The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

Abstract

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc-51-like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a-dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62-positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient-derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD-associated p62 pathology.

Keywords: C9orf72; Rab GTPase; amyotrophic lateral sclerosis; autophagy; frontotemporal dementia.

Figure 1. C9orf72 regulates the initiation of autophagy

1. A, B

   HeLa cells treated with non‐targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry‐EGFP‐LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (BafA1, 100 nM; 6 h), or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (mean ± SEM from 3 independent experiments; one‐way ANOVA with Fisher's LSD test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 μm. C9orf72 knockdown was confirmed by RT–qPCR (Appendix Fig S2).

2. C, D

   HEK293 cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1 + Torin1 (C), or BafA1 + rapamycin (D), and levels of LC3‐I and II were determined by immunoblots. Levels of LC3‐II were normalized against α‐tubulin and are shown relative to the BafA1‐treated sample (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; N = 3 experiments).

3. E, F

   Primary cortical neurons (DIV5/6) were transfected with ECFP non‐targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP‐LC3 (green). Three days post‐transfection, neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h), or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP‐LC3‐positive puncta per soma from 2 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant, *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 μm.

Source data are available online for this figure.

Figure EV1. C9orf72 regulates the initiation of autophagy

1. HEK293 cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were incubated with vehicle (Ctrl), Torin1, BafA1, or BafA1 + Torin1 for 3 h, and levels of LC3‐I and II were determined on immunoblots. Levels of LC3‐II were normalized against α‐tubulin and are shown relative to the BafA1‐treated sample (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant; *P ≤ 0.05, ****P ≤ 0.0001; N = 3 experiments). These data are also shown in Fig 1C.

2. HEK293 cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were incubated with vehicle (Ctrl), rapamycin, BafA1, or BafA1 + rapamycin for 6 h, and levels of LC3‐I and II were determined on immunoblots. Levels of LC3‐II were normalized against α‐tubulin and are shown relative to the BafA1‐treated sample (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant; **P ≤ 0.01, ***P ≤ 0.001; N = 3 experiments). These data are also shown in Fig 1D.

Figure 2. C9orf72 induces autophagy via the ULK1 complex

1. HEK293 cells treated with non‐targeting (Ctrl) or FIP200 siRNA were co‐transfected with EGFP‐LC3 and either empty vector control (EV), Myc‐C9orf72S, or Myc‐C9orf72L. 24 h post‐transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS–PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP‐LC3‐I and II. Expression of Myc‐C9orf72 was confirmed using anti‐Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti‐FIP200 antibodies. α‐tubulin was used as a loading control.

2. HeLa cells treated with non‐targeting (Ctrl) or FIP200 siRNA were co‐transfected with empty vector (EV), Myc‐C9orf72S or Myc‐C9orf72L (red), and EGFP‐LC3 (green) to label autophagosomes. As positive control, EV‐transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP‐LC3‐positive autophagosomes per cell from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 μm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2).

Source data are available online for this figure.

Figure 3. C9orf72 interacts with the ULK1 initiation complex

1. A–C

   Cell lysates of HEK293 cells co‐transfected with FLAG‐FIP200 (A), or HA‐ULK1 (B) and either empty vector control, Myc‐C9orf72S, or Myc‐C9orf72L or with Myc‐ATG13 (C) and either empty vector control, FLAG‐C9orf72S, or FLAG‐C9orf72L were subjected to immunoprecipitation with anti‐Myc (A and B) or anti‐FLAG (C) antibodies. Immune pellets were probed for Myc‐C9orf72 (A and B), FLAG‐C9orf72 (C), FLAG‐FIP200 (A), HA‐ULK1 (B), or Myc‐ATG13 (C) on immunoblots.

2. D, E

   Cell lysates of HEK293 cells transfected with empty vector control, Myc‐C9orf72S, or Myc‐C9orf72L were subjected to immunoprecipitation with anti‐Myc antibodies. Immune pellets were probed for Myc‐C9orf72 and endogenous FIP200, ULK1, and ATG13. There are multiple alternatively spliced forms of ATG13 (Jung et al, 2009; Alers et al, 2011); ATG13* is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72.

