Mitophagy regulates integrity of mitochondria at synapses and is critical for synaptic maintenance
2020; Springer Nature; Volume: 21; Issue: 9 Linguagem: Inglês
10.15252/embr.201949801
ISSN1469-3178
AutoresSinsuk Han, Yu Young Jeong, Preethi Sheshadri, Xiao Su, Qian Cai,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle6 July 2020free access Transparent process Mitophagy regulates integrity of mitochondria at synapses and is critical for synaptic maintenance Sinsuk Han Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Yu Young Jeong orcid.org/0000-0001-6786-9980 Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Preethi Sheshadri Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Xiao Su Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Qian Cai Corresponding Author [email protected] orcid.org/0000-0001-8525-2749 Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Sinsuk Han Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Yu Young Jeong orcid.org/0000-0001-6786-9980 Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Preethi Sheshadri Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Xiao Su Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Qian Cai Corresponding Author [email protected] orcid.org/0000-0001-8525-2749 Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Author Information Sinsuk Han1,‡, Yu Young Jeong1,‡, Preethi Sheshadri1, Xiao Su1 and Qian Cai *,1 1Division of Life Science, Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, NJ, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 848 445 1633; E-mail: [email protected] EMBO Rep (2020)21:e49801https://doi.org/10.15252/embr.201949801 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 Synaptic mitochondria are particularly vulnerable to physiological insults, and defects in synaptic mitochondria are linked to early pathophysiology of Alzheimer's disease (AD). Mitophagy, a cargo-specific autophagy for elimination of dysfunctional mitochondria, constitutes a key quality control mechanism. However, how mitophagy ensures synaptic mitochondrial integrity remains largely unknown. Here, we reveal Rheb and Snapin as key players regulating mitochondrial homeostasis at synapses. Rheb initiates mitophagy to target damaged mitochondria for autophagy, whereas dynein–Snapin-mediated retrograde transport promotes clearance of mitophagosomes from synaptic terminals. We demonstrate that synaptic accumulation of mitophagosomes is a feature in AD-related mutant hAPP mouse brains, which is attributed to increased mitophagy initiation coupled with impaired removal of mitophagosomes from AD synapses due to defective retrograde transport. Furthermore, while deficiency in dynein–Snapin-mediated retrograde transport recapitulates synaptic mitophagy stress and induces synaptic degeneration, elevated Snapin expression attenuates mitochondrial defects and ameliorates synapse loss in AD mouse brains. Taken together, our study provides new insights into mitophagy regulation of synaptic mitochondrial integrity, establishing a foundation for mitigating AD-associated mitochondria deficits and synaptic damage through mitophagy enhancement. Synopsis The integrity of synaptic mitochondria is maintained by a coordination of Rheb-dependent mitophagy and dynein-Snapin-mediated retrograde transport. Rheb targets damaged mitochondria for autophagy in distal axons, whereas dynein-Snapin-mediated retrograde transport promotes removal of mitophagosomes from synaptic terminals. Increased mitophagy initiation coupled with impaired retrograde transport of mitophagosomes induces mitophagy stress at Alzheimer's disease (AD) synapses. Elevated Snapin expression rescues such mitophagy failure and attenuates synaptic mitochondrial deficits, thereby ameliorating AD-linked synaptic degeneration. Introduction Neurons have high and continuous energy demands in order to maintain their proper function. Mitochondria are the main cellular energy powerhouses, supplying most of ATP by oxidative phosphorylation (OXPHOS), which is required to fuel critical neuronal functions (Sheng & Cai, 2012; Cai & Tammineni, 2016, 2017; Chamberlain & Sheng, 2019). Mitochondria also enact