IPSC-MNs at differentiation D35 were harvested in cold Hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roche)) and lysed by passing through G25 needles 25 times and then spun down at 700 x g for 10min at 4℃. The Supernatant was loaded onto pre-made 30% Percoll solution and re-centrifuged at 33,000 RPM using Beckman rotor SWI55 for 50min at 4℃. 300 ul aliquots were taken from top to bottom as fractions and all the collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot.
In advanced traditional Chinese kung fu (martial arts), Neijing (Traditional Chinese: 內勁; pinyin: nèijìng) refers to the conscious control of the practitioner's qi, or "life energy", to gain advantages in combat. Nèijìng is developed by using "Neigong" (Traditional Chinese: 內功; pinyin: nèigōng) (內功), or "internal exercises," as opposed to "wàigōng" (外功), "external exercises."
To determine if Pikfyve inhibition rescues gain-of-function processes in vivo, we measured DPR levels in C9-BAC transgenic mice 58 with or without Apilimod treatment. Although it was not previously reported 58, we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig. 6j) using a previously-validated poly(GR) antibody 11. These data are consistent with findings in another published C9-BAC mouse model 14, suggesting that poly(GR) may be a common feature of C9-BAC mice. We also detected a low level of poly(GR) in neurons from control mice (Fig. 6j), which may be derived from other repeat regions or proteins with short poly(GR) sequences. Nevertheless, GR+ punctae levels were significantly higher in C9-BAC mouse neurons than in controls (Fig. 6j). Importantly, Apilimod treatment significantly reduced the number of GR+ punctae in hippocampal neurons in C9-BAC mice after 48 hrs (Fig. 6i, j). Therefore, small molecule inhibition of Pikfyve rescues both gain- and loss-of-function disease processes induced by C9ORF72 repeat expansion in vivo.
The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045), mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S).