3. F–H

   HeLa cells transfected with HA‐ULK1 or Flag‐FIP200 and Myc‐C9orf72 or with Myc‐ATG13 and EGFP‐C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ****P ≤ 0.0001; N (cells) = (F) C9orf72S: 22; C9orf72L: 18; EV+FIP200: 18; C9orf72S+FIP200: 18; C9orf72L+FIP200: 17; (G) C9orf72S: 21; C9orf72L: 20; EV+ULK1: 20; C9orf72S+ULK1: 22; C9orf72L+ULK1: 20; (H) C9orf72S: 11; C9orf72L: 10; EV+ATG13: 11; C9orf72S+ATG13: 11; C9orf72L+ATG13: 11). Scale bar = 10 μm; see also Fig EV2.

4. I–K

   ³⁵S‐radiolabeled recombinant FIP200‐6xHis segments (I), Myc‐ATG13 (J), or HA‐ULK1 (K) were added to GST, GST‐C9orf72S, and GST‐C9orf72L immobilized on glutathione‐coated beads. ³⁵S‐radiolabeled recombinant proteins were visualized by phosphorimager (top panels). Coomassie‐stained GST, GST‐C9orf72S, and GST‐C9orf72L in the pull‐down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (^# indicates E. coli DnaK chaperonin; * indicates E. coli 60kD chaperonin; Appendix Fig S3).

Source data are available online for this figure.

Figure EV2. C9orf72 interacts with the ULK1 initiation complex

1. HeLa cells were transfected with FLAG‐FIP200, Myc‐C9orf72S and Myc‐C9orf72L or co‐transfected with FLAG‐FIP200 and either Myc‐C9orf72S or Myc‐C9orf72L as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with both anti‐FLAG and anti‐Myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = FLAG‐FIP200: 18, Myc‐C9orf72S: 22, Myc‐C9orf72L: 18, FLAG‐FIP200 + Myc‐C9orf72S: 18, FLAG‐FIP200 + Myc‐C9orf72L: 17). Scale bar = 20 μm.

2. HeLa cells were transfected with HA‐ULK1, Myc‐C9orf72S and Myc‐C9orf72L or co‐transfected with HA‐ULK1 and either Myc‐C9orf72S or Myc‐C9orf72L as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with both anti‐HA and anti‐myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = HA‐ULK1: 20, Myc‐C9orf72S: 21, Myc‐C9orf72L: 20, HA‐ULK1 + Myc‐C9orf72S: 22, HA‐ULK1 + Myc‐C9orf72L: 20). Scale bar = 20 μm.

3. HeLa cells were transfected with Myc‐ATG13, EGFP‐C9orf72S and EGFP‐C9orf72L or co‐transfected with Myc‐ATG13 and either EGFP‐C9orf72S or EGFP‐C9orf72L. The ATG13 transfection was laced with mVenus to enable detection of transfected cells for analysis (green). Transfected cells were probed with both anti‐EGFP and anti‐myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = ATG13: 11, EGFPc2‐C9orf72S: 11, EGFPc2‐C9orf72L: 10, Myc‐ATG13 + EGFPc2‐C9orf72S: 11, Myc‐ATG13 + EGFPc2‐C9orf72L: 11). Scale bar = 20 μm.

Figure 4. C9orf72 regulates translocation of the ULK1 complex

1. HEK293 cells were transfected with non‐targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho‐ULK1 (Ser757), total ULK1, and GAPDH Abs (loading control).

2. HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry‐FIP200. Twenty‐four hours post‐transfection, cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ns: not significant, ****P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

3. Primary cortical neurons (DIV5/6) were transfected with EmGFP non‐targeting (Ctrl) or C9orf72 miRNA (green) and mCherry‐FIP200 (red); for rescue experiments, the cells were additionally transfected with mCerulean‐tagged C9orf72s and C9orf72L (cyan). Three days post‐transfection, neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per soma from 2 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ns: not significant, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 μm.

4. HeLa cells were co‐transfected with mCherry‐FIP200 (red) and empty vector (EV), FLAG‐C9orf72L, or FLAG‐C9orf72S (green). As positive control, EV‐transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells) = EV: 47, EV+Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 μm.

Source data are available online for this figure.

