Induced Neuronal Progenitor Cells (iNPC’s) were generated as previously described (Meyer et al., 2014). Table 1 includes details for the patient lines used in this study. The generation and use of cells derived from these iNPC’s has been published before in Meyer et al. (2014) and Au et al. (2024). iNPC’s were maintained in DMEM: F12 with GlutaMAX (Gibco), supplemented with 1% N2 (Invitrogen), 1% B27 (Invitrogen) and 20 ng/ml FGF-Basic (PeproTech). Cells were grown on fibronectin (R&D Biosystems) coated cell culture dishes and routinely sub-cultured every 2–3 days using Accutase (Corning) to detach them.

To achieve neuronal differentiation, iNPC’s were plated into six well plates and allowed to reach ∼80% confluency. iNeuron differentiation was started by switching to DMEM: F12 media with GlutaMAX supplemented with 1% N2, 2% B27 and 2.5 μM DAPT (Sigma). After 48 h, DAPT was removed and replaced in media with 1 μM retinoic acid (Sigma), 1 μM smoothened agonist (Millipore) and 2.5 μM Forskolin (Cayman Chemical). Cells were maintained in this media for 16 days before being used in assays.

To assess dependency of neurons on OXPHOS or glycolysis for ATP production throughout differentiation, cells were treated with 10 μM oligomycin to inhibit OXPHOS, 50 mM 2-deoxyglucose to inhibit glycolysis, or both simultaneously for 30 min at 37°C, before ATP measurements were performed with the ATPlite Luminescence Assay as per the manufacturers instructions (Revvity). From this we can calculate the percentage of ATP levels, setting untreated at each time point to 100%.

For deferiprone treatments, cells were treated with either 500 μM or 1 mM for 24 h prior to fixing cells and immunostaining. For oligomycin/antimycin A treatments, cells were treated with 10 μM oligomycin and 4 μM antimycin A for 1 h before fixing cells and immunostaining. For nilotinib, BL-918 and A769662 treatments, cells were treated with 1 or 5 μM nilotinib/BL918, or 1 or 10 μM A769662, for 24 h prior to fixing cells and immunostaining. For autophagy induction, cells were treated with either 200 nM bafilomycin A1, 1 μM rapamycin, 15 μM chloroquine or 1μM rapamycin/15 μM chloroquine in combination for 5 h before fixing cells and immunostaining.

For mitochondrial membrane potential (MMP) measurements, cells were stained for 1 h with 20 μM Hoechst, 80 nM TMRM and 200 nM MitoTracker Green diluted in MEM. Cells were imaged using an Opera Phenix high content imaging system (Revvity) with a 40x water immersion objective across 15 fields of view and 6 Z stacks. Cells were maintained at 37°C and 5% CO₂ throughout imaging. Image analysis was performed using a custom protocol in Harmony 4.9 (Revvity).

For live mitophagy flux measurements, cells were stained for 1 h with 20 μM Hoechst, 80 nM tetramethylrhodamine methyl ester (TMRM), 50 nM Lysotracker DeepRed and 200 nM MitoTracker Green diluted in MEM. For induced cells, 10 μM oligomycin and 4 μM Antimycin A were added immediately before imaging. Cells were imaged using an Opera Phenix high content imaging system (Revvity) with a 40x water immersion objective across 9 fields of view and 4 Z stacks, with imaging repeated every 30 min for 90 min. Cells were maintained at 37°C and 5% CO₂ throughout imaging. Image analysis was performed using a custom protocol in Harmony 4.9 (Revvity).

iNeurons were plated at 20,000 cells per well in a fibronectin-coated 96-well Seahorse cell culture plate (Agilent) in iNeuron media and incubated overnight at 37°C and 5% CO₂. The following day, media was replaced with Seahorse media pH 7.4 (Agilent) and cells were incubated at 37°C in a non-CO₂ incubator for 60 min prior to beginning oxygen consumption readings. The effect on oxygen consumption rate (OCR) was measured 4 times for 2.5 min each in the absence and presence of 1.5 μM oligomycin (to determine coupled respiration), 1.5 μM CCCP (to determine maximal respiration and spare respiratory capacity), and finally 0.5 μM rotenone in combination with 0.5μM antimycin A (to determine proton leak and non-mitochondrial oxygen consumption). Cells were fixed in 4% PFA in PBS after the assay, stained with 10 μM Hoechst for 5 min and imaged on an InCell Analyser (GE Healthcare) to determine cell numbers per well for normalization.

