Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis
2018; Springer Nature; Volume: 10; Issue: 3 Linguagem: Inglês
10.15252/emmm.201708166
ISSN1757-4684
AutoresAmit U. Joshi, Nay L. Saw, Hannes Vogel, Anna D Cunnigham, Mehrdad Shamloo, Daria Mochly‐Rosen,
Tópico(s)Alzheimer's disease research and treatments
ResumoResearch Article15 January 2018Open Access Source DataTransparent process Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis Amit U Joshi Amit U Joshi orcid.org/0000-0002-2472-7672 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Nay L Saw Nay L Saw Behavioral and Functional Neuroscience Laboratory, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Hannes Vogel Hannes Vogel Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Anna D Cunnigham Anna D Cunnigham Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Mehrdad Shamloo Mehrdad Shamloo Behavioral and Functional Neuroscience Laboratory, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Daria Mochly-Rosen Corresponding Author Daria Mochly-Rosen [email protected] orcid.org/0000-0002-6691-8733 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Amit U Joshi Amit U Joshi orcid.org/0000-0002-2472-7672 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Nay L Saw Nay L Saw Behavioral and Functional Neuroscience Laboratory, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Hannes Vogel Hannes Vogel Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Anna D Cunnigham Anna D Cunnigham Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Mehrdad Shamloo Mehrdad Shamloo Behavioral and Functional Neuroscience Laboratory, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Daria Mochly-Rosen Corresponding Author Daria Mochly-Rosen [email protected] orcid.org/0000-0002-6691-8733 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Author Information Amit U Joshi1, Nay L Saw2, Hannes Vogel3, Anna D Cunnigham1, Mehrdad Shamloo2 and Daria Mochly-Rosen *,1 1Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA 2Behavioral and Functional Neuroscience Laboratory, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA 3Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA *Corresponding author. Tel: +1 650 724 8098; Fax: +1 650 723 4686; E-mail: [email protected] EMBO Mol Med (2018)10:e8166https://doi.org/10.15252/emmm.201708166 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Bioenergetic failure and oxidative stress are common pathological hallmarks of amyotrophic lateral sclerosis (ALS), but whether these could be targeted effectively for novel therapeutic intervention needs to be determined. One of the reported contributors to ALS pathology is mitochondrial dysfunction associated with excessive mitochondrial fission and fragmentation, which is predominantly mediated by Drp1 hyperactivation. Here, we determined whether inhibition of excessive fission by inhibiting Drp1/Fis1 interaction affects disease progression. We observed mitochondrial excessive fragmentation and dysfunction in several familial forms of ALS patient-derived fibroblasts as well as in cultured motor neurons expressing SOD1 mutant. In both cell models, inhibition of Drp1/Fis1 interaction by a selective peptide inhibitor, P110, led to a significant reduction in reactive oxygen species levels, and to improvement in mitochondrial structure and functions. Sustained treatment of mice expressing G93A SOD1 mutation with P110, beginning at the onset of disease symptoms at day 90, produced an improvement in motor performance and survival, suggesting that Drp1 hyperactivation may be an attractive target in the treatment of ALS patients. Synopsis Drp1 hyperactivation has been associated with neurodegenerative diseases, like amyotrophic lateral sclerosis (ALS). P110, an inhibitor of the mitochondrial fission protein Drp1, is shown to reduce the detrimental effects of mitochondrial dysfunction and ameliorate symptoms in an ALS mouse model. P110 suppresses mitochondrial dysfunction in patient-derived fibroblasts, SOD1 G93A NSC-34 cells and in ALS model mice. P110 reduces muscular mitochondrial pathology and oxidative stress in ALS model mice. P110 enhances motor activity in ALS model mice. Drugs that improve mitochondrial function, such as P110, may provide benefits for patients with motor neuron diseases that show mitochondrial defects. Introduction Amyotrophic lateral sclerosis (ALS), which clinically manifests by progressive muscle atrophy and paralysis, is a fatal neurodegenerative disease characterized by the death of upper and lower motor neurons (MN; Boillee et al, 2006). Individuals with ALS most commonly die of respiratory failure or pneumonia within 3–5 years from initial diagnosis (Pisa et al, 2016). Currently, the