MHY1485 induces chronic muscle fatigue in 3- to 4-week-old female B6 mice

In ME/CFS patients, ATG13 was found to be inactivated by serine phosphorylation and upregulated in the serum [7]. Since the ATG13 is a downstream target of mTOR, next, we wanted to study the role of chronic activation of mTOR in the pathogenesis of chronic muscle fatigue. To test the role of chronic activation of mTOR, we first adopted a molecular strategy to chemically induce mTOR activation in mice. In our current study, MHY1485, a selective agonist of mTOR, was suspended at 0.5% methylcellulose and orally gavaged at a dose of 2, 5, and 10 mg/kg bwt (n = 10 per group) to 3- to 4-week-old B6 mice every alternate day for 14 days (Schema: Supplementary Fig. 1A). Before feeding, mice were randomized based on gender and divided into four groups: vehicle, 2, 5, and 10 mg/kg bwt MHY1485. Health vitals such as initial body weight, body temperature, and blood pressure were recorded. After 1 week, significant mortality was noticed in the 10 mg/kg bwt group as 50% of female mice were deceased (Supplementary Fig. 1B). However, there was no mortality observed in female mice with 2 and 5 mg/kg bwt groups. These mice survived acute vital changes such as hypothermia (Supplementary Fig. 1C), reduced blood pressure (Supplementary Fig. 1D), and frequent freezing episodes until 7 days after the first dose. After that, these mice regained normal body temperature and blood pressure, even though the heart rate was observed to be lower until 2 weeks of feeding. Both 2 and 5 mg/kg groups survived until the end of the experiment timeline (1 month after the last dose) with severe fatigue. Interestingly, feeding with 5 mg/kg bwt MHY1485 resulted in significant immobility in 70% of female mice (n = 7 out of 10).

Apparently, female mice in 5 mg/kg bwt MHY1485 displayed slow movement with periodic freezing at resting conditions. These mice did not gain any body weight (Supplementary Fig. 1E) and size (Fig. 1A) when compared with vehicle-fed mice throughout the study, suggesting that MHY1485 might cause a significant deficit in muscle growth. While studying the drug metabolism, our pharmacokinetic (PK) study revealed that the serum level of MHY1485 increased as early as 15 min, achieved maximum at 45 min, and retained until 6 h after a single gavaging of MHY1485 at a dose of 5 mg/kg bwt (Fig. 1B).

Fig. 1

MHY1485 induces muscle fatigue in female B6 mice. A Three- to four-week-old B6 mice (n = 10; 5 male + 5 female/group) were orally administered with MHY1485 (labeled as “MHY”; mixed with 0.05% methylcellulose), every alternate day for 2 weeks. Representative images displayed the difference in body lengths between vehicle and 5 mg/kg MHY-fed animals. B Pharmacokinetic (PK) study to assess the bioavailability of MHY1485 in serum samples of mice (n = 3) after 0, 15, 30, 60, 90, 120, and 180 min of feeding via gavage. Results are mean ± SEM of three independent experiments. C Electrode placement strategy followed by D spontaneous EMG recording on the biceps femoris muscle of B6 mice. L stands for left, R for right, and the middle electrode is the reference electrode. The low-resolution unmagnified and high-resolution magnified EMG waves were shown for vehicles (i and ii) and (iii and iv) MHY1485-fed mice, respectively. E Reference EMG waves were observed to be consistent between groups. F Grip strength analysis in (i) the vehicle and (ii) MHY groups displayed significantly reduced (iii) latency measured on 4-, 8-, and 12-days post-drug treatment. **p < 0.001 and ***p < 0.0005 versus respective vehicle groups. The significance of the mean was tested with two-way ANOVA considering treatment and days as two effectors. Track plots represented gross motor activities of G vehicle- and H MHY-fed animals in open-field apparatus. I Total distance and J immobile time were measured in the vehicle (green dots) and MHY-fed (red dots) groups (n = 8 per group). ****p < 0.0005 and ***p < 0.001 versus MHY group compared to vehicle-fed group as derived by Mann–Whitney non-parametric test. Results are mean ± SEM of three independent experiments. K (i) Immunoblot assay of S2448phospho-mTOR (pmTOR), total mTOR (t mTOR), Raptor, and beta-actin (β-actin) were performed in muscle lysate. (ii) Relative densitometry analyses of tmTOR and pmTOR after normalizing with respective β-actin band densities (ImageJ software). **p < 0.01 versus control as derived by an unpaired t-test. L (i) Immunoblot analyses of S355phospho-ATG13 (pATG13)), total ATG13 (tATG13), and beta-actin were performed in the biceps muscle. (ii) Relative densitometry analysis indicated **p < 0.01 (= 0.0028) versus vehicle. M Representative images are WDFY3-stained muscle tissue sections of (i) vehicle and (ii) MHY1485 (5 mg/kg)-fed mice (n = 5). Insets are high-magnification images. Arrowheads indicate autophagosomes. N Dual immunofluorescence analysis of IBA1 (green) and WDFY3 (red) to detect the autophagosomes (WDFY3-ir red circles) in macrophage cells infiltrated in muscle tissue of vehicle-fed mouse. The detailed quantification analyses were performed in Supplementary Fig. 5. All raw blots were shown in Supplementary Fig. 10 and dotted histogram analysis for the quantification of autophagosome counts was shown in Supplementary Fig. 4

