The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity

Results

FGF21 is increased in human ALS muscle and spinal cord tissue

We assessed FGF21 mRNA expression in a large cohort of muscle biopsy samples of ALS patients from our clinic (Supplementary Table 2 and Figure 1). Demographics for this cohort, including age (mean 57 ± 13 y) and slight male to female predominance (1.4:1), were in line with prior epidemiological studies [1, 30]. The cohort of normal biopsies showed similar age with a slight female to male predominance (1.2:1). By qPCR, we found an ~8-fold increase in FGF21 mRNA in ALS samples (P = 0.004) compared to normal control biopsy samples (Figure 1A). There was variability in mRNA levels with several at greater than 50-fold higher than controls. We next tested post-mortem ALS muscle samples which represent more advanced (end-stage) disease as reflected by the longer disease duration (51 months versus 15 months in the biopsy group). FGF21 mRNA levels were much higher at 63-fold greater than normal control samples, indicating that FGF21 expression in muscle increases with disease progression (Figure 1B). We compared FGF21 mRNA levels with HDAC4, another known marker of muscle denervation in ALS [31], and found suggestive evidence of a correlation (Spearman rank correlation R = 0.683, P = 0.050, Supplementary Figure 1). We next assessed FGF21 protein expression by ELISA with post-mortem samples and found a ~7-fold increase in ALS samples (2463 versus 335 pg/mg of muscle tissue; P = 0.030). As with mRNA levels, there was variability among ALS samples (versus controls) with one ALS patient showing nearly 15,000 pg/mg. We next measured FGF21 mRNA levels in spinal cord tissue but observed no significant difference compared to control tissue as a whole; however, there were 4 outliers with markedly increased levels of up to 25-fold (Figure 1C). On the other hand, FGF21 protein was increased in ALS spinal cord (132 versus 29 pg/mg of spinal cord tissue; P = 0.005), but levels in spinal cord were nearly 19-fold less than ALS muscle. There was variability among the ALS patients as observed with muscle. Post-mortem spinal cord and muscle samples were matched for 18 of the ALS patients tested and they showed a positive correlation for FGF21 levels (Spearman rank correlation R = 0.49, P = 0.039) (Figure 1D). We next sought to determine whether changes in FGF21 occurred in the SOD1^G93A mouse model of ALS. This model can provide insight into temporal patterns of biomarkers identified in human ALS muscle including pre-clinical (prodromal) phases of the disease [4]. We detected a 4-fold increase in FGF21 mRNA compared to age-matched wild-type (WT) controls at 40 d post-natal (Figure 1E). Although NMJ innervation is reduced by 40% by this age, this stage is typically considered pre-symptomatic where standard testing such as rotarod and grip strength are unaffected and only subtle signs of muscle weakness can be detected by more in-depth testing [32]. In later stages, muscle FGF21 rose from ~37-fold at 60 d (pre-symptomatic) to 43-fold at 125 d (symptomatic stage). At end-stage (150 d), there was a 28-fold fold-change. FGF21 mRNA increased in spinal cord tissue in parallel with muscle FGF21. Taken together, FGF21 mRNA and protein levels were significant but variably increased in human ALS muscle and spinal cord tissues, with a disproportionate increase in muscle. Furthermore, the strong association between muscle and spinal cord FGF21 for each patient and in the SOD1^G93A mouse model suggested an underlying connectivity.