essential reactions of metabolism and Ca2+ signaling, as well as regulate cellular life/death decisions. Aged and malfunctioning mitochondria are defective in energy production and Ca2+ buffering, and also release reactive oxygen species (ROS) that are harmful at high concentrations. These defects compromise support in synaptic activities (Reddy et al, 2010; Reddy, 2011; Sheng & Cai, 2012; Cai & Tammineni, 2017). Thus, it is not unexpected that volumes of evidence suggest that mitochondrial dysfunction underlies cognitive impairment in neuronal aging and is one of the most notable hallmarks of age-related neurodegenerative diseases. Importantly, the earliest features of the onset of the highly prevalent Alzheimer's disease (AD) have been linked to mitochondrial pathology—abnormal accumulation of damaged mitochondria (Gibson & Shi, 2010; Swerdlow et al, 2010; Cai & Tammineni, 2016; Reddy & Oliver, 2019). Mitochondrial quality control, then, emerges as a central problem in the most common neurodegenerative disorders and is a clear target point for early interference in disease. Mitophagy, a specialized form of autophagy, constitutes a key mitochondrial quality control mechanism involving sequestration of damaged mitochondria into autophagosomes for subsequent degradation within lysosomes (Youle & Narendra, 2011; Sheng & Cai, 2012; Cai & Tammineni, 2016; Pickles et al, 2018). Even though some unique features of mitophagy in neurons have been described, mechanistic understanding of neuronal mitophagy and its link to pathological conditions remains very limited. Autophagocytosis of mitochondria in neurons was shown to be a feature in AD patient brains (Moreira et al, 2007a,b). Mitophagic abnormalities in AD have been demonstrated in a number of recent studies (Ye et al, 2015; Du et al, 2017; Manczak et al, 2018; Reddy et al, 2018; Cummins et al, 2019; Fang et al, 2019). Moreover, mitophagy stimulation was shown to abolish AD pathology and reverse memory impairment in AD models (Du et al, 2017; Fang et al, 2019). Therefore, there is an urgent need to investigate the detailed interaction between mitophagy deregulation and AD-linked pathological processes. Healthy mitochondria must function at synapses for neurotransmission. It has been well established that mitochondria-mediated ATP supply and Ca2+ buffering sustain various essential functions at synaptic sites (Manji et al, 2012; Sheng & Cai, 2012). However, the mechanism underlying mitochondrial maintenance at synaptic terminals is understudied in neurobiology. A major gap in our understanding is whether mitophagy serves as a key player to ensure mitochondrial integrity and function at synapses, crucial for the support of synaptic activities. Early deficit in synaptic mitochondria has been implicated in the development of synaptic pathology in AD (Reddy & Beal, 2008; Du et al, 2010, 2012; Reddy, 2011; Cai & Tammineni, 2017; Guo et al, 2017). Synaptic mitochondria are more susceptible to amyloid β (Aβ)-induced damage and display early and significant accumulation of Aβ in AD brains before extensive extracellular Aβ deposition (Reddy & Beal, 2008; Du et al, 2012; Cai & Tammineni, 2017). Early-impacted mitochondria underwent declines in respiration, complex IV activity, and Ca2+ handling capacity, as well as elevated ROS production and enhanced probability of mitochondrial permeability transition pore (MPTP) (Reddy & Beal, 2008; Reddy et al, 2010; Reddy, 2011; Du et al, 2012; Cai & Tammineni, 2017; Tonnies & Trushina, 2017). This raises an important question as to whether synaptic mitochondrial deficit is attributed to impaired mitochondrial maintenance as a result of defects in removal of damaged mitochondria from AD synapses. In the current study, we reveal that small GTPase Ras homolog enriched in brains (Rheb) initiates mitophagy and coordinates with dynein–Snapin-mediated retrograde transport, thus regulating the integrity of synaptic mitochondria. Rheb facilitates autophagic recruitment of damaged mitochondria to form mitophagosomes in distal axons, whereas dynein–Snapin-mediated retrograde transport removes nascent mitophagosomes and thus reduces