glutamate release inhibitor, riluzole, and recently approved free radical scavenger, edaravone, are the only medications approved by the FDA for ALS, and therefore, there remains a strong need for new treatment strategies (Lacomblez et al, 1996; Faes & Callewaert, 2011; Ittner et al, 2015; Hardiman & van den Berg, 2017). Furthermore, eligibility for edaravone was restricted to patients with a relatively short disease duration and preserved vital capacity, indicating a need for a more encompassing treatment (Hardiman & van den Berg, 2017; Maragakis, 2017; Sawada, 2017). For most patients, the underlying cause for ALS is not known (sporadic ALS), but over 100 different mutations in superoxide dismutase 1 (SOD1) account for < 20% of familial ALS forms (Cozzolino & Carri, 2012). Transactive response (TAR) DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) have also been genetically and pathologically linked to ALS; however, the underlying mechanisms by which these induce ALS pathology and the causal relationship between these events and the death of the motor neurons remain unclear (Mackenzie et al, 2010). Several studies suggest possible defects in mitochondrial dynamics in models of ALS, regardless of the causative mutation (Menzies et al, 2002; Ehinger et al, 2015; Tafuri et al, 2015; Sharma et al, 2016). SOD1 resides in the mitochondrial intermembrane space, and abnormal mitochondrial morphology and cristae ultrastructure have been observed in mutant SOD1 mice and in ALS patient samples, predominantly in the spinal cord (De Vos et al, 2007; Song, Song et al, 2013). Mutant SOD1G93A affects mitochondrial dynamics, resulting in a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria (Tafuri et al, 2015). Abnormal mitochondrial dynamics was also recently observed in skeletal muscle of the SOD1 G93A mice (Luo et al, 2013), together indicating the importance of mitochondrial dynamics in ALS. Mitochondria exist in the cells as highly dynamic entities, ranging from elaborate tubular networks to small organelles, through rapid and opposing processes of fission and fusion. Mitochondrial fission, the focus of our study, is mediated by the recruitment of Drp1, a cytosolic large GTPase, to the outer mitochondrial membrane by mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 kDa and 51 kDa (Mid49/Mid51) and through fission 1 (Fis1; Loson et al, 2013; Osellame et al, 2016). Whereas physiological fission is essential for maintaining mitochondrial quality (Shirihai et al, 2015), excessive Drp1-mediated fission causes mitochondrial fragmentation, mitochondrial membrane depolarization, increase in reactive oxygen production (ROS) and oxidative stress, and a decrease in ATP production and in other mitochondrial functions (Wu et al, 2011; Youle & van der Bliek, 2012; Babbar & Sheikh, 2013). Indeed, we found that inhibition of excessive Drp1 activity through blocking its interaction with Fis1 is protective in models of Parkinson's disease and Huntington's disease (Guo et al, 2013; Qi et al, 2013). Here, we determined whether Drp1 hyperactivation plays a role in the pathogenesis of ALS and whether inhibition of Drp1 hyperactivation through its interaction with Fis1 can reduce ALS pathology. Results ALS patient-derived fibroblasts show altered mitochondrial function and mitochondrial fragmentation associated with Drp1 hyperactivation We first determined whether mitochondrial dysfunction is evident in fibroblasts of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G) or in TDP43 (TAR DNA-binding protein 43; G289S) genes. As fibroblasts have a mainly glycolytic metabolism, they were cultured in galactose-containing medium for 48 h, to induce dependence on oxidative phosphorylation (OX-PHOS) for ATP production (Aguer et al, 2011). In fibroblasts derived from ALS patients, the mitochondrial network was fragmented as compared with fibroblasts from healthy subjects (control), with prevalence of round-shaped mitochondria or sphere-like clusters (Fig 1A). To quantify the mitochondrial structure change, we utilized automated image analysis and examined the effects of these ALS mutations on mitochondrial morphology. ALS patient-derived fibroblasts carrying any one of these three mutations showed a ~50% decrease in mitochondrial interconnectivity score (1.01 vs. 0.48 for control and ALS, respectively; P < 0.0001) and elongation scores (1.54 vs. 0.77 for control and ALS, respectively; P < 0.0001) (Fig 1B and C). We next determined if this mitochondrial fragmentation was mediated by Drp1/Fis1 interaction, using P110, a heptapeptide conjugated to TAT47–57 (TAT, for intracellular delivery) that selectively inhibits the interaction between Drp1 and Fis1, one of its adaptor