Figure 5. C9orf72 regulates translocation of the ULK1 complex via Rab1a

1. A, B

   HeLa cells (A) or SH‐SY5Y neuroblastoma cells (B) treated with non‐targeting (Ctrl) or Rab1a siRNA were co‐transfected with mCherry‐FIP200 (red) and empty vector (EV), Myc‐C9orf72L, or Myc‐C9orf72S (green). As positive control, EV‐transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell [A, HeLa, mean ± SEM; one‐way ANOVA with Fisher's LSD test; ns, not significant; ****P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH‐SY5Y: mean ± SEM; one‐way ANOVA with Fisher's LSD test; ns, not significant; **P ≤ 0.01; ****P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37]. Rab1a knockdown was confirmed by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

2. C

   SH‐SY5Y neuroblastoma cells treated with non‐targeting (Ctrl) or Rab1a siRNA were co‐transfected with EGFP‐LC3 (green) and empty vector (EV), Myc‐C9orf72L, or Myc‐C9orf72S (red). As positive control, EV‐transfected cells were treated for 3 h with Torin1 (250 nM). Autophagosomes were quantified as the number of EGFP‐LC3‐positive puncta per cell (mean ± SEM; one‐way ANOVA with Fisher's LSD test; ns, not significant; *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 102; Ctrl/EV/Torin1: 92; Ctrl/C9orf72L: 97; Ctrl/C9orf72S: 76; Rab1a/EV: 102; Rab1a/EV/Torin1: 107; Rab1a/C9orf72L: 101; Rab1a/C9orf72S: 87). Rab1a knockdown was confirmed by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

Figure EV3. C9orf72 regulates translocation of the ULK1 complex to the phagophore via Rab1a

1. HeLa cells were co‐transfected with empty vector (EV), FLAG‐C9orf72S or FLAG‐C9orf72S (green), Myc‐Rab1aWT or dominant negative Myc‐Rab1aS25N (DN), and mCherry‐FIP200 (red). 24 h post‐transfection, cells were treated with Torin1 (250 nM; 3 h). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 7 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant, **P ≤ 0.01, ****P ≤ 0.0001; N (cells) = WT/EV: 142; WT/EV/Torin1: 73; WT/C9orf72L: 108; WT/C9orf72S: 123; DN/EV: 159; DN/EV/Torin1: 150; DN/C9orf72L: 159; DN/C9orf72S: 123). Scale bar = 10 μm.

2. HeLa cells were co‐transfected with empty vector (EV), FLAG‐C9orf72S or FLAG‐C9orf72S (red), Myc‐Rab1aWT or dominant negative Myc‐Rab1aS25N (DN), and EGFP‐LC3 (green). 24 h post‐transfection, cells were treated with Torin1 (250 nM; 3 h). Autophagosomes were quantified as the number of EGFP‐LC3‐positive puncta per cell from 2 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant, **P ≤ 0.01, ****P ≤ 0.0001; N (cells) = WT/EV: 51; WT/EV/Torin1: 53; WT/C9orf72L: 56; WT/C9orf72S: 41; DN/EV: 53; DN/EV/Torin1: 53; DN/C9orf72L: 55; DN/C9orf72S: 42). Scale bar = 10 μm.

Figure 6. C9orf72 interacts with Rab1a

1. A human brain random cDNA library was screened in a Y2H assay using C9orf72S as bait. A clone coding for aa 46–205 of Rab1a, comprising most of the GTP‐binding G‐box domain and the C‐terminal CC‐motif was found to interact with C9orf72S.

2. Cell lysates of HEK293 cells co‐transfected with Myc‐Rab1a and either empty vector, FLAG‐C9orf72S, or FLAG‐C9orf72L were subjected to immunoprecipitation with anti‐FLAG antibody. Bound protein was eluted from beads using excess FLAG peptide. Immune eluates were probed for FLAG‐C9orf72 and Myc‐Rab1a on immunoblots. The input levels of FLAG‐C9orf72 and Myc‐Rab1a in the transfected cells are shown (Inputs). * indicates remaining Myc‐Rab1a signal after reprobing for FLAG‐C9orf72.

3. Cell lysates of HEK293 cells transfected with Myc‐C9orf72S or Myc‐C9orf72L were subjected to immunoprecipitation with anti‐Myc antibody. The resulting immune pellet was probed for endogenous Rab1a.