Figure 1. FGF21 levels are elevated in the muscle and spinal cord tissues of ALS patients and the SOD1^G93A mouse. (A) FGF21 mRNA expression was analysed in normal (n = 24) and ALS (n = 36) muscle biopsy samples via RT-qPCR. **P = 0.004, unpaired two-tailed t-test with Welch’s correction. (B) FGF21 mRNA levels were quantified in normal (n = 7) and ALS (n = 11) post-mortem muscle samples (left panel) and FGF21 protein levels (n = 13 for normal samples; n = 18 for ALS samples; right panel). **P = 0.003, ****P < 0.0001, two-tailed Mann Whitney test. (C) FGF21 mRNA levels were quantified in normal (n = 14) and ALS (n = 22) post-mortem spinal cord samples (left panel) and FGF21 protein levels (n = 12 for normal samples; n = 18 for ALS samples; right panel). **P = 0.00, two-tailed Mann Whitney test. (D) Comparison of spinal cord and muscle FGF21 protein levels for 18 ALS patients. A spearman correlation test was used for analysis. (E) FGF21 mRNA levels in the gastrocnemius muscle (left panel) and spinal cord (right panel) were quantified across different age groups (20 – 150 days; n = 4-5 per group) from SOD1^G93A mice and littermate controls. **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t-test comparing WT to SOD1^G93A. For all graphs, error bars represent SD.

FGF21 localizes to atrophic muscle fibers

Next, we determined localization of FGF21 in ALS muscle by immunohistochemistry. We observed intense intra-myofiber staining in areas of grouped atrophy (characterized by clusters of small angular fibers) which is a hallmark of denervation (Figure 2A and Supplementary Figure 7) [33]. FGF21 staining also localized to the endomysial connective tissue. On the other hand, non-atrophic fibers within the same section showed little or no staining. Five normal control muscle samples also showed no FGF21 staining (example shown in Figure 2A and Supplementary Figure 7). To confirm an association between FGF21 immunoreactivity and atrophic myofibers, we measured mean fluorescence intensity (MFI) in atrophic and non-atrophic fibers in muscle sections from five ALS patients (Figure 2B and Supplementary Figure 7). Regions of interest (ROI) for atrophic and non-atrophic myofibers were selected within the same muscle section, often in proximity, to minimize the effects of variable tissue staining (see example in Figure 2B and Supplementary Figure 7). We observed a 9-fold increase in FGF21 MFI in ROIs associated with atrophic versus non-atrophic myofibers (P < 0.0001). Taken together, these data indicate a marked increase in FGF21 expression in ALS muscle tissues, predominantly in atrophic myofibers and surrounding endomysial connective tissue.

Figure 2. FGF21 localizes to atrophic myofibers in ALS muscle. (A) Tissue sections from two ALS patients and one normal control were immunostained with an anti-FGF21 antibody and counterstained with DAPI and wheat germ agglutin (WGA). Intense immunoreactivity is observed in atrophic myofibers (asterisks) and in the endomysial space (arrows) in the ALS muscle sections. Scale bars, 100 μm in low power views and 50 μm in the enlarged views. (B) Mean fluorescence Intensity (MFI) analysis of FGF21 immunoreactivity was performed in 5 ALS patient biopsy samples and 5 normal controls. Atrophic (< 25 μm minimal Feret’s diameter) and non-atrophic myofibers were selected in the same section as shown in the micrograph (yellow outline). FGF21 MFI (per μm²) was quantitated for 46 atrophic and non-atrophic myofibers and summarized in the graph (horizontal line represents the mean). ****P < 0.0001; two-tailed Mann Whitney test. Scale bar: 50 μm.

Plasma FGF21 levels are elevated in ALS patients and correlate with enhanced survival

