C9orf72 regulates energy homeostasis by stabilizing mitochondrial complex I assembly

Abstract

The haploinsufficiency of C9orf72 is implicated in the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the full spectrum of C9orf72 functions remains to be established. Here, we report that C9orf72 is a mitochondrial inner-membrane-associated protein regulating cellular energy homeostasis via its critical role in the control of oxidative phosphorylation (OXPHOS). The translocation of C9orf72 from the cytosol to the inter-membrane space is mediated by the redox-sensitive AIFM1/CHCHD4 pathway. In mitochondria, C9orf72 specifically stabilizes translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1), a crucial factor for the assembly of OXPHOS complex I. C9orf72 directly recruits the prohibitin complex to inhibit the m-AAA protease-dependent degradation of TIMMDC1. The mitochondrial complex I function is impaired in C9orf72-linked ALS/FTD patient-derived neurons. These results reveal a previously unknown function of C9orf72 in mitochondria and suggest that defective energy metabolism may underlie the pathogenesis of relevant diseases.

Keywords: ALS; C9orf72; FTD; OXPHOS; TIMMDC1; complex I; mitochondrial import; mitochondrion; neurodegeneration; oxidative phosphorylation.

Figure 1.. C9orf72 is an Inner Mitochondrial Membrane Binding Protein.

(A) Isolated mitochondria (Mito) from WT and C9KO MEFs were subjected to digestion with proteinase K (PK). C9orf72, an IMM protein TIM23, a matrix protein NDUFB9, and OMM proteins TOM70 and TOM20 were used as controls. (B) Mitochondria isolated from WT and C9KO MEFs were either sonicated or treated with sodium carbonate to extract peripheral membrane-binding proteins from the pellet fraction (P) to the supernatant fraction (S). (C) Representative confocal images and quantification of colocalization of endogenous C9orf72 and TIM23 in WT and C9KO MEFs (n = 40 cells). Scale bars, 10 μm. (D) C9orf72 immuno-electron microscopy (EM) analysis of WT and C9KO mitochondria isolated from mouse brain cortex tissues (n = 14 images). Red arrows point to immunogold-labeled C9orf72. Scale bars, 100nm. (E and F) The gel-shift AMS-modification assay was used to measure the oxidation states of C9orf72 in cytosolic and mitochondrial fractions of HEK293 cells. R: Reduced. O(p): Partially oxidized. O: Oxidized. The oxidation states of native C9orf72 are quantified (n = 4) (F). (G) HEK293 cells with knockdown of CHCHD4 or AIFM1 were subjected to subcellular fractionation and immunoblotting of indicated proteins (n = 4). (H) Representative autoradiographs and quantitative analysis of [³⁵S]C9orf72 imported into mitochondria isolated from HEK293 cells treated with the indicated siRNAs and proteinase K (n = 3). (I) Kinetic analysis of the C9orf72 import reaction as shown in (H). A representative gel image and quantification are shown (n = 3). Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S1 and S2, and Table S1.

Figure 2.. Loss of C9orf72 Decreases Complex I-associated Mitochondrial Function.

(A) Cell death of WT and C9KO MEFs, with or without galactose treatment for the indicated time periods, was detected by annexin V staining and microscopy (n = 4). (B) Cell viability of WT and C9KO MEFs in galactose medium was examined and normalized to that under normal glucose condition (n = 3). (C) MAPR of mitochondria isolated from WT and C9KO MEFs, with or without galactose treatment, by using different substrates that enter the respiratory chain at either CI or CII (n = 3). GM: glutamate plus malate. SR: succinate plus rotenone. (D) Analysis of mitochondrial OCR in WT and C9KO MEFs. Cells were maintained in glucose or galactose medium for 16 h before OCR detection (n = 8). The dotted lines indicate the time of adding oligomycin (O), FCCP (F), and rotenone and antimycin A (R&A). (E) Intracellular ATP concentrations in MEFs, with or without galactose treatment, were determined (n = 3). (F) Representative pseudocolor ratiometric images and quantification of the ATP/ADP ratio in WT and C9KO MEFs expressing PercevalHR (n = 30 cells). Scale bars, 5 μm. (G and H) Cell death (n = 3–5) and intracellular ATP concentrations (n = 3) in WT and C9KO MEFs, after being cultured under normal or low-glucose conditions for 72 h, was analyzed. (I) Glycolysis in WT and C9KO MEFs was analyzed by measuring the concentration of its end product, L-lactate, secreted outside the cell, after culturing for 24 or 48 h (n = 3). (J) Representative images and quantification of mitochondrial membrane potential (ΔΨ_m) in WT and C9KO MEFs by TMRM staining (n > 60 cells). Scale bars, 5 μm. Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S3 and S4, and Table S2 and S3.

