PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD

C9ORF72 patient-derived neurons exhibit early ER stress-mediated adaptive response

Neurons are selectively prone to ER stress in NDs, such as ALS and FTD, influencing disease manifestation and kinetics [25, 58]. The early phase of ER stress is accompanied by an increase in mitochondrial–ER contact sites and Ca2+ uptake, thus increasing ATP production [3, 25, 56]. We longitudinally evaluated ER stress responses by qPCR in five C9ORF72-ALS/FTD patient and four control iPSC-derived MN (iMN) cell lines, two of which were corresponding isogenic iMNs (see Supplementary Fig. 1a–d, online resource for phenotypic description, quality of differentiation and pathological hallmarks). Transcripts of the major luminal ER chaperone BiP/GRP78 and its downstream effector CHOP were significantly higher in all 2-week-old C9ORF72 iMNs; these levels increased further by 4 weeks, indicating growing impairment in ER homeostasis (Fig. 1a). We next examined whether the early phase of ER stress at 2 weeks modulates or affects MAM-associated molecules. Of the four types of connectors between the ER and mitochondria, the expression of GRP75 transcript, involved in the tethering and Ca2+-signaling complex at MAMs, was consistently and significantly upregulated (two-to-fourfold higher) in all C9ORF72 iMNs. Transcripts of both GRP75 binding partners, i.e., ITPR3 and VDAC1, exhibited a significant trend toward higher expression (Fig. 1b). In contrast, other MAM connectors, MFN1, MFN2, VAPB, RMDN3, FIS1, and BAP31 remained largely unchanged compared to the healthy or isogenic control iMNs (Supplementary Fig. 2a, online resource). Importantly, the two isogenic controls had reverted the observed C9ORF72 phenotype involving ER stress and elevated GRP75 expression (Fig. 1a, b).