Since FGF21 is a secreted factor and we observed extra-myofiber immunostaining in ALS muscle tissue, we queried whether FGF21 could be detected in plasma of ALS patients. To assess this possibility, we assayed plasma samples collected from a cohort of ALS patients in our clinics who participated in a prior biomarker study (Supplementary Table 3) [9]. A set of age-matched normal controls was used for comparison. Overall, we found a significant increase in FGF21 levels in the ALS cohort (923 v. 649 pg/ml; P = 0.03), but with variability (Figure 3A). Interestingly, the FGF21 levels were more than 2-fold higher in ALS muscle versus plasma FGF21 (albeit end-stage ALS muscle; Figure 1B). None of the patients or controls had type II diabetes or known liver disease, two conditions associated with higher circulating levels of FGF21 [34]. We next examined a subset of 16 ALS patients who were followed prospectively with serial examinations and plasma sample collection. Based on calculated monthly changes in ALSFRS-R scores (∆FRS), there were 6 slow, 5 average and 5 fast progressors using previously established criteria (Figure 3B) [35]. Using the average of baseline and 3-month FGF21 plasma levels, we found that the slow progressors had more than a 4-fold increase in FGF21 compared to fast progressors (2247 v. 529 pg/ml; P = 0.003) and a ~3.5-fold increase compared to the control group (P = 0.0003; Figure 3C). The group of average progressors was intermediate between the slow and fast groups (1410 pg/ml) but did not reach statistical significance. Independent of clinical classification, there was a strong negative correlation between the ∆FRS for each subject and their respective plasma FGF21 level (R = -0.710, P = 0.003; Figure 3D). We then assessed survival of these patients based on FGF21 levels, comparing those with <1.5 fold-change (FC) over controls versus ≥ 1.5 FC (Figure 3E). The median survival for the group with low FGF21 levels was 18 months versus 75 months for the group with high FGF21 levels (P = 0.01). Three patients in the high FGF21 group are still alive at 82, 93, and 94 months after disease onset. We next looked at body mass index (BMI) and found that the <1.5 FC group had a significantly lower BMI than the >1.5 FC group (22.6 ± 3.6 v. 29.0 ± 6.6 kg/m², P = 0.04; Figure 3F). Age and duration of disease were not significantly different between the two groups (Supplementary Table 4). Interestingly, in the lower FGF21 group, 4 out of 7 patients had bulbar onset of ALS versus the high FGF21 group which all had spinal onset. Since liver is a major source of FGF21 [36], we assessed expression in the SOD1^G93A mouse at a time point of peak FGF21 expression in skeletal muscle (Supplementary Figure 2). Similar to results in Figure 1, we observed a ~150-fold increase in FGF21 mRNA in the SOD1^G93A mouse compared to WT control muscle (set at 1). In liver, the increase was significantly less at 75-fold in the SOD1^G93A mouse (P < 0.0001) although the mRNA level in WT liver was ~35-fold greater than WT muscle. Liver tissue was not available in our post-mortem repository to confirm whether this discrepancy applies more broadly to ALS patients. Taken together, ALS patients had an overall increase in plasma FGF21 and high levels correlated with slower disease progression and longer survival. While FGF21 mRNA is increased in muscle and liver tissues in the SOD1^G93A mouse, the disproportionately higher increase in muscle suggests that this tissue is a major contributor to circulating FGF21.

Figure 3. Plasma FGF21 is increased in ALS patients and high levels associated with slower disease progression and prolonged survival. (A) Plasma samples from age-matched normal controls (n = 23) and ALS patients (n = 28) and assayed by ELISA for FGF21. *P = 0.043, unpaired two-tailed t-test. (B) 16 ALS patients from a prior biomarker study were divided into slow (n = 6), average (n = 5), and fast (n = 5) progressing groups based on the average in the study monthly decline in ALSFRS-R scores. (C) Plasma FGF21 levels were measured at baseline and 3 months and averaged. The normal control values were added for comparison. **P = 0.003, ***P = 0.0003; one-way ANOVA followed by Tukey post hoc test. (D) Correlation between plasma FGF21 levels with monthly change in the ALSFRS-R scores (∆FRS) for each subject. Spearman correlation test. (E) Kaplan–Meier survival curves for study patients whose FGF21 plasma levels were < 1.5-fold-change (FC) over the normal control group versus study patients with ≥ 1.5-FC in FGF21 levels. *P = 0.015; Log-rank (Mantel-Cox) test. (F) Comparison of body mass index (BMI) between the < 1.5 FC and ≥ 1.5-FC groups. *P = 0.037; unpaired two-tailed t-test. For all graphs, error bars represent SD.