Interestingly, male mice across all different groups gained body weight and size over time. That observation intrigued us to study muscle fatigue in these female mice. Muscle fatigue can be quantitatively monitored with the EMG recording method. Therefore, next, we performed EMG recording under resting conditions (Fig. 1C). Briefly, two 2 mm electrodes were placed bilaterally on the surface of the left and right biceps femoris muscle, and a reference electrode was placed at the center position on the lower spinal cord (Fig. 1C). The EMG recording was performed at a resolution of 50 µV and the reference recording at 3 mV resolution. Vehicle-fed mice displayed characteristic normal EMG waves (Fig. 1D (i and ii)), whereas mice treated with 5 mg/kg bwt MHY1485 displayed an abnormal pattern of EMG waves with repetitive bursts followed by inactivity indicating potential myokymic changes (Fig. 1D (iii and iv)) in the muscle. Myokymic waves are characteristic of inflammatory denervation at muscle fibers. The reference electrode was placed in the spinal cord, and EMG signals through the reference electrode displayed a consistent pattern of waves in both groups (Fig. 1E). To further confirm muscle fatigue, we performed grip strength analyses on these mice (Fig. 1F; (i and ii)). Accordingly, increasing doses of MHY1485 were found to reduce grip strength latency in female mice (Fig. 1F (iii)) with a significant drop after 4 days of feeding and continued to be decreased with increasing time of feeding. Moreover, gross movement analyses in the ANY-maze open-filed arena (Stoelting Company, IL) (Fig. 1G, H) followed by the assessment of the total distance traveled (Fig. 1I) and immobility time (Fig. 1J) confirmed that mice fed with 5 mg/kg bwt MHY1485 displayed significant muscle fatigue compared to vehicle-fed mice. The movement deficit can be due to the loss of dopaminergic neuronal function in the basal ganglia. Therefore, we tested the levels of tyrosine hydroxylase (TH) enzyme in the striatal tissue of both vehicle- and MHY-fed mice (Supplementary Fig. 2A, B). The dissection strategy was previously described [17]. The result demonstrated that there was no loss of TH in MHY-fed groups, nullifying the role of dopaminergic neuronal loss in movement impairment. While monitoring mTOR activation, our immunoblot results revealed that 2 weeks of MHY feeding significantly upregulated the level of Serine 2448-phosphorylated mTOR (Fig. 1K (i and ii)), whereas no change was observed in the total level of mTOR and Raptor, an essential factor of active mTOR complex 1. Moreover, our IB results indicated that the activation of mTOR was accompanied by the upregulation of S355-phosphorylated ATG13 (Fig. 1L (i and ii)) without altering the level of total ATG13. IB results were normalized with β-actin as a loading control.

Functionally active ATG13 forms a complex with ULK1, ATG101, and FIP200 at the early stage of autophagy [20]. Therefore, to explore the inactivation of ATG13, next, we monitored the formation of a complex between ATG13 and ULK1. To evaluate that, HEK293T cells were co-transfected with FLAG-ATG13 and HA-ULK1. Autophagy was induced following 24 h of starvation under serum-free conditions. Different doses of MHY1485 were added in these cells for 2 h before IP, followed by IB analyses. Interestingly, IP with HA followed by IB with FLAG and IP with FLAG following IB with HA indicated that 5 µM of MHY significantly impaired the formation of ATG13 and ULK1 complex after 2 h of starvation (Supplementary Fig. 3A), confirming the direct role of MHY1485 in the inactivation of ATG13.