Fig. 1figure 1

C9ORF72 iMNs display ER stress-mediated increase in GRP75 expression. a qPCR analysis of IPSC-derived motoneurons (iMNs) after 1, 2 and 4 weeks of maturation from five different ALS patient lines (C9(1),(2),(3),(4),(5)), displaying increased levels of ER stress markers (BiP and CHOP) at 2 and 4 weeks, but not at 1 week compared to two healthy control lines (Ctrl(1) and (2)) or corresponding isogenic control (Iso-C9(4), Iso-C9(5)). One-way ANOVA; BiP: Controls: F = 38.94***, Sidak’s multiple comparison test: Ctrl(1) 1 week vs C9(1) 1 week n.s.; Ctrl(2) 1 week vs C9(2) 1 week n.s.; Ctrl(1) 1 week vs C9(3) 1 week n.s.; C9(1) 1 week vs C9(1) 2 and 4 weeks***; C9(2) 1 week vs C9(2) 2 and 4 weeks***; C9(3) 1 week vs C9(3) 2 weeks*; C9(3) 1 week vs C9(3) 4 week***. One-way ANOVA BiP Isogenic: F = 46.01***, Sidak’s multiple comparison test: Iso-C9(4) 1 week vs C9(4) 1 week n.s.; Iso-C9(5) 1 week vs C9(5) 1 week n.s.; C9(4) 1 week vs C9(4) 2 and 4 weeks***; C9(5) 1 week vs C9(5) 2 and 4 weeks***. One-way ANOVA; CHOP: Controls: F = 63.65***, Sidak’s multiple comparison test: Ctrl(1) 1 week vs C9(1) 1 week n.s.; Ctrl(2) 1 week vs C9(2) 1 week n.s.; Ctrl(1) 1 week vs C9(3) 1 week n.s.; C9(1) 1 week vs C9(1) 2 and 4 weeks***; C9(2) 1 week vs C9(2) 2 and 4 weeks***; C9(3) 1 week vs C9(3) 2 and 4 weeks***. One-way ANOVA CHOP Isogenic: F = 40.15***, Sidak’s multiple comparison test: Iso-C9(4) 1 week vs C9(4) 1 week n.s.; Iso-C9(5) 1 week vs C9(5) 1 week n.s.; C9(4) 1 week vs C9(4) 2 and 4 weeks***; C9(5) 1 week vs C9(5) 2 weeks**; C9(5) 1 week vs C9(5) 4 weeks***. Combined graph represents the average values for Ctrls lines (Cntrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–3–4–5), Unpaired t test; BiP: Ctrls 1 week vs C9 1 week t = 1.334, P = 0.1935, n.s.; Ctrls 2 weeks vs C9 2 weeks t = 16.59, P < 0.0001, ***; Ctrls 4 weeks vs C9 4 weeks t = 15.04, P < 0.0001, ***; CHOP: Ctrls 1 week vs C9 1 week t = 0.6029, P = 0.5512, n.s.; Ctrls 2 weeks vs C9 2 weeks t = 14.84, P < 0.0001, ***; Ctrls 4 weeks vs C9 4 weeks t = 15.40, P < 0.0001, ***. Values were normalized to relative expression of GAPDH. qPCR graphs plotted with s.d., n = 3–8 independent qPCR experiments repeated in triplicate. b qPCR analysis of mitochondria associated membrane (MAM) molecules GRP75 and its binding partners ITP3R and VDAC1 from 2-week-old iMNs. All C9ORF72 patient lines display significant increase in GRP75 transcript. (Unpaired t test; VDAC1: Ctrl(1) vs C9(1) t = 0.4234, P = 0.6896, n.s.; Ctrl(2) vs C9(2) t = 5.720, P = 0.0012**; Ctrl(1) vs C9(3) t = 6.024, P = 0.0018**; Iso-C9(4) vs C9(4) t = 1.843, P = 0.1026 n.s.; Iso-C9(5) vs C9(5) t = 3.090, P = 0.0214*. Unpaired t test; GRP75: Ctrl(1) vs C9(1) t = 12.21, P < 0.0001***; Ctrl(2) vs C9(2) t = 8.460, P < 0.0001***; Ctrl(1) vs C9(3) t = 9.333, P < 0.0001***; Iso-C9(4) vs C9(4) t = 5.128, P = 0.0006***; Iso-C9(5) vs C9(5) t = 19.32, P < 0.0001***. Unpaired t test; ITPR3: Ctrl(1) vs C9(1) t = 7.679, P = 0.0003***; Ctrl(2) vs C9(2) t = 8.262, P = 0.0002***; Ctrl(1) vs C9(3) t = 2.543, P = 0.0439*; Iso-C9(4) vs C9(4) t = 3.726, P = 0.0074**; Iso-C9(5) vs C9(5) t = 5.470, P = 0.0016**). Combined graph for Ctrls lines (Ctrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–3–4–5), Unpaired t test; VDAC1: Ctrls 2 weeks vs C9 2 weeks t = 4.026, P = 0.0003, ***; GRP75: Ctrls 2 weeks vs C9 2 weeks t = 12.06, P < 0.0001, ***; ITP3R: Ctrls 2 weeks vs C9 2 weeks t = 7.830, P < 0.0001, ***. c 1-week-old iMNs treated with TU (1 µg/mL) for 18 h display significant increase in transcripts of BiP as well as GRP75, increased expression was not only found in C9ORF72 patient lines but also in Controls and isogenic lines. Unpaired t test; BiP: Ctrl(1) vs Ctrl(1) + TU t = 13.90, P = 0.0002***; Ctrl(2) vs Ctrl(2) + TU t = 20.24, P < 0.0001***; C9(1) vs C9(1) + TU t = 33.32, P < 0.0001***; C9(2) vs C9(2) + TU t = 7.267, P = 0.0008***; C9(3) vs C9(3) + TU t = 13.29, P  = 0.0002***; Ctrl(1) + TU vs C9(1) + TU t = 6.460, P = 0.0030**; Ctrl(2) + TU vs C9(2) + TU t = 0.0038, P = 0.0038** Iso-C9(4) vs Iso-C9(4) + TU t = 7.330, P = 0.0003***; C9(4) vs C9(4) + TU t = 8.578, P = 0.0001***; Iso-C9(5) vs Iso-C9(5) + TU t = 9.384, P = 0.0007***; C9(5) vs C9(5) + TU t = 8.913, P = 0.0009***. Unpaired t test GRP75: Ctrl(1) vs Ctrl(1) + TU t = 7.643, P = 0.0016**; Ctrl(2) vs Ctrl(2) + TU t = 11.02, P = 0.0004***; C9(1) vs C9(1) + TU t = 5.953, P = 0.0040**; C9(2) vs C9(2) + TU t = 7.427, P = 0.0007***; C9(3) vs C9(3) + TU t = 18.03, P < 0.0001***; Ctrl(1) + TU vs C9(1) + TU t = 4.218, P = 0.0135*; Ctrl(2) + TU vs C9(2) + TU t = 6.485, P = 0.0013**. Iso-C9(4) vs Iso-C9(4) + TU t = 12.32, P < 0.0001***; C9(4) vs C9(4) + TU t = 3.749, P = 0.0133*; Iso-C9(5) vs Iso-C9(5) + TU t = 6.160, P = 0.0035**; C9(5) vs C9(5) + TU t = 7.524, P = 0.0017**. Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4) and C9 (C9 1–2–3–4), Unpaired t test; BiP: Ctrls vs Ctrls + TU t = 8.187, P < 0.0001, ***; C9 vs C9 + TU t = 7.885, P < 0.0001***; Ctrls + TU vs C9 + TU t = 2.995, P = 0.0057, **; GRP75 : Ctrls vs Ctrls + TU t = 11.75, P < 0.0001, ***; C9 vs C9 + TU t = 9.563, P < 0.0001,***; Ctrls + TU vs C9 + TU t = 6.187, P < 0.0001***. d Representative immunofluorescence images showing BiP and GRP75 protein expression in 1- and 2-week-old iMNs for C9ORF72 patient line 4 together with the corresponding isogenic control. Quantitative Analysis (Q.A.) of BiP and GRP75 expression reveals significantly increased levels of GRP75 and BiP in all C9ORF72 lines when compared to controls or isogenic controls at 2-week post-differentiation, but not at 1 week. Unpaired t test; BiP: 2 weeks: Ctrl(1) vs C9(1) t = 12.24, P < 0.0001***; Ctrl(2) vs C9(2) t = 5.005, P < 0.0001***; Ctrl(1) vs C9(3) t = 13.41, P < 0.0001***; Iso-C9(4) vs C9(4) t = 10.02, P < 0.0001***; Iso-C9(5) vs C9(5) t = 7.109, P < 0.0001***. Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–3–4–5), Unpaired t test; BiP: Ctrls 1 week vs C9 1 week t = 0.6497, P = 0.5167, n.s.; Ctrls 2 weeks vs C9 2 weeks t = 14.46, P < 0.0001, ***. Unpaired t test; GRP75 2 weeks: Ctrl(1) vs C9(1) t = 7.703, P < 0.0001***; Ctrl(2) vs C9(2) t = 11.08, P < 0.0001***; Ctrl(1) vs C9(3) t = 6.847, P < 0.0001***; Iso-C9(4) vs C9(4) t = 9.892, P < 0.0001***; Iso-C9(5) vs C9(5) t = 10.11, P < 0.0001***. Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–3–4–5), Unpaired t test; GRP75: Ctrls 1 week vs C9 1 week t  = 0.4153, P = 0.6788, n.s.; Ctrls 2 weeks vs C9 2 weeks t = 19.39, P < 0.0001, ***. e Representative western blot (WB) displaying increased expression of BiP and GRP75 in C9(1–2) and (4) compared to healthy control(1–2) or Iso-C9(4). (Right) Q.A. of relative BiP and GRP75 expression, normalized to GAPDH; combined graph for Ctrls(1–2–4) and C9(1–2–4). Unpaired t test; BIP: Ctrl vs C9 t = 6.142, P < 0.0001***; Unpaired t test GRP75: Ctrl vs C9 t = 6.536, P < 0.0001***. (n = 3 experiments from 3 different culture differentiations). Scale bar: d 5 μm

