Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

Abstract

Expansion of G4C2 repeats within the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Such repeats lead to decreased expression of the autophagy regulator C9ORF72 protein. Furthermore, sense and antisense repeats are translated into toxic dipeptide repeat (DPR) proteins. It is unclear how these repeats are translated, and in which way their translation and the reduced expression of C9ORF72 modulate repeat toxicity. Here, we found that sense and antisense repeats are translated upon initiation at canonical AUG or near-cognate start codons, resulting in polyGA-, polyPG-, and to a lesser degree polyGR-DPR proteins. However, accumulation of these proteins is prevented by autophagy. Importantly, reduced C9ORF72 levels lead to suboptimal autophagy, thereby impairing clearance of DPR proteins and causing their toxic accumulation, ultimately resulting in neuronal cell death. Of clinical importance, pharmacological compounds activating autophagy can prevent neuronal cell death caused by DPR proteins accumulation. These results suggest the existence of a double-hit pathogenic mechanism in ALS/FTD, whereby reduced expression of C9ORF72 synergizes with DPR protein accumulation and toxicity.

Keywords: C9ORF72; RAN translation; amyotrophic lateral sclerosis; autophagy; neurodegeneration.

Figure EV1. Expanded G4C2 repeats are translated into polyGA

1. A

   Scheme and sequence of the human C9ORF72 sense transcript with 80 G4C2 repeats fused to the eGFP in the three possible frames and cloned into the pcDNA3.1 plasmid.

2. B, C

   Fluorescence (B) and RT–qPCR (C) GFP analyses of HEK293 cells transfected for 24 h with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence and fused in all three possible frames with the GFP deleted of its ATG.

3. D, E

   GFP expression (D) and immunoblotting (E) analysis of HEK293 cells transfected for 24 h with constructs expressing either polyGA, polyGP, or polyGR expressed under an artificial ATG start codon and fused to the GFP.

4. F

   Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with either a wild‐type or a mutant (CTG into CTT) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GA frame.

5. G

   Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with either a wild‐type or a mutant (CTG into ATG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GA frame.

6. H

   RT–qPCR analysis of GFP expression of HEK293 cells transfected for 24 h with either wild‐type or mutant (CTG into CTT or ATG) constructs containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GA frame.

7. I

   Immunoblotting against the HA‐tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 20 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA‐tag in the GA frame and treated or not with MG132 and/or bafilomycin A1 for 15 h.

8. J

   RT–qPCR analysis of GFP expression of HEK293 cells transfected for 24 h with either wild‐type or mutant (AGG into CGG) constructs containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame.

Figure 1. Expanded G4C2 repeats are translated into polyGA and polyGR

1. A

   Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG.

2. B

   LC‐MS/MS spectra of the N‐terminal part of the GFP‐immunoprecipitated and LysC‐digested polyGA protein expressed as in (A).

3. C, D

   Immunoblotting against the GFP‐ (C) or the HA‐tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 3, 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame.

4. E

   Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild‐type or a mutant (AGG into CGG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame.

5. F

   Scheme of human C9ORF72 sense transcript with intron 1 retained. Expanded G4C2 repeats, CUG and AGG near‐cognate initiation codons, and polyGA and polyGR ORFs are indicated in red. C9ORF72 ORF is indicated in blue.

Figure 2. Expanded C4G2 repeats are translated into polyPG

1. A

   Immunoblotting against the HA‐tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 100 C4G2 repeats embedded in the human antisense C9ORF72 and fused in all three possible frames with a HA‐tag.

2. B

   LC‐MS/MS spectra of the N‐terminal part of the HA‐immunoprecipitated and LysC‐digested polyPG protein expressed as in (A).

3. C, D

   Immunoblotting against the GFP‐ (C) or the HA‐tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 3, 10, or 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to the GFP in the PG frame.

4. E

   Scheme of human antisense C9ORF72 transcript. Expanded C4G2 repeats, AUG initiation codon, and polyPG ORF are indicated in red.

Figure EV2. Expanded C4G2 repeats are translated into polyPG

1. A, B

   Fluorescence (A) and RT–qPCR (B) GFP analyses of HEK293 cells transfected for 24 h with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence and fused in all three possible frames with the GFP deleted of its ATG.

2. C, D

   GFP expression (C) and immunoblotting (D) analysis of HEK293 cells transfected for 24 h with constructs expressing either polyPA or polyPR expressed under an artificial ATG start codon and fused to the GFP. Fluorescence of ATG‐polyGP from Fig EV1D is shown as control.

3. E

   Immunoblotting against HA or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with either a wild‐type or a mutant (∆ATG) construct containing 100 C4G2 repeats embedded in the antisense C9ORF72 sequence fused to a HA‐tag in the PG frame.

4. F

   RT–qPCR GFP expression analysis of HEK293 cells transfected for 24 h with either a wild‐type or a mutant (∆ATG) construct containing 100 C4G2 repeats embedded in the antisense C9ORF72 sequence fused to the GFP in the PG frame.