Figure 3.. C9orf72 is Required for the Effective Assembly of Mitochondrial Complex I.

(A) Relative abundance of OXPHOS complexes in WT and C9KO MEFs was determined by immunoblotting against their respective makers (n = 3). (B) Western blotting analysis of the protein markers of OXPHOS complexes in isolated mitochondria of brain cortices and spinal cords from C9KO mice and WT littermates (n = 3). (C) BN-PAGE of the OXPHOS complexes in the isolated mitochondria from WT and C9KO MEFs (n = 4). Mature supercomplex forms of CI (SCs) were quantified by measuring the levels of NDUFB9 and NDUFB11. The CI-bound supercomplex of CIII [CIII (SCs)] or that of CIV [CIV (SCs)] as well as the free forms of CIII (CIII₂) or CIV (CIV_n) were examined and quantified using the marker for CIII or CIV, UQCRC1 or MTCO1, respectively. TIM23 was used as a loading control. (D) The rates for the assembly of the CI and CIII complexes in MEFs were analyzed by BN-PAGE after the cells were treated with chloramphenicol (CAM) to deplete CI and CIII, which were then allowed to recover (n = 4). (E) The in vitro CI assembly assay was performed by incubating the isolated mitochondria and ³⁵S-labeled CI subunit NDUFS8 for the indicated times in the presence or absence of CCCP (n = 4). The mature CI level was measured by BN-PAGE. [³⁵S]NDUFS8 import into the mitochondria, measured by SDS-PAGE, served as an internal control. Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001. See also Figures S5 and S6, and Table S4.

Figure 4.. C9orf72 Stabilizes CI Assembly Factor TIMMDC1 from Degradation.

(A and B) co-IP of TIMMDC1 with anti-C9orf72 antibody in isolated mitochondria from HEK293 cells (A), or from WT or C9KO MEFs (B). (C and D) Two-dimensional BN/SDS–PAGE analysis of isolated mitochondria from WT and C9KO MEFs, followed by immunoblotting against TIMMDC1 and C9orf72 (C). The histograms of TIMMDC1- or C9orf72-containing complexes are shown in (D), indicating the migration distance and relative abundance of the complexes. Sub-C1–3: TIMMDC1-containing subcomplex 1–3. The gray dashed line marks the comigration of C9orf72 and TIMMDC1 in the position of subcomplex 2 (Sub-C2). (E) Quantification of the relative abundance of TIMMDC1 in Sub-C1–3 and mature CI as shown in (C) (n = 5). (F) TIMMDC1 levels in isolated mitochondria from WT and C9KO MEFs were analyzed by immunoblotting (n = 3). (G) TIMMDC1 levels in isolated mitochondria from C9orf72-restored C9KO MEFs and controls were analyzed by immunoblotting (n = 3). (H) CHX chase analysis of TIMMDC1 stability in WT and C9KO MEFs (n = 3). Mitochondrial lysates were collected and TIMMDC1 levels were determined. (I and J) Two-dimensional BN/SDS–PAGE analysis of OXPHOS complexes from the mitochondria isolated from C9KO MEFs with or without restored C9orf72 protein expression (I). The relative abundance of TIMMDC1 in Sub-C1–3 and mature CI was quantified (J) (n = 3). Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S6.

Figure 5.. C9orf72 Recruits the PHB Complex to Stabilize TIMMDC1.

(A) Co-IP of PHB1 and PHB2 with anti-C9orf72 antibody was analyzed with isolated mitochondria from WT and C9KO MEFs (B) Mitochondria isolated from HEK293 cells were solubilized and subjected to immunoprecipitation with the anti-C9orf72 antibody, followed by immunodetection of PHB2. (C) Mitochondria lysates from HEK293 cells were subjected to immunoprecipitation with the anti-PHB2 antibody, followed by immunodetection of C9orf72. (D) The direct interaction of purified His-SUMO-C9orf72 and GST-PHB2 was determined by the Ni-NTA pull-down of C9orf72, followed by immunoblot analysis of the proteins. (E) WT and C9KO MEFs were transduced with lentiviral PHB2 shRNAs and the knockdown efficiency of PHBs was determined by immunoblotting. CTRL: non-targeting control shRNA. (F) CHX chase analysis of the turnover of TIMMDC1 in WT and C9KO MEFs, with or without the knockdown of PHB2. Quantifications of the turnover rates of TIMMDC1 are shown (n = 4). Data are means ± s.d., analyzed by one-way analysis of variance (ANOVA). ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S7 and Table S5.