A reduction in the number of autophagosomes does not necessarily indicate the impairment of autophagy. Induction of lysosomal function causing increased turnover of autophagosomes also displayed less numbers of autophagosomes. Therefore, next, we wanted to assess if the MHY 1485-mediated inactivation of ATG13 impaired the autophagy process [2], sparing lysosomal function. To test that, HEK293T cells, pre-transfected with eGFP-tagged LC3 (egfp-lc3) plasmid, were serum-starved for 24 h followed by 2 h of treatment with 5 µM MHY1485. Thirty minutes before plate-reading, cells were treated with 50 nM of LysoTracker™ Red DND-99 (Ex: Em = 577 nm/590 nm) and incubated at 37 °C. Accordingly, MHY1485 significantly inhibited the number of autophagosomes, as indicated by the reduced GFP signal (Supplementary Fig. 3B). However, there is no change in the lysotracker signal across different treatment groups (Supplementary Fig. 3C), suggesting that MHY1485 inhibits the formation of autophagosomes but does not alter the lysosomal function or lysosomal biogenesis. To further substantiate this, we performed fluorescence imaging of these HEK293T cells. Our results indicated that there was an induced autophagy process in starving conditions. However, 5 µM of MHY strongly inhibited the numbers of eGFP-LC3-ir autophagosomes but not lysotracker-ir lysosomes (Supplementary Fig. 3D). The total number of lysosomes remained the same. Nevertheless, the LC3 immunoblot analyses indicated that the serum starving induced the expression of LC3-II in HEK293T cells, whereas 5 µM MHY shifted the upregulation of the LC3-II isoform, indicating the impairment of the autophagy process (Supplementary Fig. 3E, F). Next, we assessed the cytotoxicity of MHY1485 by LDH assay. We observed that 5 µM of MHY did not induce any apoptotic signals in HEK293T cells, nullifying the possibility of cell death as a confounding error; however, higher doses with more than 10 µM concentration induced LDH release (Supplementary Fig. 3G).

Since the activation of mTOR followed by ATG13 phosphorylation impairs the formation of autophagosome, resulting in autophagy impairment, next, we performed immunohistochemistry analysis of WDFY3 protein, an essential marker of the autophagosome, in the muscle tissue of vehicle- (Fig. 1M (i)) and 5 mg/kg MHY (Fig. 1M (ii))-fed mice (n = 5 per group). Accordingly, we observed that MHY-feeding significantly attenuated the numbers of WDFY3-immunoreactive autophagosomes compared to the vehicle group (Supplementary Fig. 4), suggesting that the chronic activation of mTOR followed by ATG13 phosphorylation impairs the formation of autophagosomes and inhibits the autophagy process.

Collectively, our results suggest that oral gavaging of MHY1485 stimulated chronic activation of mTOR following the phosphorylation of ATG13, impairment of autophagy in muscle, and induction of significant muscle fatigue in B6 mice.

Oral administration of MHY1485 stimulated infiltration and activation of M1 macrophages in muscle tissue

Next, we wanted to study how the chronic activation of mTOR following autophagy impairment contributed to muscle pathology in female B6 mice. While examining the morphology of WDFY3-ir bodies closely (inset; Fig. 1M (i and ii)), hematoxylin-stained cells were found to be surrounded by autophagosomes in muscle parenchyma. These cells are morphologically similar to macrophages. Accordingly, we performed a dual immunostaining of macrophage marker IBA1 together with WDFY3 in vehicle- and MHY-fed mice (Supplementary Fig. 5A). Interestingly, we observed that the autophagy process was highly operative in muscle infiltrating macrophages (Fig. 1N) in vehicle-fed mice, which was strongly attenuated in muscle tissue of MHY-fed mice (Supplementary Fig. 5A). Interestingly, our correlation study (Supplementary Fig. 5B) further corroborated that there was a strong negative correlation between IBA1-ir cells and WDFY3-ir autophagosomes in skeletal muscle tissue, suggesting the autophagy impairment could play an essential role in the activation and infiltration of macrophage in muscle tissue.