To examine whether the increase in GRP75 mRNA levels was a direct response to ER stress, we treated 1-week-old iMNs, which at this point lack ER stress, with tunicamycin (TU). To exclude cell death responses, which in cultured neurons are initiated at ~ 24 h and become significant around 48 h [54], we performed a mild treatment (TU: 1 µg/mL) for 18 h. TU treatment augmented GRP75 mRNA levels in both control and mutant 1-week-old iMNs, suggesting that higher GRP75 transcripts reflected ongoing ER stress in iMNs (Fig. 1c). Of note, mutant iMNs exhibited a stronger response to TU treatment than healthy control iMNs. Moreover, other MAM molecules remained largely unaltered in response to the mild TU treatment: no significant alteration of twofold or more was observed (Supplementary Fig. 2b, online resource). We further validated GRP75 protein levels in iMNs at 1, 2, and 4 weeks by immunostaining. Corroborating the qPCR data, no change in BiP and GRP75 protein levels was observed in 1-week-old iMNs, whereas all C9ORF72-ALS/FTD patient lines exhibited higher expression of the ER stress marker BiP and GRP75 compared with healthy control iMNs and isogenic control lines after 2 weeks (Fig. 1d) and 4 weeks of differentiation (Supplementary Fig. 2c, online resource). As additional evidence, immunoblotting performed on 2-week-old iMNs confirmed elevated BiP and GRP75 expression in C9ORF72-ALS/FTD patient-derived iMNs (Fig. 1e). As shown in (Supplementary Fig. 1c, d, online resource), we detected no TDP-43 mislocalization or PolyGA aggregates in 2-week-old iMNs, suggesting that both ER stress and increased GRP75 levels are observed in iMNs before the appearance of these major C9ORF72-linked pathological hallmarks.

We next validated these observations using three C9ORF72-ALS/FTD patient-derived and three healthy control neuronal cell lines which were generated via direct conversion of fibroblasts to neurons (dNeus) [50], thereby preserving hallmarks of cellular aging (see Supplementary Fig. 3a, b, online resource for phenotypic description and quality of differentiation). Two-week-old C9ORF72 dNeus showed increased levels of BiP and CHOP mRNA, and concomitantly higher protein levels of GRP75 was detected by immunostaining Supplementary Fig. 3c, d, online resource). Comparable to iMNs, dNeus also exhibited higher expression of ITPR3 transcripts (Supplementary Fig. 3c, d, online resource), suggestive of a conserved ER stress response and associated changes in GRP75 expression in C9ORF72-ALS/FTD patient neurons.

Enhanced ER–mitochondria association are specific to C9ORF72 iMNs

GRP75 serves as a functional linker protein between the ER and the mitochondria. Thus, any changes in its expression pattern would influence both cellular compartments. Therefore, we examined whether mitochondria in C9ORF72 iMNs display early structural or functional alterations. To this end, we performed serial block-face scanning electron microscopy (SBF–SEM) on four C9ORF72, two healthy and one isogenic control iMNs. 3D reconstruction of images revealed that in all four C9ORF72 iMNs, other than in the healthy or isogenic controls, a large fraction of mitochondria were rounded in shape (Fig. 2a). To obtain a quantitative measure for the observed mitochondrial shape changes, we measured mitochondrial sphericity and plotted the values as a frequency distribution [61]. Applying a cutoff from 0.86 to 0.98, where values closer to 1.0 indicate a perfectly spherical object, all four C9ORF72 iMNs presented a significantly higher proportion of rounded mitochondria compared to healthy/isogenic controls (Fig. 2b). Subsequently, we assessed mitochondrial numbers, which were also on average higher in all four C9ORF72 iMNs compared to the control iMNs (Fig. 2c). Notably, the isogenic control Iso-C9ORF72(5) again had reverted the mitochondrial phenotype displayed by the mutant C9ORF72(5) line. Measuring the number of ER contacts that each mitochondrion made revealed that spherical mitochondria in mutant MNs on average exhibited a higher number of contact points (Fig. 2d). As GRP75 physically tethers IP3R–VDAC1, thereby promoting mitochondrial Ca2+ uptake, we quantified the IP3R–VDAC1 interaction via proximity ligation assay (PLA). All C9ORF72 iMNs at 2 weeks presented increased interaction between the two partners (Fig. 2e).