FGF21 co-receptor, β-Klotho, is altered in ALS muscle and spinal cord tissue

Given the critical role of β-Klotho (KLB) co-receptor in FGF21 signaling [36], we assessed KLB mRNA expression in muscle biopsy samples and found a ~ 4-fold increase in KLB levels in the ALS group versus controls (Figure 4A; P = 0.005). On the other hand, post-mortem ALS muscle samples had a 50% reduction in KLB mRNA levels (P = 0.03) compared to normal controls (Figure 4B). Likewise, ALS spinal cord tissue showed a significant reduction in KLB levels (P = 0.0006) compared to controls (Figure 4C). Some variability was observed with three samples having increased mRNA. Spinal cord tissue from end-stage SOD1^G93A mice paralleled these findings and showed a two-fold reduction of KLB mRNA compared to littermate controls (P = 0.008; Figure 4D). Collectively, these findings suggest a dysregulation of KLB expression in ALS muscle and spinal cord tissues that changed with disease stage and may relate to a progressive loss of motor neurons.

Figure 4. FGF21 coreceptor, β-Klotho (KLB), is dysregulated in ALS muscle and spinal cord. (A) KLB mRNA levels were measured in muscle biopsy samples from normal (n = 13) and ALS patients (n = 23). **P = 0.005, two-tailed Mann Whitney test. (B) KLB mRNA levels were measured in post-mortem muscle samples from normal controls (n = 6) and ALS (n = 11) patients. *P = 0.034, two-tailed Mann Whitney test. (C) KLB mRNA levels were measured in spinal cord samples from normal controls (n = 12) and ALS patients (n = 20). ***P = 0.0006, two-tailed Mann Whitney test. (D) KLB mRNA levels were measured in spinal cord samples from SOD1^G93A mice (n = 5) and WT controls (n = 5) at different ages. **P = 0.003; unpaired two-tailed t-test comparing WT to SOD1^G93A.

FGF21-KLB axis is dysregulated in ALS motor neurons

As we found progressive loss of KLB expression in ALS spinal cord and muscle, we assessed the FGF21-KLB axis in motor neurons. Using iPSC-derived motor neurons from ALS patients (Supplementary Table 1), we assessed the expression of FGF21 and KLB mRNAs. Compared to normal control iPSC motor neurons, there was a ~50% reduction in FGF21 mRNA levels in ALS motor neuron samples (Figure 5A; P = 0.012). On the other hand, KLB mRNA was increased by 3-fold (Figure 5B; P = 0.018). We next transfected NSC-34 motor neuron-like cells with Flag-tagged wild-type (WT) SOD1 or SOD1^G93A expression cassettes (Figure 5C) and found a similar pattern of expression with a more than 2-fold increase in KLB (Figure 5D) and a ~15% reduction in FGF21 in SOD1^G93A-expressing cells versus WT (Figure 5E). FGF21 protein measured in the CM was unchanged (Figure 5F). As FGF21-KLB signaling is a major cellular stress response [16], we assessed the effect of oxidative stress on this pathway in NSC-34 cells. When cells were exposed to oxidative stress either by MetCys deprivation which leads to depletion of the intracellular antioxidant, glutathione [37, 38], or directly with H₂O₂ [39], there was a marked induction of FGF21 and KLB mRNAs (Figure 5G, 5H). Induction of these genes was particularly pronounced with MetCys deprivation where KLB and FGF21 mRNAs increased by nearly 100-fold and 25-fold respectively. The induction by H₂O₂ was less at two to three-fold. This response was mirrored by induction of stress-response genes, ATF4 and PGC-1α, with MetCys deprivation showing the highest induction (Supplementary Figure 3A, 3B). ATF4 was not induced by H₂O₂ in NSC-34 cells.