On day 18 of differentiation, cells were fixed in 4% PFA for 30 min. After PBS washes, cells were permeabilized in PBS with 1% Tween (PBST) and 0.1% Triton X-100 for 10 min. Cells were blocked in 5% horse serum in PBST for 1 h and incubated with primary antibodies in PBST overnight at 4°C. Cells were washed in PBST and incubated with secondary antibodies for 1 h at room temperature. Nuclei were stained with 1 μM Hoechst for 5 min prior to imaging. Cells were imaged using an Opera Phenix high content imaging system (Revvity) with a 40x water immersion objective across 20 fields of view and 6 Z stacks. Details of primary and secondary antibodies are provided in Table 2.

Day 18 iNeuron pellets were resuspended in 50 μL lysis buffer consisting of RIPA buffer, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). After 30 min incubating on ice, pellets were centrifuged at 13,000 rpm for 20 min at 4°C. Supernatant was collected and protein content was determined using a Bradford assay as per the manufacturer’s instructions. All samples were denatured at 95°C for 5 min in Laemmli buffer. A 20 μg of protein was loaded on 7.5% or 12% SDS-polyacrylamide gels for resolving, with protein electrophoresis performed using Mini-PROTEAN Tetra Handcast system (Bio-Rad). Proteins were transferred to PVDF membranes (Millipore) at 250 mA for 60 min. Membranes were blocked in 5% milk or BSA in tris buffered saline with Tween20 (TBST). Details of primary and secondary antibodies are provided in Table 2.

DNA was extracted from iAstrocyte pellets using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, G1N350) following manufacturer’s instructions. A 5 μg of DNA was digested using Alul and Ddel restriction enzymes (New England Biolabs) at 37°C for 16 h and electrophoresed in a 1% agarose gel in 1 x TAE. The agarose gel was denatured in 1.5 M NaCl, 0.5 NaOH and neutralized in 3 M NaCl, 0.5 M Tris, pH 7.5. DNA was then transferred to a positively charged nylon membrane (Amersham Hybond N+) by capillary blotting overnight and cross-linked by UV irradiation. A DIG-labeled oligonucleotide probe [5’ (DIG)-(GGGGCC) × 5-(DIG) 3’] was ordered from Thermo Fisher Scientific and denatured at 95°C for 10 min and immediately quenched in ice before use. After 4 h pre-hybridization in DIG Easy Hyb™ solution (Sigma-Aldrich) at 48°C, the membrane was hybridized with 100 ng/ml of probe and fresh DIG Easy Hyb™ solution at 48°C overnight in a rotating hybridization oven. The membrane was washed with 2X SSC 0.1% SDS at 48–65°C for 10 min twice, then 0.5 X SSC 0.1% SDS for 15 min, and 0.2 X SSC 0.1% SDS for 15 min at 65°C. The membrane was then washed and blocked using the DIG Wash and Block Buffer Set (Roche) following manufacturer’s instructions, incubated with anti-DIG AP Fab fragments (Roche) at 1:20,000 for 30 min, then washed three times for 10 min in wash buffer. The membrane was then placed in a detection buffer followed by application of CSPD ready-to-use chemiluminescent substrate (Roche), sealed in a plastic sheet, and exposed for 3 h on chemiluminescent film (Amersham, Cat No. 28906837). C9ORF72-repeat expansions were quantified from the Southern blot film using an adapted semi-automated protocol (Suh et al., 2015).

Statistical analyses were performed in Prism 10.0 (GraphPad). Normality of data was confirmed with Shapiro-Wilk test. If data were normally distributed, two-tailed unpaired t-tests or two-way ANOVAs with Tukey’s multiple comparisons test were performed. A p < 0.05 was considered significant. If data were non-normally distributed, Mann-Whitney U tests were performed.