proteins on the mitochondria (Qi et al, 2013). Treatment with P110 (1 μM/day for 2 days) significantly improved mitochondrial structure and improved the interconnectivity (from 0.48 to 1.32 following P110 treatment; P < 0.0001) (Fig 1A–C). Furthermore, to confirm that Fis1 is critical for the mitochondrial structural changes observed in ALS patient-derived fibroblasts, we transiently knocked down the expression Fis1 (Fig EV1A) and observed a significant recovery in mitochondrial structure as measured by previously described methods (Figs 1A and EV1B and C). Figure 1. Mitochondrial structural and functional defects in ALS patient-derived fibroblasts are mediated by Drp1/Fis1 interaction Two healthy control-derived fibroblasts and three ALS patient-derived fibroblasts, each with a different genetic form of ALS, were treated with or without P110 (1 μM/24 h) for 48 h in defined medium or Fis1 siRNA and then stained with anti-TOM20 (a marker of mitochondria, 1:500 dilution). Side panels show enlarged areas of the white boxes. Scale bar: 0.5 μm. Mitochondrial interconnectivity in healthy control fibroblasts and ALS patient-derived fibroblasts from the stained images was quantified using a macro in ImageJ. Mitochondrial elongation in healthy control fibroblasts and ALS patient-derived fibroblasts from the stained images was quantified using a macro in ImageJ. Mitochondrial membrane potential was determined using TMRM dye after 48 h in control and ALS patient-derived fibroblasts in the presence or absence of P110 (1 μM/24 h). Mitochondrial ATP levels were in control and ALS patient-derived fibroblasts in the presence or absence of P110 (1 μM/24 h). Measurement of mitochondrial ROS production using MitoSOX in ALS patient-derived fibroblasts in the presence or absence of P110 (1 μM/24 h). Measurement of total cellular ROS production in control and ALS patient-derived fibroblasts in the presence or absence of P110 (1 μM/24 h). Levels of Drp1 were examined in mitochondrial fractions by immunoblotting; VDAC was used as a loading control. Protein levels were quantified and represented as fold change of control 1. Association of Drp1 with Fis1 was analyzed following co-immunoprecipitation by Western blot analysis in pooled total lysates of patient-derived cells treated in the presence or absence of P110 (1 μM/24 h) from three independent experiments. Protein levels were quantified and presented as ratio of Drp1 to Fis1. Data information: Mean, standard deviation, and P-values are shown. Results are presented as percent of control. n = 3 performed in (H, I) duplicate, (B, C) triplicate, or (D–G) quintuplet; probability by one-way ANOVA (with Tukey's post hoc test). At least 100 cells/group were analyzed while blinded to experimental conditions in panels (B and C). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Mitochondrial dysfunction and autophagy activation in ALS patient-derived fibroblasts Representative Western blots showing siRNA-mediated reduction in total Fis1 levels. n = 3. Mitochondrial elongation in healthy control fibroblasts and ALS patient-derived fibroblasts from the stained TOM20 images was quantified using a macro in ImageJ after Fis1 siRNA treatment. n = 3 performed in duplicate. At least 100 cells/group were analyzed while blinded to experimental conditions. Mitochondrial interconnectivity in healthy control fibroblasts and ALS patient-derived fibroblasts from the stained TOM20 images was quantified using a macro in ImageJ after Fis1 siRNA; n = 3 performed in duplicate. At least 100 cells/group were analyzed while blinded to experimental conditions. Representative Western blots showing Drp1 and p62 association with mitochondrial-enriched fractions in different ALS patient-derived fibroblasts following culturing in the presence or absence of P110 (1 μM for 24 h); VDAC was used as a loading control for mitochondrial fraction; n = 3. Levels of p62 in mitochondrial fractions were measured by immunoblotting; VDAC was used as a loading control. Protein levels were quantified and presented as fold change of control 1. Results are presented as fold of control (means ± SD). n = 3 performed in triplicate; probability by one-way ANOVA (with Tukey's post hoc test). Representative Western blots showing Drp1 and Fis1 total levels in different ALS patient-derived fibroblasts following culturing in the presence or absence of P110 (1 μM for 24 h); β-actin was used as a loading control; n = 3. Drp1 association with Fis1 was examined by Western blot following co-immunoprecipitation of pooled total lysates from three independent experiments from different ALS patient-derived fibroblasts following treatment in the presence or absence of P110 (1 μM for 24 h). n = 