Fig. 2figure 2

Mitochondrial alterations are present in C9ORF72 iMNs and iPSCs. a Representative 3D segmentation of mitochondria morphology from SBF–SEM images in 2-week-old iMNs. The Isogenic line (5) displays tubular and elongated mitochondria, while the corresponding C9ORF72 line displays small and rounded mitochondria. (no. of images: Iso-C9ORF72(5): 285; C9ORF72(5): 300). b Sphericity analysis performed on SBF–SEM image stacks, plotting sphericity values as relative frequency distribution histogram. Note the higher percentage of spherical mitochondria in mutant iMNs lines C9ORF72(1), (2), (3) and (5); (Ctrl(1) bin center 0.86 = 3.125, 0.90 = 8.333, 0.94 = 1.0412, 0.98 = 0; C9(1) bin center 0.86 = 15.464, 0.90 = 11.34, 0.94 = 6.186, 0.98 = 3.093; Unpaired t test, mitochondria numbers Ctrl(1) n = 96 vs C9ORF72(1) n = 97, t = 4.833, P < 0.0001***; Ctrl(2) bin center 0.86 = 2.597, 0.90 = 1.948, 0.94 = 0.649, 0.98 = 0; C9ORF72(2) bin center 0.86 = 7.33, 0.90 = 3.665, 0.94 = 6.806, 0.98 = 2.094; C9ORF72(3) bin center 0.86 = 8.547, 0.90 = 11.111, 0.94 = 3.419, 0.98 = 3.419; Unpaired t test, mitochondria numbers Ctrl(2) n = 154 vs C9ORF72(2) n = 191, t = 4.369, P < 0.0001***; Unpaired t test, mitochondria numbers Ctrl(2) n = 154 vs C9ORF72(3) n = 117, t = 5.148, P < 0.0001***; Iso-C9ORF72(5) bin center 0.86 = 1.25, 0.90 = 3.75, 0.94 = 2.50, 0.98 = 0; C9ORF72(5) bin center 0.86 = 16.867, 0.90 = 14.458, 0.94 = 6.024, 0.98 = 2.410; Unpaired t test, mitochondria numbers Iso-C9ORF72(5) n = 80 vs C9ORF72(5) n = 83, t = 4.206, P < 0.0001***). c SBF–SEM Q.A. of mitochondria numbers in 2-week-old iMNs, showing increased number of mitochondria in all C9ORF72 patient lines compared to controls or corresponding isogenic (Unpaired t test: Ctrl(1) vs C9ORF72(1) t = 7.556, P < 0.0001***; Ctrl(2) vs C9ORF72(2) t = 6.815, P < 0.0001***; Ctrl(1) vs C9ORF72(3), t = 6.678, P < 0.0001***; iso-C9ORF72(5) vs C9ORF72(5), t = 6.750, P < 0.0001***). Combined graph for Ctrls lines (Ctrl 1–2, Iso-C9(5)) and C9 (C9 1–2–3–5), Unpaired t test Ctrls vs C9 t = 12.53, P < 0.0001, ***. d Representative images of mitochondria contacts with ER for Iso-C9ORF72(5) and C9ORF72(5). Linear regression between the no. of contacts per mitochondria and their sphericity in 2-week-old iMNs (Ctrl(1); Y = 0.4315*X + 0.5801, P = 0.2610, n.s.; C9ORF72(1); Y = 3.315*X − 1.161, P < 0.0001***; Ctrl(2); Y = 2.092*X + 0.7326, P = 0.3874, n.s.; C9ORF72(2); Y = 1.856*X − 0.313, P < 0.0001 ***; C9ORF72(4) Y = 2.736*X − 0.9271, P < 0.0001***; Iso-C9ORF72(6): Y =  − 0.5363*X + 1.129, P = 0.1530, n.s.; C9ORF72(6):Y = 2.226*X − 0.1446, P < 0.0001 ***). e Representative images depicting increased number of PLA puncta between IP3R and VDAC1 in 2-week-old C9ORF72(1) iMNs compared to Ctrl(1) or C9ORF72(5) compared to the respective isogenic control. PLA IP3R–VDAC1: Unpaired t test: Ctrl(1), n = 21 vs C9ORF72(1), n = 21, t = 11.01, P < 0.0001***; Ctrl(2), n = 19 vs C9ORF72(2), n  = 22, t = 9.979, P < 0.0001*** Ctrl(1), n = 21 vs C9ORF72(3), n = 20, t = 8.637, P < 0.0001***; Iso-C9ORF72(4), n = 19 vs C9ORF72(4), n = 21, t = 10.55, P < 0.0001***; Iso-C9ORF72(5), n = 17 vs C9ORF72(5), n = 18, t = 11.08, P < 0.0001***, n = cell numbers. Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–3–4–5), Unpaired t test Ctrls vs C9 t = 19.60, P < 0.0001, ***. f Sphericity analysis performed on SBF–SEM image stacks of iPSCs from Ctrl 1 and 2 and C9 1 and 2, plotting sphericity values as relative frequency distribution histogram. Note the higher percentage of spherical mitochondria in mutant iPSCs lines C9ORF72(1) and (2). g Linear regression between the no. of contacts per mitochondria and their sphericity in iPSCs (Ctrl(1); Y =  − 0.2617*X + 0.8871, P = 0.6003, n.s.; C9ORF72(1); Y =  − 0.4234*X + 1.024, P = 0.3064, n.s.; Ctrl(2); Y =  − 0.1084*X + 0.8194, P = 0.7869, n.s.; C9ORF72(2); Y =  − 0.1102*X + 0.6515, P = 0.8117, n.s.). h qPCR analysis for GRP75 and CHOP transcripts from Ctrls(1–2) and C9ORF72(1–2) iPSCs lines, show no changes between Ctrls and C9ORF72 iPSCs. i Mitochondria oxygen consumption rate (OCR) analysis on iPSCs reveals impairments in basal respiration and ATP production in all C9ORF72 patients’ lines compared to Ctrls or respective isogenic. Unpaired t test basal respiration: Ctrl(1) vs C9(1) t = 7.102, P = 0.0021**; Ctrl(2) vs C9(2) t = 12.45, P = 0.0002***; Iso-C9(4) vs C9(4) t = 11.41, P = 0.0003***; Iso-C9(5) vs C9(5) t = 7.677, P = 0.0015**; ATP production: Ctrl(1) vs C9(1) t = 5.473, P = 0.0054**; Ctrl(2) vs C9(2) t = 6.401, P = 0.0031**; Iso-C9(4) vs C9(4) t = 5.488, P = 0.0054**; Iso-C9(5) vs C9(5) t = 3.708, P = 0.0207*; Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4–5) and C9 (C9 1–2–4–5), Unpaired t test, basal respiration Ctrls vs C9 t = 4.438, P = 0.0002, ***; ATP production Ctrls vs C9 t = 7.741, P < 0.0001, ***. Scale bar: d and f 0.5 μm, e 10 μm