Figure 5. FGF21-KLB signaling is dysregulated in ALS motor neurons. (A) FGF21 were measured in iPSC-derived motor neurons obtained from healthy controls and ALS patients carrying either C9orf72 or SOD1 mutations (Supplementary Table 1). *P = 0.012; two-tailed Mann Whitney test. (B) KLB mRNA levels were similarly measured in iPSC motor neurons. *P = 0.018; two-tailed Mann Whitney test. (C) NSC-34 motor neuron-like cells were transfected with FLAG-tagged WT and SOD1^G93A expression plasmids and lysates were assessed by western blot using the antibodies indicated. Bands were quantitated by densitometry and a ratio to the loading control, vinculin, was calculated (shown between the two blots). (D) KLB and (E) FGF21 mRNA levels were measured in the same lysates. *P = 0.018, ***P = 0.0002, unpaired two-tailed t-test. (F) FGF21 protein was measured in the CM of transfected NSC-34 cells. (G) FGF21 or (H) KLB mRNA levels were quantified from NSC-34 cells exposed to methionine-cystine (MetCys)-deprived media or treated with 100 mM H₂O₂ for 24 h. *P = 0.048, ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. Data points represent biological replicates and bars are the mean ± SD.

FGF21 mitigates cytotoxic stress in G93A-SOD1-expressing NSC-34 cells and iPSC-derived ALS motor neurons

We first determined the impact of SOD1^G93A on cell viability and found a 33% reduction in viability (Figure 6A) and a concomitant 50% increase in Caspase-3/7 activity consistent with apoptotic activity (Figure 6B). Next, we ectopically expressed FGF21 in NSC-34 cells co-expressing wild-type (WT) SOD1 or SOD1^G93A to determine the effect on cell viability. We assayed the CM by ELISA and western blot and found a large increase in FGF21 in transfected cells at ~35 ng/ml (Figure 6C). Cell viability increased back to control levels along with reduced Caspase-3/7 activity (Figure 6D, 6E). In a separate experiment, we added recombinant FGF21 to the culture media of SOD1^G93A-expressing NSC-34 cells and observed a similar reversal of cytotoxicity (Figure 6F). To assess further a potential paracrine effect of FGF21, NSC-34 cells expressing either (WT) SOD1 or SOD1^G93A and C2C12 cells expressing FGF21-Flag or EV were co-cultured in a transwell plate with a 3 μm porous filter (Supplementary Figure 4A). Viability was checked and found to be reduced in NSC-34 cells expressing SOD1^G93A co-cultured with C2C12-EV cells (P < 0.0001, Supplementary Figure 4B). When co-cultured with C2C12-FGF21 cells, loss of viability was blunted (P < 0.0001). We next exposed NSC-34 cells to MetCys deprivation or H₂O₂ and observed a significant loss of viability (P < 0.0001, Figure 6G) which was reversed with ectopic FGF21 (Figure 6H). We next treated human iPSC-derived motor neurons with different doses of H₂O₂ and found a difference in viability between control and ALS neurons, beginning at a dose of 6 μM and becoming significant at 12.5 μM (Figure 6I). We observed a concomitant and robust increase in KLB mRNA levels at 12.5 μM in ALS motor neurons compared to control (Supplementary Figure 5). Recombinant FGF21 treatment reversed this cytotoxicity in a dose-dependent manner (Figure 6J). In summary, SOD1^G93A or oxidative stress induced FGF21 and KLB mRNA and triggered cytotoxicity in motor neurons which could be rescued by ectopic FGF21.