5. G

   Immunoblotting against the HA or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with either a wild‐type or a Kozak consensus mutant construct containing 100 C4G2 repeats embedded in antisense C9ORF72 fused to the HA‐tag in the PG frame.

6. H

   Immunoblotting against the HA‐tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 10 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA‐tag in the PG frame and treated or not with MG132 and/or bafilomycin A1 for 15 h.

Figure 3. Decreased expression of C9ORF72 synergizes DPR protein toxicity

1. Cell viability (TO‐PRO‐3 FACS staining) of GT1‐7 neuronal cells transfected for 24 h with either wild‐type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG, or GR frame.

2. Immunoblotting against the GFP, endogenous C9ORF72, or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA.

3. Immunoblotting against the HA‐tag, endogenous C9ORF72, or GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA‐tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with bafilomycin A1 for 15 h.

4. Immunoblotting against the HA‐tag, endogenous C9ORF72, or GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA‐tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with bafilomycin A1 for 15 h.

5. Cell viability (TO‐PRO‐3 FACS staining) of GT1‐7 neuronal cells co‐transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild‐type or mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG, or GR frame.

Data information: Error bars indicate s.e.m. Student's t‐test, *P < 0.05, **P < 0.01, and ***P < 0.001. n = 5 independent transfection.

Figure EV3. Decreased expression of C9ORF72 synergizes DPR toxicity

1. A

   Immunoblotting against the HA‐tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA‐tag in the GA frame and treated or not with MG132 and/or bafilomycin A1.

2. B

   As in (A) but with cells transfected with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA‐tag in the PG frame.

3. C, D

   Left panel, representative images of immunofluorescence labeling of endogenous P62/SQSTM1 (C) or ubiquitin (D) and the GFP in GT1‐7 neuronal cells co‐transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GA frame. Right panel, quantification of the percent of co‐localization of P62 or ubiquitin with polyGA aggregates.

4. E

   Immunofluorescence labeling of M6PR in GT1‐7 neuronal cells transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA.

5. F

   Immunofluorescence labeling of EEA1 in GT1‐7 neuronal cells transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA.

Data information: Error bars indicate s.e.m. Student's t‐test, ***P < 0.001. n = 3 independent transfection. Scale bars, 10 μm. Nuclei were counterstained with DAPI.

Figure 4. Promethazine reduces DPR protein accumulation and toxicity

1. Left panel, immunoblotting against the HA‐tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 μM of the indicated compound for 15 h. Right panel, quantification of polyGA expression relative to the GAPDH.

2. Left panel, immunoblotting against the HA‐tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 h with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA‐tag in the PG frame and treated with 10 μM of the indicated compound for 15 h. Right panel, quantification of polyPG expression relative to the GAPDH.

3. Cell viability (TO‐PRO‐3 FACS staining) of GT1‐7 neuronal cells treated with 1, 3, or 10 μM of promethazine and co‐transfected for 24 h with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.

Data information: Error bars indicate s.e.m. Student's t‐test, **P < 0.01, and ***P < 0.001. n = 5 independent transfection.

Figure EV4. Promethazine reduces DPR protein accumulation and toxicity

1. Left panel, immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with a construct expressing under an artificial ATG start codon 100 GA repeats fused to the GFP (ATG (GA)100× GFP) and treated 15 h with 10 μM of the indicated drug. Right panel, quantification of polyGA expression relative to the GAPDH.

2. Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 h with ATG (GA)100× GFP and treated 15 h with 1, 3, or 10 μM of the indicated drug.

3. Left panel, GFP fluorescence of HEK293 cells transfected for 24 h with ATG (GA)100× GFP and treated with 10 μM of the indicated compound. Scale bars, 10 μm. Nuclei were counterstained with DAPI. Right panel, quantification of polyGA aggregates.

4. Cell viability (TO‐PRO‐3 FACS staining) of GT1‐7 neuronal cells treated with 1, 3, or 10 μM of promethazine and co‐transfected for 24 h with ATG (GA)100× GFP and either a control siRNA or a siRNA targeting C9orf72 mRNA.

Data information: Error bars indicate s.e.m. Student's t‐test, **P < 0.01, and ***P < 0.001. n = 5 independent transfection.

Figure 5. Model of C9ORF72 loss‐of‐function and DPR gain‐of‐function toxicity

Expanded sense G4C2 and antisense C4G2 repeats are translated into polyGA, polyGR, and polyPG DPR proteins through initiation to near‐cognate codons or a cognate ATG codon embedded in a poor Kozak sequence. Concomitantly, expanded G4C2 repeats promote epigenetic DNA changes that inhibit promoter 1b activity, ultimately resulting in decreased expression of the C9ORF72 protein. Reduced expression of C9ORF72 leads to suboptimal autophagy that promotes the toxic accumulation of polyGA, polyGR, and polyPG DPR proteins, ultimately resulting in neuronal cell death.