Figure 6.. Mitochondrial AAA Protease AFG3L2 Mediates the C9orf72-related degradation of TIMMDC1.

(A) AFG3L2 and TIMMDC1 were analyzed by immunoblotting using isolated mitochondria from HEK293 cells transfected with AFG3L2 shRNAs or non-targeting control shRNAs (n = 3). (B) HEK293 cells were transfected with AFG3L2 shRNAs or non-targeting controls, followed by treatment with increasing concentrations of chloramphenicol (CAM), and TIMMDC1 levels were analyzed in whole cell lysates (n = 3). (C) Co-IP of AFG3L2 with anti-TIMMDC1 antibody in isolated mitochondria from WT and C9KO HAP1 cells (n = 3). (D) Co-precipitation of endogenous AFG3L2 by the pull-down of TIMMDC1-His with Ni-NTA agarose beads in the presence or absence of C9orf72-Flag expressed in HEK293 cells. The expression of C9orf72-Flag decreased the interaction of AFG3L2 with TIMMDC1-His (solid arrowhead) while increasing the level of TIMMDC1-His in the input (open arrowhead), with the quantifications shown alongside (n = 3). (E) Co-IP of AFG3L2 with anti-TIMMDC1 antibody was analyzed in WT and C9KO HAP1 cells with or without PHB2 depletion (n = 3). (F) Immunoblot analysis of TIMMDC1 in mitochondria isolated from WT and C9KO MEFs expressing shRNAs against AFG3L2 or non-targeting controls (n = 3). (G and H) Two-dimensional BN/SDS–PAGE analysis of the TIMMDC1-containing subcomplexes and mature CI in isolated mitochondria from WT and C9KO MEFs expressing shRNAs against AFG3L2 (G). The relative abundance of TIMMDC1 in Sub-C1–3 and mature CI was quantified (n = 3) (H). Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S7.

Figure 7.. Dysregulated OXPHOS Activity and C9orf72 Functions in C9-ALS Patients.

(A) Immunoblot analysis and quantification of OXPHOS proteins and C9orf72 in iPSC-MNs from C9orf72-linked ALS patients (C9-ALS) and healthy controls (controls, n = 3; patients, n = 3). (B) Immunoblot analysis and quantification of OXPHOS proteins and C9orf72 in spinal cord tissues from C9-ALS patients and controls (controls, n = 5; patients, n = 3). (C) BN-PAGE analysis of intact CI levels in B lymphocytes derived from C9-ALS patients and controls via NDUFB9 immunoblotting. C9orf72 and TIMMDC1 levels were analyzed by SDS-PAGE (controls, n = 4; patients, n = 4). (D) CI NADH-ubiquinone reductase activities in the isolated mitochondria from B lymphocytes derived from healthy controls and C9-ALS patients were determined (n = 3). (E) The interaction between TIMMDC1 and AFG3L2 in mitochondria isolated from B lymphocytes of C9-ALS patients and controls was analyzed by co-immunoprecipitation with the anti-TIMMDC1 antibody (n = 3). (F and G) Analysis of cell death (F) and intracellular ATP concentrations (G) of B lymphocytes from C9-ALS patients and healthy controls, cultured in either glucose or galactose medium for 24 h (n = 3). (H) Intracellular ATP content of four iPSC-MN lines (two control lines and two C9-ALS lines) maintained under glucose conditions or treated with galactose for 24 h (n = 4). (I) Control and C9-ALS iPSC-MN lines were maintained in glucose or galactose medium for 5 days before analysis of cell viability by calcein-AM staining (n = 3–4). Representative images of calcein-AM-stained MNs (CTRL 1 and C9-ALS 1) and the quantification of the cell viability are shown. (J) Intracellular ATP concentrations of four iPSC-MN lines (two from C9-ALS patients and two from healthy controls) treated with excess glutamate for 12 h (n = 4). (K) Graphical summary of C9orf72-regulated CI assembly in mitochondria. C9orf72 is imported into the mitochondrial IMS, via an AIFM1/CHCHD4 oxidase-dependent process, during which C9orf72 forms intramolecular disulfide bonds in the cysteine-rich region. The IMS-located C9orf72 binds to TIMMDC1, an essential assembly factor of CI, specifically at the second assembly subcomplex (Sub-C2). C9orf72 promotes the stability of TIMMDC1 by recruiting PHB, which blocks the access of the AFG3L2 (m-AAA) protease to TIMMDC1. In C9-ALS patients, the haploinsufficiency of C9orf72 led to reduced CI assembly and function. Data are means ± s.d., analyzed by unpaired two-sided Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. See also Figures S7 and Table S6.