Figure 6. FGF21 mitigates cytotoxicity in NSC-34 and ALS motor neurons induced oxidative stress. (A) The viability of NSC-34 cells expressing FLAG-tagged WT-SOD1 or SOD1^G93A was determined using the Vialight assay. Viability for WT SOD1-transfected cells was set at 1. ****P < 0.0001, unpaired two-tailed t-test. (B) Caspase activation was measured in the same cells and values were normalized to activity in WT SOD1-transfected cells which was set at 1. ****P < 0.0001, unpaired two-tailed t-test. (C) FGF21 protein in the conditioned media of NSC-34 cells transfected with a FLAG-tagged FGF21 plasmid was detected by western blot (upper panel) and by ELISA (graph). ****P < 0.0001; unpaired two-tailed t-test. (D, E) NSC-34 cells expressing either WT-SOD1 or SOD1^G93A were transfected with FLAG-FGF21 and assessed for viability as in (A) and Caspase-3/7 as in (B). ****P < 0.0001, unpaired two-tailed t-test. (F) NSC-34 cells expressing either WT SOD1 or SOD1^G93A were treated with recombinant FGF21 (100 ng/ml) and assessed for viability as in (A). **P < 0.01, unpaired two-tailed t-test. (G) Cell viability was assessed in NSC-34 cells exposed to methionine-cystine (MetCys)-deprived media or treated with 100 mM H₂O₂. ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (H) NSC-34 cells transfected with FLAG-FGF21 (or empty vector) were subjected to stressors as described in (G) for 24h and then assayed for viability. **P = 0.007, ***P = 0.0002; unpaired two-tailed t-test. (I) iPSC motor neurons derived from C9-ALS patients or normal donors were exposed to different doses of H₂O₂ as indicated, and cell viability was assessed 24 h after treatment. **P < 0.01; unpaired two-tailed t-test with Welch’s correction. (J) iPSC motor neurons exposed to 12.5 μM of H₂O_2, were treated with different doses (0, 25 and 50 ng/ml) of recombinant FGF21 and cell viability was assessed as in (I). *P < 0.05, **P < 0.01; paired two-tailed t-test among the different doses of rec-FGF21 treatment in C9-ALS groups and unpaired two-tailed test in normal vs. C9-ALS groups. Data points represent biological replicates and bars are the mean ± SD.

FGF21 mitigates stress-induced toxicity in muscle cells and promotes myogenesis

Since FGF21 is known to exert autocrine effects [19], and we found that atrophic myofibers in ALS muscle express FGF21 (Figure 2), we next sought to assess the impact of FGF21 on muscle cells. Prior investigations have found that selective transgenic expression of SOD1^G93A in muscle induces oxidative stress leading to myofiber atrophy [40]. We transfected C2C12 cells with SOD1^G93A and found that FGF21 expression was significantly reduced compared to WT SOD1, with the CM showing a ~40% reduction in protein (Figure 7A, 7B). KLB mRNA was not detected, suggesting a potential β-klotho-independent function of FGF21. The pattern of FGF21 suppression resembled ALS motor neurons (Figure 5). With SOD1^G93A expression, there was a resultant loss of cell viability and an increase in Caspase-3/7 activity, each by 25%, compared to control (Figure 7C; P < 0.0001). We transfected the muscle cells with Flag-tagged FGF21 which resulted in a marked increase in protein expression detected in the CM (Figure 7D) and an attenuated loss of cell viability and apoptotic activity (Figure 7E, 7F). We next assessed the effect of oxidative stress and found an induction of FGF21 with MetCys deprivation at 3.8-fold and H₂O₂ at 2.3-fold (Figure 7G; P < 0.0001). ATF4 and PGC-1α were also induced with these stressors by 1.5 to 2.5-fold (Supplementary Figure 3C, 3D). Cell viability with MetCys deprivation led to a 75% reduction in viability and exposure to H₂O₂ resulted in a 50% reduction (Figure 7H). Ectopic FGF21 expression improved viability by 100% for MetCys deprivation and 73% for H₂O₂ (Figure 7I; P < 0.0001). In human primary myoblast cells there was a similar induction of FGF21 at 4.1-fold when treated with H₂O₂ (Figure 7J, left panel; P < 0.01). The resultant loss of viability (~25%) was reversed by recombinant FGF21 treatment back to baseline levels (Figure 7J, right panel; P < 0.05). Taken together, oxidative stress induces FGF21 in C2C12 and human primary muscle cells, and the resultant cytotoxicity can be reversed by FGF21.

