Mitochondria are defective in SMA MNs, resulting in an impaired energy homeostasis
To investigate differentially expressed proteins and discover dysregulated pathways in SMA, we used primary MNs isolated from an SMA mouse model and wild-type mice . Whole proteome analysis of 10 days in vitro (DIV) MNs detected 5165 proteins of which 681 (~ 13%) are mitochondria related (Fig. 1a and Fig. S1a, b). Among all identified proteins, 494 proteins were significantly changed in SMA compared to WT, and 61 (~ 12%) of them are localized to mitochondria based on MitoCarta2.0 database (Fig. 1a, b). Among these 61 proteins, 11 proteins are localized to the oxidative phosphorylation (OXPHOS) machinery (Fig. S1c). Interestingly, in the OXPHOS machinery, complex I seems mainly affected in SMA with 9 dysregulated proteins (Fig. S1c). To understand the biological meaning of SMA affected proteins, we analyzed gene ontology (GO) terms of all 345 significantly down-regulated and 149 significantly up-regulated proteins (Fig. 1c, d). Among the down-regulated proteins in SMA MNs we identified previously documented pathways in SMA pathology such as RNA binding, protein transport, ribonucleoprotein complexes and protein synthesis confirming the reliability of the data set (Fig. 1c) [8–11, 17]. 149 up-regulated proteins suggested mitochondrial dysfunction in ATP production (Fig. 1d). Therefore, we further investigated localization and function of mitochondria in motor axons using MitoTracker and TOM20 (Fig. 1e). Indeed, numbers of total and functional mitochondria are reduced in SMA axons (Fig. 1e). The finding of mitochondrial mis-localization is strengthened by down-regulated mitochondrial motor proteins KIF5B and KIF5BP in SMA MNs in our whole proteome analysis using mass spectrometry (MS) (Fig. S1b). In addition, mitochondria are smaller and fragmented in SMA MNs (Fig. S1d). As neurons can produce energy from glycolysis to compensate for their high energy demand , glucose uptake was monitored. Interestingly, glucose uptake is also impaired in SMA MNs (Fig. 1f). As both energy producing pathways are impaired in SMA MNs, we next measured the intracellular ATP concentrations. Indeed, ATP concentration is up to 3-fold lower in SMA compared to WT MNs (Fig. 1g). Together, these results suggest that energy homeostasis is impaired in SMA MNs due to defective mitochondria and glycolysis is unable to compensate for this defect.
Proteins are hyper-carbonylated and protein synthesis is impaired in SMA
As our MS results indicated defects in complex I of the electron transport chain and complex I is known source of reactive oxygen species (ROS) in mitochondria , we measured intracellular ROS levels using CellROX® (Fig. 2a, b). Indeed, our data indicated that ROS levels were increased in SMA compared to WT (Fig. 2a). This finding was confirmed by two independent detection methods; imaging and microplate reader (Fig. 2a and b). In heathy condition, ROS can be eliminated by oxidative stress defense proteins such as superoxide dismutase (SOD1), which clears ROS from the mitochondrial intermembrane space . Interestingly, SOD1 protein levels are down-regulated in SMA MNs, indicating defective oxidative stress defense mechanism in SMA (Fig. S1b). As oxidative stress results in carbonylation of proteins , we measured levels of carbonylated proteins in SMA MNs (Fig. 2c). Indeed, higher amounts of carbonylated proteins were detected. This data confirmed our finding that SMA MNs are under oxidative stress (Fig. 2c). Carbonylation of proteins can alter their conformation and hinder their protein synthesis [24, 47]. Furthermore, as levels of ribosomes and translation related proteins are also changed in SMA due to our MS data, we measured protein synthesis efficiency with Surface sensing of translation (SUnSET) and AHA-Click it assay in WT and SMA MNs (Fig. 2d-g). Indeed, we confirmed that SMA MNs show a reduced protein synthesis efficiency compared to WT with two independent methods (Fig. 2d-g). Taken together, these results suggest a negative correlation between ROS levels and protein synthesis.
