (SIM) images were acquired using a Zeiss Elyra PS.1 system equipped with a 100X 1.46 NA or 63X 1.4NA objective. Acquisition was performed with PCO edge sCMOS camera and image reconstruction was done with built-in structured illumination model. Confocal microscopy images were acquired using Zeiss LSM800 microcopy with 63X 1.4NA objective or Zeiss LSM780 microcopy with 40X 1.1NA objective. Z stack images were done with a step size of 2.5 um. Further image process was done with Fiji.
Live imaging of iMNs expressing a M6PR-GFP fusion protein that localizes to M6PR+ vesicles 44 confirmed that C9ORF72 patient and C9ORF72-deficient iMNs possess increased numbers of M6PR+ vesicle clusters, and that overexpression of C9ORF72 isoform A or B rescues this phenotype (Supplementary Fig. 9c-g and Supplementary Videos 5-9). Clusters did not disperse over the time course of the assay, suggesting that they are relatively stable and not in rapid flux (Supplementary Videos 5-9). In addition, M6PR+ puncta moved with a slower average speed in C9ORF72 patient and C9ORF72+/− iMNs than controls (Supplementary Fig. 9h, i). Thus, reduced C9ORF72 levels lead to fewer lysosomes in motor neurons in vitro and in vivo, and this may be due in part to altered trafficking of M6PR+ vesicles.
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).
On the other hand, the level of the Neijing force depends on the extent one can exercise over one's will power to release an inner qi energy. Within the framework of Chinese martial arts, every person is believed to possess the inborn energy of qi. Martial artists can harness the force of qi so that it is strong enough to be applied in combat. When qi is being directed by one's will, it is called Neijing.[4]
To provide a quantitative measure of (GGGGCC)n hexanuceotide expansion in C9ORF72, 100 ng of genomic DNA was amplified by touchdown PCR using primers shown in Supplementary Data Table 4, in a 28-µl PCR reaction consisting of 0.2 mM each of 7-deaza-2-deoxyguanine triphosphate (deaza-dGTP) (NEB), dATP, dCTP and dTTP, 7% DMSO, 1X Q-Solution, 1X Taq PCR buffer (Roche), 0.9 mM MgCl2, 0.7 µM reverse primer (four GGGGCC repeats with an anchor tail), 1.4 µM 6FAM-fluorescently labeled forward primer, and 1.4 µM anchor primer corresponding to the anchor tail of reverse primer (Supplementary Data Table 4). During the PCR, the annealing temperature was gradually decreased from 70 ºC and 56 ºC in 2 ºC increments with a 3 min extension time for each cycle. The PCR products were purified using the QiaQuick PCR purification kit (Qiagen) and analyzed using an ABI3730 DNA Analyzer and Peak Scanner™ Software v1.0 (Life Technologies).
Biotinylation of plasma membrane localized glutamate receptors was performed using the Piece™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Dox-NIL iMNs were incubated with 0.25mg/ml Sulfo-NHS-SS-Biotin in cold room for 1~2 hrs with end-to-end shaking. After quenching, cells were harvested by scraping and lysed with lysis buffer from the Piece™ Cell Surface Protein Isolation Kit or the M-PER™ mammilian protein extraction buffer (Thermo Fisher Scientific). Cell lysate was incubated with High Capacity NeutrAvidin™ agorase beads (Thermo Fisher Scientific), and the bound protein was eluted in 2X SDS-PAGE sample buffer supplemented with 50mM DTT for 1 hr at room temperature with end-to-end rotation, and further analyzed by western blot.
Importantly, our work establishes a new approach for suppressing DPR protein toxicity and blocking C9ORF72 pathogenesis: restoring or replacing C9ORF72 activity. Although high levels of C9ORF72 isoform A may have slightly detrimental effects on control motor neuron survival, we have only observed this in neurons without C9ORF72 repeat expansion. Thus, we would not anticipate a harmful effect of forced C9ORF72 expression in C9ORF72 patients. In addition, a better understanding of the effects of forced C9ORF72 expression could inform safe development of this therapeutic strategy. For example, determining if C9ORF72 accelerates turnover of DPR aggregates by stimulating autophagy could lead help to identify new therapeutic targets.
International Advisory Board: James Archibald (Translation Studies) - Hugo de Burgh (Chinese Media Studies) - Kristen Brustad (Arabic Linguistics) - Daniel Coste (French Language) - Luciano Curreri (Italian Literature) - Claudio Di Meola (German Linguistics) - Donatella Dolcini (Hindi Studies) - Johann Drumbl (German Linguistics) - Denis Ferraris (Italian Literature) - Lawrence Grossberg (Cultural Studies) - Stephen Gundle (Film and Television Studies) - Tsuchiya Junji (Sociology) - John McLeod (Post-colonial Studies) - Estrella Montolío Durán (Spanish Language) - Silvia Morgana (Italian Linguistics) - Samir Marzouki (Translation, Cultural Relations) - Mbare Ngom (Post-Colonial Literatures) - Christiane Nord (Translation Studies) - Roberto Perin (History) - Giovanni Rovere (Italian Linguistics) - Lara Ryazanova-Clarke (Russian Studies) - Shi-Xu (Discourse and Cultural Studies) - Srikant Sarangi (Discourse analysis) - Françoise Sabban, Centre d'études sur la Chine moderne et contemporaine (Chinese Studies) - Itala Vivan (Cultural Studies, Museum Studies)