Autophagy impairment is known to exacerbate inflammation in chronic inflammatory [21,22,23] and autoimmune diseases [24, 25]. Therefore, next, we wanted to explore if chronic mTOR activation resulting in autophagy impairment augmented inflammation in skeletal muscle tissue. At first, our hematoxylin and eosin (H&E) staining in biceps muscle tissue displayed that there was a severe infiltration of mononuclear cells in the muscle parenchyma of MHY-fed (Fig. 2B) but not vehicle-fed (Fig. 2A) B6 female mice, indicating the potential role of chronic mTOR activation in the inflammatory mononucleosis in skeletal muscle tissue. However, DAB immunostaining of CD4 (Fig. 2C) and CD8 (Fig. 2D) in serial sections of muscle tissue followed by quantification analyses (Fig. 2E, F) revealed that neither CD4+ve nor CD8+ve T cells were found to be infiltrated in muscle parenchyma through the surrounding blood vessel. Interestingly, DAB immunostaining of IBA1, a marker protein of macrophage (Mφ), in the muscle tissue (Fig. 2G, H) followed by a quantification study (Fig. 2I) revealed that these infiltrated mononuclear cells were primarily macrophages. Moreover, a dual IF analysis of CD40, the surface marker of inflammatory the M1 subtype of Mφ, along with pan-Mφ marker IBA1 (Fig. 2J) followed by a quantification (Fig. 2K) study revealed that 2 weeks of MHY-feeding strongly upregulated the expression of M1Mφ marker CD40, suggesting that the chronic activation of mTOR induced the infiltration of M1Mφ cells. Dual IF staining of IBA1 with another Mφ-derived inflammatory marker iNOS (Fig. 2L) displayed an upregulated expression of iNOS in the MHY-fed group compared to vehicle-fed groups, further suggesting that these cells were truly inflammatory M1Mφ.

Fig. 2

MHY1485 stimulates the infiltration and activation of M1 macrophage (M1Mφ) in muscle tissue. H&E staining in biceps muscle tissue of A vehicle and B MHY1485-fed mice (5 mg/kg). The infiltration of mononuclear cells (blue dots) was visible after 2 weeks of gavaging with MHY-1485 (alternative days at a dose of 5 mg/kg; n = 8/group). DAB immunostaining of serial Sects. (5 µm gap) by C CD4 and D CD8 antibodies demonstrated that these mononuclear cells were neither CD4-ir nor CD8-ir T cells. E and F are quantitative estimations of CD4- and CD8-ir cells, respectively. The quantification is done per 1 sq. mm parenchyma around blood vessels (n = 5/group). A total of 6 and 8 independent images per group were included for the quantifications of CD4 and CD8-ir cells, respectively. A non-parametric Mann–Whitney test was performed, and the resultant p-values were included on the histograms. IBA1 staining by DAB method in G vehicle- and H MHY-fed mice. I Quantification analyses of IBA1-ir cells were performed in 20 images (2 images from n = 10/group) in ImageJ software and plotted as dotted histograms in GraphPad Prism software. J Dual IF staining of IBA1 (green) and M1 Mφ marker CD40 (red) followed by K the quantification of CD40-ir cells were performed (total 20 images from two images in n = 10/group). Unpaired t-tests were performed to test the significance of means between groups. ***p < 0.0001 vs. vehicle as confirmed by parametric unpaired t-test. L Representative image of dual immunostaining of IBA1 and iNOS in the vehicle (left) and MHY-fed mice. Results are confirmed after three independent experiments

Next, we were interested in studying how these infiltrated M1Mφ cells elicited inflammatory changes in muscle cells. At first, we adopted Bielschowsky’s silver staining procedure to monitor the myelin integrity in muscle-serving nerve fibers. The horizontally sectioned muscle fibers were stained with 10% silver nitrate as discussed in the method section. The high-resolution images of nerve terminals were visualized under a phase contrast microscope and recorded at × 100 magnification (Fig. 3A). The myelin layers in vehicle-fed mice were observed to strongly adhere to muscle parenchyma. However, we observed that the oral administration of MHY1485 significantly compromised the integrity of myelin layers on muscle fibers, as indicated with lightly colored, disintegrated, and vacuolated myelin layers on muscle parenchyma. Next, a dual immunostaining method of myelin marker MBP and axonal marker TUJ1 was adopted to evaluate the myelin integrity (Fig. 3B). The presence of myelin layers as indicated with strong MBP staining was expected to mask axons as indicated with less TUJ1 signal. Accordingly, the dual immunostaining of MBP and TUJ1 in transversely sectioned muscle tissue indicated that the integrity of myelin layers in the muscle-serving nerve terminal of the MHY-fed group was severely compromised, and as a result, the TUJ1-ir axons were exposed, whereas MBP-ir myelin layers remained tightly wrapped around axonal fibers of vehicle-fed mice. The integrity of myelinated nerve fibers was further evaluated in horizontally sectioned muscle fibers by similar dual immunostaining of MBP and TUJ1 (Fig. 3C), which indicated that the oral administration of MHY, but not the vehicle, significantly damaged the myelin integrity. Moreover, the quantitative estimation of the ratio between the MFI of MBP and that of TUJ1 (Fig. 3D) confirmed that indeed there was a strong demyelinating response in the muscle fibers after chronic mTOR activation. Next, we evaluated if the chronic administration of MHY1485 caused apoptosis in muscle cells. Interestingly, our TUNEL staining identified the TUNEL+ve apoptotic signals (Fig. 3E) in the muscle cells around the blood vessel, where strong infiltration of mononuclear cells was observed. The result was further confirmed by quantification analysis (Fig. 3F). Skeletal muscle-serving efferent nerve fibers originated at the ventral horn of the spinal cord. Therefore, next, we were interested in exploring the myelin integrity in the ventral part of the spinal cord. Accordingly. LFB staining in the dorsoventrally sectioned spinal cord tissue (Fig. 3G) indicated a strong reduction of myelin staining, particularly in the ventral spinal tissue of MHY-fed mice. Accordingly, the quantitative estimation was performed to measure the volume of the LFB-stained region compared to the total volume of spinal cord tissue on a percent scale, and the resultant analyses further demonstrated that chronic MHY feeding truly generated a demyelinating response in the ventral horn region of the spinal cord (Fig. 3H). The demyelinating response in spinal cord tissue was further evaluated by IF staining with MBP (Fig. 3I). To study the role of M1Mφ cells (CNS subtype is M1 microglia) in spinal cord demyelination, a CD40 immunostaining analysis was adopted. Interestingly, our analysis identified that there was an infiltration of CD40-ir M1Mφ cells (Fig. 3J) in the demyelinated spinal cord tissue. Nevertheless, a parametric correlation analysis between the MBP-ir area of the spinal cord and the number of CD40-ir cells (Fig. 3K) further revealed that MHY feeding caused the demyelinating response in the spinal cord following the infiltration of activated M1Mφ cells. Collectively, our results suggest that the severe infiltration of M1Mφ causing the demyelinating response in muscle-serving nerves resulted in the apoptosis of muscle cells.