3. Data information: Mean, standard deviation, and P-values are shown. Source data are available online for this figure. Download figure Download PowerPoint Superoxide dismutase 1 mutation caused a ~50% decrease in mitochondrial membrane potential (MMP; measured by TMRM) and in ATP production as compared to controls cells (P < 0.0001; Fig 1D and E). This mitochondria dysfunction was associated with increased oxidative stress; mitochondrial reactive oxygen species (ROS) and total ROS levels were 186% and 250% of control, respectively, (P < 0.0001) in ALS patient-derived cells relative to control cells (Fig 1F and G). These mitochondrial defects were significantly reduced by treatment with P110 (1 μM/day for 2 days; Fig 1D–G). Under physiological conditions, minimal levels of Drp1 are associated with mitochondria to maintain physiological mitochondrial fission. Drp1 recruitment from the cytosol to the mitochondrial outer membrane, a hallmark of activated mitochondrial fission (Frank et al, 2001), was ~threefold higher in ALS patient-derived fibroblasts relative to control cells, indicating Drp1 hyperactivation; this was significantly reduced by P110 treatment, to 1.9-fold relative to control cells (P = 0.001; Figs 1H and EV1D). More importantly, we assessed the specific interaction between Drp1 and Fis1 and observed that in ALS patient-derived fibroblasts, and there was a significant increase in their association, which was reduced by P110 treatment (Fig 1I and EV1G). We did not observe any significant changes in the total protein levels of either Drp1 or Fis1 proteins in these cells (Fig EV1F). Previously, p62 (also known as SQSTM1), a protein implicated in protein aggregate formation and stalled autophagy, was shown to accumulate as the disease progresses in the G93A mouse spinal cord (Gal et al, 2007). Since p62 recruitment and accumulation at mitochondria have been associated with increased ROS production as well as mitochondrial membrane depolarization (Narendra et al, 2010), we next assessed the levels of p62 in the mitochondrial fraction in these patient-derived cells. Indicative of aberrant autophagy stall, we observed a 3.3-fold increased mitochondrial recruitment of p62, which was reduced by P110 treatment to 1.9-fold relative to controls (Fig EV1D and E), indicting an interplay between mitochondrial fragmentation via Drp1/Fis1 interaction and autophagy balance. NSC-34 cells expressing mutant SOD1 G93A exhibit mitochondrial dysfunction To further dissect the pathways involved in P110-induced benefits observed in ALS patient-derived fibroblasts, we focused on motor neurons expressing G93A SOD1 mutation using two stressors: serum starvation for 72 h or H2O2 injury. NSC34, a motor neuron-like cell line expressing human G93A SOD1, compared to cells expressing the human WTSOD1, has a significant increase in cytosolic oxidative stress. SOD1 G93A cells showed an approximately twofold increase in mitochondrial ROS (Fig 2A, P = 0.001), which was reduced when SOD1 G93A cells were treated with P110 in a dose-dependent manner (Figs 2A and EV2A; P = 0.003). The ability to concentrate the TMRM probe in mitochondria was decreased in SOD1 G93A (indicative of loss mitochondrial membrane potential; P = 0.003) that was also improved by P110 treatment in a dose-dependent manner (Figs 2A and EV2A). A causal role for endogenous nitric oxide (NO) in motor neurons and apoptosis expressing mutant SOD1 has been reported (Lee et al, 2009). As expected, we also observed a significant increase in NO levels in SOD G93A cells (under serum starvation as an additional stressor; P = 0.0395), which were significantly reduced by P110 treatment; limiting mitochondrial dysfunction was sufficient to reduce NO levels from 198% in hSOD1 G93A without to 145% with P110 (P = 0.044; Fig EV2A). This was associated with a twofold increase in cell necrosis (measured by LDH release) in mutant SOD1 cells, which was significantly reduced by P110 treatment (Fig EV2A; P = 0.0014). Similar effect was observed following acute H2O2 treatment instead of serum starvation (Fig EV2B). Figure 2. Expression of SOD1 G93A mutant in NSC-34 cells induces cellular stress in a Drp1-dependent manner Mitochondria-specific ROS levels (using indicator MitoSOX), and mitochondrial integrity (using mitochondrial membrane potential TMRM) in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells cultured under serum starvation condition in the presence or absence of P110 (0.25, 0.5, 1, 2 μM/24 h). Results are presented as percent of MOCK (empty vector). Levels of Drp1 were determined in mitochondrial fractions by immunoblotting in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells cultured under serum starvation