We next assessed whether the changes in mitochondrial structure were present within C9ORF72 iPSCs and might reflect mutation-related alterations. SBF–SEM was performed on two C9ORF72 and two healthy control iPSCs. Both C9ORF72 iPSCs displayed higher proportion of spherical mitochondria compared to controls (Fig. 2f). Notably, the number of ER–mitochondria contacts remained unchanged between mutant and control-iPSCs (Fig. 2g). Further, measurement of GRP75 and CHOP transcripts revealed no change in expression levels, suggestive of those expression changes being specific to iMNs (Fig. 2h). We evaluated key parameters of mitochondrial function using the Seahorse assay and found reduced basal respiration and consequently lower ATP production in C9ORF72 iPSCs (Fig. 2i). Taken together, these data indicate that intrinsic mitochondrial deficits are present in C9ORF72 cells and that specifically C9ORF72 iMNs undergo early increase in ER–mitochondria associations in parallel to the observed ER stress.

Increased ER–mitochondria association via GRP75 normalizes Ca2+ uptake and ATP generation in C9ORF72 iMNs

GRP75 creates a physical link between the ER membrane and the outer mitochondrial membrane by facilitating the interaction between ER-bound IP3Rs and mitochondrial VDAC1 to promote core mitochondrial Ca2+ uptake and optimal mitochondrial bioenergetics [62]. Dysregulation of mitochondrial Ca2+ homeostasis and mitochondrial Ca2+ overload has been linked to neuronal death in neurodegenerative disorders [10, 59, 60]. Therefore, we measured mitochondrial Ca2+ uptake in C9ORF72 and control iMNs after 1 and 2 weeks of differentiation. Fluo-4AM was combined with an intracellular buffer that eliminated cytosolic and ER Ca2+ signals [47], thereby enabling specifically the measurement of mitochondrial Ca2+ uptake. Unexpectedly, all 1-week-old C9ORF72 iMNs exhibited significant mitochondrial Ca2+ uptake deficits when compared with healthy or isogenic control iMNs, even though iMNs at 1 week do not yet manifest ER stress or GRP75 expression changes (Fig. 3a). Not only were Ca2+ transients lower within the mitochondria, but also the mitochondrial membrane potential, which is the driving force for Ca2+ uptake, was significantly reduced when measured simultaneously using ΔΨM probe tetramethylrhodamine methyl ester (TMRM) dye (Supplementary Fig. 4a, b, online resource). To follow up on this, we evaluated key parameters of mitochondrial function using the Seahorse assay and found reduced basal and maximal respiration, and consequently lower ATP production, suggesting that mitochondrial Ca2+ uptake deficits observed in 1-week-old C9ORF72 iMNs likely precede the ER stress response (Fig. 3b).