Fig. 3

MHY1485 augments demyelinating response in muscle and spinal cord. A Bielschowsky’s silver staining of nerve fibers in horizontally sectioned biceps muscle tissue exhibits the loss of myelin integrity (blue arrow) in MHY-fed mice (n = 5/group). B Dual IF analyses of myelin marker MBP and axonal marker TUJ-1 were performed in muscle-serving nerve bundles of the vehicle and SIM-fed mice. Transverse (dorsoventral) sectioning of the biceps muscle was performed to expose the nerve bundle. C Horizontal (anteroposterior axis) sectioning of muscle tissue followed by dual IF staining demonstrated a lateral view of MBP (green) and TUJ-1 (red) stained nerve fibers serving skeletal muscle (biceps) tissue. D MFI (mean fluorescence intensity) was calculated as described in the method section. Briefly, 10 nerve bundles were randomly selected from 5 different images with a total of 50 selections per group. MFI was calculated in the green channel for MBP and the red channel for TUJ1 in each nerve fiber. After that, the ratio was measured and plotted as a scatter histogram. An unpaired t-test was performed to test the significance of the mean between groups that resulted in ***p < 0.005 versus control. E TUNEL staining followed by F quantification studies indicate that there was moderate but significant cell loss in MHY-fed muscle tissue. ****p < 0.0001 versus vehicle as indicated by a non-parametric Mann–Whitney U test (24 images were assessed from n = 5/group). G Luxol Fast Blue (LFB) staining exhibits the loss of myelin in the ventral horn of the lumbar spinal cord tissue of MHY-fed (lower panel), but not vehicle-fed (upper panel) mice. The demyelinating region was magnified and shown inset. H Eight LFB-stained spinal cord tissue were randomly selected per group followed by measuring the area of the entire image and that of only LFB-ir region in the area measuring tool of CaptaVision + (Accu-scope INC.) software. The result was shown by a dot histogram plot. Mann–Whitney U test revealed ****p < 0.0001 versus control in 9 spinal cord images collected from n = 5 mice/group. I IF staining of myelin marker MBP (red) in the demyelinated ventral horn of the lumbar spinal cord of vehicle- (upper panel) and MHY- (lower panel) fed mice. The magnified images were shown inset. J IF analysis of M1 Mφ marker CD40 (green) in the demyelinated ventral region of the spinal cord in MHY-fed mice. Nuclei were stained with DAPI (blue). K A parametric Pearson correlation analysis was shown in a scatter plot between the % of MBP-ir area of the spinal cord (Y-axis) and CD40-ir cells (X-axis) in 27 spinal cord images. Results are confirmed after three independent experiments in n = 5 mice per group

Chronic mTOR activation is required for the expressions of IL-6 and RANTES via activation of STAT3 in muscle tissue