mitochondrial stress at synaptic terminals. Furthermore, our studies uncover that AD-related mutant hAPP mouse brains exhibit robust presynaptic retention of mitophagosomes. Such a defect is caused by increased mitophagy initiation and impaired retrograde transport of mitophagosomes. Ablation of snapin in mouse brains mimics AD-associated synaptic mitophagy stress accompanied by synaptic degeneration. In contrast, Snapin-enhanced retrograde transport attenuates synaptic mitochondrial defects through promoting removal of axonal mitophagosomes, thereby ameliorating synapse loss in AD mouse brains. Therefore, our findings provide new insights into the involvement of mitophagy failure in synaptic mitochondrial deficits and thus advance our understanding of the mechanisms underlying AD-linked synaptic deterioration, one of the earliest pathologies in stricken neurons. Results Rheb initiates mitophagy within axons upon mitochondrial damage We determined whether mitophagy is critical for maintaining mitochondrial integrity in the axons of neurons. Rheb was previously reported to be associated with mitochondria and promote autophagic engulfment of mitochondria in HeLa and muscle cells (Melser et al, 2013, 2015). We first examined whether Rheb is involved in autophagic removal of damaged mitochondria within axons. We conducted time-lapse imaging in live mature cortical neurons treated with CCCP, a mitochondrial membrane potential (Δψm) uncoupler. Under basal conditions, Rheb is mainly present in the cytoplasm of axons and displays limited association with mitochondria. Strikingly, CCCP treatment induces robust Rheb localization to depolarized mitochondria. Rheb-associated mitochondria exhibit dominant retrograde movement along axons (anterograde, 2.51% ± 0.82%; retrograde, 29.88% ± 2.37%) (Fig 1A–C). Time-lapse images have shown that Rheb co-localizes and co-migrates with mitochondria along axons treated with CCCP. Interestingly, Rheb-tagged mitochondria exhibit high motility and move exclusively in a retrograde direction (Fig 1A and B). Consistent with our previous studies (Cai et al, 2012b; Ye et al, 2015; Lin et al, 2017), CCCP treatment remarkably reduces anterograde transport, but increases retrograde transport, of mitochondria along axons (anterograde, DMSO: 24.51% ± 2.25%; CCCP: 7.66% ± 1.42%; P < 0.001; retrograde, DMSO: 13.82% ± 1.23%; CCCP: 25.72% ± 1.65%; P < 0.001) (Fig EV1A and B). It is known that Parkin-mediated mitophagy induction leads to decreased anterograde transport of mitochondria. This is caused by Parkin-triggered and ubiquitin–proteasome system (UPS)-mediated degradation of mitochondrial Rho GTPase (Miro), a component of the adaptor–motor complex essential for KIF5 motors to drive anterograde transport of mitochondria in axons (Chan & Chan, 2011; Wang et al, 2011; Liu et al, 2012; Bingol et al, 2014; Birsa et al, 2014). We hypothesize that enhanced retrograde transport could be attributed to activation of Rheb-dependent mitophagy. Figure 1. Rheb initiates mitophagy within axons upon mitochondrial damage A–C. Representative dual-channel kymographs (A) and quantitative analysis (C) showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (Δψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons was quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm). Time-lapse images (B) showing co-localization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5–7 DIV and incubated with DMSO or 10 μM CCCP for 24 h prior to imaging at 10–12 DIV. D, E. Quantitative analysis (D) and representative images (E) showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation-deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating Δψm. Mitochondria associated with Rheb were marked by white arrows. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively (D). F–H. Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon Δψm dissipation. Rheb-associated AVs move mainly in a retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively (G, H). Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (D, G) from greater than four experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t-test: ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Rheb mediates mitophagy initiation in axons that requires Nix A, B. Representative kymographs (A) and quantitative analysis (B) showing that Δψm depolarization increases retrograde transport but reduces anterograde transport of mitochondria along axons. The relative motility of mitochondria in axons treated with DMSO or CCCP was quantified (B). Data were quantified from the total number of neurons (n) indicated in parentheses (B) from more than four experiments. C, D. Quantitative analysis (C) and representative kymographs (D) showing robust recruitment of Rheb to depolarized mitochondria within axons after treatment with antimycin A (AA). The percentage of white arrow-indicated mitochondria that were targeted by Rheb in the presence or absence of AA was quantified (C). E. Representative triple-channel images showing that Rheb-associated mitochondria are targeted to autophagy after CCCP treatment. Rheb-tagged mitochondria (marked by arrows) are positive for LC3, whereas LC3 cannot be detected on mitochondria without Rheb association. F. Rab7, a late endosome/amphisome marker, is associated with Rheb-targeted mitophagosomes as indicated by white arrows within axons under CCCP treatment. G, H. Representative images (G) and quantitative analysis (H) showing that Nix RNAi reduces mitochondrial recruitment of Rheb in axons upon CCCP-induced Δψm dissipation. The percentage of mitochondria targeted by Rheb (white arrows) in axons expressing scrambled shRNA or Nix shRNA was quantified (H). I, J. Impaired biogenesis of mitophagosomes within CCCP-treated axons expressing Nix shRNA. Note that Rheb-associated mitophagosomes are markedly decreased following Nix RNAi. The number of Rheb-targeted autophagic vacuoles (AVs) (Rheb-AVs) labeled by white arrows in axons expressing scrambled shRNA or Nix shRNA was quantified (J). Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (B) or on the top of the bars (C, H, J) from at least four experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t-test: ***P < 0.001. Download figure Download PowerPoint Δψm dissipation leads to increased percentage of mitochondria targeted by Rheb (DMSO: 14.53% ± 1.21%; CCCP: 35.24% ± 1.91%, P < 0.001) (Fig 1D and E). Alternatively, we examined the effects of antimycin A (AA), a mitochondrial electron transport chain complex III inhibitor. Our previous studies have shown that a low concentration (1 μM) of AA in neurons depolarizes Δψm, which is coupled with decreased mitochondrial respiration (Cai et al, 2012b; Lin et al, 2017). In the current study, AA treatment leads to a significant increase in Rheb localization to depolarized mitochondria within axons (40.59% ± 1.98%) (Fig EV1C and D), a finding consistent with our observation in CCCP-treated axons (Fig 1D and E). Furthermore, we did not observe enhanced mitochondrial association with Rheb-SSVM (Rheb-SSVM: 3.46% ± 0.87%, P < 0.001), a farnesylation-deficient mutant of Rheb (Basso et al, 2005). Thus, our data suggest that Rheb is recruited to depolarized mitochondria in axons, which is dependent on Rheb farnesylation. We have provided additional evidence showing that Rheb is associated with autophagic vacuoles (AVs), and CCCP treatment significantly increases the number of Rheb-associated AVs (Rheb-AVs) within axons (DMSO: 2.46 ± 0.35; CCCP: 6.86 ± 0.53; P < 0.001) (Fig 1F–H). Similar to AVs, these Rheb-AVs exhibit robust retrograde movement along axons. Our triple-channel image data further demonstrated that Rheb-associated AVs in axons contain engulfed mitochondria, suggesting that they are mitophagosomes in nature (Fig EV1E). In contrast, the same treatment fails to increase the number of Rheb-SSVM-associated AVs (1.53 ± 0.19; Fig 1F and G), suggesting that Rheb localization to mitochondria is an essential step to initiate mitophagy in axons. Our data are consistent with previous studies showing that autophagosomes containing mitochondria move in a retrograde direction along axons (Maday et al, 2012; Wong & Holzbaur, 2014). This supports the possibility that mitophagy plays a role in regulating proper turnover of axonal mitochondria. Our previous