Pyruvate restores ATP levels and reduces ROS levels in SMA
Next, we pursued to restore the consequence of defective mitochondria in SMA; energy deficiency and oxidative stress. As pyruvate is known to reduce ROS in a non-enzymatic way and is a known substrate of TCA cycle , we supplemented WT and SMA MNs with 10 mM or 50 mM sodium pyruvate. Within 1 hour of treatment with 50 mM pyruvate, ATP levels were increased significantly in MN-like NSC-34 cells (Fig. S2c) and SMA MNs (Fig. 3a and Fig. S2f), while no effect was seen in WT MNs (Fig. 3a and Fig. S2f). However, lactate, which can be converted to pyruvate by lactate dehydrogenase in the cytoplasm , neither altered ATP levels in NSC-34 cell (Fig. S2d, e) nor in primary MNs (Fig. S2f). In addition, as pyruvate has been suggested as a ROS scavenger , we treated SMA MNs with pyruvate and measured ROS levels. Indeed, pyruvate could successfully reduce ROS levels in SMA MNs (Fig. 2b). Further, we confirmed pyruvate uptake and ROS reduction by treatment with 50 mM pyruvate or 10 µM of the anti-oxidant NAC in ROS induced cells by menadione treatment (Fig. S2a, g). These results suggest that pyruvate is a valuable supplement to restore ATP levels and simultaneously balance intracellular ROS levels in MNs.
Effect Of Ros On Protein Synthesis In Neurons
Based on our data, we hypothesized that the elevated ROS levels hinder protein synthesis, thus, reduction of ROS might restore impaired protein synthesis in SMA MNs. In order to further understand the effect of ROS on protein synthesis, we have modified cellular ROS levels in MNs and measure protein synthesis efficiency. We treated MNs with 10 µM NAC or 50 mM pyruvate or 100 µM menadione and performed SunSET analysis (Fig. 4a). Protein synthesis efficiency was unaltered by pyruvate or NAC in WT MNs (Fig. 4b). However, menadione-induced ROS clearly inhibited protein synthesis. As a positive control, 50 µM anisomycin was used (Fig. 4b-d). The same results were observed in NSC-34 cells (Fig. S3a-c). Interestingly, for SMA MNs, where cellular ROS levels are higher, a reduction of ROS by adding 10 µM NAC could increase protein synthesis (Fig. 4c). However, no consistent increase of protein synthesis was observed in pyruvate supplemented SMA MNs (Fig. 4c). In addition, SunSET signal was visualized and quantified by immunofluorescent staining in MNs. Indeed, these results confirmed that NAC can restore impaired protein synthesis in SMA MNs (Fig. 4d). Taken together, modulation of ROS has an effect on protein synthesis in MNs.
Whole Proteome Analysis Of Mns With Ros Manipulation
Next, we tried to obtain a systemic view of the whole proteome regulated by ROS in MNs. First, cells were incubated with 50 mM pyruvate for 1 hour, and whole proteome was analyzed by MS. We found that the levels of 122 proteins were altered in SMA MNs (Fig. 5a, c). Gene ontology (GO) analysis of altered proteins revealed proteins related to ATP production, which were enriched in all three terms (Fig. 5b). The biological process of oxidative phosphorylation was enriched, and the cellular compartment of mitochondria was affected by pyruvate supplement (Fig. 5b). In addition, ribonucleotide binding, spliceosome, RNA splicing, and mRNA processing were also affected by pyruvate. Interestingly, these terms have been reported as altered pathways in SMA (Fig. 5b). Among 122 proteins, 22 were also significantly changed by pyruvate in WT MNs, and 28 were significantly altered in pyruvate treated SMA MNs (Fig. 5c-e). Interestingly, when we compare proteins altered in SMA to pyruvate affected proteins in WT or SMA MNs, we found that the effect of pyruvate was clearer in SMA MNs (Fig. 5d, e). Next, we compared the levels of proteins altered by pyruvate in an isolated view. Among those 28 proteins, 21 proteins were down-regulated in SMA and up-regulated by pyruvate. GO analysis of these 21 proteins revealed a strong enrichment in ATP binding and mRNA processing (Fig. 5f). In contrast, pyruvate changed levels of 144 proteins in WT MNs, and among them 22 proteins were also altered in SMA compared to WT (Fig. S4a-f). However, these 22 proteins showed far less changes after pyruvate treatment (Fig. S5a, e). Due to the small changes in WT MNs, only few GO terms were identified (Fig. S4c).
Next, SMA MNs were treated with 10 µM NAC, a known antioxidant, for 1 hour. Whole proteome analysis showed that 143 proteins were significantly changed (Fig. 5g, i). GO analysis suggested these proteins have multiple functions including nucleotide binding and RNA processing (Fig. 5h). Compared with differentially expressed proteins between WT and SMA MNs, 31 proteins were common with NAC treatment in SMA MNs (Fig. 5i). Among them, 17 proteins were down-regulated in SMA compared to WT, and up-regulated by NAC treatment (Fig. 5k, l). Interestingly, these proteins also regulate RNA processing and splicing (Fig. 5l). Again, NAC showed only little effect on individual proteins in WT MNs (Fig. 5j), while it showed clear effect on SMA MNs (Fig. 5j, k, and Fig. S4g-l). ROS induction by 100 µM menadione for 1 h in WT MNs had the biggest effects on the proteome with 344 significantly altered proteins (Fig. S4m, o). In addition, menadione induced ROS also showed the biggest overlap of altered proteins with SMA affected proteins; 56 proteins (Fig. S4n-p). GO analysis revealed strong enrichment in nucleotide-binding and association with mitochondria (Fig. S4r). It is worthy to mention that pyruvate and NAC had only a small effect on WT MNs, whereas they can induce a large up-regulation of proteins in SMA MNs. This could be because basal amounts of these proteins were lower in SMA MNs compared to WT MNs.