We first sought to understand mitochondrial function in C9orf72-ALS iNeurons. We have previously shown that this iNeuron differentiation protocol yields neurons that stain positive for pan-neuronal markers β-III tubulin, MAP2 and NeuN, resulting in 83% of neurons at day18 staining positive for β-III tubulin, 75% positive for MAP2 and 52% positive for NeuN (Au et al., 2024; Supplementary Figure 1A), indeed the neurons used in Au et al. (2024) were generated at the same time from the same lines as those used in this study. Furthermore, we assessed the C9orf72 repeat expansion length in these patient iNeurons. C9-ALS lines had repeat lengths of > 1,200 (line 183), 961 (line 52), and 882 (line 78) (Supplementary Figure 1B). To assess the relative contribution of glycolysis and OXPHOS to ATP levels in the iNeurons, we measured ATP levels after treatment with inhibitors of glycolysis (2-deoxyglucose) and OXPHOS (oligomycin). In agreement with previous reports, iNPC’s were found to be heavily glycolytic, with mitochondrial inhibition only reducing ATP levels by ∼19% relative to vehicle treated controls (Schwartzentruber et al., 2020). By the end of differentiation, oligomycin led to larger reductions in ATP levels, down to 55% of UT levels in control iNeurons and 61% in C9orf72 iNeurons (Figures 1A,B). Therefore, these iNeurons have begun the metabolic switch, however, they are still reliant on glycolysis for 50% of their ATP production. No significant difference was observed in ATP levels between control and C9orf72-ALS iNeurons (Supplementary Figure 1).

Next, we stained iNeurons with TMRM and measured fluorescence intensity as an indicator of mitochondrial membrane potential (MMP). We observed a significant decrease in MMP (Figures 1C,D) (p = 0.0273). We simultaneously measured mitochondrial morphology using a membrane potential independent dye and in fixed cells using mitochondrial staining of TOM20, however, no differences in mitochondrial area, length, width: length, roundness or density were observed (Supplementary Figure 2). When comparing the distributions of mitochondrial sizes and morphologies we also found no significant differences between control and C9orf72-ALS iNeurone mitochondrial populations (Supplementary Figures 2G–J). Cell size was increased in the C9orf72 iNeurons, however, (Supplementary Figure 1C) (p = 0.0056). To further interrogate mitochondrial function, we used the Seahorse Bioanalyser to investigate mitochondrial oxygen consumption under basal and stressed conditions. Due to variability between patient lines, we observed no consistent differences in basal OCR, proton leak, coupled respiration, maximal respiration or spare respiratory capacity (Supplementary Figure 3). Given the iNeurons used in this study still relied on glycolysis for over 50% of ATP production, we also assessed extracellular acidification rate (ECAR) alongside OCR during this assay to provide an indication of glycolytic rate in these iNeurons. We found no significant differences in basal glycolytic rate, glycolytic capacity or glycolytic reserve in C9orf72-ALS iNeurons relative to controls (Supplementary Figure 3).

Initial assessments of mitophagy were performed by staining for the mitochondrial marker TOM20 and the autophagosomal marker LC3 in fixed iNeurons. A significant decrease in percentage of TOM20-positive mitochondria co-localizing with autophagosomes was observed in C9orf72-ALS iNeurons relative to controls (Figures 2A,B) (p = 0.0036). Interestingly, the C9orf72-ALS patient line with the longest repeat length of > 1,200 exhibited the most severe reduction in mitochondria co-localizing with autophagosomes. In contrast, when we investigated mitochondria-lysosome co-localization in iNeurons using live imaging, under basal conditions and after induction with oligomycin-antimycin A, we observed no differences in the number of mitochondria colocalizing with lysosomes (Figures 2C–E).