Figure 7. FGF21 mitigates cytotoxicity in C2C12 and human primary myoblasts induced by oxidative stress. (A) C2C12 myoblasts were transfected with FLAG-tagged WT and SOD1^G93A expression plasmids and lysates were assessed by western blot using the antibodies indicated. Bands were quantitated by densitometry and a ratio to the loading control, vinculin, was calculated (shown between the two blots). (B) FGF21 mRNA levels were measured in the same lysates (left panel) and FGF21 protein in the conditioned media was quantified by ELISA (right panel). *P = 0.011, **P = 0.008; unpaired two-tailed t-test. Secretory FGF21 from the conditioned media was quantified using ELISA (right panel). (C) Viability of NSC-34 cells expressing FLAG-tagged WT-SOD1 or SOD1^G93A was determined using the Vialight assay (left panel). Caspase activation was measured in the same cells and values were normalized to activity in WT SOD1-transfected cells which was set at 1 (right panel). ****P < 0.0001, unpaired two-tailed t-test. (D) FGF21 protein in the conditioned media of C2C12 cells transfected with a FLAG-tagged FGF21 was detected by western blot (upper panel) and by ELISA (graph). Estimated size of the band (kDa) is shown to the right of the blot. ****P < 0.0001; unpaired two-tailed t-test. (E, F) C2C12 myoblasts cells expressing either WT-SOD1 or SOD1^G93A were transfected with FGF21-FLAG and assessed for viability and Caspase-3/7 activity as in (C). **P = 0.003, ***P = 0.0003; unpaired two-tailed t-test. (G) FGF21 mRNA levels were quantified in C2C12 cells exposed to MetCys-deprived media or treated with 100 mM H₂O₂ for 24 h. ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (H) Cell viability was assessed in C2C12 myoblasts exposed to stressors as described in (G). ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. (I) C2C12 cells transfected with FGF21-FLAG (or empty vector) were subjected to stressors as described in (G) for 24h and then assayed for viability. ****P < 0.0001; unpaired two-tailed t-test. (J) FGF21 mRNA levels were quantified in human primary myoblast cells (HSMM) treated with 25 mM H₂O₂ for 24 h. **P = 0.0014; unpaired two-tailed t-test (left panel). HSMM cells treated with or without 25 mM H₂O₂ and recombinant FGF21 (100ng/ml) for 24h and then assayed for viability. *P < 0.05; unpaired two-tailed t-test (right panel). Data points represent biological replicates and bars are the mean ± SD.

We further investigated the impact of FGF21 on myogenesis as prior studies in preclinical models of ALS have linked myogenesis to slower disease progression [41, 42]. We induced myogenic differentiation in C2C12 cells over 96 h resulting in an increased fusion index (Figure 8A) and induction of MHC (Figure 8B). Over the same time interval, there was a progressive and significant increase in both FGF21 mRNA within cells and secreted FGF21 in the CM (Figure 8C). A similar time course of FGF21 mRNA induction was observed with myogenic differentiation of human primary muscle cells, but the magnitude of induction was more robust (Figure 8D). Ectopic expression of FGF21in C2C12 cells increased myogenic differentiation as indicated by a higher fusion index (Figure 8E; P = 0.008) and a 2.2-fold induction of MHC (Supplementary Figure 6). Likewise myogenic differentiation in human primary muscle cells was increased with recombinant FGF21 treatment as reflected by an increased myotube fusion index (Figure 8F; P = 0.028). Cell proliferation was then measured in C2C12 cells transfected with FGF21-FLAG (Figure 8G). Just prior to seeding, FGF21 was quantitated in the CM by ELISA and showed a ~7-fold increase in FGF21 in the FGF21-FLAG transfected cells versus empty vector control. We observed a significant increase in proliferation in FGF21-FLAG transfected cells at all time intervals tested, reaching 25% at 72 h (P < 0.0001). In summary, FGF21 induction occurs as part of the myogenic differentiation program, where it appears to play a positive role in myogenesis.