studies reported that nascent autophagosomes recruit dynein–Snapin, a motor–adaptor complex, to gain retrograde transport motility through fusion with late endosomes (LEs) to form amphisomes. Such a mechanism facilitates removal of AVs from distal axons for lysosomal degradation in the soma (Cheng et al, 2015a,b). Interestingly, Rheb-associated mitophagosomes are mostly positive for Rab7, a LE/amphisome marker (Fig EV1F). These data suggest that newly generated mitophagosomes fuse rapidly with LEs to form amphi-mitophagosomes, through which nascent mitophagosomes are loaded with dynein–Snapin transport machinery to enable retrograde transport motility. This result allows us to propose that Rheb-dependent mitophagy initiation coordinates with dynein–Snapin-mediated retrograde transport to remove damaged mitochondria from axonal terminals. Rheb-mediated mitophagy in axons requires Nix but not Parkin Next, we examined whether Rheb-associated mitophagy requires Nix, an outer mitochondrial membrane protein (Youle & Narendra, 2011; Cai & Jeong, 2020). Utilizing Nix shRNA that was previously described (Fei et al, 2004; Melser et al, 2013), we found that Nix RNAi results in markedly reduced localization of Rheb to axonal mitochondria upon Δψm dissipation (Scrambled shRNA: 38.83% ± 1.34%; Nix shRNA: 23.31% ± 1.26%; P < 0.001; Fig EV1G and H). Furthermore, Rheb-mediated autophagic targeting of mitochondria is impaired in axons expressing Nix shRNA, as evidenced by decreased number of Rheb-AVs (Scrambled shRNA: 8.99 ± 0.61; Nix shRNA: 1.92 ± 0.16; P < 0.001) (Fig EV1I and J). Thus, our observations collectively indicate that Nix is involved in Rheb-mediated mitophagy initiation through facilitating Rheb association with damaged mitochondria to promote mitochondrial targeting for autophagy. We next sought to address whether enhanced mitochondrial recruitment of Rheb promotes mitophagosome formation in axons. Strikingly, following dissipation of Δψm, axons expressing Rheb show a significant increase in the number of AVs and Mito-AVs/mitophagosomes (Ctrl: AVs: 7.41 ± 0.69; Mito-AVs: 3.51 ± 0.27; Rheb: AVs: 12.88 ± 0.45, P < 0.001; Mito-AVs: 6.77 ± 0.41, P < 0.001), accompanied by increased percentage of mitochondria within AVs (Ctrl: 21.99% ± 1.88%; Rheb: 42.76% ± 2.86%; P < 0.001; Fig 2A and B). Moreover, these mitophagosomes move exclusively in a retrograde direction, which is markedly enhanced in Rheb-expressed axons (Ctrl: 21.14% ± 3.52%; Rheb: 35.54% ± 2.90%; P < 0.001) (Fig 2A and C). This result supports our hypothesis that Rheb regulates mitophagosome biogenesis to remove damaged mitochondria from distal axons through retrograde transport. Figure 2. Rheb-mediated mitophagy in axons requires Nix but not Parkin A–C. Representative dual-channel kymographs (A) and quantitative analysis (B, C) showing that overexpression of Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified, respectively (B, C). D, E. Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E). F, G. Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon Δψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 h and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats. H, I. Quantitative analysis (H) and representative images (I) showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon Δψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false-colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level (I). Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H). Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (B, E) or on the top of bars (H) from at least four experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t-test: ***P < 0.001; **P < 0.01. Download figure Download PowerPoint To gain mechanistic insights into Rheb-mediated mitophagy, we performed a series of co-immunoprecipitation assays. Rheb forms a complex with LC3-II in cultured cortical neurons in the presence of CCCP (Fig EV2A). In transfected HeLa cells, the Rheb-LC3-II complex was detected upon treatment with CCCP, but not DMSO control. Moreover, Nix is associated with the Rheb-LC3-II complex (Fig EV2B and C). Our data suggest that, upon Δψm dissipation, Rheb promotes