condition in the presence or absence of P110 (0.25, 0.5, 1 μM/24 h); VDAC, a mitochondrial membrane protein, was used as a loading control. Protein levels were quantified and presented as fold change of hSOD1-WT. Levels of Drp1 phosphorylation were determined in mitochondrial fractions by immunoblotting using anti-phosphorylated-S616-Drp1 or anti-phosphorylated-S637-Drp1 antibodies in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells under serum starvation in the presence or absence of P110 (1 μM/24 h); β-actin was used as a loading control. Protein levels were quantified and presented as fold change of hSOD1-WT. Levels of Parkin and LC3BII autophagy measures were determined in mitochondrial fractions by immunoblotting in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells as in A, cultured in the presence or absence of P110 (1 μM/24 h); VDAC was used as a loading control. Protein levels were quantified and presented as fold change of MOCK (empty vector). Chymotrypsin-like activity was measured using fluorogenic substrate; Suc-LLVY-AMC to measure proteasome activity in homogenates of hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells as above in the presence or absence of P110 (1 μM/24 h). Activity levels were quantified and presented as fold change of MOCK (empty vector). Levels of phosphorylated-eIF2α, XBP1, and ATF-6 (measures of ER stress) in total fractions were measured by immunoblotting in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells, cultured as above, in the presence or absence of P110 (1 μM/24 h); β-actin was used as a loading controls. Protein levels were quantified and presented as fold change of hSOD-1 WT. Data information: Mean, standard deviation, and P-values are shown. n = 3 performed in (B, C, D, F) duplicate or (A, E) quintuplet; probability by one-way ANOVA (with Tukey's post hoc test). Source data are available online for this figure. Source Data for Figure 2 [emmm201708166-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Mitochondrial dysfunction in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells Mitochondrial health using mitochondrial membrane potential (TMRM) and mitochondria-specific ROS indicator (MitoSOX), nitric oxide levels (Griess reagent), and cell death (using LDH assay) in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells cultured in the presence or absence of P110 (1 μM for 24 h). Results are presented as percent or fold of MOCK (empty vector); n = 3 performed in quintuplet; probability by one-way ANOVA (with Tukey's post hoc test). Scale bar: 50 μm. Cell death (using LDH assay), mitochondrial-specific ROS production (MitoSOX), mitochondrial membrane potential (using TMRM), and nitric oxide levels in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells were determined following H2O2 injury in the presence or absence of P110 (1 μM/24 h); n = 3 performed in quintuplet; probability by one-way ANOVA (with Tukey's post hoc test). Co-immunoprecipitation of Drp1 with Fis1, Mff, Mid49, or Mid51 was analyzed in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells, cultured in the presence or absence of P110 (1 μM for 24 h). n = 2 performed in duplicates (pooled). Levels of Drp1 were determined in mitochondrial fractions by immunoblotting in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 cells as above, cultured in the presence or absence of P110 (0.25, 0.5, 1 μM for 24 h); VDAC, a mitochondrial membrane protein, was used as a loading control, whereas enolase, a cytosolic marker, was used to assess the mitochondrial sample purity. n = 2 performed in duplicates (pooled). Data information: Mean, standard deviation, and P-values are shown. Results are presented as percent or fold of MOCK (empty vector). Source data are available online for this figure. Download figure Download PowerPoint When cultured without serum for 72 h, Drp1 association with the Fis1 and not the other adaptors (Mff, Mid49, and Mid51) increased significantly, which was reduced by P110 treatment (Fig EV2C). Association of Drp1 with mitochondria was 2.6-fold higher in NSC-34 cells expressing SOD1 G93A mutant relative to control cells (P = 0.0008; Figs 2B and EV2D), indicating Drp1 hyperactivation, which was significantly reduced by P110 treatment (P = 0.0008). P110 treatment also significantly blocked the subsequent release of cytochrome c from the mitochondria (P = 0.0397; Fig EV3A), reduced the accumulation of active Bax on the mitochondria (P = 0.0065), and improved decreased Bcl-2 levels on the mitochondria in NSC-34 cells expressing SOD1 G93A mutant vs. control cells (P = 0.0002). Thus, P110 treatment significantly inhibited the initiation of apoptosis in these mutant cells. Click here