4. ³⁵S‐radiolabeled recombinant Myc‐Rab1a protein loaded with vehicle, GDP or GMP‐PNP was added to GST, GST‐C9orf72S, and GST‐C9orf72L immobilized on glutathione‐coated beads. ³⁵S‐radiolabeled recombinant Myc‐Rab1a protein was visualized by phosphorimager (top panel). Coomassie‐stained GST, GST‐C9orf72S, and GST‐C9orf72L in the pull‐down samples are shown (bottom panel). The identity of the Coomassie protein bands was confirmed by mass spectrometry (^# indicates E. coli DnaK chaperonin; * indicates E. coli 60kD chaperonin; Appendix Fig S3). Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test; ns, not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

5. Increasing volumes of ³⁵S‐radiolabeled recombinant Myc‐C9orf72L protein were incubated with equal amounts of GST‐Rab1a immobilized on glutathione‐coated beads in an equilibrium binding experiment. 8 μl of ³⁵S‐radiolabeled recombinant Myc‐C9orf72L protein was incubated with GST as a negative control. Bound ³⁵S‐radiolabeled Myc‐C9orf72L protein was visualized by phosphorimager. Coomassie‐stained GST‐Rab1a and GST in the pull‐down samples are shown. Densitometry analysis of the amount of ³⁵S‐radiolabeled recombinant Myc‐C9orf72L protein bound to GST‐Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola (R ² = 0.94).

Source data are available online for this figure.

Figure 7. C9orf72 mediates interaction of Rab1a with the ULK1 complex

1. HEK293 cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were transfected with HA‐ULK1, Myc‐Rab1a, or HA‐ULK1 + Myc‐Rab1a as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with anti‐HA and anti‐Myc antibodies and processed for PLA. PLA proximity signals (red) per cell were determined from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ****P ≤ 0.0001; N (cells) = Ctrl/HA‐ULK1: 125, Ctrl/Myc‐Rab1a: 149, Ctrl/HA‐ULK1 + Myc‐Rab1a: 163, C9orf72/HA‐ULK1: 136, C9orf72/Myc‐Rab1a: 133, C9orf72/HA‐ULK1 + Myc‐Rab1a: 155). Scale bar = 20 μm.

2. HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were co‐transfected with mCherry‐FIP200 (red) and empty vector (EV) or Myc‐Rab1a(Q70L) (green). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 4 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, *P ≤ 0.05, ****P ≤ 0.0001; N (cells) = Ctrl/EV: 101; Ctrl/Q70L: 106; C9orf72/Q70L: 42). C9orf72 knockdown was determined by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

3. HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were co‐transfected with EGFP‐LC3 (green) and empty vector (EV) or Myc‐Rab1a(Q70L) (red). Autophagosomes were quantified as the number of EGFP‐LC3‐positive puncta per cell (mean ± SEM; one‐way ANOVA with Fisher's LSD test, **P ≤ 0.01, ****P ≤ 0.0001; N (cells) = Ctrl/EV: 44; Ctrl/Q70L: 32; C9orf72/Q70L: 47). C9orf72 knockdown was determined by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

Figure 8. Loss of C9orf72 induces p62 accumulation

1. HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were immunostained for endogenous p62. Accumulation of p62 was quantified by counting p62‐positive puncta per cell from 3 independent experiments (mean ± SEM; unpaired t‐test, ****P ≤ 0.0001; N (cells) = Ctrl: 74, C9orf72: 55). C9orf72 knockdown was confirmed by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.

2. Primary cortical neurons (DIV5) were transduced with 4TU/cell EmGFP non‐targeting control miRNA (Ctrl) or C9orf72 miRNA (green); for rescue experiments, the cells were additionally transduced with 4TU/cell mVenus‐tagged C9orf72s and C9orf72L (verified by immunoblot, Appendix Fig S2). Neurons were immunostained for endogenous p62 3 days post‐transduction. Accumulation of p62 was quantified by counting p62‐positive puncta per soma from 2 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, **P ≤ 0.01; N (cells) = Ctrl miRNA: 80; C9orf72 miRNA: 80; C9orf72 miRNA+C9orf72L+C9orf72S: 75). Scale bar = 10 μm.

Figure 9. C9ALS/FTD iNeurons exhibit autophagy deficits

1. A, B

   Two C9ALS/FTD iNeuron cultures (201 in A and 183 in B) and their matching controls (209 in A and 155 in B) were treated with vehicle (Ctrl) or bafilomycin A1 (BafA1, 100 nM; 6 h) and processed for immunoblot detection of LC3. LC3‐II levels were normalized to α‐tubulin (mean ± SEM; one‐way ANOVA with Fisher's LSD test; ns, not significant; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; Ctrl 209/Pat 201, n = 4; Ctrl 155/Pat 183, n = 6).

Source data are available online for this figure.