So far, our results suggest that chronic administration of MHY1485 following mTOR activation recruited inflammatory M1Mϕ cells and implemented a demyelinating response in muscle tissue. Next, we wanted to explore the effect of chronic mTOR activation on the expressions of inflammatory cytokines in these cell types. First, a cytokine array of 40 different cytokines revealed that 5 µM MHY-1485, but not solvent control (DMSO), significantly induced the expressions of IL6 and RANTES (Fig. 4A), but not other inflammatory cytokines such as IL-1β, TNFα, IL12, or IFN-γ in human C20 microglial cells. Upregulations of inflammatory chemokines such as MCP-1, MIP-1α, and eotaxin were also not observed. The result was further confirmed by quantitative real-time PCR analyses of IL6 (Fig. 4B) and RANTES (Fig. 4C) and IB of IL6 (Fig. 4D), followed by relative densitometric quantification (Fig. 4E). Next, a similar cytokine array in the serum samples of mice dosed with 5 mg/kg bwt MHY1485 resulted in similar upregulations in both IL6 and RANTES but not other cytokines (Fig. 4F). Subsequent ELISA analyses of IL6 (Fig. 4G) and RANTES (Fig. 4H) in the plasma of MHY1485-treated mice (n = 5) further confirmed that both cytokines were strongly upregulated after 2 weeks of feeding with 5 mg/kg bwt MHY1485. Apart from myeloid-lineage macrophage or microglia cells, both these cytokines are also found to be profusely expressed in muscle cells and therefore called myokines. Interestingly, mRNA analyses revealed that upon 2 weeks of feeding with 5 mg/kg bwt MHY1485, gene expressions of IL6 (Fig. 4I) and RANTES (Fig. 4J) were significantly upregulated in muscle tissue. To explore the clinical relevance of these findings, plasma samples of eight control and eight age-matched ME/CFS patients were analyzed for ELISA analyses of IL6 (Fig. 4K) and RANTES (Fig. 4L). Interestingly, strong elevations of IL6 and RANTES were observed in the plasma samples of ME/CFS patients, suggesting that MHY1485-induced upregulations of IL6 and RANTES are pathologically relevant to ME/CFS. To explore the direct role of mTOR in the upregulations of IL6 and RANTES, the de novo expression of mTOR was attenuated by mTOR siRNA (Supplementary Fig. 6A, B) in MHY-treated C20 microglial cells, followed by IL6 (Supplementary Fig. 6C) and RANTES (Supplementary Fig. 6D) protein expressions. MHY-1485 failed to upregulate IL6 and RANTES once the expression of mTOR was attenuated in microglial cells, suggesting the direct role of mTOR in the upregulations of IL6 and RANTES in microglial cells.

Fig. 4

MHY1485 upregulates IL6 and RANTES. A An antibody array of 40 inflammatory cytokines (RayBiotech) was performed in the supernatants of C20 human microglial cells treated with 5 µM of MHY1485 for 48 h. Control sup was collected from microglia treated with DMSO for 48 h. The red arrow shows the IL6 expression, whereas the blue arrow indicates the RANTES expression. B Realtime mRNA expressions of IL6 were monitored in microglia treated with 1, 2, and 5 µM of MHY for 5 h under serum-free conditions. **p < 0.01 versus control as measured with the Mann–Whitney non-parametric test. C Realtime mRNA expressions of RANTES were monitored in human microglial cells treated with 1, 2, and 5 µM of MHY1485 for 5 h under serum-free conditions. **p < 0.01 versus control as measured with the Mann–Whitney test. D Immunoblot analysis of IL6 (~ 20 kDa) was performed in human microglial cells after treating increasing doses of MHY1485 for 24 h. The resultant expression was normalized with respective β-actin expressions followed by analyzing E relative density. **p < 0.01 versus control (Mann–Whitney test). F At 14 days, after the last dose of MHY1485 feeding blood collection was carried out via cardiac puncture method, serum was isolated in heparin tube, and then performed a similar antibody array method of 40 inflammatory cytokines. Red and blue arrows indicate IL6 and RANTES expressions, respectively. ELISA analyses of G IL6 and H RANTES in the serum samples were performed (n = 8/group). Non-parametric Mann–Whitney test represents ***p < 0.005 (= 0.0002) versus vehicle. Realtime mRNA expressions of I IL6 and J RANTES were performed in muscle tissue of vehicle and MHY-fed mice (n = 5 per group). The non-parametric Mann–Whitney tests represent **p < 0.01 and *p < 0.05 versus vehicle. Results are mean ± SEM of three different experiments. K IL6 and L RANTES concentrations were also measured by quantitative ELISA methods in sera of n = 8 healthy and age-matched n = 8 ME/CFS patients. Mann–Whitney tests represent *p < 0.05 (= 0.0407) and ***p < 0.005 (= 0.0002) versus the control group. Results are mean ± SEM of three different experiments