Fig. 3figure 3

Enhanced ER–mitochondria coupling via GRP75 promotes optimal mitochondrial calcium uptake and bioenergetics. a Baseline (0–60 s) and stimulated mitochondrial calcium (Ca2+) uptake (80 s onward) traces from Ctrls(1 and 2) and C9ORF72(1,2,3) iMNs and Iso-C9ORF72(4 and 5) and C9ORF72(4 and 5) iMNs at 1-week post-differentiation when there is no ER stress or GRP75 upregulation. All patient lines exhibited decreased mitochondrial Ca2+ transients compared to controls or respective isogenic controls. (No. of iMNs: Ctrl(1): 38, Ctrl(2): 22, C9(1) 47, C9(2) 20; C9(3): 22, Iso-C9(4): 40, C9(4): 38, Iso-C9(5): 19, C9(5): 20, from 3–4 independent cultures). Multiple t test at 80 s: Ctrl(1) mean = 1.97, C9(1) mean = 1.56, P = 0.0046; Ctrl(2) mean = 1.94, C9(2) mean = 1.30, P < 0.0001; Ctrl(2) mean = 1.94, C9(3) mean = 1.40, P = 0.0002; Iso-C9(4) mean = 2.18, C9(4) mean = 1.49, P < 0.0001, iso-C9(5) mean = 2.00, C9(5) mean = 1.57, P = 0.004). b Mitochondria oxygen consumption rate analysis (Seahorse) on 1-week-old iMNs reveals impairments in basal respiration, ATP production and maximal respiration in all C9ORF72 patients lines compared to control or respective isogenic lines. (Paired t test: basal respiration: Ctrl(1) vs C9(1) t = 7.756, P = 0.0045**; Ctrl(2) vs C9(2) t = 11.33, P = 0.0015**; Ctrl(2) vs C9(3) t = 7.043, P = 0.0059**; Iso-C9(4) vs C9(4) t = 4.99, P = 0.0155*; ATP production: Ctrl(1) vs C9(1) t = 26.13, P = 0.0001***; Ctrl(2) vs C9(2) t = 6.916, P = 0.0062**; Ctrl(2) vs C9(3) t = 6.924, P = 0.0062**; Iso-C9(4) vs C9(4) t = 4.432, P = 0.0213*; maximal respiration: Ctrl(1) vs C9(1) t = 30.19, P < 0.0001***; Ctrl(2) vs C9(2) t = 18.27, P = 0.0004***; Ctrl(2) vs C9(3) t = 14.10, P = 0.0008***; Iso-C9(4) vs C9(4) t = 4.544, P = 0.020*). Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4) and C9 (C9 1–2–3–4), Unpaired t test basal respiration Ctrls vs C9 t = 10.47, P < 0.0001, ***; ATP production Ctrls vs C9 t = 13.79, P < 0.0001, ***; maximal respiration Ctrls vs C9 t = 10.26, P < 0.0001, ***. c Baseline (0–60 s (s)) and stimulated mitochondrial Ca2+ uptake (80 s onward) traces from Ctrls(1 and 2) and C9(1,2,3) iMNs and Iso-C9(4 and 5) and C9(4 and 5) iMNs at 2-week post-differentiation. All the patients’ lines show comparable mitochondria Ca2+ transient to controls or respective isogenic controls. (Number of iMNs: Ctrl(1): 38, Ctrl(2): 22, C9(1) 47, C9(2) 20; C9(3): 22, Iso-C9(4): 40, C9(4): 38, Iso-C9(5): 19, C9(5): 20, from 3–4 independent cultures). Multiple t test at 120 s: Ctrl(1) mean = 1.93, C9(1) mean = 1.83, P = 0.577; Ctrl(1) mean = 1.93, C9(2) mean = 1.81, P = 0.198; Ctrl(2) mean = 1.91, C9(3) mean = 1.75, P = 0.416; Multiple t test at 100 s: Iso-C9(4) mean = 1.84, C9(4) mean = 1.63, P = 0.218, iso-C9(5) mean = 1.80, C9(5) mean = 1.86, P = 0.776). (Right) Treatment with GRP75 inhibitor MKT-077 completely abolished Ca2+ transients in C9ORF72 patient iMNs. Control and Isogenic lines also reveal reduction in Ca2+ transients after MKT-077 treatment. MKT-077 curves are plotted as dotted lines (Number of iMNs: Ctrl(1) + MKT-077: 39, Ctrl(2) + MKT-077: 13, C9(1) + MKT-077: 40, C9(2) + MKT-077: 21; C9(3) + MKT-077: 11, Iso-C9(4) + MKT-077: 39, C9(4) + MKT-077: 39, Iso-C9(5) + MKT-077: 21, C9(5) + MKT-077: 21, from 5–6 independent cultures). Multiple t test at 100 s: Ctrl(1) mean = 2.008, Ctrl(1) + MKT-077 mean = 1.26, P < 0.0001. Ctrl(2) mean = 1.89, Ctrl(2) + MKT-077 mean = 1.104, P = 0.0003; C9(1) mean = 1.64, C9(1) + MKT-077, mean = 1.14, P < 0.0001; C9(2) mean = 1.75, C9(2) + MKT-077 mean = 0.98, P < 0.0001; C9(3), mean = 1.74, C9(3) + MKT-077, mean = 1.07, P = 0.005; Iso-C9(4), mean = 1.84, Iso-C9(4) + MKT-077, mean = 1.30, P = 0.0002; C9(4), mean = 1.63, C9(4) + MKT-077, mean = 0.999, P < 0.0001; Iso-C9(6), mean = 1.80, Iso-C9(6) + MKT-077 mean = 0.93, P < 0.0001; C9(6), mean = 1.86, C9(6) + MKT-077,mean = 0.83, P < 0.0001). d Mitochondria oxygen consumption rate analysis (OCR) via Seahorse assay in 2-week-old iMNs. When GRP75 is upregulated patient lines show improvement in basal respiration, ATP production and maximal respiration compared to the 1-week condition. (Paired t test basal respiration: Ctrl(1) vs C9(1) t = 2.064, P = 1309 n.s.; Ctrl(2) vs C9(2) t = 5.624, P = 0.0111*; Ctrl(2) vs C9(3) t = 4.417, P = 0.0215*; Iso-C9(4) vs C9(4) t = 2.741, P = 0.0713 n.s.; ATP production: Ctrl(1) vs C9(1) t = 2.138, P = 0.122 n.s.; Ctrl(2) vs C9(2) t = 4.371, P = 0.0222*; Ctrl(2) vs C9(3) t = 4.183, P = 0.0249*; Iso-C9(4) vs C9(4) t = 0.9993, P = 0.3913 n.s.; maximal respiration: Ctrl(1) vs C9(1) t = 14.82, P = 0.0007***; Ctrl(2) vs C9(2) t = 3.547, P = 0.0382*; Ctrl(2) vs C9(3) t = 8.631, P = 0.0033**; Iso-C9(4) vs C9(4) t = 2.982, P = 0.0585 n.s.). Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4) and C9 (C9 1–2–3–4), Unpaired t test basal respiration Ctrls vs C9 t = 6.807, P < 0.0001***; ATP production Ctrls vs C9 t = 3.865, P = 0.0007***; maximal respiration Ctrls vs C9 t = 6.613, P < 0.0001***. e qPCR analysis of CHOP and GRP75 transcripts after 48 h of 15 μM Salubrinal treatment. Salubrinal treatment suppress ER stress and reduce GRP75 transcript levels to controls in C9ORF72 patients lines but no change was detected in control or isogenic control lines. Unpaired t test CHOP: Ctrl(1) vs Ctrl(1) + Sal t = 0.379, P = 0.7177 n.s.; Ctrl(2) vs Ctrl(2) + Sal t = 1.289, P = 0.2448 n.s.; C9(1) vs C9(1)  + Sal t = 4.975, P = 0.0025**; C9(2) vs C9(2) + Sal t = 6.161, P = 0.0008***; C9(3) vs C9(3) + Sal t = 6.702, P = 0.0005***; Iso-C9(4) vs Iso-C9(4) + Sal t = 1.004, P = 0.3542 n.s.; C9(4) vs C9(4) + Sal t = 3.876, P = 0.0082**; Combined graph for control lines (Ctrl 1–2, Iso-C9 4) and C9 (C9 1–2–3–4): unpaired t test CHOP: Ctrls vs C9 t = 8.571, P < 0.0001***; Ctrls vs Ctrls + Sal t = 1.507, P = 0.1459, n.s.; C9 vs C9 + Sal t = 10.10, P < 0.0001, ***. Unpaired t test GRP75: Ctrl(1) vs Ctrl(1) + Sal t = 0.7898, P = 0.4738 n.s.; Ctrl(2) vs Ctrl(2) + Sal t = 2.356, P = 0.078 n.s.; C9(1) vs C9(1) + Sal t  = 5.251, P = 0.0063**; C9(2) vs C9(2) + Sal t = 14.76, P = 0.0001***; C9(3) vs C9(3) + Sal t = 5.881, P = 0.00042***; Iso-C9(4) vs Iso-C9(4) + Sal t = 1.289, P = 0.2670 n.s.; C9(4) vs C9(4) + Sal t = 4.754, P = 0.0089***). Combined graph for Ctrl lines (Ctrl 1–2, Iso-C9 4) and C9(C9 1–2–3–4): unpaired t test GRP75: Ctrls vs C9 t = 7.922, P < 0.0001***; Ctrls vs Ctrls + Sal t = 0.111, P = 0.9130, n.s.; C9 vs C9 + Sal t = 9.617, P < 0.0001, ***. qPCR graphs plotted with s.d., n = 3–4 independent qPCR experiments repeated in triplicate. f Baseline (0–60 s) and stimulated mitochondrial Ca2+ uptake (80 s onward), traces from Ctrl(2) and C9ORF72(1,2,3) iMNs and Iso-C9ORF72(4 and 5) and C9ORF72(4 and 5) iMNs after Salubrinal treatment are shown. All patient lines show reduced mitochondria Ca2+ transients compared to controls or respective isogenic controls. (Number of iMNs: Ctrl(2): 10, Ctrl(2) + Sal: 13, C9(1): 10, C9(1) + Sal: 12, C9(2): 10, C9(2) + Sal: 13, Iso-C9(4): 10, Iso-C9(4) + Sal: 11, C9(4): 10, C9(4) + Sal: 12, Iso-C9(5): 10, Iso-C9(5) + Sal: 11, C9(5): 11, C9(5) + Sal: 12). Multiple t test at 80 s: Ctrl(2) mean: 2.00, Ctrl(2) + Sal mean 2.08, P = 0.55; C9(1) mean: 1.79, C9(1) + Sal mean:1.26, P < 0.0001, C9(2) mean: 1.87, C9(2) + Sal mean:1.33, P < 0.0001; Iso-C9(4) mean: 1.96, Iso-C9(4) + Sal mean: 1.85, P = 0.354, C9(4) mean: 1.87, C9(4) + Sal mean: 1.35, P < 0.0001, Iso-C9(5) mean: 1.94, Iso-C9(5) + Sal mean: 1.98, P = 0.632; C9(5) mean: 1.81, C9(5) + Sal mean: 1.30, P < 0.0001. Scale bar: e 10 μm