There have been multiple reports indicating a role for the C9orf72 protein and DPR’s on autophagy initiation and autophagosome-lysosome fusion. We stained cells with the lysosomal marker LAMP2 and autophagosome marker LC3. We observed no differences in autophagosome-lysosome co-localization in iNeurons (Figures 3A,B), however, we did observe a reduction in autophagosome density in C9orf72-ALS iNeurons (Figure 3C) (p = 0.0056). Once again, the C9orf72-Als line with the most severe reduction is the line with the longest repeat length. We sought to confirm whether autophagy induction was impaired in these iNeurons by manipulating autophagy with well characterized autophagy modulators (Klionsky et al., 2021). iNeurons were treated with bafilomycin A1 or chloroquine, both of which block lysosome degradation of autophagosomes and their contents. Rapamycin was also used as an inhibitor of mTOR, to induce autophagy and autophagosome production, however, in these cells under these conditions rapamycin alone was not sufficient to induce, autophagosome production. There is some debate around the ability of rapamycin to induce autophagy in neurons (Tsvetkov et al., 2010; Rubinsztein and Nixon, 2010). However, treatment with bafilomycin and chloroquine did produce alterations in autophagic flux as expected. Treatments with bafilomycin and chloroquine led to significant increases in autophagosomes in control, but not C9orf72-ALS iNeurons (Figure 3D) (p = 0.0004 for bafilomycin treatments and p = 0.0095 for chloroquine treatments in Control iNeurons). We observed no differences in lysosomal area or density in C9orf72-ALS iNeurons relative to controls (Supplementary Figures 4A–C).

We next sought to establish whether common mitophagy initiating pathways upstream of autophagosomal engulfment were disrupted in C9orf72-ALS. Previous studies have shown disruptions to PINK1/Parkin, BNIP3 and BNIP3L-dependent mitophagy in SOD1-ALS, TDP43-ALS, FUS-ALS and sporadic ALS (sALS) neurons (Lagier-Tourenne et al., 2012; Palomo et al., 2018; Rogers et al., 2017; Stribl et al., 2014). We observed no differences in Parkin co-localization with mitochondria in C9orf72-ALS iNeurons relative to controls, although there was variation between the C9orf72-ALS patient lines. Downstream of Parkin, we also observed no differences in phospho-ubiquitin accumulation at mitochondria in C9orf72-ALS iNeurons (Figure 4). The recruitment of Sequestosome-Like Receptor proteins NDP52 and P62 were also not disrupted in C9orf72-ALS iNeurons (Supplementary Figure 5). Co-localization of BNIP3, BNIP3L and phospho-BNIP3L with mitochondria basally showed a trend toward being reduced, however, due to large variability between both controls and patients this did not reach statistical significance (Figure 5 and Supplementary Figure 6). After induction with the iron chelator deferiprone (DFP), BNIP3 and BNIP3L localization at the mitochondria was significantly increased in control and C9orf72-ALS iNeurons (Figure 5) (p = 0.0017 in control iNeurons, p = 0.0011 in C9orf72-ALS iNeurons). Moreover, no changes in expression of BNIP3 and BNIP3L were observed via immunoblot (Supplementary Figures 7, 8). Interestingly, we also observed a significant negative correlation between mitochondrial co-localization and protein expression levels in BNIP3L but not BNIP3 in our iNeuron lines (Supplementary Figures 7E,F), (p = 0.0014). Interestingly, as with the mitochondria colocalizing with mitochondria and autophagosome density, the line with the longest C9orf72 repeat expansion showed the lowest BNIP3L levels at mitochondria and highest BNIP3L expression in total.

C9orf72 has previously been shown to mediate the recruitment of the ULK1 complex to the early autophagosome (Sellier et al., 2016; Webster et al., 2016). We investigated co-localization of mitochondria with ULK1 to investigate whether its recruitment to mitochondria prior to mitophagy was disrupted. We observed a significant reduction in ULK1 puncta co-localizing with mitochondria (Figure 6) (p = 0.0402). However, we observed no differences in ULK1 puncta density or expression via immunoblot (Figure 6C and Supplementary Figures 9E,F). We also investigated whether increasing ULK1 activity would rescue autophagy deficits caused by this impaired recruitment. Treatment with the AMPK activator A769662 and nilotinib, a drug that has been previously shown to activate ULK1, had no impact on mitophagy or autophagosome production in control or C9orf72 iNeurons. The ULK1 activator BL-918 was the only compound that appeared to have any ability to increase mitophagy in both control and C9orf72-ALS neurons at the highest concentration (increasing mitophagy to 140.7% in control neurons relative to DMSO and increasing mitophagy from 46.3 to 61.7% in C9orf72 neurons relative to DMSO-treated control neurons) although this did not reach statistical significance due to a variable response across the various lines (Supplementary Figure 9). It should be noted here, however, we have not confirmed specific target engagement of these compounds in our model system. Moreover, we show no significant changes in C9orf72 expression in C9orf72-ALS iNeurons (Supplementary Figure 1).