mitophagy by forming a complex with Nix and LC3-II. Given that Parkin is not present in HeLa cells, this result indicates that such a mechanism is independent of the Parkin pathway. We further conducted imaging studies to address whether Rheb and Nix, but not Parkin, are important for targeting damaged mitochondria for autophagy in axons. After CCCP treatment, the number of mitophagosomes and the percentage of mitochondria within AVs are remarkably reduced in axons expressing Rheb shRNA and Nix shRNA, but not Parkin shRNA and scrambled shRNA (Fig 2D and E). We and others previously demonstrated the knockdown efficiency of Parkin shRNA (Cai et al, 2012b; Rojansky et al, 2016). Together with our biochemical results, these findings collectively suggest that initiation of mitophagy in axons requires Rheb and Nix, but not Parkin. Click here to expand this figure. Figure EV2. Rheb is involved in mitophagy but not autophagy in axons A. Rheb-LC3-II complex was immunoprecipitated by the antibody against Rheb, but not normal IgG control, from the lysates of cortical neurons treated with CCCP. B. Rheb-LC3-II complex was detected in the lysates of transfected HeLa cells treated with CCCP, but not DMSO control. C. Immunoprecipitation of Myc-Rheb with LC3-II and HA-Nix from HeLa cell lysates with an anti-Myc antibody or normal IgG as a control in the presence of CCCP. D–G. Representative images (E, F) and quantitative analysis (D, G) showing that Rheb RNAi has no detectable effects on trehalose (Tre)-induced autophagy in axons. The numbers of axonal autophagosomes (E) and mitophagosomes (white arrows in F) in neurons expressing scrambled shRNA or Rheb shRNA were quantified in the presence of DMSO or trehalose, respectively (D, G). H. Rheb is enriched in cytoplasm under basal conditions and is not associated with either autophagosomes or mitochondria in axons upon trehalose treatment. I, J. Representative images (I) and quantitative analysis (J) showing that Rheb is not required for Parkin-dependent mitophagy in the soma upon Δψm dissipation. The percentage of total neurons displaying Parkin localization to mitochondria indicated by white arrows in the soma of CCCP-treated neurons expressing scrambled shRNA or Rheb shRNA was quantified (J). K, L. Quantitative analysis (K) and representative blots (L) showing knockdown efficiency of Rheb shRNA (small hairpin RNA, (shRNA) #1 and shRNA #2), and silencing effects on mitochondrial degradation respective to control (scrambled shRNA) in HEK293 cells. Data were quantified from four independent repeats. Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (D, G, J) from at least four independent experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t-test: ***P < 0.001; *P < 0.05. Download figure Download PowerPoint We next determined whether the role of Rheb is specific to mitophagy, but not other forms of autophagy. We and others have shown that trehalose induces non-selective autophagy in neurons (Kruger et al, 2012; Feng et al, 2017). Consistently, trehalose treatment markedly increases the number of AVs within axons, compared to that of control axons treated with (DMSO: 0.25 ± 0.13; Tre: 9.82 ± 0.75, P < 0.001) (Fig EV2D and E). Importantly, Rheb RNAi has no detectable impact on trehalose-induced autophagy, as evidenced by the unaltered number of axonal AVs (9.74 ± 0.58; P > 0.05). There is no significant difference in the number of mitophagosomes within axons expressing scrambled shRNA and Rheb shRNA (Fig EV2F and G). In addition, Rheb is not associated with autophagosomes and mitochondria in axons upon autophagy induction by trehalose (Fig EV2H). Thus, these results suggest that Rheb plays a critical role in mitophagy, but not in non-selective autophagy. We and others have demonstrated that Parkin-mediated mitophagy primarily occurs in the soma of neurons, where degradative lysosomes are highly enriched (Cai et al, 2010, 2012b; Devireddy et al, 2015; Xie et al, 2015; Ye et al, 2015; Maday & Holzbaur, 2016; Sung et al, 2016; Lin et al, 2017; Tammineni et al, 2017a; Cheng et al, 2018; Lee et al, 2019). We next ex
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