We next measured mitochondrial Ca2+ uptake in 2-week-old C9ORF72 iMNs, which exhibit ER stress and increased GRP75 expression. Upon Ca2+ release from the ER, the maximum mitochondrial Ca2+ uptake and mitochondrial membrane potentials were not significantly altered in these neurons compared with their healthy/isogenic controls (Fig. 3c and Supplementary Fig. 4c, online resource). The mitochondrial Ca2+ transients thus detected are a cumulative readout of the release of Ca2+ from ER stores not only by IP3R, but also via ryanodine receptors and sphingolipid Ca2+ release-mediating proteins of the ER. Therefore, to reveal the contribution of increased GRP75 to mitochondrial Ca2+ uptake, iMNs were treated with MKT-077, an established pharmacological inhibitor of GRP75 [26], at different concentrations to establish an optimum dosage curve for the inhibition of mitochondrial Ca2+ uptake specifically in iMNs (Supplementary Fig. 4d, online resource). 5 µM MKT-077 treatment led to reduced mitochondrial Ca2+ transients within both healthy and isogenic controls. However, in all C9ORF72 iMNs mitochondrial Ca2+ transients were significantly impaired (Fig. 3c), suggesting that elevated GRP75 levels likely promoted optimal ER–mitochondrial association, thereby reversing mitochondrial Ca2+ uptake deficits in C9ORF72 iMNs. Notably, the ameliorated mitochondrial Ca2+ uptake within 2-week-old iMNs was also reflected by almost normalized mitochondrial respiration and ATP production in C9ORF72 iMNs (Fig. 3d and Supplementary Fig. 4e, online resource).

As further evidence for the involvement of ER stress-mediated GRP75 increase in normalizing mitochondrial Ca2+ uptake, we treated 2-week-old iMNs harboring higher GRP75 levels with 15 μM ER stress inhibitor salubrinal (Sal) for 48 h. This abrogated ER stress and normalized GRP75 expression in C9ORF72 iMNs (Fig. 3e). Sal treatment led to reduced mitochondrial Ca2+ transients within C9ORF72 iMNs, but had no effect on control/isogenic control iMNs (Fig. 3f). Our data suggest that C9ORF72 iMNs exhibit early mitochondrial impairments, which are neutralized by ER stress-mediated elevated GRP75 expression, suggestive of an early adaptive response crucial for sustaining mitochondrial function.