Next, we were interested in exploring how MHY1485 stimulated the expressions of IL6 and RANTES. Upon activation, mTOR activates STAT3 via phosphorylation at its Serine 727 and Tyrosine 705 residues [26, 27]. To test these possibilities, human microglial cells were stimulated with 1, 2, and 5 µM of MHY1485 for 2 h, then fractionated for nuclear extract, and finally analyzed for IB. Interestingly, MHY1485 dose-dependently induced the phosphorylation and subsequent nuclear translocation of Y705P STAT3, but not S727P STAT3 (Fig. 5A). The result was normalized with H3 histone. This result indicates the possible role of MHY1485-induced Y705 phosphorylation of STAT3 in the transcriptional regulations of IL6 and RANTES. To further confirm, these nuclear extracts were probed with an oligonucleotide probe of STAT3 and run for an EMSA gel-shift assay. Based on that result, MHY1485 dose-dependently induced the DNA-binding of STAT3 (Fig. 5B), suggesting that MHY1485 induced the transcriptional activity of STAT3. Interestingly, subsequent promoter analysis of IL6 at chromosome 7 detected a consensus STAT3 response element (Fig. 5C). A chromatin immunoprecipitation (ChIP) by STAT3 antibody study followed by real-time PCR with IL6 promoter-specific primers indicated that MHY1485 directly induced the recruitment of STAT3 at the IL6 promoter (Fig. 5D, E). The specificity of ChIP was validated by the IP with IgG (Fig. 5F). Similarly, a canonical STAT3 binding site was also observed in the RANTES promoter (Fig. 5G), and the subsequent ChIP analyses revealed that upon MHY treatment, there was an augmented recruitment of STAT3 (Fig. 5H, I) in the RANTES promoter of C20 microglial cells. The specificity of STAT3 recruitment was further confirmed by the negative PCR data in the IgG-pulled fraction (Fig. 5J). To evaluate the role of STAT3 in inducing the transcription of IL6, next, we performed a GFP reporter assay, in which IL6 promoter with STAT3 response element was cloned upstream of GFP reporter (pwtIL6). The GFP construct was then transfected to human microglial cells, followed by treatment with different doses of MH1485 for 5 h. Interestingly, increasing doses of MHY1485 stimulated GFP reporter activity (Fig. 5K), as shown by a fluorometric assay measured with Ex: Em = 480 nm/535 nm signal ratio. To further validate the result, we performed a site-directed mutagenesis of the STAT3 response element at the IL6 promoter (p∆IL6) followed by the transfection in human microglial cells (Fig. 5L). Subsequent fluorimetric analysis revealed that increasing doses of MHY1485 were unable to induce the GFP reporter activity once cells were transfected with the p∆IL6 promoter. While exploring the effect of MHY1485 feeding on the activation of STAT3 in skeletal muscle, our DAB staining indicated that the oral administration of MHY significantly stimulated the levels of Y705PSTAT3 (Fig. 5M) in the epithelium of biceps muscle tissue compared to control. Further analysis revealed that these Y705PSTAT3-ir cells were mostly IBA1-ir macrophages (Fig. 5N). Collectively, our results suggest that the chronic mTOR activation followed by the STAT3 phosphorylation at its Tyr 705 is critical for the upregulation of IL6 and RANTES in infiltrated M1Mϕ cells, leading to the chronic inflammation and demyelinating response in muscle tissue.