Ros Regulates Initiation Of Mrna Translation
Due to GO analysis, a major biological process, mRNA translation is dysregulated in SMA MNs. The volcano plot illustrates 360 proteins found in our MS data related to mRNA translation based on the MGI database (Fig. 6a). Among them, 40 out of 47 are significantly down-regulated in SMA MNs compared to WT ones (Fig. 6a). mRNA translation is tightly regulated in eukaryotic cells by two major processes; initiation and elongation. As we already know that efficiency of protein synthesis is impaired in SMA MNs [9, 10, 17], we deciphered further mechanisms to determine which step is disrupted by SMN loss. To distinguish between the effects of initiation and elongation, we measured both processes in MNs. First, to assess the rate of protein elongation, we used the SunRiSE assay. In brief, translation initiation was blocked by 2 µg/ml harringtonine at different time intervals, before newly synthesized peptides were tagged with 10 µg/ml puromycin (Fig. 6b). Puromycin tagged proteins were detected by Western blot with anti-puromycin antibody. Data analysis did not show any clear changes in the elongation rate in neither WT nor SMA MNs after supplementation of NAC or pyruvate (Fig. 6c, d).
As translational elongation is unaltered by SMA or ROS, we next focused on translational initiation. One of the well described translational initiation mechanisms is cap-dependent translation initiation by 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1). Therefore, we measured the phosphorylation status of 4E-BP1 after modulation of ROS levels. Reduced phosphorylation status of 4E-BP1 indicates that initiation of mRNA translation is indeed impaired in SMA MNs (Fig. 6e). No significant difference was observed by pyruvate or NAC in WT cells (Fig. 6f). While induction of ROS by 100 µM menadione resulted in a reduction of 4E-BP1 phosphorylation, suggesting excessive ROS can inhibit mRNA translation at the initiation step (Fig. 6f). Furthermore, pyruvate and NAC increased the phosphorylation of 4E-BP1 in SMA MNs (Fig. 6g). Taken together, this unprecedented data reveals that mRNA translation is impaired at the initiation step in SMA MNs, while elongation is unaltered. Moreover, initiation of mRNA translation is regulated by ROS via 4E-BP1.
SMN protein levels are regulated by pyruvate and ROS via mTOR
Most interestingly, pyruvate supplement did not only increase ATP levels and reduce ROS, but also increased SMN levels in WT and SMA MNs as well as NSC-34 cells (Fig. 7a, b, and Fig. S5a, e). To understand the molecular mechanism underlying increased SMN levels, we measured Smn mRNA levels in WT MNs and NSC-34 cells after pyruvate treatment (Fig. S5b). No significant increase of Smn mRNA levels were observed, suggesting post transcriptional regulation of SMN levels (Fig. S5b). Next, protein synthesis inhibitor, anisomycin prevented the pyruvate induced increase of SMN protein levels (Fig. S5c). This data confirms that protein synthesis of SMN is regulated by pyruvate. In addition, 10 µM NAC increased SMN levels in MNs (Fig. 7c, d and Fig. S5e). As mTOR, especially mTORC1 is a major regulator of protein synthesis , we tested whether increase of SMN protein levels is mTOR dependent. We treated MNs with water-soluble mTOR blocker, 100 nM WYE-687 dihydrochloride and supplied 50 mM pyruvate. Indeed, pyruvate increased SMN levels in WT MNs and this effect was abolished by WYE-687 dihydrochloride (Fig. 7e). In addition, pyruvate increased mTORC1 activity measured by phosphorylation status of the S6K as well as its target ribosomal S6 protein (S6) in WT MNs (Fig. 7f, g and Fig. S5d). Further, reduction of ROS by NAC treatment in SMA MNs increased the mTORC1 activity (Fig. 7h). Taken together, our data showed that cellular ROS and ATP levels regulate SMN protein synthesis via regulating mTORC1. Furthermore, re-balancing ROS levels with anti-oxidant in SMA MNs can increase SMN protein synthesis.