Reduced GRP75 levels in C9ORF72-ALS/FTD post-mortem tissue and C9-500 rodent neurons

We next examined lumbar spinal cord specimens of four C9ORF72-ALS/FTD and four control cases by immunofluorescence and DAB immunohistochemistry. We found homogeneous, strong cytoplasmic GRP75 immunoreactivity of numerous large and small anterior horn neurons in the control cases. In contrast, a large fraction of the remaining neurons in C9ORF72-ALS/FTD cases showed a considerable reduction in average GRP75 immunoreactivity (Fig. 4a and Supplementary Fig. 5a, online resource). Many α-MNs in the lumbar spinal cord of the C9ORF72-ALS/FTD patients contained characteristic pTDP-43 aggregates of varying morphology (dash- or dot-like/granular, skein-like, dense/globular, Supplementary Fig. 5b, online resource), probably depending upon their stage of maturation [52]. We consistently observed that α-MNs harboring large, compact globular or skein-like pTDP-43 aggregates showed reduced cytoplasmic GRP75 immunoreactivity in comparison to the adjacent α-MNs with high levels of GRP75 that were either completely devoid of pTDP-43 aggregates or harbored only minor amounts of small, dispersed, granular pTDP-43 microaggregates. Of note, we also observed rare α-MNs, in which intense GRP75 immunoreactivity coincided with larger amounts of mostly granular pTDP-43 (Fig. 4b, and Supplementary Fig. 5c, online resource, bottom image).

Fig. 4figure 4

Age-dependent reduction in GRP75 expression in human and rodent C9ORF72 CNS tissue. a Representative images showing differential GRP75 immunoreactivity in human lumbar spinal cord anterior horns of C9ORF72 ALS patients compared to the normal controls. The variable immunofluorescence intensity was further scored as low, normal, medium and high as depicted. Images are shown from two C9ORF72 patients and rendered for expression intensity. Right: Q.A. of GRP75 average fluorescence intensity: unpaired t test Control vs C9ORF72, t = 11.61, P < 0.0001***; Q.A. of GRP75 immunoreactivity: normal (C9ORF72 18%), moderate reduction (C9ORF72 40%), strong reduction (C9ORF72 42%), Chi-square analysis P < 0.0001***. (Number of MNs analyzed, Control: 29; C9ORF72: 77), n = 4 C9ORF72-fALS patients and n = 4 age-matched controls. Note that in Control α-MNs, GRP75 immunoreactivity was uniform, whereas more α-MNs from C9ORF72-ALS/FTD cases were examined in detail to account for the high degree of variability in GRP75 staining. b Representative double immunofluorescence labeling of human C9ORF72-ALS/FTD α-MNs within the lumbar spinal cord using antibodies against GRP75 and pTDP-43. Arrow: pTDP-43-positive inclusion. c Representative double immunofluorescence labeling of C9ORF72-ALS/FTD patient hippocampal dentate gyrus neurons compared to normal control. Note the overall reduced labeling of GRP75 and its focal sequestration with PolyGA aggregates in C9ORF72-ALS/FTD patient dentate gyrus hippocampal neurons, where 71.50% ± 3.6 of the neurons showed GRP75 sequestration within PolyGA aggregates. Sixty PolyGA aggregate bearing neurons were analyzed. d Representative confocal images of spinal MNs stained for GRP75 from WT and C9-500 animals at different ages. 3D rendering of MNs based on the intensity level of GRP75 staining show increased levels of GRP75 expression at P80 and decreased levels of GRP75 expression in P200 C9-500 animals compared to WT. e Longitudinal Q.A. of GRP75 expression in WT and C9-500 animals showing MNs with high GRP75 levels at P80 and P120 (41–48%) with subsequent drop in expression after P150, where GRP75 levels are below WT expression levels. f Representative confocal immunofluorescence imaging of GRP75 expression in WT and C9-500 cortex showing increased GRP75 levels at P150 and below WT levels of expression at P240. g Representative WB of brain extracts from WT and C9-500 animals at P240. MAM isolation using sucrose gradient revealed decreased GRP75 levels in C9-500 animals compared to WT, while no difference was detected for the loading controls MFN2. Quantitative analysis of the intensity of GRP75 at the MAM compared to WT (Unpaired t test: t = 6.263, P = 0.0033**). N = 3 mice, repeated thrice as separate experiments. Scale bars: a 15 µm, b 60 µm, c 8 µm, d 30 and 10 µm (zoom), f 30 µm

Similarly, an overall reduction in cytoplasmic GRP75 immunoreactivity of hippocampal dentate gyrus neurons in the same C9ORF72-ALS/FTD patients as studied above was observed (Supplementary Fig. 5d, online resource). Notably, this was prominent in nearly all pTDP-43 aggregate-bearing dentate gyrus neurons (Supplementary Fig. 5d, online resource, red arrows). In line with this observation, PolyGA aggregate-bearing dentate gyrus neurons generally showed reduced GRP75 immunoreactivity in comparison to the adjacent neurons devoid of PolyGA aggregates (Fig. 4c). On the other hand, in those neurons which harbored PolyGA aggregates, the remaining GRP75 immunoreactivity often showed a tendency to preferentially co-localize with PolyGA accumulations (Fig. 4c).

Reprogramming resets the epigenetic age of induced pluripotent stem cells (iPSCs), [49]; therefore, we assume that iPSC-derived neurons used in this study most closely resemble those in “young” mutation carriers. This renders them suitable tools for identifying the molecular mechanism by which “young” MNs might be still able to protect themselves against abnormal ALS proteins. Thus, for the assessment of GRP75 expression relevant to the chronic course and later stages of the disease, we examined GRP75 levels in the C9-500 BAC mouse model. These mice display RNA foci, pTDP43

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