Figure 8. FGF21 is upregulated during myogenesis and facilitates myogenic differentiation of C2C12 and human primary muscle cells (HSMM). (A) C2C12 myoblasts were treated with DM for various time intervals and immunostained with an anti-MHC antibody followed by DAPI counterstaining. Myotube formation was detected by MHC-positive staining. Scale bars, 100 μm. The fusion index (%) was quantified as described in the methods (right panel). (B) C2C12 myoblasts treated with DM were lysed at specific time intervals and immunoblotted with antibodies against MHC and vinculin. Densitometry values (24 h time interval was set at 1) are shown. (C) FGF21 mRNA levels were quantified from the lysates and FGF21 protein from conditioned media. ***P = 0.0005 comparing to the 24 h time interval, ^####P < 0.0001 comparing to the 24 and 48 h time intervals; one-way ANOVA followed by Tukey’s multiple comparisons test. (D) HSMM cells were treated with DM for various time intervals and FGF21 mRNA levels were quantified from the lysates as described in (C). **P < 0.01, ****P < 0.0001; unpaired two-tailed t-test. (E) C2C12 myoblasts were transfected with an FGF21-FLAG plasmid and cultured in DM for 96 h. Myotube formation was assessed by MHC-positive staining as in (A). Scale bar, 100 μm (left panel). Fusion index for transfected C2C12 cells. **P = 0.008; unpaired two-tailed t-test (right panel). (F) HSMM cells were treated with recombinant FGF21 (100ng/ml) and cultured in DM for 96 h. Myotube formation was assessed by MHC-positive staining as in (A, E). Scale bar, 100 μm (left panel). Fusion index for transfected HSMM cells. *P = 0.02; unpaired two-tailed t-test (right panel). (G) FGF21 levels in the conditioned media from C2C12 myoblasts transfected with FGF21-FLAG or empty vector control were quantified by ELISA (upper graph). ****P < 0.0001; unpaired two-tailed t test. Cells were reseeded and cultured in growth medium (GM) for 72 h. Cell proliferation was assessed at indicated time intervals using MTS (lower graph). **P = 0.007, ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparisons test. Data points represent biological replicates and bars are the mean ± SD.

Author Contributions

A.G. and P.H.K. contributed to the conception and design of the study. P.H.K, Y.S., M.K., and N.J. developed the ALS and control tissue sample repository at UAB. A.G, Y.S., R.S., B.K.S., B.Z., A.Y., M.K., N.J., R.H., J.D.T.D.S.P., J.S.S., M.A., E.H.V., and P.H.K. contributed to data acquisition. A.G., Y.S., R.S., K.S., A.T.M., S.A.A., and P.H.K. contributed to data analysis and interpretation; A.G., and P.H.K. contributed to drafting the text and preparing the figures.

Acknowledgments

Some of the ALS tissue specimens were provided by the Department of Veterans Affairs Biorepository (VA Merit review BX002466). Normal control tissues were provided by the Tissue Procurement Shard Resource and the UAB Tissue Biorepository. We are also grateful to our patients who courageously agreed to donate tissue samples post-mortem to advance ALS research.

Conflicts of Interest

The authors report no conflicts of interest.

Ethical Statement and Consent

Collection of all human tissue and blood samples was approved by the following Institutional Review Boards: UAB (091222037 and X100908007), Birmingham VA Medical Center (151217001) and Cedars-Sinai (21505).

Written informed consent was obtained from patients who participated in these studies. For post-mortem tissue collection, informed consent was also obtained from the next-of-kin at the time of death. All animal procedures were approved by the UAB Institutional Animal Care and Use Committee (APN 22755).

Funding

This work was supported by NIH Grants R01NS092651 and R21NS111275-01 (PHK), and by the Department of Veterans Affairs Merit Review BX006231-01(PHK).

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