to expand this figure. Figure EV3. Increases apoptotic markers in hSOD1-G93A-expressing NSC-34 differentiated cells relative to hSOD1-WT Representative Western blots showing levels of cytochrome c, BAX, and Bcl-2 were examined in mitochondrial fractions by immunoblotting in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells under serum starvation in the presence or absence of P110 (1 μM/24 h); VDAC. Protein levels were quantified and presented as fold change of hSOD-1 WT. Representative Western blots showing levels of autophagy-associated proteins; Parkin and LC3BII examined in mitochondrial fractions. Representative Western blots showing phosphorylated-JNK, total JNK, p62, and LC3BII total levels in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells under serum starvation in the presence or absence of P110 (1 μM/24 h); VDAC and β-actin were used as a loading control for mitochondrial fraction (top panels) and total fraction (lower panels), respectively. Protein levels were quantified and presented as fold change of hSOD-1 WT. Representative Western blots showing levels of ER stress-associated protein, GRP78, CHOP, in hSOD1-WT- and hSOD1-G93A-expressing NSC-34 differentiated cells under serum starvation in the presence or absence of P110 (1 μM/24 h); β-actin was used as a loading control. Protein levels were quantified and presented as fold change of hSOD-1 WT. Data information: Mean, standard deviation, and P-values are shown. n = 3 performed in (A) duplicates or (B, C) quintuplet; probability by one-way ANOVA (with Tukey's post hoc test). Source data are available online for this figure. Download figure Download PowerPoint Phosphorylation of Drp1 at Ser-616 by cyclin-dependent kinase (CDK) 1/cyclin B or CDK5 promotes mitochondrial fission, whereas dephosphorylation of Drp1 at Ser-637 by calcineurin facilitates its translocation to mitochondria and subsequently increases mitochondrial fission (Liesa et al, 2009; Campello & Scorrano, 2010). Therefore, a balance between Drp1 Ser-616/Ser-637 phosphorylation ratio reflects Drp1 activity. Western blot analysis of total protein lysates showed a significant increase in Drp1 phosphorylation at Ser-616 combined with a decrease in phosphorylation at Ser-637 in NSC-34 SOD1 G93A cells (Fig 2C, P = 0.0002). These results indicate that Drp1 hyperactivation and phosphorylation occur in NSC-34 cells expressing SOD1 G93A and that treatment with P110 inhibits this hyperactivation (P = 0.0012). The ubiquitin-proteasomal system, important for maintaining protein quality control, is also compromised in experimental models of familial ALS (Cheroni et al, 2009; Dantuma & Bott, 2014; Scotter et al, 2014), and increased levels of autophagy/mitophagy markers, LC3BII and p62, have been reported in ALS models (Soo et al, 2015; Goode et al, 2016; Oakes et al, 2017). Isolated mitochondria from NSC-34 SOD1 G93A cells also had higher levels of LC3BII (P = 0.0005) and p62 (P = 0.0073) and that P110 treatment significantly reduced stalled mitophagy (Fig 2D; P = 0.0146 & P = 0.0216, respectively). Furthermore, increased c-Jun N-terminal kinase (JNK) phosphorylation, which indicates increased cellular stress, and increased LC3BII conversion and p62-enhanced accumulation in total lysates, which demonstrate altered autophagic flux, were all significantly normalized by P110 treatment in SOD1 G93A cells (Fig EV3B). Mutant SOD1 is degraded by the proteasome and partial inhibition of proteasome activity leads to the formation of large SOD1-containing aggregates, which is thought to contribute to neuropathology (Hyun et al, 2003). Recent report indicates that the levels of proteasomal 20S constitutive catalytic subunits are significantly reduced in the spinal cord of SOD1G93A mice at an advanced stage of the disease (Kabashi et al, 2012). Similarly, we observed a decreased chymotrypsin-like proteasomal activity in NSC-34 SOD1 G93A cells (P = 0.0087) that was restored by P110 treatment (P = 0.0065; Fig 2E). Since activated JNK is one of the mediators of ER stress-induced apoptosis (Szegezdi et al, 2006), we also determined the levels of markers of ER stress, XBP1, and ATF6α and the extent of eIF2α phosphorylation, in SOD1G93A NSC34 cells. The levels of these ER stress markers in the SOD mutant cells, which were higher than in WT cells, were significantly reduced following P110 treatment, thus indicating a functional connection between Drp1 hyperactivation and ER stress (Figs 2F and EV3C). Other ER stress markers, GRP78 and CHOP, increased by serum starvation in G93A expressing in motor neuron-like cells as compared to the WT (Fig EV3C), were significantly normalized by P110 t
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