Fig. 5

The essential role of STAT3 in MHY 1485-mediated expression of IL6 and RANTES. A Immunoblot analyses of mTOR with phosphorylated tyrosine 705 (pY705) and phosphorylated Serine727 (pS727) in the nuclear extracts of human C20 microglial cells treated with 1, 2, and 5 µM of MHY1485. Histone 3 (H3) immunoblot analysis was performed as a control. (Inset) Densitometric analyses were done in ImageJ followed by normalization with respective H3 bands. B EMSA analysis of STAT3 (probed with STAT3 oligonucleotide; Li-Cor Bioscience) in the nuclear extracts of human microglial cells treated with 1 and 2 µM MHY1485 (NC = negative control). The raw blot is shown in Supplementary Fig. 10. C The promoter analysis of the human IL6 promoter at chromosome 7 displays the detailed location and sequence of consensus STAT3 responsive element. D Chromatin immunoprecipitation (ChIP) analysis of STAT3- and IgG-pulled DNA (product length = 123 bp) surrounding STAT3 responsive element of IL6 promoter. Chromosomal DNA was isolated from blue = input; green = control; and red = 5 µM MHY1485-treated microglial cells, performed with ChIP, and the resultant real-time PCR amplification data suggests increased binding of STAT3 at IL6 promoter. Fold increases in E anti-STAT3- and F IgG-pulled down DNA were displayed after normalizing the Ct value of MHY-pulled down DNA with the Ct value of the input. *p < 0.05 versus control and ns = no significance. Results were mean ± SD of three different experiments. G Human RANTES promoter map at chromosome 17 with location and sequence. H Realtime PCR amplification of human RANTES promoter pulled down by anti-STAT3 antibody in human microglial cells treated with 5 µM MHY1485 (red). Control cells were treated with DMSO (green), and input (blue) was no antibody-treated group. Fold-increase of chromosomal DNA surrounding the STAT3-responsive element of the RANTES promoter was quantified after pulling down with I STAT3 antibody and J IgG. The result was further quantified with relative Ct analyses in (K) anti-STAT3 and (L) IgG-pulled DNA after normalizing with input Ct. *p < 0.05 versus control and ns = no significance. Results were mean ± SD after three different experiments. K GFP-reporter assay of wild-type IL6 promoter cloned at STAT3 response element tagged GFP at the N-terminus region (pwtIL6). The promoter clone was transfected in HEK293T cells followed by stimulation with 1, 2, and 5 µM of MHY1485, and the resultant GFP signal was quantified with Ex: Em = 485 nm:535 nm. Results are mean ± SD of three different experiments. *p < 0.05 versus control and ns = no significance. L Site-directed mutagenesis of STAT3-response element of human IL6 promoter followed by the construction of a GFP reporter clone was performed. A dose-dependent effect of MHY1485 on the expression of GFP reporter was evaluated in HEK293T cells. ns = no significance. M DAB immunostaining of Y705PSTAT3 in skeletal muscle tissue (biceps muscle biopsies) of vehicle- and 5 mg/kg MHY1485-fed mice (n = 5/group). The representative image was derived from muscle epithelium. N Dual IF analysis of Y705PSTAT3 and macrophage-marker IBA1 in epithelial tissue of vehicle- and MHY-fed mice (n = 5/per group). Insets are magnified images of respective enclosed areas. Nuclei were stained with DAPI

Suppression of ATG13 in ATG13 repressor (Tg-ATG13) mice showed severe but transient post-exertional fatigue

A pathological hallmark of chronic fatigue syndrome is post-exertional fatigue. Does chronic mTOR activation feeding exacerbate muscle fatigue after treadmill exercise? To answer that question, we performed a single-session treadmill exercise trial in vehicle- (Fig. 6A) and MHY1485 (5 mg/kg)-fed (Fig. 6B) mice. Mice were fed with MHY1485 (5 mg/kg) on alternative days for 2 weeks, rested for 1 week, and then subjected to treadmill exercise for monitoring the post-exertional fatigue. The overall movement was monitored 1 day before, immediately after, and 2 days after treadmill exercise in an ANY-maze infrared sensor-controlled open-field arena. Animals were subjected to running on the treadmill at a speed of 14 rpm for 15 min following 1 min of acclimatization at a speed of 8 rpm. The baseline movement recorded one day before the treadmill exercise was found to be moderate but significantly diminished (Supplementary Fig. 7A) in MHY-fed mice (Fig. 6B; first row) compared to vehicle-fed mice (n = 5 per group) (Fig. 6A; first row). The treadmill exercise implemented acute but temporary fatigue immediately after treadmill exercise in both vehicles- (Fig. 6A; second row) and MHY-fed (Fig. 6B; second row) groups, as shown by track-plot analyses. However, quantitative measurement of average speed (Supplementary Fig. 7A) indicated that MHY-fed mice displayed severe acute fatigue with significantly less physical movement compared to vehicle-fed mice. Interestingly, 2 days of complete rest completely recovered the vehicle-fed mice (Fig. 6A; third row). However, MHY-fed mice continued to display severe impairment of physical movement even after 2 days of complete rest, as indicated by our track-plot analysis (Fig. 6B; third row), followed by quantitative measurement of average speed (Supplementary Fig. 7A) in the arena. Since our results indicate that the functional inactivation of ATG13 implements muscle fatigue, next we wanted to develop a unique transgenic mouse model to study the direct role of ATG13 in post-exertional fatigue. Embryonic ablation of the atg13 gene is lethal in the fetal stage. Therefore, we generated a knock-out first ATG13-repressor mouse (Tg-ATG13) in which a lacZ repressor element was inserted upstream of exon 5 of its atg13 gene (Fig.